Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user

on 04 June 2024
 NeoReviews
ARTICLES Editor-in-Chief: Dara Brodsky, Boston, MA
Associate Editor: Josef Neu, Gainesville, FL
796 Primary Mitochondrial Disorders in the Neonate Associate Editor, CME: Henry C. Lee, Palo Alto, CA
Associate Editor, NeoQuest: Rita Dadiz, Rochester, NY
Rodrigo Tzovenos Starosta, Marwan Shinawi Associate Editor, Perspectives: Mamta Fuloria, Bronx, NY
Associate Editor, Maternal-Fetal Medicine: Brett Young, Boston, MA
813 Congenital Disorders of Red Blood Cells Associate Editor, Video Corner: Akshaya Vachharajani, Columbia, MO
Rhucha Joshi, Erin Myers, Artemiy Kokhanov Assistant Editor, CME: Santina A. Zanelli, Charlottesville, VA
Early Career Physician:
Theodore De Beritto, Los Angeles, CA
829 Evaluating Genetic Disorders in the Neonate: The Role of •
••
•••
•
Editorial Fellow:

•
•
Exome Sequencing in the NICU
T. Niroshi Senaratne, Sulagna C. Saitta
Colby Day, Jacksonville, FL
EDITORIAL BOARD
MOC

•
•
• •
••
•••
Carl Backes, Columbus, OH
Anisha Bhatia, Canton, OH
Alison Chu, Los Angeles, CA
INDEX OF SUSPICION IN THE NURSERY Corinne L. Leach, Buffalo, NY
Krithika Lingappan, Houston, TX
841 Increased Nuchal Translucency Without Aneuploidy in a Sai Mukthapuram, Cincinnati, OH
Zeynep Salih, Carmel, IN
Fetus: Abnormal Postnatal Radiograph and a Novel Mutation Elizabeth Schulz, Bethesda, MD
Niraj Kumar Dipak, Mamta Muranjan, Shilpa Pandya, Thomas E. Wiswell, Honolulu, HI
Clyde Wright, Aurora, CO
Manjusha Bhikurao Naik
Managing Editor: Heidi Willis
Manager, Journal Publishing: Josh Sinason
845 Jaundice in a 10-hour-old Infant Medical Copy Editor: Beena Rao
Elizabeth A. Hagan, Abdul-Azeez Abdullahi, Issam Halabi,
Publisher: American Academy of Pediatrics
Akshaya Vachharajani President: Moira A. Szilagyi
Chief Executive Officer/Executive Vice President:
849 Term Neonate with Hematochezia Mark Del Monte
Chief Product and Services Officer/SVP Membership, Marketing,
Mariha Khan, Thomas G. Boyce, Aditya Joshi and Publishing:
Mary Lou White
Vice President, Publishing: Mark Grimes
Director, Journal Publishing: Joseph Puskarz
MATERNAL-FETAL CASE STUDIES NeoReviews™
(ISSN 1526-9906) is owned and controlled by the American Academy of Pediatrics. It is
852 Neonatal Implications of Maternal Thrombocytopenia published monthly by the American Academy of Pediatrics, 345 Park Blvd., Itasca, IL 60143.
Statements and opinions expressed in NeoReviews™ are those of the authors and not
during Pregnancy necessarily those of the American Academy of Pediatrics or its Committees. Recommendations
Arlin Delgado, Stephanie Ros included in this publication do not indicate an exclusive course of treatment or serve as a
standard of medical care. Subscription price for NeoReviews™ for 2022: AAP/CPS Member, $150;
AAP In-Training/National A_liate Member, $115; Nonmember, $190; Nonmember In-Training /
Allied Health, $135;AAP Perinatal Section Member, $135.
Institutions call for pricing (866-843-2271).
VISUAL DIAGNOSIS © AMERICAN ACADEMY OF PEDIATRICS, 2022. All rights reserved. Printed in USA. No part
may be duplicated or reproduced without permission of the American Academy of
856 Early Term Infant with Prenatal Brain Abnormalities and Pediatrics.
Decreased Oral Intake POSTMASTER: Send address changes to NEOREVIEWS™, American Academy
of Pediatrics, Member and Customer Care, 345 Park Blvd., Itasca, IL 60143.
Parvathi Nataraj, Dhanashree Rajderkar, Diomel de la Cruz, Michael D. Weiss
CME/MOC Information:
The American Academy of Pediatrics (AAP) is accredited by the Accreditation Council
for Continuing Medical Education (ACCME) to provide continuing medical education for
physicians.
COMPLEX FETAL CARE The AAP designates this journal-based CME activity for a maximum of 24.0 AMA PRA
Category 1 Credit(s)™. Physicians should claim only the credit commensurate with
861 Fetal Hepatomegaly the extent of their participation in the activity.
In order to earn CME credits and/or ABP MOC Part 2 points, you must participate in
Jina Park, Devlynne Sasha Ondusko, Bill H. Chang, Emily A. Edwards, this activity online at www.neoreviews.org, where you will view additional information,
Sylvia Doan, Ken Gatter, Ibrahim Hajjali, Amanda Kim complete your CME quizzes, and claim credit online.
NeoReviews™ and NeoReviewsPlus™ are supported, in part, through an educational grant
from Abbott Nutrition, a proud supporter of the American Academy of Pediatrics.
The American Academy of Pediatrics is committed to principles of equity, diversity,
and inclusion in its publishing program.

Answer Key appears on page e860.

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 ARTICLE

Primary Mitochondrial Disorders in the

Neonate
Rodrigo Tzovenos Starosta, MD, PhD,* Marwan Shinawi, MD*
*Washington University School of Medicine, Saint Louis, MO

EDUCATION GAPS
AUTHOR DISCLOSURES Drs Starosta and 1. The diagnosis of a primary mitochondrial disorder (PMD) is complex and
Shinawi have disclosed no financial
relationships relevant to this article. This often requires a combination of biochemical, radiologic, histologic, and
commentary does not contain a molecular tests.
discussion of an unapproved/
investigative use of a commercial 2. Most PMDs in the neonate are caused by pathogenic variants in nuclear
product/device. genes.
3. PMDs have several possible neonatal presentations that are frequently
ABBREVIATIONS nonspecific and can be confused with clinical findings of common
ATP adenosine triphosphate neonatal medical complications.
CoQ10 coenzyme Q10
COXPD combined oxidative 4. Management of mitochondrial diseases is still mainly supportive, though
phosphorylation deficiency promising novel treatment modalities are currently being studied.
CSF cerebrospinal fluid
ES exome sequencing
ETC electron transport chain
GRACILE growth retardation (currently OBJECTIVES After completing this review, readers should be able to:
called “growth delay”),
aminoaciduria, cholestasis,
iron overload, lactic acidosis, 1. Explain basic mitochondrial biology, structure, and main functions.
and early death
HCM hypertrophic cardiomyopathy 2. Explain that most proteins required for mitochondrial assembly,
L/P lactate-to-pyruvate maintenance, and execution of numerous mitochondrial activities are
MCLC/MC monolysocardiolipin-to-
encoded by nuclear genes.
cardiolipin
MDDS mtDNA depletion syndrome 3. Identify distinct modes of inheritance associated with PMDs.
MELAS mitochondrial
encephalomyopathy, lactic 4. Describe the different diagnostic approaches for the neonate with a
acidosis, and stroke-like
suspected PMD.
episodes
MERRF myoclonic epilepsy with 5. Describe common mitochondrial presentations and PMDs in the neonate.
ragged red fibers
MNGIE mitochondrial 6. Explain the rationale and evidence behind commonly used therapeutic
neurogastrointestinal interventions for PMDs.
encephalomyopathy
MR magnetic resonance
mtDNA mitochondrial DNA
NARP neuropathy, ataxia, and ABSTRACT
retinitis pigmentosa
Primary mitochondrial disorders (PMDs) are a heterogeneous group of
nDNA nuclear DNA
NGS next generation sequencing disorders characterized by functional or structural abnormalities in the
PMD primary mitochondrial mitochondria that lead to a disturbance of cellular energy, reactive oxygen
disorder
species, and free radical production, as well as impairment of other
rRNA ribosomal RNA
SNHL sensorineural hearing loss intracellular metabolic functions, causing single- or multiorgan dysfunction.
VUS variant of uncertain PMDs are caused by pathogenic variants in nuclear and mitochondrial
significance genes, resulting in distinct modes of inheritance. Onset of disease is
WGS whole-genome sequencing

e796 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 variable and can occur in the neonatal period, with a high morbidity and mortality. In this article, we review the most
common methods used for the diagnosis of PMDs, as well as their prenatal and neonatal presentations. We highlight
the shift in the diagnostic approach for PMDs since the introduction of nontargeted molecular tests into clinical
practice, which has significantly reduced the use of invasive studies. We discuss common PMDs that can present in
the neonate, including general, nonsyndromic presentations as well as specific syndromic disorders. We also review
current treatment advances, including the use of mitochondrial “cocktails” based on limited scientific evidence and
theoretical reasoning, as well as the impending arrival of personalized mitochondrial-specific treatments.

INTRODUCTION numerous mitochondrial activities, are encoded by nuclear

The mitochondria are intracellular double-membrane organ- genes, translated in the cytoplasm, and then targeted into the
elles important for the production of most of the energy mitochondria. Consequently, primary mitochondrial disorders
required by cells in the form of adenosine triphosphate (PMDs) can be caused by pathogenic variants in nDNA or
(ATP) through 5 multimeric electron transport chain (ETC) mtDNA, resulting in distinct modes of inheritance. (8) In pe-
complexes. In addition, fatty acid oxidation, ketogenesis, diatric patients, approximately 80% of causative variants are
ketolysis, multiple reactions of the Krebs (citric acid) cycle, found in the nDNA. (8) Although pathogenic variants in
catabolism of some amino acids (eg, branched chain, lysine, mtDNA exhibit maternal inheritance, genetic defects in nu-
glycine, ornithine, and proline), and part of the urea cycle clear genes are inherited in autosomal (recessive or dominant)
reactions all take place in the mitochondrial matrix. Further- or X-linked patterns. (1) In both circumstances, the genetic ab-
more, mitochondria are involved in the production of pyrimi- normality can also be de novo. During germline or somatic
dine, heme, and estrogen/testosterone, as well as cholesterol cell division, mitochondria and mtDNA molecules are distrib-
metabolism, calcium homeostasis at synapse neurotransmis- uted stochastically into each daughter cell. During this replica-
sion, angiogenesis, immunologic and inflammatory responses, tive segregation process, a pathogenic variant in mtDNA will
and apoptosis. (1)(2)(3)(4)(5) Not unexpectedly, impaired mito- be randomly distributed in cells, resulting in heterogeneous
chondrial activity has serious implications and causes multiple populations of normal and mutant mitochondria in each cell,
symptoms with multisystemic manifestations. The pathome- a phenomenon known as heteroplasmy, which reflects the
chanisms underlying these disorders reflect disturbances in 1 mutation load in each cell type or tissue. Homoplasmy means
or more mitochondrial functions such as insufficient ATP pro- that all mtDNA copies are identical. These processes explain
duction through oxidative phosphorylation, the production of the variation in the percentage level of mutant mtDNA
reactive oxygen species and free radicals, and the impairment between siblings and between organs and tissues within the
of intracellular calcium homeostasis, all of which lead to same person. The cell or tissue will manifest a mitochondrial
apoptosis and target organ damage. An inventory of the mito- pathology only beyond a certain threshold level of mutant mi-
chondrial proteins, which are encoded by nuclear and mito- tochondria. (8)(9) This threshold level varies between tissues
chondrial DNA (mtDNA) genes, and their metabolic pathways depending on their energy demand, which can explain the
are compiled and continuously updated at the Human Mito- phenotypic variability among patients with the same mtDNA
Carta website. (6) pathogenic variant. (1)
mtDNA is a double-stranded circular molecule of ap- Because of their phenotypic heterogeneity, genetic vari-
proximately 16.5 Kb long that encompasses 37 genes: 2 ri- ability, clinical overlap with nonmitochondrial disorders,
bosomal RNA (rRNA), 22 transfer RNA, and 13 subunits lack of specific and sensitive diagnostic biomarkers, and
of the ETC. In this regard, mtDNA varies from nuclear rarity, diagnosing PMDs can be very challenging. (8)
DNA (nDNA) in its structure, size, gene regulation, and
content of genes. mtDNA is exclusively maternally inher- PRIMARY MITOCHONDRIAL DISORDERS
ited. Each mitochondrion has 5 to 10 copies of mtDNA, PMDs (also known as mitochondriopathies or mitochondrial
and each cell contains hundreds to thousands of copies of cytopathies) are a clinically diverse category of progressive
mtDNA. (7) The quantity of mitochondria in each cell is diseases characterized by a malfunction of 1 or more ETC
determined by the cell type's energy needs and can in- complexes or other disturbances of mitochondrial function
crease in response to aberrant function or demand. and/or structure and associated with high rates of morbid-
Most ETC subunits, as well as over 1,300 proteins required ity and mortality. (10) Approximately 1 in 6,000 children
for mitochondrial assembly, maintenance, and execution of are affected by a PMD. (1) PMDs preferentially affect

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e797

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 tissues and organs with high energy demands (eg, brain, elevated blood and CSF lactate-to-pyruvate (L/P) ratio is re-
skeletal muscle, eye, and heart), can present at any age, and liably used to distinguish ETC diseases from pyruvate me-
display a wide range of symptoms and signs. (1) The tabolism disorders. Some of the excess pyruvate is
presentation of PMDs is pleiotropic. Common presenting converted to alanine, which may be increased in the
scenarios include progressive multisystem involvement, plasma of patients with PMDs. Elevations of lactate, the L/
complex neurologic phenotypes (eg, encephalopathy, intrac- P ratio, and alanine can be seen in approximately 86%,
table epilepsy, stroke-like episodes, movement disorders, 45%, and 42%, respectively, of patients with PMD, and
ataxia, neuropathy), developmental regression, myopathy, are used as relatively sensitive initial tests for patients with
cardiomyopathy, hepatopathy, ocular abnormalities, and re- suspected PMDs. (15)(16) Importantly, false-positive ele-
nal dysfunction. (11) There are additional syndromic presen- vated lactate levels can be related to poor collection or
tations with a group of recurrent clinical features, which sample handling techniques. The measurement of pyru-
include mitochondrial encephalomyopathy, lactic acidosis, vate levels can be technically challenging and may be sub-
and stroke-like episodes (MELAS); myoclonic epilepsy with ject to several preanalytical and analytical limiting factors.
ragged red fibers (MERRF); neuropathy, ataxia, and retinitis Elevation in glycine, proline, and threonine as well as hy-
pigmentosa (NARP); Pearson syndrome; and mitochondrial pocitrullinemia can also suggest PMDs. (14)(17) The detec-
neurogastrointestinal encephalomyopathy (MNGIE). Updated tion of aminoaciduria in urine amino acid analysis may
diagnostic criteria of these syndromic PMDs were recently suggest renal tubulopathy, which is known to be associ-
published. (11) In addition to their classic multisystemic ated with PMDs. Besides lactate, analysis of urine organic
($3 organs) presentations, PMDs might also affect only 1 or- acids may also reveal elevations of intermediates of the
gan, resulting in a limited presentation, such as hearing loss Krebs cycle (such as succinate, fumarate, a-ketoglutarate)
or blindness. For example, pathogenic variants in the mito- in approximately 31% of patients, which can be nonspe-
chondrial rRNA coding gene MT-RNR1 predispose patients cific markers of mitochondrial dysfunction. (16)(17) An el-
to aminoglycoside ototoxicity and late onset of sensorineural evated methylmalonic acid concentration in a patient
hearing loss (SNHL), and pathogenic variants of MT-TS1 suspected to have a PMD may be an indication of SU-
cause childhood-onset SNHL. CLA1- or SUCLG1-associated disease, but methylmalonic
acidurias, cobalamin defects, and vitamin B12 deficiency
LABORATORY TESTS FOR DIAGNOSING PMDs need to be ruled out in such cases. (15) Elevations in urine
The identification of clinical findings that suggest a PMD is 3-methylglutaconic acid may indicate Barth syndrome, (18)
usually the first step in the diagnostic evaluation. However, which can be confirmed biochemically by a monolysocar-
PMDs cannot be diagnosed only on the basis of phenotypic diolipin-to-cardiolipin (MCLC/MC) ratio. (19) There are no
findings, and various diagnostic studies are needed to con- data supporting the usefulness and diagnostic value of
firm the diagnosis. (12) There is no single diagnostic test measurements of carnitine levels and acylcarnitine profile.
that can definitively confirm or rule out PMDs. Instead, (14) However, these measurements are commonly ordered
clinicians use various diagnostic methods, including meta- to rule out secondary carnitine deficiency, secondary acyl-
bolic analyses of body fluids, different imaging modalities, carnitine changes due to mitochondrial dysfunction, and
and molecular studies (Table 1). inborn errors of fatty acid oxidation metabolism, all of
The diagnostic evaluation of a patient with a clinical which can cause phenocopies of PMDs. Finally, mitochon-
suspicion of PMDs usually starts with noninvasive bio- drial dysfunction can lead to a disruption of the urea cycle
chemical tests on blood, urine, and cerebrospinal fluid (part of which takes place in the mitochondrial matrix),
(CSF) specimens. These studies include measurements of leading to secondary hyperammonemia.
lactate and pyruvate in blood, ammonia in blood, urine All patients suspected of having a PMD should also be
and serum amino acids, urine organic acids, carnitine lev- comprehensively evaluated for electrolyte abnormalities and
els, and acylcarnitine profile in blood. (13)(14) Pyruvate is a metabolic acidosis, which can be associated with an in-
the end-product of the glycolytic pathway. In aerobic con- creased anion gap secondary to lactate accumulation or with
ditions it is largely converted to acetyl-CoA, which can en- a normal anion gap due to renal tubular dysfunction. They
ter the Krebs cycle; in anaerobic conditions or when the should also be tested for liver dysfunction via a hepatic
mitochondrial oxidative phosphorylation cascade is dis- panel (including alanine and aspartate aminotransferases,
rupted, pyruvate is converted to lactate in the cytosol. Ele- g-glutamyl transferase, direct and indirect bilirubin levels),
vated lactate can be detected in blood, CSF, and urine. An hypoglycemia, hematologic abnormalities (complete blood

e798 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Table 1. Diagnostic Evaluation for Patients with Primary Mitochondrial Disorders
Biochemical studies in all patients
 Complete metabolic panel (CMP)
 Hepatic panel (if liver enzymes elevated in CMP)
 Blood gas (if bicarbonate is low in CMP and/or lactate is elevated)
 Complete blood count
 Lactate-pyruvate ratio
 Ammonia
 Creatine kinase
 Plasma amino acids
 Plasma total and free carnitine plus acylcarnitine profile
 Urine amino acids
 Urine organic acids
 GDF15 and FGF21a
Biochemical studies in CSFb (for patients with neurologic presentation)
 Chemistry and cell count
 Lactate
 Amino acids
Molecular testing for all patients
 mtDNA sequencing and deletion/duplication studies
 Nuclear DNA testing (targeted vs panels vs ES/WGS)
Muscle biopsyc (performed only if molecular testing is negative or inconclusive)
 Routine histology
 Electron microscopy
 Histochemistry (eg, for ragged red fibers)
 Electron transport chain activities
 Ubiquinone (ie, coenzyme Q10) measurement
 mtDNA sequencing and deletion/duplication studies
 Quantitative analysis of mtDNA
Studies to determine extent of disease and multisystem involvement (based on presentation)
 Brain magnetic resonance imaging (brain magnetic resonance spectroscopy, if available)
 Electrocardiography
 Echocardiography
 Ophthalmologic examination
 Audiology
 Neurodiagnostics (EEG, EMG/NCS)

CSF=cerebrospinal fluid; EEG=electroencephalography; EMG/NCS=electromyography/nerve conduction studies; FGF21=fibroblast growth fac-

tor 21; GDF15=growth differentiation factor 15; mtDNA=mitochondrial DNA; ES/WGS=exome sequencing/whole-genome sequencing.
a
These studies are not commonly available for clinical testing and their usefulness is under clinical research investigation.
b
Additional studies may be performed (eg, neurotransmitters) based on clinical presentation.
c
Some functional and morphologic studies can be performed on other biopsy specimens (eg, liver or skin).

cell count), and elevated creatine phosphokinase, which can homeostasis. (21)(22)(23)(24) These biomarkers are most
reflect muscle involvement. Patients with neurologic symp- sensitive when used concomitantly as first-tier diagnostic
toms such as seizures, encephalopathy, or abnormal move- tests for myopathic PMDs caused by mitochondrial transla-
ments should have a lumbar puncture and CSF collection tion defects or mtDNA deletions. (25) They do not appear
for lactate, L/P ratio, and routine CSF studies (cell count, to be as sensitive for PMDs without a major component of
glucose, and culture). Measurement of CSF amino acids musculoskeletal tissue damage. (20)(25) We anticipate that
and neurotransmitters can help rule out nonmitochondrial the role of these biomarkers will be further expanded in the
diagnoses. future as additional clinical studies evaluate their useful-
Growth differentiation factor 15 and fibroblast growth ness and validity in clinical practice.
factor 21 are 2 novel biomarkers of mitochondrial dysfunc- Electrocardiography and echocardiography are essential
tion that are being studied and increasingly used in clinical in all neonates suspected of having a PMD, as cardiomy-
practice. (20) Growth differentiation factor 15 is a cytokine opathy is one of the most common manifestations. (9)
secreted in response to tissue injury, whereas fibroblast Ophthalmologic and audiologic evaluations should also be
growth factor 21 is a component of the mitochondrial inte- considered for all patients, as the optic nerves, retinas,
grated stress response signaling. (20)(21) However, these and inner ear are commonly affected in PMDs. (26)(27)
biomarkers are nonspecific indicators of mitochondrial dys- Brain magnetic resonance (MR) imaging is used to inves-
function, as predicted, given their extensive roles in tissue tigate aberrant signals in the white matter, basal ganglia

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e799

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 and brain stem, as well as nonvascular infarcts (“metabolic sequencing and deletion/duplication analysis has been
strokes”), and should be considered in all patients suspected shown to have a higher yield than targeted gene panels
of having a PMD with neurologic symptoms. (13)(28) It has when investigating patients suspected of having a PMD,
been shown that the most significant positive predictive (32)(33)(34) with a diagnostic yield ranging from 22% to
value of a PMD in a neonate is the finding of characteristic 60% in different studies. (29)(31)(32)(33)(35)(36) NGS has
MR imaging abnormalities in the basal ganglia. (29) When been proven to be the most reliable mtDNA sequencing
available, brain MR spectroscopy should be performed con- technology that improves the sensitivity and detection of
currently to search for lactate peaks in the brain, which indi- point mutations, low-level heteroplasmy, and deletions/
cate local lactic acidosis, raising suspicion for a PMD. Based duplications. (37) It is important to keep in mind, how-
on the clinical picture, further neurodiagnostic procedures ever, that ES has a lower coverage of base pairs for genes
may be performed, such as electroencephalography if the pa- of interest than do panels, which may especially limit the
tient exhibits seizures or encephalopathy. Electromyography/ usefulness of a negative result. (8) Nonetheless, unless
nerve conduction studies are recommended if there are the clinical picture fits a very specific recognizable mito-
concerns for myopathy and/or neuropathy, which are not chondrial phenotype or if there is a strong family history
common presentations in neonates with PMDs. Muscle of a PMD with a known familial pathogenic variant, ES
ultrasonography has a low sensitivity but high specificity for plus mtDNA analysis should be considered the first-tier
PMDs and may be used as an additional test for patients molecular test in patients suspected of having a PMD.
with a suspected PMD when an experienced sonographer is (8)(16)(38)
available. (30) Finally, medical genetics and child neurology, Whole-genome sequencing (WGS) entails sequencing
and other specialties as necessary, should be consulted early not only the protein-coding regions but also noncoding re-
in the evaluation process when there is a suspicion or a diag- gions both within the genes (ie, the introns) and between
nosis of a PMD. genes (eg, promoter and other regulatory and nonprotein
The discovery of multiple nuclear genes that cause coding regions). WGS can reliably analyze copy number
PMDs over the last few decades, as well as the introduc- variants (ie, deletions and duplications) and has the poten-
tion of high-throughput, massively parallel sequencing tial to be used for repeat expansion analysis. Although few
(also known as next generation sequencing [NGS]) into clini- WGS studies on the diagnostic yield for PMDs have been
cal practice, which resulted in cost and turnaround time published to date, there is evidence that WGS has a higher
reductions, dramatically changed the diagnostic approach diagnostic yield than ES, (39)(40) and that WGS may be
for patients with PMDs. If the patient exhibits certain clin- more precise in assessing heteroplasmy rates than tradi-
ical characteristics that are compatible with one of the syn- tional PCR-based mtDNA testing. (41)(42) The expanded
dromic forms of PMDs, a targeted test can be performed use of ES and WGS in the last decade revealed several
using a single gene or a small number of genes related to nonmitochondrial disorders that create phenocopies of mi-
the suspected disease. However, because the presentation tochondrial presentations and are associated with second-
of PMDs is typically nonspecific, a more gene-inclusive ary mitochondrial dysfunction. (12)(43)(44) Rapid ES and
strategy is preferable. Gene panels, that is, sequencing WGS, with turnaround times ranging from 1 to 7 days,
of a predetermined set of genes known to cause the ob- have been found to be effective in diagnosing PMDs and
served phenotype, are the traditional diagnostic strategy other rare genetic diseases, (45)(46) and should be consid-
used in NGS. The number of genes included in these pan- ered in critically ill neonates.
els varies among laboratories, making it difficult to select All molecular studies discussed herein are routinely per-
the appropriate one for each patient. formed on DNA extracted from peripheral blood. However,
Exome sequencing (ES) is an NGS-based assay that if the results are negative and there is a high index of suspi-
amplifies and sequences all known protein-coding genes. cion for a PMD due to a mtDNA mutation, testing of an-
ES can be combined with mitochondrial genome se- other tissue or body fluid (eg, urine, muscle tissue, or buccal
quencing and deletion/duplication studies using NGS. swab) for mtDNA mutations and heteroplasmy is needed.
ES data analysis is phenotype-driven and diagnostic labo- (13)(47) Maternal testing for the proband’s identified mtDNA
ratories must be provided with phenotypic information mutation is required to determine whether the mutation is
about the patient before starting analysis to facilitate an- de novo, and, if present, its degree of heteroplasmy. This in-
notation, curation, and interpretation of results in a formation is important for genetic counseling and variant in-
meaningful way. (31) ES in conjunction with mtDNA terpretation. For example, the presence of a homoplasmic

e800 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 variant of uncertain significance (VUS) in an asymptomatic arrhythmias are seen. (51) Hydrops fetalis can also occur. (52)
mother suggests that the variant is not pathogenic. For example, hydrops fetalis in Barth syndrome is a result of
Before the widespread availability of molecular studies, fetal dilated cardiomyopathy, endocardial fibroelastosis, and/
muscle (skeletal or cardiac), liver, and/or skin biopsies or left ventricular noncompaction. (19)(53) In exceptionally
were extensively used for functional, histologic, and ge- rare cases, fetal anemia requiring in utero transfusions may
netic studies. However, these invasive tests are currently be a result of Pearson syndrome, an mtDNA deletion syn-
performed only if molecular testing yields negative or in- drome. (54)(55) A variety of fetal malformations and other
conclusive results (eg, detection of VUSs or low level of congenital anomalies have also been reported (49)(56) but
heteroplasmy in blood) and there is a high index of suspi- their association with PMDs and their pathomechanisms are
cion for a PMD or a neuromuscular disease that can be uncertain (Table 3).
confirmed with a muscle biopsy. (38)(48) Muscle tissue is
typically used, but it is recommended to perform a biopsy NEONATAL PRESENTATIONS OF PMDs
of the most affected organ. For example, if the main pre- PMDs can present at essentially any age, with 5% to 30%
sentation is hepatopathy (eg, liver failure), a common sce- of patients experiencing symptoms in the first month of
nario in pediatric patients with PMDs, a liver biopsy age, mostly in the first 2 days after birth. (15)(34)(56)
should be considered and carefully planned with a liver One study showed that 60% of patients with neonatal
specialist. Occasionally, a skin biopsy or buccal swab may PMDs presented at birth or within 24 hours after birth.
be performed for ETC studies if other tissues are not ac- (51) The high energy demands of labor, delivery, and the
cessible or available for testing. (13) Specimens should be early neonatal period are thought to make newborns
investigated with routine histologic examination, electron with impaired energy production more vulnerable. (51)
microscopy (shape, structure, and number of mitochon- Signs and symptoms of PMDs in neonates are typically
dria, intramitochondrial inclusions), histochemistry (histo- ambiguous and nonspecific and sometimes confounded
chemical stains for succinate dehydrogenase, cytochrome with clinical findings of neonatal distress such as birth
C oxidase, and the modified Gomori trichrome stain for the asphyxia, neonatal sepsis, and cardiorespiratory failure. (52)
pathognomonic ragged red fibers), ETC enzyme activity Furthermore, elevated lactate levels in the neonate can
and western blot analysis, CoQ10 quantification, mtDNA se- be caused by conditions other than PMDs, including re-
quencing and deletion/duplication studies, and quantitative spiratory failure with hypoxia, hemodynamic instability,
analysis of mtDNA. (7)(12) In addition to their invasive na- heart failure, necrotizing enterocolitis, and sepsis.
ture, which can be even more challenging in a critically ill Therefore, the diagnosis of PMDs can be very challeng-
neonate, these studies have other limitations such as difficul- ing and requires a high degree of clinical suspicion.
ties in distinguishing between PMDs and secondary mito- Table 4 lists common neonatal presentations of PMDs
chondrial dysfunction and low sensitivity of histopathologic in neonates.
findings (such as ragged red fibers) in neonates. Table 2 lists Many neonates with PMDs present with nonspecific
the different testing modalities commonly used and their signs such as poor feeding, hypotonia, seizures, liver dys-
benefits and caveats. function (with or without cholestasis and hypoglycemia),
respiratory insufficiency, and in extremis circulatory col-
PRENATAL PRESENTATIONS OF PMDs lapse. (51)(56)(57) The respiratory symptoms may reflect a
Prenatal manifestations of PMDs are typically nonspecific compensatory mechanism for metabolic acidosis, cardiac
and do not raise diagnostic suspicion for this category of dysfunction, and/or brain depression. In these cases, espe-
illnesses unless there is a family history of a sibling af- cially when there is cardiac involvement, the mortality
fected with a PMD (Table 2). Nonetheless, roughly one- may be as high as 50% in the first 3 months of age.
third of patients with PMDs may have some prenatal man- (15)(51)(56) One study that included 129 patients with neo-
ifestations, with fetal growth restriction, small for gesta- natal-onset PMDs found hypotonia in 90%, apneic events
tional age, poly- or oligohydramnios, and reduced fetal in 69%, cardiomyopathy in 40%, and liver impairment in
movements being the most prevalent. (49)(50)(51) One 35% of the neonates. The same study found elevated lac-
study reported prematurity and fetal growth restriction in tate in 87% and hyperammonemia in 42% of patients. (15)
30% and 35%, respectively, of patients with PMDs. (15) Another study found that the most common symptoms in
Occasionally, fetal cardiac abnormalities such as antenatal neonates with PMDs after the first day after birth were
onset of hypertrophic cardiomyopathy (HCM) and fetal poor feeding, recurrent emesis, and poor weight gain, and

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e801

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Table 2. Accuracy, Benefits, and Caveats of Common Testing Modalities for PMDs
Test Accuracy Benefits Caveats
Lactate Relatively sensitive marker for Noninvasive Nonmitochondrial disorders can
certain PMDs Easy and fast to obtain cause elevated lactate
Very low specificity Lactate elevation can be related to
poor collection or handling
techniques
Normal lactate levels do not rule
out PMDs
Lactate-pyruvate ratio Moderate sensitivity and Noninvasive Pyruvate is unstable and needs to
specificity Easy and fast to obtain be collected and immediately
transferred to a tube containing
perchlorate on ice
Postprandial values can be
elevated
GDF15 and FGF21 Under investigation Potentially higher specificity for Unclear sensitivity and specificity
some mitochondrial Limited availability
conditions
Urine organic acids Elevations of Krebs cycle Noninvasive Interpretation can be difficult in
intermediates in urine have Easy and fast to obtain the ill neonate, especially if
low sensitivity and specificity An elevation of there is a liver dysfunction or
for PMDs 3-methylglutaconic acid may multisystemic organ failure
point to Barth syndrome
Serum amino acids Low sensitivity and specificity Noninvasive Interpretation can be difficult in
Easy and fast to obtain the ill neonate, especially if
there is liver dysfunction or
parenteral nutrition provided
Muscle biopsy Low sensitivity and specificity May clarify functional aspects of Invasive
VUSs or establish a diagnosis Nonspecific for a genetic etiology;
secondary mitochondrial
dysfunctions are common
NGS panel testing Moderate sensitivity, high Points to specific diagnosis May not include important genes
specificity May include genes associated associated with the presenting
with nonmitochondrial phenotype
diseases that are in the Detection and reporting variants of
differential diagnosis uncertain significance
Negative results do not rule out
PMDs
ES/WGS High sensitivity and specificity Points to specific diagnosis. Requires extensive consenting
Includes genes associated including for secondary findings
with diseases that are in the Potential for nonpaternity/
differential diagnosis nonmaternity disclosure
Detection and reporting variants of
uncertain significance
Negative results do not rule out
PMDs

FGF21=fibroblast growth factor 21; GDF15=growth differentiation factor 15; NGS=next generation sequencing; PMDs=primary mitochondrial
disorders; VUSs=variants of uncertain significance; ES/WGS=exome sequencing/whole-genome sequencing.

many patients exhibited more than 1 symptom. (51) Car- determining the prognosis in neonates with PMDs: pa-
diac involvement usually takes the form of HCM. (9)(58) tients with cardiomyopathy have a 10-year survival rate of
The presence of cardiomyopathy is an important factor in 18%, compared with 67% in patients without cardiomyop-
athy. (9) Although there is no difference in overall survival
Table 3. Common Prenatal Presentations of Primary between patients with pathogenic mtDNA variants and
Mitochondrial Disorders those with pathogenic nDNA variants, higher rates of het-
 Fetal growth restriction with low birthweight eroplasmy (or homoplasmy) are known to cause a more
 Hydrops fetalis severe phenotype in patients with a pathogenic mtDNA
 Poly- or oligohydramnios
variant. (9)
 Reduced fetal movements
 Prematurity PMDs in neonates frequently present with involvement
 Fetal cardiomyopathy of multiple organs. In fact, multisystemic disease is the
 Fetal arrhythmia
most common presentation and was found in 69% of

e802 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Table 4. Common Neonatal Presentations of Primary However, it is easiest to think of MDDSs as a continuum
Mitochondrial Disorders of manifestations centered mainly on encephalic, muscu-
Neurologic
lar, and hepatic involvement with a spectrum of clinical
 Hypotonia severity. (60)(61)
 Poor feeding (with or without emesis) Encephalomyopathic MDDSs are associated with bial-
 Apneic spells
 Encephalopathy lelic pathogenic variants in FBXL4, RRM2B, SUCLA2,
 Neonatal seizures and SUCLG1. Affected patients present in the neonatal
Cardiorespiratory
 Respiratory insufficiency
period with hypotonia, encephalopathy, and feeding diffi-
 Cardiomyopathya culties associated with lactic acidosis. Seizure, movement
 Circulatory collapse disorders, and autonomic dysfunction can also occur. As
Hepatic
 Hepatomegaly in other forms, further systemic manifestations can also
 Cholestasis occur in FBXL4-related MDDSs, such as cardiomyopathy,
 Acute liver failure
arrhythmia, liver dysfunction, ocular abnormalities, hear-
 Hypoglycemia
Other ing loss, and neutropenia. (62) Proximal renal tubulop-
 Tubulopathy/nephropathy athy is more commonly found in patients with RRM2B-
 Small for gestational age
 Neonatal death related MDDSs. (63) Pathogenic variants in SUCLA2 or
SUCLG2 should be suspected when a phenotype of ence-
Multiple symptoms can arise in the same patient with some mito-
chondrial illnesses that have multisystemic involvement. phalomyopathic MDDS is associated with elevated urine
a
Typically, hypertrophic and commonly associated with hypotonia methylmalonic acid levels. (59)
and/or encephalopathy. Pathogenic variants in POLG cause a spectrum of over-
lapping phenotypes, including myocerebrohepatopathy
patients in 1 study and it predicts higher mortality rate that can present in the first few months of age. (4) This is
within 2 days after birth. (56) a dramatic presentation with severe hypotonia, encepha-
lopathy, and hepatic dysfunction that normally manifests
COMMON PMDs WITH NEONATAL PRESENTATIONS
first as hypoglycemia and lactic acidosis; it progresses to
mtDNA Depletion Syndromes hepatic dysfunction with fibrosis, cirrhosis, and liver fail-
The mtDNA depletion syndromes (MDDSs) are a group ure, with death typically occurring before age 1 year.
of disorders caused by pathogenic variants in nuclear (4)(59)(64)(65)(66) Seizures are uncommon in myocerebro-
genes that encode proteins involved in the synthesis and hepatopathy. In contrast, another severe POLG-related
maintenance of the pool of mitochondrial nucleotides, or MDDS, Alpers-Huttenlocher syndrome, presents later in in-
in the replication of mtDNA, and thus are important for fancy with developmental regression, intractable seizures,
mtDNA integrity. (59) These genes include (but are not and liver failure. (64) Death usually occurs in early to mid-
limited to) POLG, TWNK, TK2, DGUOK, ANT1, MPV17, childhood due to neurologic complications.
RRM2B. (59) mtDNA content in affected tissues in Hepatocerebral MDDSs are caused by pathogenic var-
MDDSs, which can be confirmed using quantitative iants in DGUOK, MPV17, POLG, and TWNK. Most pa-
mtDNA analysis, is typically less than 20% of matched tients with DGUOK-related MDDS exhibit a multisystem
controls. When mtDNA is depleted, the components of disorder that becomes evident in the first weeks after
the oxidative phosphorylation chain cannot be synthe- birth, and includes hepatic disease with cholestasis and
sized properly, leading to an impairment in ATP synthe- progressive liver dysfunction as well as neurologic abnor-
sis and end-organ dysfunction. (60) Clinically, MDDSs malities. MPV17-related MDDS is typically associated with
are usually divided into 1 of 3 forms: encephalomyo- cholestatic liver failure, with onset occurring from birth
pathic, hepatocerebral, and myopathic forms. Some geno- throughout childhood (most patients presenting in the
type-phenotype correlation is known, where patients with first year of age). Patients also exhibit neurologic mani-
POLG pathogenic variants have more neurologic symp- festations, including hypotonia, microcephaly, seizures, white
toms, whereas those with pathogenic variants in DGUOK matter abnormalities, motor and sensory peripheral neuropa-
or MPV17 have more hepatic involvement, which is com- thy, as well as gastrointestinal abnormalities (dysmotility,
monly associated with severe neonatal metabolic acidosis, feeding difficulties, and poor weight gain). (67)(68)(69) Most
severe and recurrent hypoglycemia, rapidly progressive affected individuals with MPV17 and DGUOK pathogenic
liver failure leading to cirrhosis, and early death. (59) variants die of liver failure in infancy or early childhood. (68)

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e803

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 The mortality rate in patients with MPV17-related MDDS af- death in the neonatal period, with the most common causes
ter liver transplantation is 55% to 60%, with most patients of death being respiratory failure, intractable neurologic
also exhibiting neurologic features before transplantation and compromise, and secondary infections (most commonly as-
before death. (67)(68)(70) piration pneumonia). (74)
The myopathic form of MDDSs, which is most com-
monly caused by variants in TK2, is rare and severe in the Pearson Syndrome
neonatal period, presenting with hypotonia and myopathy Pearson syndrome is a rare multisystemic PMD caused by
that lead to respiratory insufficiency in most patients. (4)(71) a single large mtDNA deletion (1.1–10 Kb) with high degree
Concomitant neurologic involvement (seizures, encephalopa- of heteroplasmy. (83)(84) Patients with Pearson syndrome
thy, optic atrophy) has been found in 30% of cases. (4)(72) commonly present in the neonatal period with pancytope-
The median survival after the beginning of symptoms nia, which leads to transfusion-dependent anemia and op-
is approximately 1 year. (71) TK2 encodes thymidine ki- portunistic infections due to neutropenia. (83)(85) The bone
nase 2, a mitochondrial matrix enzyme that is essential marrow typically shows hypocellularity, vacuoles in hemato-
for the salvage pathway of deoxynucloside triphosphate poietic precursors, and ringed sideroblasts, though not all
synthesis. patients display these findings. (86) Exocrine pancreatic in-
sufficiency is also a common feature of Pearson syndrome
Leigh Syndrome and may lead to steatorrhea, malabsorption, and poor
Leigh syndrome is a general description of subacute or chronic weight gain beginning in early infancy. (87) Approximately
progressive necrotizing encephalopathy caused by mitochondrial one-fourth of the patients will develop secondary diabetes
dysfunction. (73) The hallmark of Leigh syndrome is the pres- mellitus. (83) Hepatomegaly, and less often splenomegaly,
ence of bilateral, often symmetrical lesions in deep gray matter with or without hepatic dysfunction can occur and renal tu-
structures such as the basal ganglia, the thalami, and the brain- bulopathy and different endocrinopathies are observed in
stem, which consist of areas of necrosis, gliosis, and vascular some patients. (85) Although the mortality rate in infancy is
proliferation. (59) The mean age at onset is 7 months, ranging high, aggressive management of anemia and pancreatic in-
from birth to adulthood. (74) Clinically, these patients present sufficiency of affected patients has been proven effective in
with poor weight gain, recurrent emesis, seizures, movement increasing survival. (88)(89) Some patients with Pearson
disorders (dystonia, chorea), and respiratory failure. (59)(73) syndrome who survive past early childhood may develop
(75)(76) Recurrent metabolic decompensations during acute in- the related Kearns-Sayre syndrome, a progressive neuro-
fectious illnesses lead to developmental regression and additional muscular disorder of childhood onset. (85)(87)(90) The di-
neurologic deterioration. Although most patients present in in- agnosis of Pearson syndrome is confirmed with mtDNA
fancy, mostly between 3 and 10 months of age or childhood, the deletion studies in blood, which is a relatively easy and
onset of neurologic symptoms such as lethargy, autonomic dys- reliable test for this disorder. In fact, detection of mtDNA
function, hypo- or hypertonia, and abnormalities of the respira- deletions in peripheral blood is more reliable than their de-
tory drive in the first month of age can occur in about 15% of tection in muscle or liver tissue, to the contrary of most
patients, and usually results in a poor prognosis. (73)(74)(77) other PMDs. (86)
The pathophysiology of Leigh syndrome includes a wide-
spread disturbance of the ETC complexes, which results in Barth Syndrome
energy deficit in neurons. (78)(79) Laboratory studies usu- Barth syndrome is an X-linked PMD characterized by cardio-
ally show elevated lactate and L/P ratio (59)(76) and CSF lac- myopathy, cyclic neutropenia, and skeletal myopathy. It is
tate levels correlate with earlier disease onset and poorer caused by pathogenic variants in TAZ, which encodes for
outcomes. (74) Almost 100 different nDNA and mtDNA tafazzin, a transacylase located on the inner mitochondrial
genes have been implicated so far in the etiology of Leigh membrane that converts monolysocardiolipin into cardiolipin
syndrome. (73)(80) Although there are some established ge- at the mitochondrial membranes. Cardiolipin is a phospho-
notype-phenotype correlations—such as higher occurrence lipid essential for the maintenance of the special properties of
of cardiac and ocular manifestations in patients with var- the mitochondrial membranes. Carrier females with normal
iants in MT-ND and NDUF genes (81) and earlier-onset karyotype and X inactivation studies are typically asymptom-
disease in patients with higher mtDNA heteroplasmy rates atic and have no biochemical abnormalities. Approximately
(82)—it is not possible to predict outcome and prognosis 70% of patients with Barth syndrome have cardiomyopathy
based solely on the genotype. Leigh syndrome can cause in infancy, and in approximately 40%, the age at diagnosis of

e804 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 cardiomyopathy is between birth and 1 month. (91) The most extremely brittle hair and alopecia) and profound congeni-
common cardiomyopathy patterns are dilated cardiomyopathy tal bilateral SNHL. (96) No other organ involvement has
(with or without endocardial fibroelastosis) and left ventricu- been described in the classic Bj€
ornstad syndrome. (96)
lar noncompaction, both of which often lead to neonatal-
onset heart failure. (19)(51) HCM has been reported but is Combined Oxidative Phosphorylation Deficiency
less common. (19) Infants can also present with sudden ven- Combined oxidative phosphorylation deficiency (COXPD) is
tricular arrhythmias; sudden cardiac death in infancy without characterized by a concomitant decrease in the activity of 2 or
a history of cardiomyopathy has also been reported. (18) Neu- more of the 5 ETC complexes. (99) Several mtDNA and
tropenia affects 90% of patients with Barth syndrome, how- nDNA genes have been implicated in COXPD, with 55 OMIM
ever, the onset is not always in infancy; during nadirs, entries for COXPD currently known. Although the phenotypic
infectious complications are common and can be fatal. spectrum is broad, these conditions commonly manifest in the
Hypotonia/muscle weakness is very common and is mostly neonatal or early infantile periods with significant end-organ
proximal in infants. (19) Infants can have lactic acidosis and dysfunction, particularly hepatopathy, cardiomyopathy, enceph-
hypoglycemia, and several cases of acute metabolic decom- alopathy, and neuromuscular damage. (99)(100)(101)(102) For
pensations leading to death have been described in neonates. example, patients with COXPD23 caused by pathogenic var-
(19) Biochemical findings highly supportive of the diagnosis iants in GTPBP3 exhibit a severe neonatal-onset presentation
are hyperexcretion of 3-methylglutaconic acid in urine and associated with lactic acidosis, acute encephalopathy, and
the pathognomonic elevation of MCLC/MC ratio. (18) HCM. (103) On the other hand, some types of COXPD can
present with a tissue-specific phenotype, for example, pa-
BCS1L-related Mitochondrial Diseases tients with pathogenic variants in MIPEP, which cause
BSC1L is a nuclear gene that encodes for mitochondrial com- COXPD31 can present with severe cardiomyopathy in the
plex III assembly factor. (59)(92) Three main autosomal reces- first months of age, which proceeds to encephalopathy
sive disease phenotypes have been described in association months or years later (104); those with pathogenic variants
with pathogenic variants in BCS1L that cause isolated complex in VARS2 that cause COXPD20 can present with retinal
III deficiency. (59) The most extensively reported phenotype is dystrophy and ophthalmoplegia, with a relatively milder
GRACILE syndrome, which stands for growth retardation (cur- central nervous system involvement. (105) Such genotype-
rently called “growth delay”), aminoaciduria, cholestasis, iron phenotype correlations can help guide prognosis and
overload, lactic acidosis, and early death. This is a severe syn- counseling for patients and families with COXPD.
drome with prenatal-onset fetal growth restriction and postnatal
presentation with lactic acidosis as well as renal and hepatic in- Primary CoQ10 Deficiency
volvement leading to multiorgan failure. (92) The mean age at Coenzyme Q10 (also known as ubiquinone, hereby referred to
death for patients with GRACILE syndrome is 27 days, though as CoQ 10) is an important electron carrier in the ETC, respon-
reports describe patients surviving into childhood. (92) GRAC- sible for transferring electrons from complexes I and II to
ILE syndrome is most commonly associated with compound complex III. CoQ10 is synthesized from derivates of tyrosine
heterozygosity or homozygosity for the c.232A>G (p.Ser78Gly) and mevalonate in a multistep enzymatic pathway; pathogenic
variant, which is common in the Finnish population due to a variants in 10 genes encoding proteins important for CoQ10
founder effect (92)(93); however, there are rare reports of biosynthesis cause primary CoQ10 deficiency. (106)(107) Pri-
GRACILE syndrome caused by other variants in different pop- mary CoQ10 deficiency conditions have heterogenous clinical
ulations. (92)(94)(95) The intermediate phenotype associated presentations and age at onset varies from birth to late adult-
with BCS1L is a Leigh-like presentation that begins often in hood. Pathogenic variants in COQ4 and COQ 9 are typically
the neonatal period and progresses in severity. (59)(96) This associated with severe early neonatal onset multisystemic pre-
disease can present at any age (including the neonatal pe- sentation with a high mortality rate. (107) Clinical symptoms
riod) and can be associated with renal tubulopathy and hep- of these entities can be seen on the first day after birth though
atopathy, which range from mild to severe. (59)(95)(97)(98) later presentations have been reported. The most common
There is currently no clear genotype-phenotype correlation findings include encephalopathy, cardiomyopathy (hypertro-
to predict this intermediate phenotype. (96) The mildest phic or dilated), seizures, hypotonia, cerebellar atrophy, and se-
phenotype caused by pathogenic variants in BCS1L is vere lactic acidosis. (108)(109) Lung hypoplasia and dysplastic
Bj€ornstad syndrome, which is characterized by pili torti kidneys have also been reported in patients with pathogenic
(a congenital twisting of the hair shafts that leads to variants in COQ 7. (110) Outcomes of patients presenting in

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e805

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 the neonatal period are generally poor, with most dying in the antioxidants, vitamins, mitochondrial cofactors, and other
first months of age. (108)(109) The identification of biallelic supplements, which is hypothesized to maximize residual
pathogenic variants in CoQ10 biosynthesis genes can establish mitochondrial complex function and reduce oxidative
the diagnosis of primary CoQ10 deficiency. When the molecu- stress and redox imbalance in the mitochondria. (123)(124)
lar results are inconclusive (VUSs in any of these genes) or Common constituents of the mitochondrial cocktail include
negative with high index of clinical suspicion, CoQ10 measure- antioxidants and oxidative stress-quenching agents such as
ment on muscle tissue or fibroblasts is needed to establish the N-acetylcysteine, sulfolipid a-lipoic acid, CoQ10 in the form of
diagnosis. Importantly, high-dose CoQ10 supplementation has ubiquinone or ubiquinol (the reduced and active form of
been reported to be beneficial in some patients with primary CoQ10), and a-tocopherol (vitamin E) (124)(125)(126)(127); co-
CoQ10 deficiency, especially in those with later-onset presenta- factors involved in mitochondrial function such as thiamine
tions; lack of efficacy in patients with neonatal onset disease (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3),
has been documented. (108)(111)(112)(113) Given the excellent pantothenic acid (vitamin B5) (1)(124)(126)(127); and other nu-
safety profile of CoQ10 supplementation and the lack of other tritional supplements such as L-citrulline, (1)(126)(127) L-carni-
therapeutic options, it is reasonable to attempt this treatment tine, and creatine. (1)(126) A clinical trial and many open-label
for patients presenting in the neonatal period despite the pau- studies have not shown a clear benefit of specific formula-
city of evidence of efficacy. tions of the mitochondrial cocktail in clinical outcomes, but
some biochemical markers have been shown to improve.
Aminoglycoside Toxicity (128)(129) Currently there is no strong evidence to suggest
Mitochondrial nonsyndromic SNHL is a spectrum of predis- that mitochondrial cocktails are helpful in mitochondrial dis-
position to hearing loss with or without aminoglycoside ex- eases. (61)(124)(127)(128) However, these treatments are rec-
posure. In the neonate, the most common presentation is ommended by many clinicians and the use of mitochondrial
SNHL, which develops within a few days or weeks after cocktails is currently based on clinicians’ clinical judgment on
aminoglycoside treatment commonly used for neonatal sep- a case-by-case basis after an open discussion with the family,
sis. (114) Pathogenic variants in MT-RNR1 (m.1494C>T and which includes disclosure of the lack of proven benefit.
m.1555A>G) predispose neonates to aminoglycoside-related For certain PMDs, an individualized treatment is recom-
SNHL. (114)(115) Hearing loss resulting from aminoglyco- mended based on some scientific evidence and theoretical
side toxicity is usually bilateral, nonprogressive, moderate to reasoning. (125)(130) Patients with primary CoQ10 deficiency
profound, and more pronounced at the higher frequencies. should be treated with ubiquinone or preferably ubiquinol.
(116)(117) Hearing loss caused by aminoglycoside ototoxicity L-arginine is used for the treatment of acute metabolic
happens through the destruction of cochlear hair cells and stroke and its prevention in patients with MELAS. In vitro
is irreversible. (118)(119) It is worth noting that some people studies showed efficacy of specific amino acid supplementa-
with these pathogenic variants can still have late-onset pro- tion in certain types of transfer RNA synthetase deficiency.
gressive hearing loss even if they have not been exposed to (131) We anticipate that this personalized treatment approach
aminoglycosides, and these patients often have a family his- will dominate the field as our understanding of the molecu-
tory of hearing loss. (114)(120)(121) Aminoglycosides are lar and biochemical mechanisms of PMDs increases.
contraindicated for patients with the aforementioned geno- A ketogenic diet has been hypothesized as a potential treat-
types unless the risk of SNHL is balanced by the severity of ment for PMDs by promoting a shunt in energy production
the illness and lack of alternative therapies. (122) from glycolysis to b-oxidation, leading to increased flow of elec-
trons through complex II and essentially bypassing complex I.
MANAGEMENT OF PMDs (126) Outside of the scope of seizure and PDH deficiency
Despite the fact that PMDs are among the most common in- treatment, (132)(133)(134) despite isolated reports of the keto-
herited metabolic disorders, currently no effective, validated genic diet leading to symptomatic improvement in patients
treatments (based on randomized, double-blind, controlled with PMDs, this diet has not been shown unequivocally to be
clinical trials) are available for these conditions. In clinical efficacious as an intervention for PMDs; a high rate of side ef-
practice, many treatments with hypothetical efficacy and high fects has been reported in several studies. (126)(135)(136)(137)
safety profile are used empirically, especially given that the ne- Thus, it is not currently possible to recommend a ketogenic
onate with a PMD is frequently in critical condition. diet as a universal treatment strategy for PMDs.
A common proposed treatment for PMDs is the mito- Additional treatments for PMDs are being studied in clin-
chondrial cocktail. This is a variable compound of several ical trials. Two especially promising compounds include

e806 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 sonlicromanol and elamipretide. Sonlicromanol is a scaven- References
ger of reactive oxygen species and was designed to restore re-
1. Gorman GS, Chinnery PF, DiMauro S, et al. Mitochondrial
dox balance in the mitochondrial matrix, aiming to limit the
diseases. Nat Rev Dis Primers. 2016;2:16080
cascade of oxidative stress. (138)(139)(140) Elamipretide is a
2. Das M, Alzaid F, Bayry J. Regulatory T cells under the mercy of
small peptide that penetrates the mitochondria and stabilizes mitochondria. Cell Metab. 2019;29(2):243–245
cardiolipin against oxidative stress, which helps preserve the 3. Naon D, Scorrano L. At the right distance: ER-mitochondria
inner mitochondrial structure in a context of increased reac- juxtaposition in cell life and death. Biochim Biophys Acta.
tive oxygen species. (141)(142) Both sonlicromanol and elami- 2014;1843(10):2184–2194

pretide are currently being studied in clinical trials. 4. Wang H, Han Y, Li S, et al. Mitochondrial DNA depletion
syndrome and its associated cardiac disease. Front Cardiovasc Med.
(138)(139)(143)(144)(145)(146)(147)
2022;8:808115
5. Madeira VMC. Overview of mitochondrial bioenergetics. Methods
CONCLUSIONS Mol Biol. 2018;1782:1–6
PMDs are clinically and genetically heterogeneous diseases 6. Rath S, Sharma R, Gupta R, et al. MitoCarta3.0: an updated
caused by pathogenic variants in mtDNA or nDNA and are mitochondrial proteome now with sub-organelle localization
and pathway annotations. Nucleic Acids Res. 2021;49(D1):
associated with high morbidity and mortality. They can pre-
D1541–D1547
sent at any age, and many patients with PMD exhibit symp-
7. DiMauro S, Schon EA, Carelli V, Hirano M. The clinical maze
toms in the neonatal period. Manifestations of PMDs are of mitochondrial neurology. Nat Rev Neurol. 2013;9(8):
pleiotropic and diverse, and they preferentially affect tissues 429–444
and organs with high energy demands such as brain, skeletal 8. Gusic M, Prokisch H. Genetic basis of mitochondrial diseases.
muscles, eyes, heart, liver, and kidneys. Symptoms and signs FEBS Lett. 2021;595(8):1132–1158

of PMD in neonates are typically ambiguous and nonspecific 9. Imai-Okazaki A, Kishita Y, Kohda M, et al. Cardiomyopathy in
children with mitochondrial disease: prognosis and genetic
and can frequently be confused with nonmitochondrial dis-
background. Int J Cardiol. 2019;279:115–121
eases and other neonatal complications. Diagnosis of PMDs
10. Rahman J, Rahman S. Mitochondrial medicine in the omics era.
can be difficult and cannot be done solely based on pheno- Lancet. 2018;391(10139):2560–2574
typic findings. In this review, we presented the main diagnos- 11. Emmanuele V, Ganesh J, Vladutiu G, et al; North American
tic approaches used in clinical practice, along with their Mitochondrial Disease Consortium (NAMDC). Time to harmonize
strengths and weaknesses. The increasing use of NGS for mitochondrial syndrome nomenclature and classification: A
consensus from the North American Mitochondrial Disease
nontargeted molecular tests has revolutionized the diagnostic
Consortium (NAMDC). Mol Genet Metab. 2022;136(2):125–131
approach for patients with PMDs, and therefore, invasive stud-
12. Parikh S, Karaa A, Goldstein A, et al. Diagnosis of ‘possible’
ies are rarely used. We also reviewed the most common syn- mitochondrial disease: an existential crisis. J Med Genet.
dromic and nonsyndromic presentations of PMDs seen in 2019;56(3):123–130
neonates. We briefly reviewed the current treatment ap- 13. Parikh S, Goldstein A, Koenig MK, et al. Diagnosis and
proaches used in clinical practice. As more studies are con- management of mitochondrial disease: a consensus statement
from the Mitochondrial Medicine Society. Genet Med.
ducted in PMDs with special focus on neonatal presentations,
2015;17(9):689–701
we anticipate the diagnosis and treatment of these disorders
14. Haas RH, Parikh S, Falk MJ, et al; Mitochondrial Medicine
to become more straightforward and effective. Subsequently Society’s Committee on Diagnosis. The in-depth evaluation of
the prognosis for these disorders will improve. suspected mitochondrial disease. Mol Genet Metab.
2008;94(1):16–37
15. Honzik T, Tesarova M, Magner M, et al. Neonatal onset of
mitochondrial disorders in 129 patients: clinical and laboratory
American Board of Pediatrics characteristics and a new approach to diagnosis. J Inherit Metab
Dis. 2012;35(5):749–759
Neonatal-Perinatal Content 16. Witters P, Saada A, Honzik T, et al. Revisiting mitochondrial
Specifications diagnostic criteria in the new era of genomics. Genet Med.
2018;20(4):444–451
• Know the etiology, clinical manifestations, laboratory
17. Murayama K, Shimura M, Liu Z, Okazaki Y, Ohtake A. Recent
features, and management of infants with lysosomal,
topics: the diagnosis, molecular genesis, and treatment of
peroxisomal, and mitochondrial disorders.
mitochondrial diseases. J Hum Genet. 2019;64(2):113–125
• Know the causes and differential diagnosis of meta-
18. Imai-Okazaki A, Kishita Y, Kohda M, et al. Barth syndrome:
bolic encephalopathy. different approaches to diagnosis. J Pediatr. 2018;193:256–260
19. Clarke SL, Bowron A, Gonzalez IL, et al. Barth syndrome.
Orphanet J Rare Dis. 2013;8:23

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e807

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 20. Maresca A, Del Dotto V, Romagnoli M, et al. Expanding and value of muscle and skin biopsies in the genetics era.
validating the biomarkers for mitochondrial diseases. J Mol Med Neuropediatrics. 2017;48(4):309–314
(Berl). 2020;98(10):1467–1478 39. Riley LG, Cowley MJ, Gayevskiy V, et al. The diagnostic utility of
21. Forsstr€
om S, Jackson CB, Carroll CJ, et al. Fibroblast growth factor genome sequencing in a pediatric cohort with suspected
21 drives dynamics of local and systemic stress responses in mitochondrial disease. Genet Med. 2020;22(7):1254–1261
mitochondrial myopathy with mtDNA deletions. Cell Metab.
40. Rius R, Compton AG, Baker NL, et al. Application of genome
2019;30(6):1040–1054.e7
sequencing from blood to diagnose mitochondrial diseases. Genes
22. Johann K, Kleinert M, Klaus S. The role of GDF15 as a (Basel). 2021;12(4):607 10.3390/genes12040607
myomitokine. Cells. 2021;10(11):2990 10.3390/cells10112990
41. Raymond FL, Horvath R, Chinnery PF. First-line genomic
23. Chung HK, Ryu D, Kim KS, et al. Growth differentiation factor 15 diagnosis of mitochondrial disorders. Nat Rev Genet.
is a myomitokine governing systemic energy homeostasis. J Cell 2018;19(7):399–400
Biol. 2017;216(1):149–165
42. Husami A, Slone J, Brown J, Bromwell M, Valencia CA, Huang T.
24. Tezze C, Romanello V, Sandri M. FGF21 as modulator of Clinical utility of whole genome sequencing for the detection of
metabolism in health and disease. Front Physiol. 2019;10:419 mitochondrial genome mutations. J Genet Genomics.
25. Lehtonen JM, Auranen M, Darin N, et al. Diagnostic value of 2020;47(3):167–169
serum biomarkers FGF21 and GDF15 compared to muscle sample
43. Schon KR, Horvath R, Wei W, et al; Genomics England Research
in mitochondrial disease. J Inherit Metab Dis. 2021;44(2):469–480
Consortium. Use of whole genome sequencing to determine
26. Zeviani M, Carelli V. Mitochondrial retinopathies. Int J Mol Sci. genetic basis of suspected mitochondrial disorders: cohort study.
2021;23(1):210 10.3390/ijms23010210 BMJ. 2021;375:e066288
27. Hutchin TP, Cortopassi GA. Mitochondrial defects and hearing 44. Turro E, Astle WJ, Megy K, et al; NIHR BioResource for the
loss. Cell Mol Life Sci. 2000;57(13-14):1927–1937 100,000 Genomes Project. Whole-genome sequencing of patients
28. Alves CAPF, Gonçalves FG, Grieb D, Lucato LT, Goldstein AC, with rare diseases in a national health system. Nature.
Zuccoli G. Neuroimaging of mitochondrial cytopathies. Top Magn 2020;583(7814):96–102
Reson Imaging. 2018;27(4):219–240 45. Ouyang X, Zhang Y, Zhang L, et al. Clinical utility of rapid exome
29. Tolomeo D, Orsucci D, Nesti C, et al. The diagnostic approach to sequencing combined with mitochondrial DNA sequencing in
mitochondrial disorders in children in the era of next-generation critically ill pediatric patients with suspected genetic disorders.
sequencing: A 4-year cohort study. J Clin Med. 2021;10(15):3222 Front Genet. 2021;12:725259
10.3390/jcm10153222
46. Akesson LS, Eggers S, Love CJ, et al. Early diagnosis of Pearson
30. Pillen S, Morava E, Van Keimpema M, et al. Skeletal muscle syndrome in neonatal intensive care following rapid mitochondrial
ultrasonography in children with a dysfunction in the oxidative genome sequencing in tandem with exome sequencing. Eur J Hum
phosphorylation system. Neuropediatrics. 2006;37(3):142–147 Genet. 2019;27(12):1821–1826
31. Wortmann SB, Koolen DA, Smeitink JA, van den Heuvel L, 47. de Laat P, Koene S, van den Heuvel LP, Rodenburg RJ, Janssen
Rodenburg RJ. Whole exome sequencing of suspected MC, Smeitink JA. Clinical features and heteroplasmy in blood,
mitochondrial patients in clinical practice. J Inherit Metab Dis. urine and saliva in 34 Dutch families carrying the m.3243A > G
2015;38(3):437–443
mutation. J Inherit Metab Dis. 2012;35(6):1059–1069
32. Lieber DS, Calvo SE, Shanahan K, et al. Targeted exome sequencing
48. Hardy SA, Blakely EL, Purvis AI, et al. Pathogenic mtDNA
of suspected mitochondrial disorders. Neurology.
mutations causing mitochondrial myopathy: The need for muscle
2013;80(19):1762–1770
biopsy. Neurol Genet. 2016;2(4):e82
33. Pronicka E, Piekutowska-Abramczuk D, Ciara E, et al. New
49. von Kleist-Retzow JC, Cormier-Daire V, Viot G, et al. Antenatal
perspective in diagnostics of mitochondrial disorders: two years’
manifestations of mitochondrial respiratory chain deficiency. J
experience with whole-exome sequencing at a national paediatric
Pediatr. 2003;143(2):208–212
centre. J Transl Med. 2016;14(1):174
50. Tavares MV, Santos MJ, Domingues AP, et al. Antenatal
34. Hu C, Li X, Zhao L, et al. Clinical and molecular characterization
manifestations of mitochondrial disorders. J Inherit Metab Dis.
of pediatric mitochondrial disorders in south of China. Eur J Med
2013;36(5):805–811
Genet. 2020;63(8):103898
51. Gibson K, Halliday JL, Kirby DM, Yaplito-Lee J, Thorburn DR,
35. Ohtake A, Murayama K, Mori M, et al. Diagnosis and molecular
basis of mitochondrial respiratory chain disorders: exome Boneh A. Mitochondrial oxidative phosphorylation disorders
sequencing for disease gene identification. Biochim Biophys Acta. presenting in neonates: clinical manifestations and enzymatic and
2014;1840(4):1355–1359 molecular diagnoses. Pediatrics. 2008;122(5):1003–1008

36. Taylor RW, Pyle A, Griffin H, et al. Use of whole-exome 52. Gire C, Girard N, Nicaise C, Einaudi MA, Montfort MF, Dejode
sequencing to determine the genetic basis of multiple JM. Clinical features and neuroradiological findings of
mitochondrial respiratory chain complex deficiencies. JAMA. mitochondrial pathology in six neonates. Childs Nerv Syst.
2014;312(1):68–77 2002;18(11):621–628
37. Cui H, Li F, Chen D, et al. Comprehensive next-generation 53. Steward CG, Newbury-Ecob RA, Hastings R, et al. Barth syndrome:
sequence analyses of the entire mitochondrial genome reveal new an X-linked cause of fetal cardiomyopathy and stillbirth. Prenat
insights into the molecular diagnosis of mitochondrial DNA Diagn. 2010;30(10):970–976
disorders. Genet Med. 2013;15(5):388–394 54. Chung J, Lee MY, Chung JH, Won HS. Extremely Rare Case of
38. Wortmann SB, Mayr JA, Nuoffer JM, Prokisch H, Sperl W. A Fetal Anemia Due to Mitochondrial Disease Managed with
guideline for the diagnosis of pediatric mitochondrial disease: the Intrauterine Transfusion. Medicina (Kaunas). 2022;58(3):328

e808 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 55. Wild KT, Goldstein AC, Muraresku C, Ganetzky RD. Broadening 73. Bakare AB, Lesnefsky EJ, Iyer S. Leigh syndrome: a tale of two
the phenotypic spectrum of Pearson syndrome: Five new cases and genomes. Front Physiol. 2021;12:693734
a review of the literature. Am J Med Genet A. 2020;182(2):365–373 74. Sofou K, De Coo IF, Isohanni P, et al. A multicenter study on
56. Ebihara T, Nagatomo T, Sugiyama Y, et al. Neonatal-onset Leigh syndrome: disease course and predictors of survival.
mitochondrial disease: clinical features, molecular diagnosis and Orphanet J Rare Dis. 2014;9:52
prognosis. Arch Dis Child Fetal Neonatal Ed. 2022;107(3):329–334 75. Malfatti E, Bugiani M, Invernizzi F, et al. Novel mutations of ND
57. del Mar O’Callaghan M, Emperador S, Lopez-Gallardo E, et al. genes in complex I deficiency associated with mitochondrial
New mitochondrial DNA mutations in tRNA associated with three encephalopathy. Brain. 2007;130(Pt 7):1894–1904
severe encephalopamyopathic phenotypes: neonatal, infantile, and 76. Chang X, Wu Y, Zhou J, Meng H, Zhang W, Guo J. A meta-
childhood onset. Neurogenetics. 2012;13(3):245–250 analysis and systematic review of Leigh syndrome: clinical
58. Ware SM, El-Hassan N, Kahler SG, et al. Infantile cardiomyopathy manifestations, respiratory chain enzyme complex deficiency, and
caused by a mutation in the overlapping region of mitochondrial gene mutations. Medicine (Baltimore). 2020;99(5):e18634
ATPase 6 and 8 genes. J Med Genet. 2009;46(5):308–314 77. Leshinsky-Silver E, Lev D, Tzofi-Berman Z, et al. Fulminant
59. Uziel G, Ghezzi D, Zeviani M. Infantile mitochondrial neurological deterioration in a neonate with Leigh syndrome due to
encephalopathy. Semin Fetal Neonatal Med. 2011;16(4):205–215 a maternally transmitted missense mutation in the mitochondrial
60. El-Hattab AW, Craigen WJ, Scaglia F. Mitochondrial DNA ND3 gene. Biochem Biophys Res Commun. 2005;334(2):582–587
maintenance defects. Biochim Biophys Acta Mol Basis Dis. 2017; 78. Bolea I, Gella A, Sanz E, et al. Defined neuronal populations drive
1863(6):1539–1555 fatal phenotype in a mouse model of Leigh syndrome. Elife. 2019;
61. Ram on J, Vila-Julia F, Molina-Granada D, et al. Therapy prospects 8:e47163
for mitochondrial DNA Maintenance Disorders. Int J Mol Sci. 79. Uittenbogaard M, Sen K, Whitehead M, et al. Genetic and
2021;22(12):6447 mitochondrial metabolic analyses of an atypical form of Leigh
62. Dai H, Zhang VW, El-Hattab AW, et al. FBXL4 defects are common syndrome. Front Cell Dev Biol. 2021;9:767407
in patients with congenital lactic acidemia and encephalomyopathic 80. Lake NJ, Compton AG, Rahman S, Thorburn DR. Leigh syndrome:
mitochondrial DNA depletion syndrome. Clin Genet. 2017;91(4): one disorder, more than 75 monogenic causes. Ann Neurol. 2016;
634–639 79(2):190–203
63. Keshavan N, Abdenur J, Anderson G, et al. The natural history of 81. Sofou K, de Coo IFM, Ostergaard E, et al. Phenotype-genotype
infantile mitochondrial DNA depletion syndrome due to RRM2B correlations in Leigh syndrome: new insights from a multicentre
deficiency. Genet Med. 2020;22(1):199–209 study of 96 patients. J Med Genet. 2018;55(1):21–27
64. Rahman S, Copeland WC. POLG-related disorders and their 82. Honzik T, Tesarova M, Vinsova K, et al. Different laboratory and
neurological manifestations. Nat Rev Neurol. 2019;15(1):40–52 muscle biopsy findings in a family with an m.8851T>C mutation
65. Nogueira C, de Souza CF, Husny A, Derks TG, Santorelli FM, in the mitochondrial MTATP6 gene. Mol Genet Metab. 2013;
Vilarinho L. MPV17: fatal hepatocerebral presentation in a Brazilian 108(1):102–105
infant. Mol Genet Metab. 2012;107(4):764 83. Farruggia P, Di Marco F, Dufour C. Pearson syndrome. Expert Rev
66. Franklin AD, Chaudhari BP, Koboldt DC, Machut KZ. Polymerase Hematol. 2018;11(3):239–246
gamma mitochondrial DNA depletion syndrome initially 84. Broomfield A, Sweeney MG, Woodward CE, et al. Paediatric single
presenting as disproportionate respiratory distress in a moderately mitochondrial DNA deletion disorders: an overlapping spectrum of
premature neonate: a case report. Front Genet. 2021;12:664278 disease. J Inherit Metab Dis. 2015;38(3):445–457
67. Shimura M, Kuranobu N, Ogawa-Tominaga M, et al. Clinical and 85. Farruggia P, Di Cataldo A, Pinto RM, et al. Pearson syndrome: a
molecular basis of hepatocerebral mitochondrial DNA depletion retrospective cohort study from the Marrow Failure Study Group of
syndrome in Japan: evaluation of outcomes after liver A.I.E.O.P. (Associazione Italiana Emato-Oncologia Pediatrica).
transplantation. Orphanet J Rare Dis. 2020;15(1):169 JIMD Rep. 2016;26:37–43
68. El-Hattab AW, Wang J, Dai H, et al. MPV17-related mitochondrial 86. Tadiotto E, Maines E, Degani D, Balter R, Bordugo A, Cesaro S.
DNA maintenance defect: New cases and review of clinical, Bone marrow features in Pearson syndrome with neonatal onset: A
biochemical, and molecular aspects. Hum Mutat. 2018;39(4): case report and review of the literature. Pediatr Blood Cancer. 2018;
461–470 65(4) 10.1002/pbc.26939
69. Mahjoub G, Habibzadeh P, Dastsooz H, et al. Clinical and 87. Crippa BL, Leon E, Calhoun A, Lowichik A, Pasquali M, Longo N.
molecular characterization of three patients with Hepatocerebral Biochemical abnormalities in Pearson syndrome. Am J Med Genet
form of mitochondrial DNA depletion syndrome: a case series. A. 2015;167A(3):621–628
BMC Med Genet. 2019;20(1):167 88. Son JS, Seo GH, Kim YM, et al. Clinical and genetic features of
70. Huang AC, Ebel NH, Romero D, et al. Outcomes after liver four patients with Pearson syndrome: An observational study.
transplantation in MPV17 deficiency (Navajo neurohepatopathy): a Medicine (Baltimore). 2022;101(5):e28793
single-center case series. Pediatr Transplant. 2022;26(5):e14274 89. Collin-Ducasse H, Maillotte AM, Monpoux F, Boutte P, Ferrero-
71. Garone C, Taylor RW, Nascimento A, et al. Retrospective natural Vacher C, Paquis V. Neonatal Pearson syndrome: two case studies
history of thymidine kinase 2 deficiency. J Med Genet. [article in French]. Arch Pediatr. 2010;17(1):38–41
2018;55(8):515–521 90. Goldstein A, Falk MJ. Mitochondrial DNA Deletion Syndromes.
72. Berardo A, Domınguez-Gonzalez C, Engelstad K, Hirano M. 2003 Dec 17 [Updated 2019 Jan 31]. In: Adam MP, Everman DB,
Advances in thymidine kinase 2 deficiency: clinical aspects, Mirzaa GM, et al., editors. GeneReviewsVR [Internet]. Seattle (WA):

translational progress, and emerging therapies. J Neuromuscul Dis. University of Washington, Seattle; 1993–2022. Available from:
2022;9(2):225–235 https://www.ncbi.nlm.nih.gov/books/NBK1203/

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e809

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 91. Roberts AE, Nixon C, Steward CG, et al. The Barth Syndrome neonatal mitochondrial encephalomyopathy. J Med Genet. 2015;
Registry: distinguishing disease characteristics and growth data 52(9):627–635
from a longitudinal study. Am J Med Genet A. 2012;158A(11): 109. Smith AC, Ito Y, Ahmed A, et al; Care4Rare Canada Consortium.
2726–2732 A family segregating lethal neonatal coenzyme Q10 deficiency
92. Hikmat O, Isohanni P, Keshavan N, et al. Expanding the caused by mutations in COQ9. J Inherit Metab Dis. 2018;41(4):
phenotypic spectrum of BCS1L-related mitochondrial disease. Ann 719–729
Clin Transl Neurol. 2021;8(11):2155–2165
110. Kwong AK, Chiu AT, Tsang MH, et al. A fatal case of COQ7-
93. Fellman V, Lemmel€a S, Sajantila A, Pihko H, J€arvel€a I. Screening associated primary coenzyme Q10 deficiency. JIMD Rep.
of BCS1L mutations in severe neonatal disorders suspicious for 2019;47(1):23–29
mitochondrial cause. J Hum Genet. 2008;53(6):554–558
111. Hashemi SS, Zare-Abdollahi D, Bakhshandeh MK, et al. Clinical
94. Serdaro
glu E, Takcõ Ş, Kotarsky H, et al. A Turkish BCS1L spectrum in multiple families with primary COQ10 deficiency. Am
mutation causes GRACILE-like disorder. Turk J Pediatr. 2016;58(6): J Med Genet A. 2021;185(2):440–452
658–661
112. Drovandi S, Lipska-ZieR tkiewicz BS, Ozaltin F, et al; PodoNet
95. Olahova M, Ceccatelli Berti C, Collier JJ, et al. Molecular genetic Consortium; mitoNET Consortium; CCGKDD Consortium.
investigations identify new clinical phenotypes associated with Oral coenzyme Q10 supplementation leads to better
BCS1L-related mitochondrial disease. Hum Mol Genet. 2019;28(22): preservation of kidney function in steroid-resistant nephrotic
3766–3776
syndrome due to primary coenzyme Q10 deficiency. Kidney Int.
96. Baker RA, Priestley JRC, Wilstermann AM, Reese KJ, Mark PR. 2022;102(3):604–612
Clinical spectrum of BCS1L mitopathies and their underlying
113. Lu M, Zhou Y, Wang Z, Xia Z, Ren J, Guo Q. Clinical phenotype,
structural relationships. Am J Med Genet A. 2019;179(3):373–380
in silico and biomedical analyses, and intervention for an East
97. de Lonlay P, Valnot I, Barrientos A, et al. A mutant mitochondrial Asian population-specific c.370G>A (p.G124S) COQ4 mutation in
respiratory chain assembly protein causes complex III deficiency in a Chinese family with CoQ10 deficiency-associated Leigh
patients with tubulopathy, encephalopathy and liver failure. Nat syndrome. J Hum Genet. 2019;64(4):297–304
Genet. 2001;29(1):57–60
114. Nguyen T, Jeyakumar A. Genetic susceptibility to aminoglycoside
98. Kanako KI, Sakakibara N, Murayama K, et al. BCS1L mutations ototoxicity. Int J Pediatr Otorhinolaryngol. 2019;120:15–19
produce Fanconi syndrome with developmental disability. J Hum
115. Fischel-Ghodsian N, Prezant TR, Fournier P, Stewart IA, Maw M.
Genet. 2022;67(3):143–148
Mitochondrial mutation associated with nonsyndromic deafness.
99. Coenen MJ, Antonicka H, Ugalde C, et al. Mutant mitochondrial Am J Otolaryngol. 1995;16(6):403–408
elongation factor G1 and combined oxidative phosphorylation
116. Rizzi MD, Hirose K. Aminoglycoside ototoxicity. Curr Opin
deficiency. N Engl J Med. 2004;351(20):2080–2086
Otolaryngol Head Neck Surg. 2007;15(5):352–357
100. Smits P, Mattijssen S, Morava E, et al. Functional consequences of
mitochondrial tRNA Trp and tRNA Arg mutations causing 117. Men M, Jiang L, Wang H, et al. Unique penetrance of hearing loss
combined OXPHOS defects. Eur J Hum Genet. 2010;18(3):324–329 in a five-generation Chinese family with the mitochondrial 12S
rRNA 1555A > G mutation. Acta Otolaryngol. 2011;131(9):970–975
101. Paramasivam A, Meena AK, Venkatapathi C, Pitceathly RDS,
Thangaraj K. Novel biallelic NSUN3 variants cause early-onset 118. Diepstraten FA, Hoetink AE, van Grotel M, et al. Aminoglycoside-
mitochondrial encephalomyopathy and seizures. J Mol Neurosci. and glycopeptide-induced ototoxicity in children: a systematic
2020;70(12):1962–1965 review. JAC Antimicrob Resist. 2021;3(4):dlab184

102. Diodato D, Tasca G, Verrigni D, et al. A novel AIFM1 mutation 119. O’Sullivan ME, Perez A, Lin R, Sajjadi A, Ricci AJ, Cheng AG.
expands the phenotype to an infantile motor neuron disease. Eur J Towards the prevention of aminoglycoside-related hearing loss.
Hum Genet. 2016;24(3):463–466 Front Cell Neurosci. 2017;11:325
103. Kopajtich R, Nicholls TJ, Rorbach J, et al. Mutations in GTPBP3 120. Zhao H, Li R, Wang Q, et al. Maternally inherited aminoglycoside-
cause a mitochondrial translation defect associated with induced and nonsyndromic deafness is associated with the novel
hypertrophic cardiomyopathy, lactic acidosis, and encephalopathy. C1494T mutation in the mitochondrial 12S rRNA gene in a large
Am J Hum Genet. 2014;95(6):708–720 Chinese family. Am J Hum Genet. 2004;74(1):139–152
104. Eldomery MK, Akdemir ZC, V€ ogtle FN, et al. MIPEP recessive 121. Lanvers-Kaminsky C, Ciarimboli G. Pharmacogenetics of drug-
variants cause a syndrome of left ventricular non-compaction, induced ototoxicity caused by aminoglycosides and cisplatin.
hypotonia, and infantile death. Genome Med. 2016;8(1):106 Pharmacogenomics. 2017;18(18):1683–1695
105. Abu Diab A, AlTalbishi A, Rosin B, et al. The combination of 122. McDermott JH, Wolf J, Hoshitsuki K, et al. Clinical
whole-exome sequencing and clinical analysis allows better pharmacogenetics implementation consortium guideline for the
diagnosis of rare syndromic retinal dystrophies. Acta Ophthalmol. use of aminoglycosides based on MT-RNR1 genotype. Clin
2019;97(6):e877–e886 Pharmacol Ther. 2022;111(2):366–372
106. Acosta MJ, Vazquez Fonseca L, Desbats MA, et al. Coenzyme Q 123. Bottani E, Lamperti C, Prigione A, Tiranti V, Persico N, Brunetti
biosynthesis in health and disease. Biochim Biophys Acta. 2016; D. Therapeutic approaches to treat mitochondrial diseases: “one-
1857(8):1079–1085 size-fits-all” and “precision medicine” strategies. Pharmaceutics.
107. Alcazar-Fabra M, Trevisson E, Brea-Calvo G. Clinical syndromes 2020;12(11):E1083
associated with coenzyme Q10 deficiency. Essays Biochem. 2018; 124. Tragni V, Primiano G, Tummolo A, et al. Personalized medicine in
62(3):377–398 mitochondrial health and disease: molecular basis of therapeutic
108. Chung WK, Martin K, Jalas C, et al. Mutations in COQ4, an approaches based on nutritional supplements and their analogs.
essential component of coenzyme Q biosynthesis, cause lethal Molecules. 2022;27(11):3494

e810 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 125. Barcelos I, Shadiack E, Ganetzky RD, Falk MJ. Mitochondrial 138. Smeitink J, van Maanen R, de Boer L, Ruiterkamp G, Renkema
medicine therapies: rationale, evidence, and dosing guidelines. H. A randomised placebo-controlled, double-blind phase II study
Curr Opin Pediatr. 2020;32(6):707–718 to explore the safety, efficacy, and pharmacokinetics of
126. Rinninella E, Pizzoferrato M, Cintoni M, Servidei S, Mele MC. sonlicromanol in children with genetically confirmed
Nutritional support in mitochondrial diseases: the state of the art. mitochondrial disease and motor symptoms (“KHENERGYC”).
Eur Rev Med Pharmacol Sci. 2018;22(13):4288–4298 BMC Neurol. 2022;22(1):158
127. Karaa A, Kriger J, Grier J, et al. Mitochondrial disease patients’ 139. Singh A, Faccenda D, Campanella M. Pharmacological advances in
perception of dietary supplements’ use. Mol Genet Metab. 2016; mitochondrial therapy. EBioMedicine. 2021;65:103244
119(1-2):100–108 140. Beyrath J, Pellegrini M, Renkema H, et al. KH176 safeguards
128. Tarnopolsky MA. The mitochondrial cocktail: rationale for mitochondrial diseased cells from redox stress-induced cell death
combined nutraceutical therapy in mitochondrial cytopathies. Adv by interacting with the thioredoxin system/peroxiredoxin enzyme
Drug Deliv Rev. 2008;60(13-14):1561–1567 machinery. Sci Rep. 2018;8(1):6577
129. Rodriguez MC, MacDonald JR, Mahoney DJ, Parise G, Beal MF, 141. Birk AV, Liu S, Soong Y, et al. The mitochondrial-targeted
Tarnopolsky MA. Beneficial effects of creatine, CoQ10, and lipoic compound SS-31 re-energizes ischemic mitochondria by interacting
acid in mitochondrial disorders. Muscle Nerve. 2007;35(2):235–242 with cardiolipin. J Am Soc Nephrol. 2013;24(8):1250–1261
130. Distelmaier F, Haack TB, Wortmann SB, Mayr JA, Prokisch H. 142. Allen ME, Pennington ER, Perry JB, et al. The cardiolipin-binding
Treatable mitochondrial diseases: cofactor metabolism and beyond. peptide elamipretide mitigates fragmentation of cristae networks
Brain. 2017;140(2):e11 following cardiac ischemia reperfusion in rats. Commun Biol.
131. Friederich MW, Timal S, Powell CA, et al. Pathogenic variants in 2020;3(1):389
glutamyl-tRNAGln amidotransferase subunits cause a lethal mitochondrial 143. Koene S, Spaans E, Van Bortel L, et al. KH176 under development
cardiomyopathy disorder. Nat Commun. 2018;9(1):4065 for rare mitochondrial disease: a first in man randomized
132. Zimmern V, Minassian B, Korff C. A Review of targeted therapies controlled clinical trial in healthy male volunteers. Orphanet J Rare
for monogenic epilepsy syndromes. Front Neurol. 2022;13:829116 Dis. 2017;12(1):163
133. Rotondo E, Riva A, Graziosi A, et al. Non-pharmacological 144. Janssen MCH, Koene S, de Laat P, et al. The KHENERGY Study:
treatments for pediatric refractory epilepsies. Expert Rev Neurother. Safety and efficacy of KH176 in mitochondrial m.3243A>G
2022;22(4):337–349 spectrum disorders. Clin Pharmacol Ther. 2019;105(1):101–111
134. Inui T, Wada Y, Shibuya M, et al. Intravenous ketogenic diet 145. Reid Thompson W, Hornby B, Manuel R, et al. A phase 2/3
therapy for neonatal-onset pyruvate dehydrogenase complex randomized clinical trial followed by an open-label extension to
deficiency. Brain Dev. 2022;44(3):244–248 evaluate the effectiveness of elamipretide in Barth syndrome, a
135. Zweers H, van Wegberg AMJ, Janssen MCH, Wortmann SB. genetic disorder of mitochondrial cardiolipin metabolism. Genet
Ketogenic diet for mitochondrial disease: a systematic review on Med. 2021;23(3):471–478
efficacy and safety. Orphanet J Rare Dis. 2021;16(1):295 146. Karaa A, Haas R, Goldstein A, Vockley J, Cohen BH. A
136. Ahola S, Auranen M, Isohanni P, et al. Modified Atkins diet randomized crossover trial of elamipretide in adults with
induces subacute selective ragged-red-fiber lysis in mitochondrial primary mitochondrial myopathy. J Cachexia Sarcopenia Muscle.
myopathy patients. EMBO Mol Med. 2016;8(11):1234–1247 2020;11(4):909–918
137. Qu C, Keijer J, Adjobo-Hermans MJW, et al. The ketogenic diet as 147. El-Hattab AW, Zarante AM, Almannai M, Scaglia F. Therapies for
a therapeutic intervention strategy in mitochondrial disease. Int J mitochondrial diseases and current clinical trials. Mol Genet Metab.
Biochem Cell Biol. 2021;138:106050 2017;122(3):1–9

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e811

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 NEOREVIEWS QUIZ

NEO
QUIZ
1. Primary mitochondrial disorders (PMDs) are a heterogeneous group of diseases
that can present in the neonatal period. Pathogenic variants in both nuclear
DNA (nDNA) and mitochondrial DNA can cause PMD. What proportion of
PMDs found in pediatric patients are caused by nDNA mutations?

A. 20%.
B. 35%.
C. 50%.
D. 65%
E. 80%.
2. PMDs are among the most common inherited metabolic disorders and have
a variable age onset across the lifespan. Up to 30% of PMDs present in the REQUIREMENTS: Learners can
neonatal period, most commonly within the first 48 hours after birth. What is take NeoReviews quizzes and
the prevalence of PMDs in children? claim credit online only at:
https://publications.aap.org/
A. 1 in 2,500 children. neoreviews.
B. 1 in 6,000 children.
C. 1 in 25,000 children. To successfully complete 2022
NeoReviews articles for AMA PRA
D. 1 in 50,000 children.
Category 1 Credit™, learners
E. 1 in 100,000 children. must demonstrate a minimum
3. The diagnosis of PMDs can be challenging because of nonspecific signs and performance level of 60% or
symptoms that can overlap with other common conditions including higher on this assessment. If
you score less than 60% on the
hypoxic-ischemic encephalopathy, sepsis, and cardiorespiratory failure as
assessment, you will be given
well as lack of specific and sensitive diagnostic biomarkers. Which of the additional opportunities to
following findings should alert clinicians of the possibility of a PMD? answer questions until an
overall 60% or greater score is
A. A high blood and cerebrospinal fluid lactate-to-pyruvate ratio.
achieved.
B. A low blood alanine level.
C. The presence of N-acetyl-aspartate peak on magnetic resonance spectroscopy. This journal-based CME activity
D. A prenatal history of severe hydronephrosis. is available through Dec. 31,
E. A low urine 3-methylglutaconic acid level. 2024, however, credit will be
recorded in the year in which
4. A 2-week-old term neonate is hospitalized for poor weight gain and recurrent the learner completes the quiz.
emesis. A significant physical examination finding is hypotonia. Brain magnetic
resonance imaging reveals symmetric basal ganglia and thalami lesions. What
is this neonate’s most likely diagnosis?
A. Pearson syndrome.
B. Barth syndrome.
C. Leigh syndrome. 2022 NeoReviews is approved
for a total of 30 Maintenance of
D. Encephalomyopathic mitochondrial DNA depletion syndrome.
Certification (MOC) Part 2
E. Combined oxidative phosphorylation deficiency. credits by the American Board
5. A 40-week-gestation male infant is admitted to your NICU with heart failure of Pediatrics (ABP) through the
AAP MOC Portfolio Program.
secondary to cardiomyopathy. You suspect Barth syndrome. Which of the
NeoReviews subscribers can
following statements regarding Barth syndrome is correct? claim up to 30 ABP MOC Part 2
A. Most patients with Barth syndrome present with cardiomyopathy between points upon passing 30 quizzes
birth and 1 month of age. (and claiming full credit for
each quiz) per year. Subscribers
B. Hypertrophic cardiomyopathy is the most common form of cardiomyopathy
can start claiming MOC credits
reported in Barth syndrome. as early as October 2022. To
C. Barth syndrome is inherited as an autosomal recessive disorder. learn how to claim MOC points,
D. Neutropenia affects 90% of patients with Barth syndrome. go to: https://publications.aap.
E. The presence of distal hypotonia is typical. org/journals/pages/moc-credit.

e812 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 ARTICLE

Congenital Disorders of Red Blood Cells

Rhucha Joshi, MD,* Erin Myers, MD,† Artemiy Kokhanov, MD‡
*
Division of Neonatal Medicine, Department of Pediatrics, University of California Irvine, Irvine, CA
†
Department of Pediatrics, University of California Irvine, Irvine, CA
‡
Department of Neonatology, MemorialCare Miller Children’s and Women’s Hospital Long Beach, Long Beach, CA

PRACTICE GAPS

Normal hematopoiesis and red blood cell physiology are fundamental to

neonatal health. Neonatal clinicians should understand physiologic fetal
and neonatal hematopoiesis and be aware of the various disorders
associated with abnormal structure and function of red blood cells.

AUTHOR DISCLOSURES Drs Joshi, Myers,

OBJECTIVES After completing this review, readers should be able to: and Kokhanov have disclosed no financial
relationships relevant to this article. This
commentary does not contain a
1. Explain the process of red blood cell hematopoiesis in the fetus and neonate. discussion of an unapproved/
investigative use of a commercial
2. Describe the clinical features of various inherited disorders of red blood product/device.
cells as they relate to the neonate.
3. Recognize the importance of timely recognition and prompt management ABBREVIATIONS
of red blood cell disorders. ALAS2 d-aminolevulinate synthase 2
ATP adenosine triphosphate
4. Recognize the role of genetic counseling in inherited disorders of red DBA Diamond-Blackfan anemia
blood cell production. DC dyskeratosis congenita
EMA eosin-50 -maleimide
EPO erythropoietin
FA Fanconi anemia
ABSTRACT G6PD glucose-6-phosphate
Understanding the physiologic process of red blood cell development dehydrogenase
G-CSF granulocyte–colony-stimulating
in utero and subsequent erythropoiesis in the neonate is crucial as
factor
this determines red blood cell structure and therefore function, Hb hemoglobin
which is vital to neonatal health. Infants frequently experience anemia, HE hereditary elliptocytosis
and special consideration must be given to the evaluation of these infants HPP hereditary pyropoikilocytosis
HS hereditary spherocytosis
to determine the correct etiology. Traditionally, anemia is conceptualized NADH nicotinamide adenine
in terms of inadequate red blood cell production, increased red blood cell dinucleotide (reduced)
destruction, or whole blood loss. This framework translates well to NADPH nicotinamide adenine
dinucleotide phosphate
inherited red blood cell defects, which include genetic abnormalities in (reduced)
bone marrow productivity or structure of the red blood cell membrane, OMIM Online Mendelian Inheritance in
enzymes, or hemoglobin. This article highlights fetal and neonatal Man
PK pyruvate kinase
erythropoiesis and the underlying etiologies of the inherited red blood cell
RBC red blood cell
disorders, as well as reviews the appropriate diagnostic evaluation and SCD sickle cell disease
next steps in management. It is imperative that neonatal clinicians remain SDS Shwachman-Diamond
informed about these disorders to enable early recognition and treatment, syndrome
TAR thrombocytopenia absent
and ultimately to improve outcomes in affected infants. radius
XLSA X-linked sideroblastic anemia

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e813

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 INTRODUCTION and later moving to the liver (6–30 weeks’ gestation) and
Red blood cell (RBC) integrity and function are vital to spleen (9–28 weeks’ gestation). At 28 weeks’ gestation,
neonatal health because RBCs enable oxygen delivery to the bone marrow becomes the primary site for erythropoi-
tissues, and therefore support the aerobic metabolism of esis. (4) The formation of RBCs in the early embryo be-
every organ system in the neonate. Furthermore, anemia gins with yolk sac mesodermal cells and is referred to as
is the most common hematologic abnormality in the primitive erythropoiesis. All subsequent RBC formation be-
NICU. (1) Preterm infants in particular are at high risk of gins with hematopoietic stem cells and is known as defini-
developing anemia for various reasons, including anemia tive erythropoiesis. Hematopoietic stem cells are differentiated
of prematurity, iatrogenic blood loss, suboptimal nutrition, into erythrocytes in multiple stages (Figure 1). Mature eryth-
and losses associated with clinical decompensation such rocytes do not contain nuclei or organelles, so hemoglobin
as sepsis. (2) Anemia in the neonate is defined as a hemoglo- synthesis occurs in the precursor cells. Maturation of erythro-
bin value or hematocrit concentration greater than 2 standard cyte precursors involves proliferation through cell division, in-
deviations below the normal mean for postnatal age. While crease in hemoglobin synthesis, decrease in cell size,
physiologic anemia of the newborn and anemia of prematu- degeneration of nuclei, and decrease in cytoplasmic RNA. (5)
rity are the most common causes of neonatal anemia, neona-
tal clinicians must remain vigilant for additional causes of Hemoglobin Synthesis
anemia and other RBC disorders caused by associated co- Hemoglobin is a tetramer composed of 4 globin chains,
morbidities and optimization of treatment. (3) It is prudent each containing a heme molecule. Each heme molecule is
for NICU clinicians to be familiar with fetal hematopoiesis a protoporphyrin ring with iron (Fe21) in the center,
and its regulation as well as inherited disorders of RBCs. which facilitates reversible binding of oxygen. (6) There
Conceptually, anemia can be divided into instances of are 6 types of globin chains. Genes that code for a-globin
inadequate RBC production, increased RBC destruction, (HBA1, HBA2) and z-globin (HBZ) are located on chromo-
or whole blood loss (the last etiology being outside the some 16. Genes that code for b-globin (HBB), g-globin
scope of this article). Anemia secondary to suboptimal pro- (HBG1, HBG2), d-globin (HBD), and e-globin (HBE1) are
duction of red cells is often due to congenital genetic dis- located on chromosome 11. Expression of these genes
orders. This disturbance can be with either progenitor varies during embryonic and fetal development and gives
cells causing pancytopenia (eg, Fanconi anemia [FA], rise to different types of hemoglobin, in a process known
Shwachman-Diamond syndrome (SDS), thrombocytopenia as hemoglobin switching (Table 1). HbF is the primary fetal
absent radius syndrome [TAR]) or in the specific erythro- hemoglobin. In late fetal gestation, HbF production de-
cyte cell line (eg, Diamond-Blackfan anemia [DBA]). Ab- creases and HbA production begins. HbA becomes the
normal structure of RBCs (eg, hereditary spherocytosis dominant hemoglobin by 6 months after birth. (7)
[HS], hereditary elliptocytosis [HE], sickle cell anemia) or
abnormal enzymatic function (eg, glucose-6-phosphate de- Regulation of Erythropoiesis
hydrogenase [G6PD] deficiency, pyruvate kinase [PK] defi- Erythropoietin (EPO) is a hormone produced by the kid-
ciency) result in anemia due to malformed or maladapted ney that acts on the bone marrow to upregulate erythro-
RBCs being removed more rapidly from the circulation. poiesis. Hypoxia causes increased EPO production. EPO
The variations in the underlying etiology of RBC malfunc- binds to its receptor, EPOR, leading to a cascade of events
tion have different associated pathologies as well as treat- that allows early release of reticulocytes from bone mar-
ment prognoses. row, decreases the time needed for RBC maturation, and
The goal of this article is to review normal neonatal prevents RBC death. Many transcription factors play key
erythropoiesis and its genetic regulation, highlight key roles in the regulation of erythropoiesis. Some of these in-
characteristics in the underlying genetic etiologies, sum- clude GATA1, LDB1, FOG1, SCL/TAL1, and LMO2. (5)(8)
marize the approach to diagnosis of inherited RBC disor-
ders, and outline appropriate steps in management. REVIEW OF INDIVIDUAL CONDITIONS/
SYNDROMES
OVERVIEW OF ERYTHROPOIESIS Congenital disorders of RBCs can be divided into bone
Erythropoiesis is the process by which RBCs, or erythro- marrow failure syndromes, hemoglobinopathies, sideroblastic
cytes, are formed. Sites of erythropoiesis vary throughout anemia, RBC membrane defects, and RBC enzyme defects
gestation, starting in the yolk sac (3–8 weeks’ gestation) (Figure 2).

e814 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Figure 1. An overview of erythropoiesis. In late fetal gestation and postnatal life, most of this process occurs in the bone marrow. The final two stages,
reticulocyte and erythrocyte, are found in circulating blood.

Bone Marrow Failure Syndromes DNA damage in the presence of DNA cross-linking agents.
Bone marrow failure syndromes are disorders that lead to Many patients with FA develop malignancies in the first 3 deca-
decreased production of 1 or more cell lines. All of the disor- des of life and most have bone marrow failure by 40 years of
ders discussed here involve reduced production of RBCs, age. Supportive management includes transfusions, granulocy-
manifesting as anemia. These syndromes increase predispo- te–colony-stimulating factor (G-CSF), and granulocyte-macro-
sition to malignancies later in life, so early diagnosis is key phage–colony-stimulating factor. Curative treatment is with
to ensuring appropriate surveillance of affected individuals. hematopoietic stem cell transplantation. (11)

Fanconi Anemia Diamond-Blackfan Anemia

Fanconi anemia (FA) (Online Mendelian Inheritance in Man Diamond-Blackfan anemia (DBA) (OMIM: 105650, 612562,
[OMIM]: 227650, 300514, 227645, 614082, 617244, and 613309, and others) is a pure red cell aplasia characterized by
others) is a disorder of chromosomal instability. About one- a decrease in erythrocyte precursors with otherwise normal
third of patients with FA do not display phenotypic features, bone marrow. About 50% of patients have craniofacial abnor-
which can present a challenge for diagnosis. In patients who malities (eg, cleft lip and palate), thumb anomalies, and car-
do have clinical features, the expression can be variable. Ab- diac defects. (12) DBA is caused by mutations in genes that
normalities can include intrauterine growth restriction, skele- code for ribosome proteins. At least 17 genes are implicated,
tal anomalies (eg, thumb malformations, scoliosis), skin including RPS19 (most common), RPL11, and RPS26. Inheri-
dyspigmentation, genitourinary anomalies, cardiac defects, in- tance is mostly autosomal dominant and rarely X-linked reces-
testinal atresias, microcephaly, and hydrocephalus. (9) Muta- sive. Laboratory findings include macrocytic anemia and
tions occur in 1 of 21 genes, including FANCA (most reticulocytopenia. Bone marrow evaluation can differentiate
common), FANCC, and FANCG. Inheritance is autosomal re- DBA from other bone marrow failure syndromes because of
cessive, except for FANCB (which is X-linked recessive) and the normal amounts of myeloid cells (white blood cell precur-
FANCR (which is autosomal dominant). The mechanism by sors) and megakaryocytes (platelet precursors). Management
which FA gene mutations cause disease is still being investi- involves RBC transfusions and corticosteroids. Bone marrow
gated. (10) Laboratory findings include pancytopenia, reticulo- transplantation is curative and can improve outcomes. (11)
cytopenia, hypocellular bone marrow, and sometimes elevation
of hemoglobin (Hb)F. Diagnosis of FA is made through chro- Shwachman-Diamond Syndrome
mosomal breakage analysis, which demonstrates increased Shwachman-Diamond syndrome (SDS) (OMIM: 260400,
617941) is a rare congenital disorder described by Shwach-
Table 1. Hemoglobin Types man et al in 1964 as the syndrome of pancreatic insuffi-
Hemoglobin (Hb) Globin ciency and bone marrow dysfunction. (13) This disorder is
Type Composition Production characterized by genetic heterogeneity with 2 main forms
Hb Gower 1 f2-e2 Embryonic (yolk sac) currently distinguished. (14)(15) SDS is characterized by multior-
Hb Gower 2 a2-e2 Embryonic (yolk sac)
gan and system involvement with predominant manifes-
Hb Portland f2-c2 Embryonic (yolk sac)
HbF a2-c2 Fetal (liver) tations being exocrine pancreatic insufficiency, skeletal
HbA2 a2-d2 Adult (bone marrow) abnormalities, and bone marrow failure. (16) Other
HbA a2-b2 Adult (bone marrow)
manifestations of SDS include skeletal abnormalities

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e815

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Figure 2. Various inherited red blood cell disorders and their associated genes. Created with BioRender.com.

such as thoracic dystrophy, neurocognitive problems, absence of the radii. (24) The disease is inherited in an au-
and hepatomegaly. (17) Although neutropenia is the tosomal recessive manner and is caused by compound het-
most common hematologic abnormality in SDS, aplastic erozygosity for a rare null mutation in the gene RBM8A
anemia and macrocytosis can be among the presenting on chromosome 1, region q21. (25) Aplastic anemia is a
symptoms as well. (18)(19) SDS rarely presents in neonates. possible erythroid lineage manifestation of TAR syn-
In those rare cases, typical neonatal manifestations include drome; however, it does not frequently occur in neonates.
acute severe infections, aplastic anemia, and growth failure Presence of thumbs aids in differentiating TAR syndrome
secondary to exocrine pancreatic insufficiency. (18)(20)(21) from FA. (26)
Fecal fat levels may be increased due to malabsorption. De-
creases in other biomarkers such as serum trypsinogen and Dyskeratosis Congenita
isoamylase may be seen later in life. (22) Specific SDS Dyskeratosis congenita (DC; OMIM: 127550, 613989, 613990,
growth charts for affected patients have been developed. (23) 615190, 616553, 613987, 224230, 616353, 613988, 305000) is
The anemia in SDS may range from intermittent or clinically a rare disorder characterized by mucocutaneous abnormalities
asymptomatic to severe, requiring transfusion of RBCs. Patients and pancytopenia from bone marrow failure. The classic triad
with SDS are at lifelong risk for malignant transformation seen is abnormal skin pigmentation, dystrophic nails, and oral
involving myelodysplasia or acute myelogenous leukemia. (18) leukoplakia. (27) Patients with DC have mutations in 1 of the
Diagnosis is made based on the presence of clinical features various genes. Inheritance is X-linked recessive (DKC1 gene),
and can be confirmed using gene-targeted or comprehensive autosomal dominant (TERC, TERT, or TINF2 genes), or auto-
genomic testing. (17)(18) Management involves G-CSF for neu- somal recessive (TERT, RTEL1, ACD, NHP2, NOP10, PARN,
tropenia, RBC transfusion for anemia, and pancreatic enzyme or WRAP53 genes). These mutations involve the telomerase
replacement. (11) Hematopoietic stem cell transplantation is the complex, which is required for synthesis and maintenance of
sole treatment for severe pancytopenia, myelodysplastic syndrome, telomere repeats at chromosome ends to ensure cell survival.
or leukemic transformation. (16) Laboratory findings include pancytopenia, reticulocytopenia,
macrocytosis, and sometimes increased HbF. Diagnosis can
Thrombocytopenia Absent Radius Syndrome be made with genetic testing for known mutations or flow cy-
Thrombocytopenia absent radius (TAR) syndrome tometry fluorescence in situ hybridization of white blood cell
(OMIM: 274000) is a rare genetic disorder characterized, subsets, which will show shortened telomeres. Most patients
as the name implies, by reduced platelet count and with DC die due to complications of bone marrow failure.

e816 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Hematopoietic stem cell transplantation is not necessarily cu- complete blood cell count, peripheral blood smear, and
rative given the high risk of fatal pulmonary fibrosis and vas- iron studies (to rule out iron deficiency anemia). Further
cular complications. (11) testing can be performed using hemoglobin electrophore-
The genetic features of the most common bone marrow sis. Definitive diagnosis can be made with genetic test-
failure syndromes are summarized in Table 2. ing. (27)

Hemoglobinopathies b-Thalassemia. In b-thalassemia (OMIM: 613985), excess

Hemoglobinopathies are caused by mutations in genes in- a chains precipitate and form inclusion bodies. This leads
volved in hemoglobin synthesis. They are divided into 2 to oxidative stress, damage to cell membranes, and eventu-
main groups: thalassemia syndromes and abnormal he- ally apoptosis of erythrocyte precursors. Death of these
moglobins (also referred to as structural hemoglobin var- precursors is termed ineffective erythropoiesis. The bone
iants). In thalassemia syndromes, mutations lead to a marrow attempts to increase RBC production, but the
decreased rate of hemoglobin synthesis but the structure
RBCs that are created contain inclusion bodies and are se-
of hemoglobins is normal. In abnormal hemoglobins, mu-
questered and destroyed by the spleen.
tations cause changes in hemoglobin structure.
Patients with b-thalassemia can be asymptomatic in the
fetal period and up to 6 months postnatally due to predomi-
Thalassemias
nance of HbF (a2–g2). Around 6 months of age, g-globin
Thalassemias are caused by decreased or absent synthesis
production decreases and the inability to synthesize b-globin
of globin chains and are named according to the type of
chain that is affected. Impaired globin chain synthesis due to mutations leads to symptoms of anemia. g and d glo-
leads to reduced hemoglobin synthesis. Unaffected glo- bins are upregulated, but this is not sufficient to offset the
bins are synthesized at a normal rate and the imbalance deficiency of HbA (a2–b2). Anemia prompts an increase in
between a and b chains can cause damage to erythrocyte EPO and the resultant ineffective erythropoiesis leads to
precursors. The details of a- and b-thalassemia are dis- bone marrow expansion, thinning of bone cortex, frontal
cussed below. Screening tests for thalassemias include a bossing, and extramedullary hematopoiesis.

Table 2. Bone Marrow Failure Syndromes

Phenotype
Disorder Type/Group OMIM No. Gene Chromosome Position Mode of Inheritance
Fanconi anemia Estren-Dameshek type 227650 FANCA 16 q24.3 Autosomal recessive
(Group A)
Type 2 (Group B) 300514 FANCB X p22.2 X-linked recessive
Type 3 (Group C) 227645 FANCC 9 q22.32 Autosomal recessive
Group G 614082 FANCG (XRCC9) 9 p13.3 Autosomal recessive
Group R 617244 FANCR (RAD51) 15 q15.1 Autosomal recessive
Diamond-Blackfan 1 105650 RPS19 19 q13.2 Autosomal dominant
anemia 7 612562 RPL11 1 p36.11 Autosomal dominant
10 613309 RPS26 12 q13.2 Autosomal dominant
Shwachman- 1 260400 SBDS 7 q11.21 Autosomal recessive
Diamond 2 617941 EFL1 15 q25.2 Autosomal recessive
syndrome
Thrombocytopenia – 274000 RBM8A 1 q21.1 Autosomal recessive
absent radius
syndrome
Dyskeratosis 1 (AD) 127550 TERC 3 q26.2 Autosomal dominant
congenital 2 (AD), 4 (AR) 613989 TERT 5 p15.33 Autosomal dominant,
Autosomal recessive
3 (AD) 613990 TINF2 14 q12 Autosomal dominant
4 (AD), 5 (AR) 615190 RTEL1 20 q13.33 Autosomal dominant,
Autosomal recessive
6 (AD), 7 (AR) 616553 ACD 16 q22.1 Autosomal dominant,
Autosomal recessive
2 (AR) 613987 NHP2 5 q35.3 Autosomal recessive
1 (AR) 224230 NOP10 15 q14 Autosomal recessive
6 (AR) 616353 PARN 16 p13.12 Autosomal recessive
3 (AR) 613988 WRAP53 17 p13.1 Autosomal recessive
X-linked 305000 DKC1 X q28 X-linked recessive

AD=autosomal dominant, AR=autosomal recessive, OMIM=Online Mendelian Inheritance in Man.

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e817

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 b-thalassemia is inherited in an autosomal recessive a-thalassemia occurs when no a-globins are produced and is
manner. Nearly 300 mutations have been discovered in called Hb Bart hydrops fetalis syndrome. Only fetuses with
the b-globin gene, and additional mutations in d and g Hb Bart develop hypoxia, heart failure, and hydrops fetalis;
globin genes. those who survive to birth usually require long-term transfu-
The following conventions are used to describe b-thal- sions. (28)(30)
assemia genes: b1 (decreased globin production), b0
(absent globin production), and b (normal globin produc- Abnormal Hemoglobins
tion). b-thalassemia can be categorized using 2 different Abnormal hemoglobins, or structural hemoglobin variants,
classification systems. The first system takes into consid- are caused by mutations that lead to changes in hemoglobin
eration the severity of phenotype based on the amount of structure. Mutations are inherited in an autosomal codomi-
functional b-globins (Table 3). (28)(29) The second classi- nant manner. This group of disorders can be divided into 4
fication system is based on transfusion requirements: categories: (1) sickle cell disorders, (2) hemoglobins with de-
transfusion-dependent thalassemia and non–transfusion- creased stability (eg, Brockton, Philly, Peterborough variants
dependent thalassemia. leading to hemolytic anemia; Hirosaki and Terre Haute var-
Patients who require long-term transfusions are at risk iants leading to hemolytic anemia and Heinz body forma-
for iron overload. Accumulation of iron leads to deposition tion), (3) hemoglobins with altered oxygen affinity (eg, high-
in the liver, heart, and pancreas, causing organ damage. affinity variants Kempsey, Hiroshima, York; low-affinity var-
Patients must undergo iron chelation therapy in addition iants Kansas, Beth Israel, St. Mand
e), and (4) hemoglobins
to receiving transfusions. Deferoxamine is a commonly that cause methemoglobinemia (eg, variants M-Iwate,
used agent. Hematopoietic stem cell transplantation can M-Saskatoon). (31)(32) This review discusses sickle cell dis-
cure thalassemia major. (28) orders, the most common category of abnormal hemoglobins.

a-Thalassemia. In a-thalassemia (OMIM: 604131), the in- Sickle Cell Disease. Sickle cell disease (SCD; OMIM:
ability to synthesize a-globin can cause in utero effects be- 603903) is a group of disorders characterized by HbS pro-
cause the primary intrauterine hemoglobin type is HbF duction, sickle cell formation, and the effects of sickling on
(a2–g2). Accumulation of g-globins does not lead to pre- various organs. HbS occurs due to mutation in the b-globin
cipitation. In fact, g-globins can form tetramers called Hb gene HBB on chromosome 11p15.4. The mutation leads to
Bart (g4). As b-globins are produced later in gestation, they a substitution of glutamic acid with valine for amino acid
can also form tetramers called HbH (b4). HbH can eventually 6. The mode of inheritance is autosomal recessive. (33)
precipitate and form inclusion bodies, leading to RBC destruc- Patients who are homozygous for HbS (SS) have the most
tion. Hb Bart and HbH have a high affinity for oxygen and severe disease phenotype. Patients who are heterozygous,
therefore lead to decreased oxygen delivery to tissues. (28) with 1 HbS allele in combination with another b-globin
There are several forms of a-thalassemia. a-thalassemia mutation (such as HbC or b-thalassemia) have less severe
minor (trait) results from 2 dysfunctional genes and causes disease manifestations.
mild anemia. a-thalassemia intermedia, also known as HbH When HbS is deoxygenated, a hydrophobic area of the
disease, results from 2 dysfunctional genes with diminished hemoglobin chain is exposed. Other HbS molecules bind
function. HbH disease usually does not require transfusions to hide the hydrophobic areas, thus creating HbS poly-
except in cases of hemolytic crisis. The most severe form of mers. As these polymers elongate, they distort the RBC

Table 3. b-Thalassemia Types

Type of b-Thalassemia Genotype Laboratory Findings Clinical Findings and Management
Thalassemia Major b0/ b0 HbA decreased or absent Anemia, hepatosplenomegaly, iron overload, bone abnormalities
b1/ b0 HbA2 variable Chronic transfusion requirement
b1/ b1 HbF increased
Thalassemia Intermedia b1/ b0 HbA decreased or absent Presents at later age (2-6 years), mild anemia
b1/ b1 HbA2 increased May need blood transfusion
HbF increased
Thalassemia Minor (trait) b1/ b HbA decreased but predominant Normal to mild anemia, presents during states of stress
b0/ b HbA2 increased Transfusion not needed
HbF normal or increased

Hb=hemoglobin.

e818 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 membrane and cause sickling. Sickle cells are unable to the mainstay of therapy. RBC transfusion can decrease the
change shape and therefore, increase blood viscosity and amount of circulating sickle cells and improve blood flow. Single
obstruct capillaries. (34) Polymerization and sickling can transfusions are given for splenic sequestration, aplastic crises,
be reversible with reoxygenation of HbS. Cycles of sickling and acute chest syndrome. Patients with high risk for or history
and unsickling can damage RBC membranes and lead to of stroke are treated with long-term transfusions. Patients who
chronic hemolytic anemia. (31) require long-term transfusions need iron chelation therapy to
The classic presentation of SCD is vaso-occlusive crisis, prevent iron overload. Antibiotic prophylaxis with penicillin and
which most often occurs in bones, lungs, liver, spleen, eyes, early initiation of antibiotics in cases of suspected infection are
central nervous system, and the urogenital tract. It can be crucial. Hematopoietic stem cell transplantation is the only cura-
triggered by acidosis, hypoxia, dehydration, infection, fever, tive therapy, but its use is limited by difficulty in finding human
and cold temperatures. Infants can have pain and swelling leukocyte antigen–matched family donors. (35)(36)(38)
in the hands and feet, known as hand-foot syndrome. Trap-
ping of blood in the spleen can lead to splenic sequestration Sickle Cell Trait. Patients who are heterozygous, with 1
and loss of splenic function over time. (35) This predisposes HbS allele and a normal a-globin allele, have sickle cell
patients to infections with encapsulated organisms, such as trait. Individuals with sickle cell trait are usually asymp-
tomatic but can have mild symptoms during times of ex-
Pneumococcus, Haemophilus, and Salmonella. (36) Infections
treme exertion. (35) Sickle cell trait provides protection
are the most common cause of death in SCD, especially in
against severe Plasmodium falciparum malaria. This ex-
the first few years of age. Patients can also develop acute
plains why the HbS allele has high prevalence in sub-
chest syndrome, defined by fever or respiratory symptoms
Saharan Africa, the Mediterranean, the Middle East, and
along with lung infiltrates noted on chest radiography. Other
India. (38)
serious complications of SCD include fat embolisms from
bone marrow necrosis, pulmonary hypertension, aplastic epi-
Sideroblastic Anemia
sodes (especially with parvovirus infection), cardiomegaly,
Sideroblastic anemia refers to a subset of inherited and ac-
avascular necrosis of the hip, retinopathy, and stroke. Find-
quired disorders of the bone marrow wherein iron overac-
ings of SCD on laboratory evaluation include normocytic
cumulates in the mitochondria of erythrocyte precursors.
anemia, poikilocytosis, and anisocytosis (with sickle cells, tar-
The pathophysiologic impact of this defect is abnormal
get cells, nucleated RBCs, and Howell-Jolly bodies), and re-
iron metabolism by the mitochondria. This ultimately re-
ticulocytosis. (35) Sickle cell disease infrequently manifests
sults in a large increase in reactive oxygen species causing
in the newborn population; this is largely because HbF can-
cellular damage and eventual cell death. (41)
not copolymerize with HbS. (37)
Early screening and diagnosis of SCD is essential for X-linked Sideroblastic Anemia
disease recognition and prompt intervention as sickling X-linked sideroblastic anemia (XLSA; OMIM: 300751) is an X-
events and complications can begin at 3 months of age. It linked recessive disorder that was first described by Thomas
is important in light of the fact that the initial presentation Cooley in 1945. (42) It is caused by a mutation in the X-linked
might be a fatal infection or an acute splenic sequestration gene ALAS2 on chromosome Xp11.21 that encodes erythroid
crisis. (38) In the United States, all newborns have under- specific enzyme d-aminolevulinate synthase 2 (ALAS2) (43)
gone screening for SCD in all states since 2006. (39) This enzyme catalyzes the first and rate-limiting reaction of
Three different technologies—high-performance liquid heme biosynthesis—condensation of glycine with succinyl-
chromatography, isoelectric focusing, and capillary electro- CoA to yield 5-aminolevulic acid in the presence of pyridoxal
phoresis—can be used to separate and quantify hemoglo- phosphate as a cofactor. (44) This enzymatic defect results
bin types from dried spot blood or cord blood samples. in ineffective heme synthesis, which in turn leads to inef-
Newborn samples contain mostly HbF and some HbA. fective erythropoiesis and systemic iron overload. (43) Clini-
Identification of HbS in the sample is suggestive of SCD cal features include anemia, formation of ring sideroblasts
and can be confirmed with other testing modalities. (40) (erythroid precursors with perinuclear ring formed by iron
Treatment of SCD includes measures to prevent vaso- laden mitochondria) in the bone marrow, and systemic iron
occlusive crises, such as hydration and avoiding low-oxygen envi- overload. (Figure 3). (45) The anemia in XLSA is highly var-
ronments (eg, high altitude and strenuous exercise). Hydroxyurea iable and typically hypochromic microcytic with increased
can increase HbF production and decrease the number of RBC distribution width. At times, prominent dysmorphism
vaso-occlusive crises. During a vaso-occlusive crisis, analgesia is (micro-, normo-, and even macrocytic) of erythrocytes has

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e819

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 cytochrome b, the inner mitochondrial membrane proteins
which are parts of the electron transport chain. (51)(52) Muta-
tions in these genes result in disruption of oxidative phos-
phorylation, leading to deficient adenosine triphosphate (ATP)
production, which in turn leads to an increase in anaerobic
glycolysis and lactic acidosis. Clinical features include macro-
cytic anemia and exocrine pancreatic insufficiency, as well as
hepatic and renal impairment. (50) Anemia is the most prom-
inent clinical feature and typically is aregenerative, with low
reticulocyte count and refractory in nature, requiring frequent
blood transfusions. (49)(53) Bone marrow features suggestive
of Pearson syndrome include hypocellularity and the presence
of cytoplasmic vacuolization of myeloid and erythroid precur-
sors, as well the presence of ringed sideroblasts. (54)(55) Other
hematologic findings include neutropenia and thrombocyto-
penia. (50) Exocrine pancreatic insufficiency is caused by fi-
brosis and can lead to malabsorption and failure to thrive. (53)
Figure 3. Ringed sideroblasts, typical of X-linked sideroblastic anemia. Besides lactic acidosis, other biochemical abnormalities that
Created with BioRender.com.
can be present in patients with Pearson syndrome are eleva-
tion of plasma alanine and low levels of plasma citrulline and
been observed. (46) Similar to most X-linked recessive dis-
arginine. The latter is thought to be due to decreased activity
eases, most heterozygous females do not exhibit clinical
of ornithine transcarbamylase. (56) Management is supportive
symptoms. Nonetheless, occasionally heterozygous females
care with blood transfusions and G-CSF as well as correction
may be affected due to skewed lyonization or severe loss-of-
of metabolic acidosis. (50) The prognosis is poor, with chil-
function mutations in the ALAS2 gene resulting in apopto-
dren typically dying in infancy or early childhood. (57) Among
sis of mutated erythroid precursors. (47) Management in-
the causes of death are multiorgan system failure, persistent
cludes high-dose pyridoxine supplementation and iron
reduction therapies. Pyridoxine (vitamin B6), which is con- metabolic acidosis, and septicemia. (58) Bone marrow dys-
verted into the ALAS2 cofactor pyridoxal phosphate, acts by function resolves in survivors, but they develop Kearns-Sayre
prolonging the half-life of ALAS2. However, the response is syndrome characterized by external ophthalmoplegia, retinitis
variable. (48) The main goal of iron reduction therapies, pigmentosa, and cardiac conduction abnormalities. (53)(59)
such as phlebotomy and iron chelation, is to prevent the de- The genetic features of sideroblastic anemia are sum-
velopment of hepatic cirrhosis, hepatocellular carcinoma, marized in Table 4. (60)(61)
diabetes, and heart failure from iron overload. (43)

Pearson Syndrome RBC Membrane Defects

Pearson marrow-pancreas syndrome (OMIM: 557000) is a The erythrocyte membrane requires a unique composition
rare mitochondrial disorder first described by Howard Pear- of lipids and proteins to achieve a flexible biconcave shape,
son and colleagues in 1979. (49) To date, only approximately which enables these cells to withstand the stress of trans-
150 cases have been reported. (50) This syndrome results port via the circulatory system. Abnormalities in any of
from sporadic deletions of mitochondrial genes ND4, ND5, these key membrane components can lead to malforma-
ND6, and CYTB. These genes encode the subunits of nicotin- tion, with resultant membrane instability and increased
amide adenine dinucleotide (NADH) dehydrogenase and erythrocyte lysis.

Table 4. Sideroblastic Anemia

Disorder Phenotype OMIM No. Gene(s) Chromosome Position Mode of Inheritance
X-linked sideroblastic anemia 300751 ALAS2 X p11.21 X-linked recessive
Pearson syndrome 557000 ND4, ND5, ND6, CYTB – – Mitochondrial

OMIM=Online Mendelian Inheritance in Man.

e820 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Hereditary Spherocytosis caused by alterations in the EPB41, SPTA1, SPTB, and
Hereditary spherocytosis (HS) (OMIM 182900, 616649, SLC4A1 genes. These genes encode protein 4.1, a-spectrin,
270970, 612653, 612690) constitutes a heterogenous b-spectrin, and band 3 protein, respectively. Another form
group of congenital RBC disorders described by Oskar of HE is called hereditary pyropoikilocytosis (HPP; OMIM
Minkowski in 1900. (62) These disorders stem from alter- 266140) and is caused by a defect in the SPTA1 gene,
ations in the genes ANK1, SPTB, SPTA1, SLC4A1 and which encodes the protein spectrin. (72)(73)(74)(75)(76) As
EBB42, which encode RBC membrane proteins ankyrin, can be seen, the genes and proteins affected in HE overlap
b-spectrin, a-spectrin, band 3, and protein 4.2, respec- those affected in HS. As such, the 2 groups of disorders
tively. (63) Defects of a-spectrin have been reported to share many clinical features. The hallmark of elliptocytosis is
cause severe anemia whereas defects of ankyrin, band 3, the presence of oval or elliptical-shaped RBCs on peripheral
and protein 4.2 have been associated with the develop- blood smear. (77) Newborns typically present with transient
ment of mild to moderate anemia. (64) Altered structure hemolytic anemia and jaundice. Packed RBC transfusion
of membrane proteins leads to imperfect connection of and phototherapy may be indicated in cases of severe anemia
the inner membrane cytoskeleton to the outer lipid bi- and hyperbilirubinemia. (78) HPP is the most severe form of
layer. (65) As a result, RBCs lose their ability to maintain HE. It typically presents in neonates with severe hemolytic
the distinctive biconcave shape and assume the spherical anemia that persists throughout the life span. (79) Complica-
shape instead. (66) Other characteristics of spherocytes tions of hemolysis, such as splenomegaly and cholelithiasis
include decrease in mean corpuscular volume, increase in are common, which may require splenectomy. (80)
mean corpuscular hemoglobin concentration, absence of The genetic features of different types of RBC membrane
central pale region, and increased osmotic fragility. The defects are summarized in Table 5. (72)(73)(74)(75)(76)(81)
tests that measure hemolysis—osmotic fragility test, acidi-
fied glycerol test and Pink test—can be used as first-line RBC Enzyme Defects
diagnostic means, however, the sensitivity of these tests is Although erythrocytes lack a nucleus and other organelles,
low. (65) The flow cytometric eosin-50 -maleimide (EMA) the rich enzyme composition is vital for proper function.
test has been used as an alternative test for diagnosis of The primary form of metabolism in the erythrocyte is the
HS with high sensitivity and specificity. This test is based Embden-Meyerhof pathway, which requires functional PK
on labeling RBCs with the fluorescent dye EMA. The for the cell to generate energy stored as ATP, glutathione,
spherocytes have decreased binding of the dye compared and pyridine nucleotides (NADH and nicotinamide ade-
to normal RBCs and, thus, decreased fluorescence inten- nine dinucleotide phosphate [NADPH]). Subsequently,
sity. (65)(67) Typical clinical manifestations of HS include most of the NADPH in RBCs is formed via metabolism
anemia with hemolysis, reticulocytosis, jaundice due to of glucose-6-phosphate, enabled by G6PD. Both of these
unconjugated hyperbilirubinemia, cholelithiasis, and spleno- metabolic processes must be intact for adequate cellular
megaly. (64) The most common presenting sign in neonates energy formation. NADPH formation also maintains ade-
with HS is jaundice. Anemia is present in fewer than half of quate levels of reduced glutathione, which protects against
neonates affected by HS whereas splenomegaly is rarely oxidative damage. (82)
detected. Management includes control of hyperbilirubinemia
with phototherapy and an exchange transfusion, if needed. G6PD Deficiency
Clinically significant anemia can benefit from packed RBC G6PD deficiency (OMIM: 300908) was first described by
transfusions. (68) The use of recombinant EPO has been Alving et al in 1956 in patients who had hemolytic anemia
reported with varying success. (69)(70)(71) Folic acid should induced by primaquine. (83) The disorder has X-linked dom-
be given to patients with moderate or severe disease. Splenec- inant inheritance and is caused by mutations in the G6PD
tomy is very infrequently performed during the first year of gene located at the X-chromosome in the q28 region. (84)
age. (68) G6PD is a housekeeping enzyme that is present in all cells
of the human body. It participates in the pentose phosphate
Hereditary Elliptocytosis pathway and plays a key role in the defense against cellular
Hereditary elliptocytosis (HE) (OMIM 611804, 130600, damage from oxidative stress. Reduced NADPH that is gen-
617948, 166900) comprises a heterogeneous group of erated in the pentose phosphate pathway is then used to
congenital RBC disorders characterized by elongated, oval, convert oxidized glutathione into its reduced state. (85) RBCs
or elliptical-shaped erythrocytes. These conditions are are particularly vulnerable to reactive oxidation because of

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e821

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Table 5. Red Blood Cell Membrane Defects
Phenotype RBC
OMIM membrane
Disorder Type Number Gene Chromosome Position Mode of Inheritance Protein
Hereditary 1 182900 ANK1 8 p11.2 Autosomal dominant, Ankyrin
spherocytosis Autosomal recessive
2 616649 SPTB 14 q23-24.1 Autosomal dominant b-spectrin
3 270970 SPTA1 1 q22-23 Autosomal recessive a-spectrin
4 612653 SLC4A1 17 q21-22 Autosomal dominant Band 3
5 612690 EPB42 15 q15-q21 Autosomal recessive Protein 4.2
Hereditary 1 611804 EPB41 1 p35.3 Autosomal dominant, Protein 4.1
elliptocytosis Autosomal recessive
2 130600 SPTA1 1 p23.1 Autosomal dominant a-spectrin
3 617948 SPTB 14 p23.3 Autosomal dominant b-spectrin
4 166900 SLC4A1 17 q21.31 Autosomal dominant Band 3
HPP 266140 SPTA1 1 q23.1 Autosomal recessive a-spectrin

HPP=hereditary pyropoikilocytosis; OMIM=Online Mendelian Inheritance in Man.

their exposure to large amounts of oxygen as well as their inabil- PK catalyzes the last step of glycolysis—the conversion of
ity to synthetize new proteins as mature cells. (86) G6PD defi- phosphoenolpyruvate to pyruvate with a yield of ATP. A
ciency leads to a state of NADPH deficiency, inability to reduce decrease in ATP production creates a deficit of energy re-
oxidized glutathione and, consequently, to severe oxidative dam- quired to maintain cellular structural and functional integ-
age. Hemolytic anemia follows as a result. (87) The hemolysis rity with subsequent premature destruction of cells in the
can be provoked by stress, certain foods rich in oxidative substan- spleen or liver. (98) A deficiency in PK also leads to the
ces (eg, fava beans), and medications (eg, quinine, sulfa drugs), build-up of 2,3-diphosphoglycerate, the upstream metabo-
as well as certain environmental exposures (eg, naphthalene lite of glycolysis, which leads to a shift of the oxygen-he-
from mothballs). (88)(89) In neonates, G6PD deficiency poses a moglobin dissociation curve to the right. (99) Clinical
risk for developing indirect hyperbilirubinemia and the need for features in neonates include anemia and hyperbilirubine-
phototherapy. Liu et al reported that in G6PD-deficient neonates mia. Symptoms can be heterogenous, depending on the
the risk of hyperbilirubinemia is 3.92 times higher and the risk degree of enzyme deficiency and hemolysis. Severe defi-
of phototherapy is 3.01 times higher compared to newborns with ciency can lead to fetal anemia and development of hy-
normal G6PD. (90) G6PD deficiency also increases the risk of drops fetalis. (100) On peripheral blood smear,
kernicterus. Thus, 20.8% of infants in the kernicterus registry erythrocytes are typically normochromic. Polychromasia
had G6PD deficiency as opposed to the general prevalence of and presence of echinocytes (RBCs with small horny pro-
G6PD deficiency in the United States of 4% to 7%. (91)(92) The jections) can be observed. (101)(102) Definitive diagnosis
diagnosis can be made with quantitative spectrophotometric anal- is made using the spectrophotometric assay of erythrocyte
ysis or a rapid fluorescent spot test detecting the generation of PK activity. (103) False-negative results can be seen after re-
NADPH from NADP. During the period of acute hemolysis, re- cent transfusions or incomplete removal of white blood cells
sults of G6PD deficiency testing can be falsely negative due to or platelets from the specimen. Also, given the higher levels
preferential lysis of older cells with the lowest G6PD levels. New- of PK in reticulocytes, false-negative results can be seen in
borns are not routinely screened for G6PD deficiency in the some patients due to reticulocytosis. (98) Treatment includes
United States. The mainstay of G6PD deficiency management is management of hyperbilirubinemia and anemia. Photother-
avoidance of oxidative stressors. (93) In neonates, it is important apy or, in severe cases, exchange transfusion may be re-
to manage jaundice and prevent kernicterus using phototherapy quired. (100) Severe anemia may require blood transfusions
and, in severe cases, exchange transfusion. (93)(94)(95) and splenectomy. Frequent blood transfusions might lead to
iron overload and the need for iron chelation. (104)
Pyruvate Kinase Deficiency The genetic features of different RBC enzyme defects
Pyruvate kinase (PK) deficiency (OMIM: 266200) is an in- are summarized in Table 6.
herited metabolic disorder first reported by Valentine et al in
1961. (96) This disorder has autosomal recessive inheritance CONCLUSION
and is caused by homozygous or compound heterozygous mu- Inherited disorders of RBCs can present at different peri-
tation in the gene PKLR on chromosome 1, region q22. (97) ods of life ranging from in utero to adulthood. The

e822 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Table 6. Red Blood Cell Enzyme Defects
Disorder Phenotype OMIM Number Gene Chromosome Position Mode of Inheritance
Glucose-6-phosphate dehydrogenase deficiency 300908 G6PD X q28 X-linked dominant
Pyruvate kinase deficiency 266200 PKLR 1 q22 Autosomal recessive

OMIM=Online Mendelian Inheritance in Man.

presentation severity can also vary widely from asymptom- in preterm infants: a systematic review. Front Pediatr.
2021;9:644462
atic to severe multisystem dysfunction requiring immedi-
2. Bain A, Blackburn S. Issues in transfusing preterm infants in the
ate interventions. Important focus should be placed on
NICU. J Perinat Neonatal Nurs. 2004;18(2):170–184
genetic diseases with resultant decreased RBC production
3. Aher S, Malwatkar K, Kadam S. Neonatal anemia. Semin Fetal
such as FA, DBA, SDS, and DC as these also have associ- Neonatal Med. 2008;13(4):239–247
ated anatomic abnormalities and associated comorbidities 4. Letterio J, Pateva I, Petrosiute A, Ahuja S. Hematologic and
that require further specialized treatments. Determining oncologic problems in the fetus and neonate. In: Martin RJ,
the underlying etiology via timely diagnosis and prompt Fanaroff AA, Walsh MC. Fanaroff and Martin’s Neonatal-Perinatal
Medicine, 2-Volume Set: Diseases of the Fetus and Infant. 11th ed.
management are key to improving outcomes in newborns
Philadelphia, PA: Elsevier; 2019:1416–1503
affected with these diseases. Genetic counseling should be
5. Palis J. Primitive and definitive erythropoiesis in mammals. Front
provided to the parents of affected children to estimate the Physiol. 2014;5:3
risk of recurrence and future pregnancy planning. A high 6. Otto CN. Hemoglobin metabolism. In: Keohane EM, Otto CN,
index of suspicion and broad medical knowledge will aid Walenga JM, eds. Rodak’s Hematology: Clinical Principles and
Applications. 6th ed. Philadelphia, PA: Elsevier; 2020:
physicians in diagnosing the congenital RBC disorders in
91–103
due course and take appropriate steps in management to
7. Farid Y, Bowman NS, Lecat P. Biochemistry, hemoglobin
reduce morbidity and mortality. synthesis. In: StatPearls. Treasure Island, FL: StatPearls
Publishing; 2022. Available at: https://www.ncbi.nlm.nih.gov/
books/NBK536912/. Accessed September 11, 2022
American Board of Pediatrics 8. Butina M. Erythrocyte production and destruction. In: Keohane
EM, Otto CN, Walenga JM, eds. Rodak’s Hematology: Clinical
Neonatal-Perinatal Content Principles and Applications. 6th ed. Philadelphia, PA: Elsevier;
Specifications 2020:62–77
• Know the biochemical characteristics of fetal 9. Auerbach AD. Fanconi anemia and its diagnosis. Mutat Res.
hemoglobin. 2009;668(1-2):4–10

• Know the developmental biology of hemoglobin 10. Elghetany MT, Punia JN, Marcogliese AN. Inherited bone marrow
types. failure syndromes. Clin Lab Med. 2021;41(3):417–431

• Know the clinical and laboratory features of neonatal he- 11. Lo C, Glader B, Sakamoto KM. Bone marrow failure. In: Keohane
EM, Otto CN, Walenga JM, eds. Rodak’s Hematology: Clinical
moglobinopathies, including the thalassemias.
Principles and Applications. 6th ed. Philadelphia, PA: Elsevier;
• Know the indications for and approaches to screening 2020:299–315
for hemoglobinopathies in the newborn population.
12. Kim HY, Kim HJ, Kim SH. Genetics and genomics of bone
• Know normal erythropoiesis in the fetus and neonate. marrow failure syndrome. Blood Res. 2022;57(S1):S86–S92
• Know the factors regulating erythropoiesis in the fetus 13. Shwachman H, Diamond LK, Oski FA, Khaw KT. The syndrome
and neonate including erythropoietin. of pancreatic insufficiency and bone marrow dysfunction. J
• Know the causes of and diagnostic approach to an in- Pediatr. 1964;65:645–663
fant who is anemic at birth. 14. OMIM. # 260400: Shwachman-Diamond syndrome 1; SDS1.
2022. Available at: https://www.omim.org/entry/260400. Ac-
cessed September 11, 2022
15. OMIM. # 617941: Shwachman-Diamond syndrome 2; SDS2.
Acknowledgment 2022. Available at: https://www.omim.org/entry/617941. Accessed
We thank Alison Chu, MD, for her help with this article. September 11, 2022
16. Bezzerri V, Cipolli M. Shwachman-diamond syndrome: molecular
mechanisms and current perspectives. Mol Diagn Ther. 2019;23(2):
References 281–290
1. Kalteren WS, Verhagen EA, Mintzer JP, Bos AF, Kooi EMW. 17. Nelson AS, Myers KC. Diagnosis, treatment, and molecular
Anemia and red blood cell transfusions, cerebral oxygenation, pathology of Shwachman-Diamond syndrome. Hematol Oncol Clin
brain injury and development, and neurodevelopmental outcome North Am. 2018;32(4):687–700

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e823

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 18. Nelson A, Myers K. Shwachman-Diamond syndrome. In: Adam 37. de Montalembert M. Sickle cell disease in the neonatal period
MP, Mirzaa GM, Pagon RA, et al, eds. GeneReviewsV R . University [article in French]. J Gynecol Obstet Biol Reprod (Paris). 2004;
of Washington, Seattle; 1993. Available at: https://www.ncbi.nlm. 33(1 suppl):S12–S14
nih.gov/books/NBK1756/. Accessed September 11, 2022 38. Kato GJ, Piel FB, Reid CD, et al. Sickle cell disease. Nat Rev Dis
19. Saito-Benz M, Miller HE, Berry MJ. Shwachman-Diamond Primers. 2018;4:18010
syndrome (SDS) in a preterm neonate. J Paediatr Child Health. 39. Monus T, Howell CM. Current and emerging treatments for
2015;51(12):1228–1231 sickle cell disease. JAAPA. 2019;32(9):1–5
20. Black LV, Soltau T, Kelly DR, Berkow RL. Shwachman-Diamond 40. Fr€
ommel C. Newborn screening for sickle cell disease and other
syndrome presenting in a premature infant as pancytopenia. hemoglobinopathies: a short review on classical laboratory
Pediatr Blood Cancer. 2008;51(1):123–124 methods—isoelectric focusing, HPLC, and capillary electrophoresis.
21. Todorovic-Guid M, Krajnc O, Marcun Varda N, et al. A case of IJNS. 2018;4(4):39
Shwachman-Diamond syndrome in a male neonate. Acta Paediatr. 41. Camaschella C. Hereditary sideroblastic anemias: pathophysiology,
2006;95(7):892–893 diagnosis, and treatment. Semin Hematol. 2009;46(4):371–377
22. Burroughs L, Woolfrey A, Shimamura A. Shwachman-Diamond 42. Cooley TB. A severe type of hereditary anemia with elliptocytosis:
syndrome: a review of the clinical presentation, molecular interesting sequence of splenectomy. Am J Med Sci. 1945;209:561
pathogenesis, diagnosis, and treatment. Hematol Oncol Clin North
43. Donker AE, Raymakers RA, Nieuwenhuis HK, et al. X-linked
Am. 2009;23(2):233–248
sideroblastic anaemia due to ALAS2 mutations in the
23. Cipolli M, Tridello G, Micheletto A, et al. Normative growth charts Netherlands: a disease in disguise. Neth J Med. 2014;72(4):210–217
for Shwachman-Diamond syndrome from Italian cohort of 0-8
44. Phillips JD. Heme biosynthesis and the porphyrias. Mol Genet
years old. BMJ Open. 2019;9(1):e022617
Metab. 2019;128(3):164–177
24. Toriello HV. Thrombocytopenia absent radius syndrome. In:
45. Fujiwara T, Harigae H. Molecular pathophysiology and genetic
Adam MP, Mirzaa GM, Pagon RA, et al., eds. GeneReviewsV R.
mutations in congenital sideroblastic anemia. Free Radic Biol Med.
University of Washington, Seattle; 1993. Available at: https://www.
2019;133:179–185
ncbi.nlm.nih.gov/books/NBK23758/. Accessed September 11, 2022
46. M
endez M, Moreno-Carralero MI, Morado-Arias M, et al.
25. OMIM. # 274000: Thrombocytopenia-absent radius syndrome;
Sideroblastic anemia: functional study of two novel missense
TAR. 2022. Available at: https://www.omim.org/entry/274000.
mutations in ALAS2. Mol Genet Genomic Med. 2016;4(3):273–282
Accessed September 11, 2022
47. Aivado M, Gattermann N, Rong A, et al. X-linked sideroblastic
26. Khincha PP, Savage SA. Neonatal manifestations of inherited
anemia associated with a novel ALAS2 mutation and unfortunate
bone marrow failure syndromes. Semin Fetal Neonatal Med. 2016;
skewed X-chromosome inactivation patterns. Blood Cells Mol Dis.
21(1):57–65
2006;37(1):40–45
27. Walne AJ, Dokal I. Advances in the understanding of dyskeratosis
48. May A, Bishop DF. The molecular biology and pyridoxine
congenita. Br J Haematol. 2009;145(2):164–172 responsiveness of X-linked sideroblastic anaemia. Haematologica.
28. Keohane EM. Thalassemias. In: Keohane EM, Otto CN, Walenga 1998;83(1):56–70
JM, eds. Rodak’s Hematology: Clinical Principles and Applications. 49. Pearson HA, Lobel JS, Kocoshis SA, et al. A new syndrome of
6th ed. Philadelphia, PA: Elsevier; 2020:424–444 refractory sideroblastic anemia with vacuolization of marrow
29. Ali S, Mumtaz S, Shakir HA, et al. Current status of beta- precursors and exocrine pancreatic dysfunction. J Pediatr. 1979;
thalassemia and its treatment strategies. Mol Genet Genomic Med. 95(6):976–984
2021;9(12) 50. Farruggia P, Di Marco F, Dufour C. Pearson syndrome. Expert Rev
30. Farashi S, Harteveld CL. Molecular basis of a-thalassemia. Blood Hematol. 2018;11(3):239–246
Cells Mol Dis. 2018;70:43–53 51. Rotig A, Colonna M, Bonnefont JP, et al. Mitochondrial DNA
31. Forget BG, Bunn HF. Classification of the disorders of deletion in Pearson’s marrow/pancreas syndrome. Lancet. 1989;
hemoglobin. Cold Spring Harb Perspect Med. 1(8643):902–903
2013;3(2):a011684–a011684 52. Khasawneh R, Alsokhni H, Alzghoul B, et al. A novel
32. Thom CS, Dickson CF, Gell DA, Weiss MJ. Hemoglobin variants: mitochondrial DNA deletion in patient with Pearson syndrome.
biochemical properties and clinical correlates. Cold Spring Harb Med Arch. 2018;72(2):148–150
Perspect Med. 2013;3(3):a011858 53. Pronman L, Rondinelli M, Burkardt DD, et al. Pearson syndrome:
33. OMIM. # 603903: Sickle cell anemia. 2022. Available at: https:// a rare cause of failure to thrive in infants. Clin Pediatr (Phila).
www.omim.org/entry/603903. Accessed September 11, 2022 2019;58(7):819–824
34. Sundd P, Gladwin MT, Novelli EM. Pathophysiology of sickle cell 54. Tadiotto E, Maines E, Degani D, et al. Bone marrow features in
disease. Annu Rev Pathol Mech Dis. 2019;14(1):263–292 Pearson syndrome with neonatal onset: a case report and review
35. Randolph TR. Hemoglobinopathies (structural defects in of the literature. Pediatr Blood Cancer. 2018;65(4)
hemoglobin). In: Keohane EM, Otto CN, Walenga JM, eds. Rodak’s 55. Falcon CP, Howard TH. An infant with Pearson syndrome: a rare
Hematology: Clinical Principles and Applications. 6th ed. Philadelphia, cause of congenital sideroblastic anemia and bone marrow failure.
PA: Elsevier; 2020:394–423 Blood. 2017;129(19):2710
36. Kohne E. Hemoglobinopathies: clinical manifestations, diagnosis, 56. Crippa BL, Leon E, Calhoun A, et al. Biochemical abnormalities in
and treatment. Dtsch Arztebl Int. 2011;108(31-32):532–540 Pearson syndrome. Am J Med Genet A. 2015;167A(3):621–628

e824 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 57. Atale A, Bonneau-Amati P, R€otig A, et al. Tubulopathy and 76. OMIM. # 266140: Pyropoikilocytosis, hereditary; HPP. 2022.
pancytopaenia with normal pancreatic function: a variant of Available at: https://www.omim.org/entry/266140. Accessed
Pearson syndrome. Eur J Med Genet. 2009;52(1):23–26 September 11, 2022
58. Nilay M, Phadke SR. Pearson syndrome: spontaneously recovering 77. Soderquist C, Bagg A. Hereditary elliptocytosis. Blood. 2013;
anemia and hypoparathyroidism. Indian J Pediatr. 2020;87(12): 121(16):3066
1070–1072 78. Liu C, Eun HS, Nah H, et al. Newborn hereditary elliptocytosis
59. Lee HF, Lee HJ, Chi CS, et al. The neurological evolution of confirmed by familial genetic testing. Int J Lab Hematol. 2020;
Pearson syndrome: case report and literature review. Eur J 42(1):e20–e22
Paediatr Neurol. 2007;11(4):208–214 79. Niss O, Chonat S, Dagaonkar N, et al. Genotype-phenotype
60. OMIM. #300751: Anemia, sideroblastic. Available at: https://www. correlations in hereditary elliptocytosis and hereditary
omim.org/entry/300751. Accessed 30 June 2022 pyropoikilocytosis. Blood Cells Mol Dis. 2016;61:4–9
61. OMIM. #557000: Pearson marrow-pancreas syndrome. 2022. 80. Pincez T, Guitton C, Landman-Parker J, et al. Subtotal and total
Available at: https://www.omim.org/entry/557000. Accessed splenectomy for hereditary pyropoikilocytosis: benefits and
September 11, 2022 outcomes. Am J Hematol. 2018;93(10):E340–E342
62. Minkowski O. Ueber eine hereditare,unter dem Bilde eines 81. Wu Y, Liao L, Lin F. The diagnostic protocol for hereditary
chronischen Ikterus mit urobilinurie, Splenomegalie and spherocytosis: 2021 update. J Clin Lab Anal. 2021;35(12):e24034
Nieronsiderosis verlaufende Affektion. Verhandl. d. Kongr. f. inn. 82. Steiner LA, Gallagher PG. Erythrocyte disorders in the perinatal
Med. 1900;18:316 period. Semin Perinatol. 2007;31(4):254–261
63. He BJ, Liao L, Deng ZF, et al. Molecular genetic mechanisms of 83. Alving AS, Carson PE, Flanagan CL, Ickes CE. Enzymatic
hereditary spherocytosis: current perspectives. Acta Haematol. deficiency in primaquine-sensitive erythrocytes. Science. 1956;
2018;139(1):60–66 124(3220):484–485
64. Perrotta S, Gallagher PG, Mohandas N. Hereditary spherocytosis. 84. OMIM. # 300908: Anemia, nonspherocytic hemolytic, due to
Lancet. 2008;372(9647):1411–1426 G6PD deficiency. 2022. Available at: https://www.omim.org/
65. Da Costa L, Galimand J, Fenneteau O, Mohandas N. Hereditary entry/300908. Accessed September 11, 2022
spherocytosis, elliptocytosis, and other red cell membrane 85. Karafin MS, Francis RO. Impact of G6PD status on red cell
disorders. Blood Rev. 2013;27(4):167–178 storage and transfusion outcomes. Blood Transfus. 2019;17(4):
66. Jacob HS, Ruby A, Overland ES, Mazia D. Abnormal membrane 289–295
protein of red blood cells in hereditary spherocytosis. J Clin Invest. 86. Luzzatto L, Nannelli C, Notaro R. Glucose-6-phosphate
1971;50(9):1800–1805 dehydrogenase deficiency. Hematol Oncol Clin North Am. 2016;
67. Manciu S, Matei E, Trandafir B. Hereditary spherocytosis: 30(2):373–393
diagnosis, surgical treatment and outcomes—a literature review. 87. Luzzatto L, Arese P. Favism and glucose-6-phosphate
Chirurgia (Bucur). 2017;112(2):110–116 dehydrogenase deficiency. N Engl J Med. 2018;378(1):60–71
68. Christensen RD, Yaish HM, Gallagher PG. A pediatrician’s 88. Prins TJ, Trip-Hoving M, Paw MK, et al. A survey of practice and
practical guide to diagnosing and treating hereditary spherocytosis knowledge of refugee and migrant pregnant mothers surrounding
in neonates. Pediatrics. 2015;135(6):1107–1114 neonatal jaundice on the Thailand-Myanmar border. J Trop
69. Tchernia G, Delhommeau F, Perrotta S, et al. Recombinant Pediatr. 2017;63(1):50–56
erythropoietin therapy as an alternative to blood transfusions 89. Lee SWH, Chaiyakunapruk N, Lai NM. What G6PD-deficient
in infants with hereditary spherocytosis. Hematol J. 2000;1(3): individuals should really avoid. Br J Clin Pharmacol. 2017;83(1):
146–152 211–212
70. Morrison JF, Neufeld EJ, Grace RF. The use of erythropoietin- 90. Liu H, Liu W, Tang X, Wang T. Association between G6PD
stimulating agents versus supportive care in newborns with deficiency and hyperbilirubinemia in neonates: a meta-analysis.
hereditary spherocytosis: a single centre’s experience. Eur J Pediatr Hematol Oncol. 2015;32(2):92–98
Haematol. 2014;93(2):161–164 91. Johnson L, Bhutani VK, Karp K, Sivieri EM, Shapiro SM. Clinical
71. Farruggia P, Puccio G, Ramenghi U, et al. Recombinant report from the pilot USA Kernicterus Registry (1992 to 2004).
erythropoietin vs. blood transfusion care in infants with hereditary J Perinatol. 2009;29(suppl 1):S25–S45
spherocytosis: a retrospective cohort study of A.I.E.O.P. patients. 92. Nkhoma ET, Poole C, Vannappagari V, Hall SA, Beutler E. The
Am J Hematol. 2017;92(6):E103–E105 global prevalence of glucose-6-phosphate dehydrogenase
72. OMIM. # 611804: Elliptocytosis 1; EL1. 2022. Available at: https:// deficiency: a systematic review and meta-analysis. Blood Cells Mol
www.omim.org/entry/611804. Accessed September 11, 2022 Dis. 2009;42(3):267–278
73. OMIM. # 130600: Elliptocytosis 2; EL2. 2022. Available at: 93. Frank JE. Diagnosis and management of G6PD deficiency. Am
https://www.omim.org/entry/130600. Accessed September 11, Fam Physician. 2005;72(7):1277–1282
2022 94. Wong FL, Ithnin A, Othman A, Cheah FC. Glucose-6-phosphate
74. OMIM. # 617948: Elliptocytosis 3; EL3. 2022. Available at: https:// dehydrogenase (G6PD)-deficient infants: enzyme activity and gene
www.omim.org/entry/617948. Accessed September 11, 2022 variants as risk factors for phototherapy in the first week of life.
75. OMIM. # 166900: Ovalocytosis, Southeast Asian; SAO. 2022. J Paediatr Child Health. 2017;53(7):705–710
Available at: https://www.omim.org/entry/166900. Accessed 95. Dey SK, Jahan S, Jahan I, et al. Exchange transfusion for
September 11, 2022 hyperbilirubinemia among term and near term in NICU of a

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e825

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 tertiary care hospital of Bangladesh: Findings from a prospective 100. Grace RF, Bianchi P, van Beers EJ, et al. Clinical spectrum of
study. Euroasian J Hepatogastroenterol. 2021;11(1):21–26 pyruvate kinase deficiency: data from the Pyruvate Kinase
96. Valentine WN, Tanaka KR, Miwa S. A specific erythrocyte Deficiency Natural History Study. Blood. 2018;131(20):2183–2192
glycolytic enzyme defect (pyruvate kinase) in three subjects with 101. Chilcote RR, Jones B. Shape change (echinocytosis) in pyruvate
congenital non-spherocytic hemolytic anemia. Trans Assoc Am kinase deficient red cells is mediated by the coupled bilayer
Physicians. 1961;74:100–110 hypothesis. Pediatr Res. 1984;18:238A
97. OMIM. # 266200: Pyruvate kinase deficiency of red cells. 2022. 102. Grace RF, Zanella A, Neufeld EJ, et al. Erythrocyte pyruvate kinase
Available at: https://www.omim.org/entry/266200. Accessed deficiency: 2015 status report. Am J Hematol. 2015;90(9):825–830
September 11, 2022 103. Bianchi P, Fermo E, Glader B, et al. Addressing the diagnostic
98. Grace RF, Barcellini W. Management of pyruvate kinase gaps in pyruvate kinase deficiency: Consensus recommendations
deficiency in children and adults. Blood. 2020;136(11):1241–1249 on the diagnosis of pyruvate kinase deficiency. Am J Hematol.
99. Lakomek M, Winkler H, Pekrun A, et al. Erythrocyte pyruvate 2019;94(1):149–161
kinase deficiency: the influence of physiologically important 104. Grace RF, Mark Layton D, Barcellini W. How we manage patients
metabolites on the function of normal and defective enzymes. with pyruvate kinase deficiency. Br J Haematol. 2019;184(5):
Enzyme Protein. 1994;48(3):149–163 721–734

e826 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 NEOREVIEWS QUIZ

NEO
QUIZ
1. Erythropoiesis occurs in various sites throughout gestation, with the bone
marrow eventually becoming the primary site at 28 weeks’ gestation.
Erythropoietin (EPO) is a key hormone in the regulation of erythropoiesis.
Which of the following statements regarding EPO is correct?

A. Hyperoxia leads to increased EPO production.

B. EPO is primarily produced in the fetal yolk sac and liver.
C. EPO leads to a longer duration of the red blood cell maturation process.
D. The binding of EPO to the receptor, EPOR, leads to early release of
reticulocytes from bone marrow.
REQUIREMENTS: Learners can
E. Red blood cell death increases in the presence of EPO.
take NeoReviews quizzes and
2. An infant is noted to have cleft lip and palate and absent thumbs. claim credit online only at:
Laboratory evaluation is notable for macrocytic anemia and https://publications.aap.org/
neoreviews.
reticulocytopenia. Bone marrow evaluation reveals normal amounts of
myeloid cells and megakaryocytes. Which of the following is the most likely To successfully complete 2022
diagnosis? NeoReviews articles for AMA PRA
Category 1 Credit™, learners
A. Fanconi anemia must demonstrate a minimum
B. Diamond-Blackfan anemia performance level of 60% or
C. Shwachman-Diamond syndrome higher on this assessment. If
D. Thrombocytopenia absent radius syndrome you score less than 60% on the
assessment, you will be given
E. Dyskeratosis congenita
additional opportunities to
3. A fetus is noted to have hydrops and further evaluation reveals that there answer questions until an
are family members with thalassemia trait. If this case of hydrops is caused overall 60% or greater score is
achieved.
by a form of thalassemia, what is the most likely condition?
A. Hemoglobin Bart hydrops fetalis syndrome This journal-based CME activity
is available through Dec. 31,
B. b1 thalassemia major
2024, however, credit will be
C. Hemoglobin H disease recorded in the year in which
D. a-thalassemia intermedia the learner completes the quiz.
E. Hemoglobin b1 inclusion disease
4. A male neonate presents with jaundice and hyperbilirubinemia. The family
history includes jaundice and anemia in the neonatal period in the father
and a sibling. The complete blood cell count reveals decreased mean
corpuscular volume and increase in mean corpuscular hemoglobin
2022 NeoReviews is approved
concentration. Further examination reveals absence of central pale region
for a total of 30 Maintenance of
and increased osmotic fragility. Result of a flow cytometric eosin-50 - Certification (MOC) Part 2
maleimide test is positive. Which of the following is the most likely credits by the American Board
condition? of Pediatrics (ABP) through the
AAP MOC Portfolio Program.
A. Pearson syndrome NeoReviews subscribers can
B. X-linked sideroblastic anemia claim up to 30 ABP MOC Part 2
C. Sickle cell trait points upon passing 30 quizzes
(and claiming full credit for
D. a-thalassemia
each quiz) per year. Subscribers
E. Hereditary spherocytosis can start claiming MOC credits
as early as October 2022. To
learn how to claim MOC points,
go to: https://publications.aap.
org/journals/pages/moc-credit.

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e827

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 5. A neonate presents with hyperbilirubinemia and anemia. Hemolysis is noted
without obvious cause. Exchange transfusion is performed. On peripheral
blood smear, erythrocytes are normochromic. Further evaluation leads to
suspicion for pyruvate kinase deficiency. Which of the following statements
regarding pyruvate kinase deficiency is correct?
A. Hemolysis is caused by a structural defect in erythrocyte membranes
leading to an elliptical enlarged shape.
B. The most common inheritance pattern is X-linked dominant.
C. A decrease in adenosine triphosphate production creates a deficit of
energy required to maintain cellular structural and functional integrity.
D. Red blood cells are destroyed prematurely in the capillaries of the
kidneys and lungs, also leading to renal and pulmonary compromise.
E. Those with the heterozygous variant are protected against malaria.

e828 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 ARTICLE

Evaluating Genetic Disorders in the

Neonate: The Role of Exome Sequencing
in the NICU
T. Niroshi Senaratne, PhD,* Sulagna C. Saitta, MD, PhD†
*Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA
†
Division of Clinical Genetics, Department of Pediatrics, Division of Reproductive Genetics, Department of Obstetrics and Gynecology,
David Geffen School of Medicine at UCLA, Los Angeles, CA

PRACTICE GAPS

Understanding the strengths and limitations of various genetic tests is key

to developing an efficient diagnostic algorithm and providing appropriate
pre- and post-test genetic counseling. This review aims to improve the
understanding of exome sequencing among nongeneticists and promote
appropriate use of this diagnostic method.

OBJECTIVES After completing this article, readers should be able to:

1. Identify clinical scenarios where testing for single gene disorders is indicated.
2. Understand the distinction between next-generation sequencing panels
and exome sequencing.
3. Identify limitations of next-generation sequencing–based testing.
AUTHOR DISCLOSURES Drs Senaratne
and Saitta have disclosed no financial
relationships relevant to this article. This
ABSTRACT commentary does not contain a discussion
of an unapproved/investigative use of a
With recent advances in the technologies used for genetic diagnosis as well as commercial product/device.
our understanding of the genetic basis of disease, a growing list of options is
available for providers when caring for a newborn with features suggesting an
ABBREVIATIONS
underlying genetic etiology. The choice of the most appropriate genetic test
for a specific situation includes clinical considerations such as the phenotypic ACMG American College of Medical
Genetics and Genomics
features and type of genetic abnormality suspected, as well as practical
AMP Association for Molecular
considerations such as cost and turnaround time. In this review, we discuss Pathology
clinical exome sequencing in the context of genetic evaluation of newborns, CMA chromosomal microarray
including technical considerations, variant interpretation, and incidental/ analysis
CNVs copy number variants
secondary findings. Strengths and limitations of exome sequencing are
FISH fluorescence in situ
discussed and compared with those of other commonly known tests such as hybridization
karyotype analysis, fluorescence in situ hybridization, chromosomal microarray, NGS next-generation sequencing
and sequencing panels, along with integration of results from prenatal testing NIPS noninvasive prenatal screen
PPV positive predictive value
if available. We also review future directions including genome sequencing and
SNVs single nucleotide variants
other emerging technologies that are starting to be used in clinical settings. VUS variants of uncertain clinical
significance

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e829

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 INTRODUCTION In this review, we discuss the changing landscape of
Congenital anomalies, cardiorespiratory decompensation, or genetic testing relevant to NICU practice, with a focus on
neurologic features such as seizures or hypotonia can pre- sequencing-based testing, particularly the use of clinical
sent in the neonatal period and are often an indication for exome sequencing which is especially helpful when a dif-
admission to the NICU. Many of these conditions have an ferential diagnosis is expansive or not well defined.
underlying genetic etiology. Defining and confirming a diag-
nosis typically requires the use of cytogenetic and molecular BACKGROUND: EXOME SEQUENCING
genetic techniques. Early diagnosis leads to early adoption of NGS refers to massively parallel DNA sequencing which
a tailored treatment plan and enhances the accuracy of infor- allows interrogation of multiple targets simultaneously.
mation provided to the family. Most neonatal clinicians are NGS may be performed in a targeted manner, for exam-
familiar with the recognizable features of well-established ple, by sequencing multiple exons of a single gene or set
syndromes such as trisomies 13, 18, or 21. These chromo- of genes relevant to a specific clinical indication (sequenc-
somal disorders have been defined for several decades, and ing panel). Examples of NGS panels include testing for
most practitioners in NICU settings are aware of the clinical genes associated with congenital hearing loss, cardiomyopa-
features and further evaluation needs of such infants when thy, and infantile epilepsy. Alternatively, it can occur in a
a trisomy is suspected. In the past decade, the application of nontargeted, genome-wide fashion, which allows the identi-
next-generation sequencing (NGS) has allowed the early fication of new genetic disorders or unusual presentations
identification of many new disorders in which a single gene of known single-gene disorders. The application of NGS to
rather than a chromosomal region is disrupted, resulting in the coding regions of all genes, corresponding to approxi-
the patient’s clinical findings or phenotype. Often, the clini- mately 1% to 2% of the genome, is referred to as exome
cal diagnosis is not well-recognized and the care team is not sequencing (Fig). (1)(2) In contrast, genome sequencing also
able to immediately send testing for a specific disorder as includes noncoding regions both within and between
they might for trisomy 21, for example. genes. Currently, exome sequencing is more commonly

Figure. Example workflow for exome sequencing. Following DNA extraction from the patient specimen, the genomic DNA is fragmented by sonication or di-
gested with enzymes (dark blue sections represent coding regions or exons, whereas light blue sections represent noncoding regions including introns and
areas between genes). Library preparation includes ligation or attachment of adaptor sequences (green) to the DNA fragments. This will now allow for the
downstream sequencing steps. A target enrichment step is performed to enrich for coding regions, which is often performed by hybridization with complemen-
tary bait sequences. Then this library of target molecules is subjected to massively parallel DNA sequencing where each target molecule is sequenced many
times (red lines represent sequencing reads). The sequencing reads are then aligned to the appropriate position in the reference genome, and any positions
where the sequence reads differ from the reference are identified as sequence variants. These variants are then filtered and classified according to American Col-
lege of Medical Genetics and Genomics/Association for Molecular Pathology criteria, to identify DNA variants that may be clinically relevant for the individual.

e830 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 used than genome sequencing in clinical practice (see fur- gene-specific coverage information, if there are specific
ther discussion below). NGS is now routinely used in test- genes of interest for a given case.
ing for single-gene disorders, in particular, for disorders in
which single nucleotide variants (SNVs) are a common ge- VARIANT CLASSIFICATION AND
netic mechanism, as opposed to larger copy number vari- INTERPRETATION
ants (CNVs) in which larger stretches of a chromosome are Given the large number of sequence variants identified during
involved and typically detected on chromosomal microar- an exome sequencing study, the classification and clinical in-
rays. SNVs include variants that result in single amino acid terpretation of these variants can be a challenge for the report-
changes (missense variants) as well as variants that result ing laboratory as well as for ordering clinicians. Guidelines
in premature termination of protein translation (nonsense from the American College of Medical Genetics and Geno-
variants). NGS can also detect small insertion/deletion mics (ACMG) and the Association for Molecular Pathology
variants (indels) on the order of 5 to 10 base pairs that can (AMP) recommend a 5-tier system for the classification of
result in a frameshift, which can disrupt protein translation germline variants: 1) pathogenic, 2) likely pathogenic, 3) uncer-
and often result in premature termination of the nascent tain significance, 4) likely benign, and 5) benign. (4) Evidence
protein. Although exome sequencing is focused on the cod- supporting a benign or likely benign classification can include
ing regions of genes or exons, most assays also include a high prevalence in unaffected individuals, functional studies
small part of the adjacent introns, making it possible to iden- showing no damaging effect on protein function, or a variant
tify variants at the exon/intron boundaries, which can affect type that is not known to have a disease association for a spe-
RNA splicing, the resulting mRNA, and its translated protein. cific gene. In contrast, evidence supporting a pathogenic or
Multiple NGS technologies are available, which use dif- likely pathogenic classification includes reports of a specific
ferent sequencing chemistries. (3) The technologies most variant in similarly affected unrelated individuals, functional
commonly used in the clinical setting are short-read se- studies that show a damaging effect on protein function, or a
quencing methods, which are able to sequence relatively variant type expected to be damaging, such as nonsense var-
short fragments of DNA (100–500 base pairs). Technolo- iants in a gene in which loss-of-function is a known disease-
gies that are able to sequence longer DNA molecules are causing mechanism. This leaves a large number of variants of
also now available (see discussion under Future Directions). uncertain clinical significance (VUS), which cannot be definitely
Each target molecule is sequenced multiple times, resulting classified as disease-causing but also cannot be ruled out as
in multiple sequencing “reads,” and the number of reads benign. Therefore, the possible outcomes of exome sequenc-
covering a specific position is referred to as the depth of ing can include a diagnostic positive finding, a negative result
sequencing or coverage. In general, variant calls are more reli- (which does not rule out a genetic basis for the disease but
able as depth of coverage increases. (2) This is important in simply means that a causative variant has not been identified),
particular for mosaic variants that may only be present in a or a result of uncertain significance. It is important for pa-
small proportion of cells in the individual. It is important to tients to be informed about these possibilities during pretest
note that because exome sequencing involves sequencing of genetic counseling. (5)
a targeted portion of the entire genome (the exons), the pro- The development of large databases of genetic variation in
cedure requires a target enrichment step, and ultimately, healthy individuals such as the Exome Aggregation Consor-
the sequencing coverage is not uniform across the exome. tium (ExAC), (6) subsequently renamed the Genome Aggre-
Here, some genes will have much higher or lower coverage gation Database (gnomAD) (7) (https://gnomad.broadinstitute.
than others. It has been suggested that a minimum cover- org/) has greatly facilitated variant interpretation. (8) The fre-
age of 10× is required to identify heterozygous variants in quency of variants in gnomAD allows laboratories to distin-
the constitutional DNA, but this may not be sufficient for guish common benign/likely benign variations from rare
all regions. This is particularly true for genes in which variants that may be disease-causing, and also allows genes/
mosaic variants (not present in all cells) may cause disease. regions to be identified which are depleted from variation in
(2) In addition, due to variable coverage across the the general population and may be candidate disease loci.
exome/genome, to attain a minimum 10× coverage, the However, it is important to note that there may be a pop-
mean coverage required will be much higher (75–100× ulation variation not adequately captured by these data-
for exome sequencing, and 30× for genome sequenc- bases, and rarity/absence of a variant in gnomAD does
ing). (2) As coverage can vary depending on the assay not necessarily correlate to pathogenicity. Often more
used and other factors, laboratories can typically provide VUS are identified in individuals of ethnic groups that

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e831

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 are not well-represented in the study populations, and condition (carrier status) is reported. In addition, even when
the potential for misclassification of variants is also in- applying ACMG–AMP criteria, variants may be classified
creased. (5)(9) In addition, while variants present in data- differently between laboratories. (16)(17) This may become
bases such as gnomAD are unlikely to play a role in relevant if exome sequencing is performed in addition to
early-onset high-penetrance disorders, it is possible that other NGS testing such as a gene panel; some variants may
some of these variants are associated with incomplete be reported by the laboratory performing panel testing but
penetrance (wherein the clinical features may not be pre- not by the laboratory performing exome sequencing (or vice
sent in all individuals carrying the variant) or late-onset versa), and/or classified differently by the different laborato-
phenotypes. These scenarios would explain the presence of ries. These discrepancies in reporting can occur particularly
these variants in unaffected individuals. In genetic testing of when parental samples are available for a laboratory to help
newborns, when the main concern is to identify variants asso- with exome interpretation, while they may not have been in-
ciated with congenital disorders, databases such as gnomAD cluded for panel testing. In addition, with the growing appli-
can be extremely helpful to rule out benign/likely benign var- cation of prenatal exome sequencing (18)(19)(20)(21)(22) for
iants. Additional resources such as the ClinVar database (10) specific clinical indications, many laboratories have different
(https://www.ncbi.nlm.nih.gov/clinvar/), Online Mendelian reporting criteria for postnatal and prenatal cases. In some
Inheritance in Man (11) (https://www.omim.org/), and the cases, VUS may not be reported prenatally because it may
Human Gene Mutation Database (12) (http://www.hgmd.cf. not be possible to discern an associated phenotype in utero,
ac.uk/) are also helpful to identify known disease-causing var- for example, in a primarily neurodevelopmental gene. In a
iants in a particular gene. case with a history of prenatal exome sequencing but with-
In addition to the classification of variants according to out a diagnostic finding, one option is to order a postnatal
the ACMG-AMP criteria, laboratories interpret variants in reanalysis in which the same data are reanalyzed without re-
the context of patient phenotype, inheritance pattern, and peating the sequencing itself. Any additional phenotypic in-
additional evidence about the variant. For example, al- formation that may now be available postnatally as well as
though dozens of VUS may be identified in an individual, less stringent postnatal reporting criteria may reveal addi-
typically only those most relevant to the clinical indication tional variants.
are reported. Providing as much detail as possible regarding Reanalysis is also an option for postnatal exome sequenc-
patient phenotype, family history, and differential diagnoses ing if the initial test did not identify a diagnostic finding,
to the testing laboratory will facilitate the reporting of the as there will always be new genes with disease associations
most clinically relevant variants. In addition, many laborato- identified in the literature, and there is potential for new phe-
ries will prefer if parental samples can be submitted in addi- notypes to manifest in the patient, which can inform the
tion to the proband’s (“trio” exome sequencing if both exome reanalysis. Studies have shown that periodic reanalysis
biological parents are available or “duo” if only 1 parent is can increase the diagnostic yield of exome sequencing, rang-
available) which are then sequenced as comparators. Trio ing from a 5% to 26% increase in diagnostic yield. (23) In a
exome sequencing is usually preferred because having infor- series of 250 patients with initial exome analysis performed
mation about inheritance patterns facilitates variant filtering in 2012, comprehensive manual reanalysis in 2017 increased
and increases diagnostic yield. (13)(14)(15) Variants identified the diagnostic yield from 24.8% to 46.8%, most of which
as de novo (not inherited) in an individual with no family his- was due to identification of new disease genes. (24) Although
tory of the disease, or variants inherited from an affected par- there is no standard practice for the frequency of postnatal
ent when there is a family history, are strong candidates for exome reanalysis, (25) most studies reported in the literature
positive diagnostic findings. Depending on the clinical situa- were performed after 1- to 2-year intervals, (23)(26) which
tion, it may also be useful to include a sibling in the exome allows time for new genes and phenotypes to be reported in
sequencing test; for example, in 2 similarly affected siblings, the literature and in clinical databases. Exome reanalysis may
the laboratory can prioritize shared variants, or if 1 or both pa- be initiated by the clinician, or in some cases, may be initi-
rents are not available, the laboratory can infer an inheritance ated by the laboratory. In addition, if enough time has passed
pattern for shared variants seen in an unaffected sibling. since the initial exome analysis that significant improvements
It is important to keep in mind that each laboratory has have been made in the technology used for sequencing, re-
its own reporting criteria, which can affect which types of peat sequencing may be a better option compared with rean-
VUS a laboratory reports, or whether or not a single alysis. Consultation with the laboratory that performed the
heterozygous variant associated with an autosomal recessive initial testing is recommended when considering reanalysis.

e832 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 CONSENT FOR EXOME SEQUENCING AND algorithm, the exome test may not be able to identify dele-
INCIDENTAL FINDINGS tions or duplications at the level of single or multiple exons,
As described before, it is essential that patient families be which will be stated in their reporting criteria. In contrast,
appropriately counseled regarding the potential outcomes NGS panels, which typically provide higher coverage for the
of exome sequencing for their child. (27) This should in- selected genes of interest, may have greater power to detect
clude the potential for the exome test to identify so-called CNVs, as well as greater analytical sensitivity compared with
“incidental findings.” Unlike panel tests, which are focused exome sequencing. This is an important consideration, par-
on genes related to a specific indication, the exome test as- ticularly when testing for a very specific indication such as
sesses all genes, thus there is a chance to identify variants congenital hearing loss. However, in cases with a broad dif-
that are unrelated to the primary clinical indication. The ferential diagnosis, exome sequencing is expected to have a
ACMG has curated and regularly updates a list of genes higher overall diagnostic yield and is preferred. Thus, there
that should be evaluated for pathogenic/likely pathogenic is a trade-off between more expansive coverage and depth of
variants and reported even when unrelated to the reason coverage. Here, a medical genetics consultation can be help-
for referral. (28)(29)(30) (ACMG Secondary Findings or SF ful in selecting an appropriate testing platform.
gene list; see https://www.ncbi.nlm.nih.gov/clinvar/docs/ Another important consideration is the fact that certain
acmg/.) This list includes genes associated with hereditary regions of the genome are technically more difficult to se-
predisposition to cancer or cardiomyopathies, whereby re- quence, such as regions with high GC content, resulting
porting disease-causing variants can help identify individu- in lower coverage. Regions that contain repetitive DNA se-
als who will benefit from more frequent screening or other quences, including genes that share high sequence homo-
interventions specific to a certain disorder (ie, the genetic logy (pseudogenes), create challenges with alignment of
finding is associated with medical actionability). If trio short DNA reads, and result in poor coverage of these
exome sequencing is performed, the report will include in- genes. There are many examples of well-known disease genes
heritance information for these variants when found in the that may not be amenable to NGS testing due to high se-
patient, and thus has the potential to diagnose a parent at quence homology or other issues with sequence coverage;
the same time as a child. Even if family members do not these genes include PKD1 (autosomal dominant polycystic
undergo sequencing, the identification of an incidental kidney disease), STRC (autosomal recessive sensorineural
finding in a child can have implications for the entire fam- hearing loss), SMN1 (spinal muscular atrophy), CYP21A2 (con-
ily, because cascade testing may be recommended for un- genital adrenal hyperplasia), and CHEK2 and PMS2 (cancer
affected family members. Most laboratories provide an option predisposition), among others. (33) Consultation with testing
for families to opt out of receiving incidental findings includ- laboratories to confirm the coverage of specific genes in their
ing those in the ACMG secondary findings gene list, and the exome assay or on their curated panels may be indicated.
family should be informed of this option and the possible ad- Other types of variants that are not detectable with
vantages/disadvantages during the pretest genetic counseling. exome sequencing include triplet repeat expansions (such
In addition, because exome sequencing has the potential to as those associated with myotonic dystrophy or fragile
identify unexpected familial relationships, such as consan- X syndrome) or variants associated with imprinting disor-
guinity (31) or nonpaternity, (32) these outcomes should also ders, such as Beckwith-Weidemann syndrome, which are
be discussed with the family and alternative testing options often caused by DNA methylation changes (epigenetic var-
considered if needed. iants) rather than genetic variants. In cases with clinical
suspicion for a disorder in one of these categories, a tar-
LIMITATIONS OF EXOME SEQUENCING geted assay that can detect the types of alterations relevant
Although exome sequencing is a powerful technique, the as- to the disease is recommended.
say has certain limitations that must be kept in mind. First,
the exome test focuses on coding variation, and is not de- APPLICATION OF EXOME SEQUENCING IN THE NICU
signed to detect pathogenic variants that may be present in In newborns with clinical concern for a genetic disorder,
noncoding regions. Second, the variable coverage associated exome sequencing can be a powerful diagnostic tool. Exome
with exome sequencing creates challenges for the detection sequencing is often performed after other tests have ruled
of larger CNVs, which are typically detected using other diag- out other larger variant types, which may include karyotype
nostic methods such as chromosomal microarray (see further analysis, fluorescence in situ hybridization (FISH), and/or
discussion below). Depending on the laboratory’s assay and chromosomal microarray analysis (CMA). A description of

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e833

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Table. Comparison of Various Technologies Commonly Used for Genetic Diagnosis, Including Strengths and
Limitations of Each Technique
Common Applications/
Method Description Resolution Strengths and Limitations
Chromosome analysis Preparation of set of 5-10 Mb (in high-resolution Recognizable aneuploidy
(karyotype study) chromosomes from dividing studies, 3-5 Mb) syndromes, such as trisomy
cells, allowing the In practice, resolution varies (3 copies) of 13, 18, and 21,
identification of numerical depending on quality of or monosomy (only a single
and structural abnormalities. chromosome preparation copy) of the X chromosome
After staining with special Karyotype can also identify
dyes, each pair of structural abnormalities such
chromosomes shows a as larger deletions/
distinctive size, centromeric duplications as well as
position, and banding pattern translocations and
inversions, and is the study
of choice for this
Karyotype is often necessary to
determine the underlying
structural basis of
abnormalities identified with
other methods eg, to
identify cases of trisomy 21
resulting from a
Robertsonian translocation
Fluorescence in situ Uses fluorescently labeled DNA As small as 100–200 kb, Targeted assessment of the
hybridization (FISH) molecules (“probes”) that depending on probe design copy number of specific
bind to a specific region of chromosomal regions (eg,
interest in the genome, rule out common
followed by visualization of aneuploidies X, Y, 13, 18,
the hybridization patterns and 21, determine presence
using fluorescence or absence of SRY)
microscopy FISH has many advantages
including rapid turnaround
time (same day or overnight
for certain specimen types),
improved resolution
compared to karyotype,
high sensitivity for
mosaicism, and the ability to
interrogate nondividing
tissues, which means FISH
may be performed on a
wide variety of specimen
types
However, a limitation of FISH
is that it is only informative
for the specific regions
being targeted, and
therefore requires prior
knowledge of specific
conditions to test for, and
even then, may miss
atypical rearrangements of
these regions that are
outside the region targeted
by the FISH probes
Chromosomal Patient DNA is fragmented and 25–50 kb (depends on array CMA can detect copy number
microarray analysis fluorescently labeled before platform and laboratory variants in a genome-wide
(CMA) being hybridized to an array reporting criteria, which may approach, revealing
of DNA probes. Microarray vary between prenatal and disorders usually identified
testing can be performed for postnatal analysis) by cytogenetic analysis and
either a targeted region, or multiple individual FISH
more commonly, in a studies
genome-wide fashion CMA can detect changes too
small to be observed by
karyotype analysis, including
microdeletion/microduplication
syndromes, and those with
atypical breakpoints which may
be missed by FISH
Continued

e834 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Table. (Continued)
Common Applications/
Method Description Resolution Strengths and Limitations
Structural rearrangements that do
not affect copy number such as
balanced translocations or
inversions are not detectable
with this method
Next-generation Allows multiplexed DNA Single nucleotide variants (SNVs) Detection of single-gene
sequencing (NGS) sequencing of different target as well as small insertions/ disorders, which may be
regions, eg, set of genes duplications (indels) 5–10 bp done in a targeted manner
related to a specific (NGS panels) or by
indication (panel), exome interrogation of the coding
(coding regions of all genes), regions of all genes (exome
or genome (coding and non- sequencing) in cases with a
coding regions) broader differential diagnosis
Mainly used to detect SNVs
(missense and nonsense
variants) as well as small
insertions/duplications
(frameshifts)
Depending on the assay and
variant calling algorithms
that are used, certain NGS
assays can also detect
exon-level deletions/
duplications

these methods, common applications, and strengths and that led to the genomic imbalances identified on array. For
limitations of each technique are given in the Table. For example, although CMA (or FISH) can identify the gain of
cases with high clinical suspicion for well-recognized whole chromosome 21 associated with Down syndrome, these
chromosome disorders (aneuploidy), a karyotype is recom- methods cannot distinguish Down syndrome cases due to a
mended. Depending on the clinical scenario, FISH can pro- translocation from regular trisomy 21, with 3 freestanding
vide added benefits. For example, FISH provides a rapid copies of the chromosome. Knowing if a translocation is the
turnaround time, is relatively sensitive for mosaicism, and underlying mechanism for Down syndrome in an infant can
can detect changes too small to be observed on karyotype; have important implications for the family’s recurrence risk
therefore FISH can be used in cases in which a sex chromo- in future pregnancies. CMA may also identify copy number
some abnormality is suspected to rapidly confirm the sex changes suggestive of an underlying structural abnormality,
chromosome complement (XX or XY), to detect mosaicism such as a gain or duplication involving a terminal segment
(eg, mosaic Turner syndrome, in which both 45,X and of one chromosome and a loss or deletion involving a termi-
46,XX cell populations are noted), or to detect small dele- nal segment of another chromosome. This scenario is sug-
tions involving the SRY gene on the Y chromosome. A dis- gestive of an unbalanced translocation. In these cases, a
advantage of FISH is that because it is a targeted assay, it karyotype test is indicated to confirm the underlying genetic
can miss atypical rearrangements, such as cases of Turner mechanism. Again, structural analysis is typically needed for
syndrome caused by structural abnormalities of the X chro- accurate recurrence risk counseling for the parents of a child
mosome rather than loss of the entire X chromosome, so affected with a chromosomal disorder.
FISH is usually recommended in conjunction with karyotyp- Tests such as karyotyping, FISH, and CMA can iden-
ing. Over the past decade, FISH testing for other situations tify relatively large-scale chromosome abnormalities, which
has been largely replaced by CMA when there is clinical con- are important to rule out disorders associated with loss or
cern for a microdeletion/microduplication disorder. With a gain of multiple genes. Clinical scenarios raising concern
single test, microarray can detect CNVs in a genome-wide for a chromosome abnormality include involvement of multi-
approach, revealing disorders usually identified by cytoge- ple organ systems and/or recognizable phenotypes associated
netic analysis and multiple individual FISH studies. It is im- with a known deletion or duplication syndrome. Examples of
portant to note that in certain cases, microarray will still recognizable syndromes include deletions in chromosomes
require follow-up testing with karyotype and/or FISH, which 4p (Wolf-Hirschhorn syndrome), 5p (cri-du-chat), 22q11.2
can provide information about the underlying mechanism (DiGeorge/velocardiofacial syndrome), or 1p36, among others.

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e835

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 CMA is recommended as a first-tier test in the genetic result has the potential to influence patient care, (43) exome
evaluation of patients with unexplained developmental sequencing can also be a more efficient strategy than per-
delay/intellectual disability, autism spectrum disorders, forming multiple single-gene tests or NGS panels. However,
or multiple congenital anomalies, and has been shown to NGS panels are still useful when there are commercial panels
have a diagnostic yield of 15% to 20% for these indica- available which cover the genes of interest for a specific case.
tions. (34) Advantages of performing NGS panel testing include lower
Exome sequencing is mainly used for detection of SNVs, cost and reduced potential for incidental/secondary find-
and can be very useful in testing for disorders caused by ings. Because panel-based tests often have higher sequenc-
pathogenic variants in single genes. This is especially true ing depth and coverage, they have the potential to detect
after a chromosomal abnormality has been ruled out using deletions/duplications that may have been missed with
CMA and/or karyotyping and FISH. However, it is impor- exome sequencing. This becomes particularly useful for
tant to note that in some clinical scenarios, a single-gene CNVs that are too small to observe with CMA (eg, single
disorder may be considered more likely than a chromo- exon-level deletions or duplications).
somal abnormality, and exome sequencing or other NGS With increasing use of prenatal genetic testing, it is pos-
assay may be performed as the first test. Examples include sible that some of the genetic evaluation, in particular for
infantile epileptic encephalopathy, inborn errors of metabo- ruling out chromosome abnormalities, may have been com-
lism, Noonan syndrome spectrum disorders (RASopathies), pleted prenatally. A common indication for prenatal diag-
and certain cardiac malformations such as heterotaxy. Even nostic testing is an abnormal noninvasive prenatal screen
for indications such as developmental delay/intellectual dis- (NIPS). Also known as noninvasive prenatal testing or cell-free
ability and multiple congenital anomalies when CMA has DNA screening, these assays analyze the cell-free fetal DNA
typically been performed first, recent reviews suggest that fraction (derived from placental trophoblasts) circulating in
exome and genome sequencing is very useful and can be maternal peripheral blood, and can be offered starting at
cost-effective when used early in the diagnostic evaluation, 9 to 10 weeks of gestation. (44)(45)(46) Although NIPS was
suggesting that exome or genome sequencing may be con- initially recommended for women at high risk of carrying a
sidered as a first-tier or second-tier test. (35) Therefore, it is fetus with a chromosomal abnormality, recent guidelines
likely that the genetic evaluation of these cases will change recommend that it should be offered to all pregnant women
in the future, especially as technological improvements con- regardless of age or other risk factors. (46) The technical ap-
tinue to expand the types of variants that can be identified proaches and analyses vary by platform, but in general,
by sequencing-based assays. NIPS uses NGS to assess the copy number of different chro-
Studies of unselected case cohorts have reported the diag- mosome regions in cell-free DNA. It is performed as an ini-
nostic yield of exome sequencing to range from 25% to 32%, tial screen for common aneuploidy conditions, with some
(15)(36)(37)(38)(39) with higher yields reported for specific laboratories screening for rarer aneuploidies as well as
clinical indications. (5) For example, one study reported a microdeletion/microduplication syndromes and other seg-
yield of 36% for patients with multiple congenital anomalies, mental aneuploidies. An important metric to consider when
(40) while another group reported a yield of 56% for individ- counseling patients with abnormal NIPS results is the posi-
uals with laterality disorders (eg, heterotaxy) and associated tive predictive value (PPV), which is the probability that a
congenital heart defects. (41) The different estimates for diag- positive screening result represents a true positive. Although
nostic yield reported in the literature should be interpreted in NIPS has high PPV for common aneuploidies such as tri-
the context of patient selection criteria as well as differences somy 21, the PPV is significantly lower for rarer aneuploi-
in variant interpretation/definition of a diagnostic finding dies and microdeletions, and appropriate follow-up testing
among laboratories. and genetic counseling are critical for an accurate diagnosis.
Because exome sequencing is a wide-ranging approach, it (46)(47)(48)(49) In addition, because the risk of trisomy in-
is particularly useful in cases in which the differential diagno- creases with maternal age, the PPV of NIPS is age depen-
sis is broad, and there is suspicion for a genetic disorder with- dent. For patients in whom NIPS shows a high risk of a
out a clear candidate gene or genes to test. It is also useful for chromosomal abnormality in the fetus, or with atypical/
disorders that may have genetic heterogeneity (ie, multiple inconclusive results, diagnostic testing such as karyotyping
causative genes associated with a certain phenotype) or for or CMA of amniotic fluid or chorionic villus specimens is
which other tests have failed to identify a causative genetic recommended. (46) Because the range of disorders accurately
variant. (42) In the NICU setting, where obtaining a rapid identified with NIPS is limited, and because it represents a

e836 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 screening technology rather than diagnostic testing, it cannot the vast majority of noncoding variation, the impact on
replace diagnostic testing at this time. gene expression and function is uncertain. The develop-
Another common indication for prenatal testing is the ment of splice prediction algorithms (59)(60) has facilitated
identification of structural anomalies during fetal ultraso- the interpretation of noncoding variation by identifying var-
nography. Depending on the types of ultrasound abnormal- iants that may disrupt proper splicing of RNA. However,
ities observed, and often after ruling out a chromosomal functional validation of these predictions through RNA se-
abnormality, some cases may also receive prenatal NGS quencing may be necessary. (61) In spite of these chal-
panels and/or exome sequencing. (18)(19)(20)(21)(22) Re- lenges, the advantages of detecting multiple variant types
sults from prenatal diagnostic testing, if available, can through a single assay will likely lead to increased applica-
help inform the most efficient diagnostic algorithm and tion of genome sequencing in years to come.
even affect the delivery plan and immediate clinical Genome sequencing is also not without its limitations.
plan in the newborn setting. However, prenatal exome For example, most NGS-based methods do not have robust
sequencing is also associated with several challenges, in- performance in repeat-rich and other difficult-to-sequence re-
cluding limited availability of phenotypic information gions, as is also seen in exome sequencing. A growing num-
needed for interpreting variants, as well as lack of data in ber of technological developments facilitate the interrogation
the literature regarding the prenatal presentation associated of such difficult-to-sequence regions. For example, long-read
with variants in certain genes. (50) Prenatal exome se- sequencing methods, which allow for sequencing of longer
quencing is generally recommended for cases with fetal target DNA molecules, (62) have recently facilitated the se-
anomalies suggestive of a genetic etiology with no genetic quencing of a complete human genome, (63)(64) and have
diagnosis after CMA, and/or with a history of recurrent also been shown to be clinically useful for identifying CNVs.
pregnancy loss or similarly affected pregnancies. (50) (65) Another emerging technology that provides an alternative
to sequencing is optical genome mapping, in which DNA is
UTILITY OF GENOME SEQUENCING AND FUTURE labeled at specific sequence motifs and then linearized and
DIRECTIONS passed through nanochannels, which allows large molecules
Genome sequencing is also being increasingly used for clini- of DNA to be imaged. This technology has been shown to be
cal diagnosis, and has been shown to have utility in the new- particularly effective in detecting structural rearrangements
born/pediatric intensive care settings (43)(51)(52)(53); however, and CNVs in various clinical settings. (66)(67)(68) Thus it is
the relative increase in yield from genome sequencing (ie, likely that the landscape of genetic testing available and the
the proportion of diagnostic findings that would not have types of genetic variants identified will continue to evolve
been detected with exome sequencing) is still unclear. Ge- rapidly.
nome sequencing has several advantages over exome se-
quencing, including more uniform sequencing coverage CONCLUSIONS
and the ability to detect disease-causing variation in non- With the growing list of diagnostic methods available in the
coding regions. This may include variants in introns that af- clinical setting as well as the ever-expanding knowledge of
fect RNA splicing (while exome sequencing can typically the genetic basis of disease, genetic testing approaches are
identify variants near the intron/exon boundaries, genome continually evolving. Understanding the types of variants
sequencing is required to identify deep intronic variants), that can be detected with each assay and the strengths and
CNVs near genes that affect their expression (position limitations of each technique are important for developing
effects), or transposable element insertions in noncoding a plan for genetic testing as well as genetic counseling for
regions. (54)(55)(56) Genome sequencing has also been patients and their families. Exome sequencing is a powerful
shown to be capable of accurately detecting CNVs for clini- technique that allows coding variation to be interrogated in
cal diagnosis (57)(58) and therefore has the potential to a genome-wide fashion, which is particularly useful when a
replace exome sequencing and CMA with a single test. Fur- single-gene disorder is suspected but the differential diag-
ther, it could increase diagnostic yield by identifying dele- nosis is broad. Due to limitations in the types of variants
tions and duplications too small to be detected with CMA that can be detected, exome sequencing may be used in
but not detectable with exome sequencing. Currently, sig- conjunction with other tests, though this depends on the
nificant challenges are associated with the implementation clinical scenario and may change in the future as diagnostic
of genome sequencing in the clinical setting, including modalities continue to evolve. Ultimately, the identification
issues with reimbursement and variant interpretation. For of a diagnostic genetic finding has important implications,

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e837

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 including developing a care plan for the individual needs of 13. Carneiro TN, Krepischi AC, Costa SS, et al. Utility of trio-based
exome sequencing in the elucidation of the genetic basis of isolated
the patient, in some cases with implications for treatment,
syndromic intellectual disability: illustrative cases. Appl Clin Genet.
surveillance, and/or family planning, and also in providing 2018;11:93–98
a much-needed answer for patient families. 14. Wright CF, Fitzgerald TW, Jones WD, et al; DDD study. Genetic
diagnosis of developmental disorders in the DDD study: a scalable
analysis of genome-wide research data. Lancet. 2015;385(9975):
1305–1314
American Board of Pediatrics 15. Lee H, Deignan JL, Dorrani N, et al. Clinical exome sequencing for
Neonatal-Perinatal Content genetic identification of rare Mendelian disorders. JAMA. 2014;
312(18):1880–1887
Specification 16. Amendola LM, Jarvik GP, Leo MC, et al. Performance of ACMG-
• Know the disorders for which molecular genetic stud- AMP variant-interpretation guidelines among nine laboratories in
ies are clinically indicated, such as cystic fibrosis, and the Clinical Sequencing Exploratory Research Consortium. Am J
how to interpret test results. Hum Genet. 2016;98(6):1067–1076
17. Amendola LM, Muenzen K, Biesecker LG, et al; CSER Sequencing
and Diagnostic Yield Working Group. Variant classification
concordance using the ACMG-AMP variant interpretation guidelines
across nine genomic implementation research studies. Am J Hum
References Genet. 2020;107(5):932–941

1. Seaby EG, Pengelly RJ, Ennis S. Exome sequencing explained: a 18. Lord J, McMullan DJ, Eberhardt RY, et al; Prenatal Assessment
practical guide to its clinical application. Brief Funct Genomics. 2016; of Genomes and Exomes Consortium. Prenatal exome
15(5):374–384 sequencing analysis in fetal structural anomalies detected by
ultrasonography (PAGE): a cohort study. Lancet. 2019;393(10173):
2. Rehder C, Bean LJH, Bick D, et al. Next-generation sequencing for
747–757
constitutional variants in the clinical laboratory, 2021 revision: a
technical standard of the American College of Medical Genetics and 19. Petrovski S, Aggarwal V, Giordano JL, et al. Whole-exome
Genomics (ACMG). Genet Med. 2021;23(8):1399–1415 sequencing in the evaluation of fetal structural anomalies: a
prospective cohort study. Lancet. 2019;393(10173):758–767
3. Slatko BE, Gardner AF, Ausubel FM. Overview of next-generation
sequencing technologies. Curr Protoc Mol Biol. 2018;122(1):e59 20. Monaghan KG, Leach NT, Pekarek D, Prasad P, Rose NC; ACMG
Professional Practice and Guidelines Committee. The use of fetal
4. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the
exome sequencing in prenatal diagnosis: a points to consider
interpretation of sequence variants: a joint consensus recommendation document of the American College of Medical Genetics and
of the American College of Medical Genetics and Genomics and Genomics (ACMG). Genet Med. 2020;22(4):675–680
the Association for Molecular Pathology. Genet Med. 2015;17(5):
21. Sparks TN, Lianoglou BR, Adami RR, et al. Exome sequencing for
405–424
prenatal diagnosis in nonimmune hydrops fetalis. N Engl J Med.
5. O’Donnell-Luria AH, Miller DT. A clinician’s perspective on clinical 2020;383(18):1746–1756.
exome sequencing. Hum Genet. 2016;135(6):643–654
22. Li R, Fu F, Yu Q, et al. Prenatal exome sequencing in fetuses with
6. Lek M, Karczewski KJ, Minikel EV, et al; Exome Aggregation congenital heart defects. Clin Genet. 2020;98(3):215–230
Consortium. Analysis of protein-coding genetic variation in 60,706
23. Ji J, Leung ML, Baker S, Deignan JL, Santani A. Clinical exome
humans. Nature. 2016;536(7616):285–291
reanalysis: current practice and beyond. Mol Diagn Ther. 2021;25(5):
7. Karczewski KJ, Francioli LC, Tiao G, et al. The mutational constraint 529–536
spectrum quantified from variation in 141,456 humans. Nature.
24. Liu P, Meng L, Normand EA, et al. Reanalysis of clinical exome
2020;581(7809):434–443
sequencing data. N Engl J Med. 2019;380(25):2478–2480
8. Gudmundsson S, Singer-Berk M, Watts NA, et al; Genome
25. Deignan JL, Chung WK, Kearney HM, Monaghan KG, Rehder
Aggregation Database Consortium. Variant interpretation using
CW, Chao EC; ACMG Laboratory Quality Assurance Committee.
population databases: lessons from gnomAD. Hum Mutat. 2022;
Points to consider in the reevaluation and reanalysis of genomic
43(8):1012–1030
test results: a statement of the American College of Medical
9. Manrai AK, Funke BH, Rehm HL, et al. Genetic misdiagnoses and Genetics and Genomics (ACMG). Genet Med. 2019;21(6):
the potential for health disparities. N Engl J Med. 2016;375(7): 1267–1270
655–665
26. Ewans LJ, Schofield D, Shrestha R, et al. Whole-exome sequencing
10. Landrum MJ, Lee JM, Benson M, et al. ClinVar: improving access to reanalysis at 12 months boosts diagnosis and is cost-effective when
variant interpretations and supporting evidence. Nucleic Acids Res. applied early in Mendelian disorders. Genet Med. 2018;20(12):
2018;46(D1):D1062–D1067 1564–1574
11. Amberger JS, Bocchini CA, Scott AF, Hamosh A. OMIM.org: 27. ACMG Board of Directors. Points to consider for informed
leveraging knowledge across phenotype-gene relationships. Nucleic consent for genome/exome sequencing. Genet Med. 2013;15(9):
Acids Res. 2019;47(D1):D1038–D1043 748–749
12. Stenson PD, Ball EV, Mort M, et al. Human Gene Mutation 28. Green RC, Berg JS, Grody WW, et al. ACMG recommendations for
Database (HGMD): 2003 update. Hum Mutat. 2003;21(6): reporting of incidental findings in clinical exome and genome
577–581 sequencing. Genet Med. 2013;15(7):565–574

e838 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 29. Kalia SS, Adelman K, Bale SJ, et al. Recommendations for reporting 44. Wong FC, Lo YM. Prenatal diagnosis innovation: genome
of secondary findings in clinical exome and genome sequencing, sequencing of maternal plasma. Annu Rev Med. 2016;67:
2016 update (ACMG SF v2.0): a policy statement of the American 419–432
College of Medical Genetics and Genomics. Genet Med. 2017;19(2): 45. Hui L, Bianchi DW. Noninvasive prenatal DNA testing: the
249–255 vanguard of genomic medicine. Annu Rev Med. 2017;68:
30. Miller DT, Lee K, Gordon AS, et al; ACMG Secondary Findings 459–472
Working Group. Recommendations for reporting of secondary 46. American College of Obstetricians and Gynecologists’ Committee on
findings in clinical exome and genome sequencing, 2021 Practice Bulletins—Obstetrics; Committee on Genetics, Society for
update: a policy statement of the American College of Medical
Maternal-Fetal Medicine. Screening for fetal chromosomal
Genetics and Genomics (ACMG). Genet Med. 2021;23(8):
abnormalities: ACOG practice bulletin, number 226. Obstet Gynecol.
1391–1398
2020;136(4):e48–e69
31. Gonzales PR, Andersen EF, Brown TR, et al; on behalf of the
47. Gregg AR, Skotko BG, Benkendorf JL, et al. Noninvasive prenatal
ACMG Laboratory Quality Assurance Committee. Interpretation and
screening for fetal aneuploidy, 2016 update: a position statement of
reporting of large regions of homozygosity and suspected
the American College of Medical Genetics and Genomics. Genet
consanguinity/uniparental disomy, 2021 revision: a technical
Med. 2016;18(10):1056–1065
standard of the American College of Medical Genetics and
Genomics (ACMG). Genet Med. 2022;24(2):255–261 48. Schwartz S, Kohan M, Pasion R, Papenhausen PR, Platt LD. Clinical
experience of laboratory follow-up with noninvasive prenatal testing
32. Eno C, Bayrak-Toydemir P, Bean L, et al. Misattributed parentage as
using cell-free DNA and positive microdeletion results in 349 cases.
an unanticipated finding during exome/genome sequencing: current
Prenat Diagn. 2018;38(3):210–218
clinical laboratory practices and an opportunity for standardization.
Genet Med. 2019;21(4):861–866 49. Martin K, Iyengar S, Kalyan A, et al. Clinical experience with a
single-nucleotide polymorphism-based non-invasive prenatal test for
33. Mandelker D, Schmidt RJ, Ankala A, et al. Navigating highly
five clinically significant microdeletions. Clin Genet. 2018;93(2):
homologous genes in a molecular diagnostic setting: a resource for
293–300
clinical next-generation sequencing. Genet Med. 2016;18(12):
1282–1289 50. International Society for Prenatal Diagnosis; Society for
Maternal and Fetal Medicine; Perinatal Quality Foundation.
34. Miller DT, Adam MP, Aradhya S, et al. Consensus statement:
Joint position statement from the International Society for
chromosomal microarray is a first-tier clinical diagnostic test for
Prenatal Diagnosis (ISPD), the Society for Maternal Fetal
individuals with developmental disabilities or congenital anomalies.
Am J Hum Genet. 2010;86(5):749–764 Medicine (SMFM), and the Perinatal Quality Foundation (PQF)
on the use of genome-wide sequencing for fetal diagnosis.
35. Manickam K, Mcclain MR, Demmer LA, et al. Exome and genome
Prenat Diagn. 2018;38(1):6–9
sequencing for pediatric patients with congenital anomalies or
intellectual disability: an evidence-based clinical guideline of the 51. Sanford EF, Clark MM, Farnaes L, et al; RCIGM Investigators.
American College of Medical Genetics and Genomics (ACMG). Rapid whole genome sequencing has clinical utility in children in
Genet Med. 2021;23(11):2029–2037 the PICU. Pediatr Crit Care Med. 2019;20(11):1007–1020

36. Yang Y, Muzny DM, Xia F, et al. Molecular findings among patients 52. Dimmock D, Caylor S, Waldman B, et al. Project Baby Bear: rapid
referred for clinical whole-exome sequencing. JAMA. 2014;312(18): precision care incorporating rWGS in 5 California children’s
1870–1879 hospitals demonstrates improved clinical outcomes and reduced
costs of care. Am J Hum Genet. 2021;108(7):1231–1238
37. Iglesias A, Anyane-Yeboa K, Wynn J, et al. The usefulness of whole-
exome sequencing in routine clinical practice. Genet Med. 2014; 53. Mestek-Boukhibar L, Clement E, Jones WD, et al. Rapid
16(12):922–931 paediatric sequencing (RaPS): comprehensive real-life workflow
for rapid diagnosis of critically ill children. J Med Genet. 2018;
38. Valencia CA, Husami A, Holle J, et al. Clinical impact and cost-
55(11):721–728
effectiveness of whole exome sequencing as a diagnostic tool: a
pediatric center’s experience. Front Pediatr. 2015;3:67 54. Vaz-Drago R, Cust

odio N, Carmo-Fonseca M. Deep intronic
mutations and human disease. Hum Genet. 2017;136(9):
39. Farwell KD, Shahmirzadi L, El-Khechen D, et al. Enhanced
1093–1111
utility of family-centered diagnostic exome sequencing with
inheritance model-based analysis: results from 500 unselected 55. Zhang F, Lupski JR. Non-coding genetic variants in human disease.
families with undiagnosed genetic conditions. Genet Med. 2015; Hum Mol Genet. 2015;24(R1):R102–R110
17(7):578–586 56. Payer LM, Burns KH. Transposable elements in human genetic
40. Retterer K, Juusola J, Cho MT, et al. Clinical application of whole- disease. Nat Rev Genet. 2019;20(12):760–772
exome sequencing across clinical indications. Genet Med. 2016;18(7): 57. Gross AM, Ajay SS, Rajan V, et al. Copy-number variants in clinical
696–704 genome sequencing: deployment and interpretation for rare and
41. Bolkier Y, Barel O, Marek-Yagel D, et al. Whole-exome sequencing undiagnosed disease. Genet Med. 2019;21(5):1121–1130
reveals a monogenic cause in 56% of individuals with laterality 58. Chaubey A, Shenoy S, Mathur A, et al. Low-pass genome
disorders and associated congenital heart defects. J Med Genet. 2022; sequencing: validation and diagnostic utility from 409 clinical cases
59(7):691–696 of low-pass genome sequencing for the detection of copy number
42. ACMG Board of Directors. Points to consider in the clinical variants to replace constitutional microarray. J Mol Diagn. 2020;
application of genomic sequencing. Genet Med. 2012;14(8):759–761 22(6):823–840
43. Farnaes L, Hildreth A, Sweeney NM, et al. Rapid whole-genome 59. Jian X, Boerwinkle E, Liu X. In silico prediction of splice-altering
sequencing decreases infant morbidity and cost of hospitalization. single nucleotide variants in the human genome. Nucleic Acids Res.
NPJ Genom Med. 2018;3:10 2014;42(22):13534–13544

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e839

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 60.Jaganathan K, Kyriazopoulou Panagiotopoulou S, McRae JF, et al. 65. Magini P, Mingrino A, Gega B, et al. Third-generation cytogenetic
Predicting splicing from primary sequence with deep learning. Cell. analysis. J Mol Diagn. 2022;24(7):711–718
2019;176(3):535–548.e24 66.Lam ET, Hastie A, Lin C, et al. Genome mapping on nanochannel
61. Lee H, Huang AY, Wang L-K, et al. Diagnostic utility of transcriptome arrays for structural variation analysis and sequence assembly. Nat
sequencing for rare Mendelian diseases. Genet Med. 2020;22(3): Biotechnol. 2012;30(8):771–776
490–499 67. Mantere T, Neveling K, Pebrel-Richard C, et al. Next generation
cytogenetics: genome-imaging enables comprehensive structural
62. Logsdon GA, Vollger MR, Eichler EE. Long-read human genome
variant detection for 100 constitutional chromosomal aberrations in
sequencing and its applications. Nat Rev Genet. 2020;21(10):
85 samples. bioRxiv. 2020:2020.07.15.205245
597–614
68. Sahajpal NS, Barseghyan H, Kolhe R, Hastie A, Chaubey A. Optical
63. Zahn LM. Filling the gaps. Science. 2022;376(6588):42–43
genome mapping as a next-generation cytogenomic tool for
64. Nurk S, Koren S, Rhie A, et al. The complete sequence of a human detection of structural and copy number variations for prenatal
genome. Science. 2022;376(6588):44–53 genomic analyses. Genes (Basel). 2021;12(3):398

e840 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 INDEX OF SUSPICION IN THE NURSERY

Increased Nuchal Translucency Without

Aneuploidy in a Fetus: Abnormal
Postnatal Radiograph and
a Novel Mutation
Niraj Kumar Dipak, NKD,* Mamta Muranjan, MM,† Shilpa Pandya, SP,‡ Manjusha Bhikurao Naik, MBN‡
*Department of Pediatrics & Neonatology, Motherland Hospital, Noida, Uttar Pradesh, India
†
Department of Pediatrics, Genetic Division, Seth GS Medical College, KEM Hospital, Parel, Mumbai, Maharashtra, India
‡
Department of Pediatrics & Neonatology, Dr LH Hiranandani Hospital, Powai, Mumbai, Maharashtra, India

PRESENTATION
A full-term (40 1/7 weeks) male infant with a birthweight of 3.630 kg is delivered
via lower-segment cesarean section by a 31-year-old primigravida woman. Prenatal
screening was unremarkable and showed her to be VDRL nonreactive, HBsAg nega-
tive, rubella immune, and human immunodeficiency virus nonreactive. The preg-
nancy is complicated by increased nuchal translucency at 13 weeks which is followed
by amniocentesis revealing normal karyotype and polyhydramnios by 32 weeks. The
infant cries immediately after birth with normal Apgar scores noted upon birth.
Physical examination shows generalized floppiness (Fig 1) with multiple facial dys-
morphisms, ie, high forehead, depressed nasal bridge, hypertelorism, bilateral dys-
plastic ears, high-arched palate, and micrognathia (Fig 2). In addition, a plethora of
abnormal physical findings are noted, including wide open anterior fontanelle, large
posterior fontanelle with separated sutures, bilateral corneal edema/cloudiness,
redundant fold of skin over neck, widely spaced nipples, bilateral undescended testis,
and bilateral congenital talipes equinovarus. Over the first several postnatal days, he
requires noninvasive respiratory support with intermittent mechanical ventilation
initially for retained lung fluids, and later, for inability to maintain airway and gener-
alized hypotonia. Chest radiography performed for respiratory distress is marked by
epiphyseal stippling at the hip joint (head of femur) bilaterally (Fig 3).
Ultrasonography reveals no structural anomaly of the skull, but in the abdomen
and kidney, ureter, and bladder region, bilateral echogenic kidneys are noted, sug-
gesting cortical cysts with normal corticomedullary differentiation.
In view of normal karyotype found via amniocentesis, generalized hypotonia, and epiph-
AUTHOR DISCLOSURES Drs Dipak,
yseal stippling on radiography, a range of tests is requested. Plasma very long chain and Muranjan, Pandya, and Naik have
branched chain fatty acids reveal elevated C26:0- 0.92 (0.231/ 0.09 microg/ml) disclosed no financial relationships
and C26/C22: 0.05 (0.01±0.004), whereas serum phytanic acid, pristanic acid, C22 0, and relevant to this article. This commentary
does not contain a discussion of an
C24 0 are within normal limits. Further, serum pipecolic acid level is 376.65 mmol/L unapproved/investigative use of a
(range, 0.7–2.5 mmol/L) and plasmalogen-to-fatty acid ratio indicates normal C16:0 commercial product/device.

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e841

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Figure 3. Epiphyseal stippling at hip joint bilaterally.

117x) resulting in a frameshift and premature truncation of

the protein 14 amino acids downstream to codon 789
(p.Phe789CysfsTer14; ENST00000304611) is detected. Sequencing
analysis of the parents shows heterozygous state for this novel
mutation of c.42933525_42933526delACof PEX6.

DISCUSSION
Differential Diagnosis
Increased nuchal translucency is considered to be a marker
of chromosomal anomalies. But the same without aneu-
ploidy is not completely innocuous. These cases may present
with fetal akinesia, intrathoracic and extrathoracic compres-
sive syndromes (ie, if there is abnormally narrow thoracic
Figure 1. Generalized floppiness with bilateral congenital talipes cage or an intrathoracic lesion such as a diaphragmatic her-
equinovarus.
nia), orofacial cleft defects, body stalk anomalies, and struc-
tural anomalies, including cardiac defects. (1)(2)
DMA/C16:0 fatty acid 0.054 (0.021–0.070) and low C18:0
However, a substantial number of such fetuses have no
DMA/C18:0 fatty acid ratios 0.022 (0.027– 0.120).
anomalies and good neonatal outcome. Vieira et al (3)
Molecular genetic testing study reveals a novel mutation
found normal karyotype in 54.4% of 116 cases of nuchal
of PEX6 gene. A homozygous 2–base pair deletion in exon
13 of the PEX6 gene (chr6:42933525_42933526delAC; Depth: translucency above the 95th percentile (only 68% had con-
ventional karyotype available). Normal births and immedi-
ate postnatal development were observed in 93% of these
fetuses. Socolov et al (4) reported that of 58 euploid cases,
40 (69%) had normal outcome and 18 (31%) had adverse
outcomes (miscarriages, cardiac abnormalities, or compli-
cations due to prematurity).
Generally, syndromes that result in profound neonatal
hypotonia, (ie, Down syndrome, Prader Willi syndrome, Lowe
syndrome) do not cause epiphyseal stippling. Stippled epiphy-
sis is seen in rhizomelic chondrodysplasia punctata (RCDP),
Zellweger syndrome, Conradi-H€ unermann syndrome, warfa-
rin embryopathy, alcohol-related embryopathy, maternal
mixed connective tissue disorder, and maternal systemic lu-
pus erythematosus (SLE). (5) Cases are described in which
mothers with high-titer antiribonucleoprotein antibodies and
Figure 2. Low-set ears with deformity, redundant fold of skin at neck. maternal SLE have fetuses with chondrodysplasia punctata

e842 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 and no identifiable genetic disorder, biochemical deviation, or Patient Course
history of teratogen exposure. (5) On day 19 after birth, following some improvement in
tone, the infant was weaned off respiratory support, but
Actual Diagnosis he remained dependent on tube feeds and was discharged
With normal karyotype in amniocentesis, neonatal hypotonia, on day 26 with feeding tube in situ.
characteristic facial features, and epiphyseal stippling on radio- At 2 months, the infant was admitted again for an episode
graphy, peroxisomal disorders are considered. The diagnosis of fever with urinary tract infection (Klebsiella pneumonia).
in the current case was established based on suggestive clini- Later he succumbed to an episode of severe pneumonia at
cal and biochemical features and identification of a novel mu- 5 months of age.
tation of the PEX6 gene. Biallelic pathogenic variants in 1 of
13 known PEX genes are responsible for peroxisomal disor- Lessons for the Clinician
ders, of which PEX1, PEX6, and PEX12 account for the major- • Fetuses with increased nuchal translucency without aneu-
ity, whereas PEX11B is least frequent. (6) Peroxisome ploidy, may have fetal akinesia, cardiac defects, intrathoracic
biogenesis disorder 4A, also known as Zellweger syndrome and extrathoracic compression and body stalk anomaly.
(OMIM#614862) and peroxisome biogenesis disorder 4B • Sometimes initial radiographs obtained for clinical man-
(OMIM#614863) are caused by homozygous or compound agement reveal the diagnosis in floppy neonates.
heterozygous mutations in the PEX6 gene (OMIM*601498). • In neonatal presentation of ZSDs, serum phytanic and pris-
The in-silico prediction of the variant is damaging by tanic acid levels remain normal because of minimal dietary
MutationTaster2. intake of phytanic acid in formula and breast-fed infants.
This novel mutation of chr6:42933525_42933526de-
lAC is analyzed as the likely cause of the clinical and bio-
chemical features with in silico analysis. This PEX6
variation has not been reported in the 1,000 Genomes
American Board of Pediatrics
database and has a minor allele frequency of 0.002% in Neonatal-Perinatal Content
the ExAC database. Specification
• Know how age at presentation (in utero, neonate, in-
The Condition fancy or later) affects the differential diagnosis of the
Peroxisomal biogenesis disorder is divided into 2 clinical presentation of genetic disorders.
subtypes: 1) Zellweger spectrum disorder (ZSD), and • Recognize the diagnostic implications of single vs.
multiple anomalies.
2) RCDP spectrum. ZSD includes Zellweger syndrome,
• Recognize the clinical features and know how to diag-
which is the most severe; neonatal adrenoleukodystrophy
nose craniofacial anomalies.
(ALD), of intermediate severity; and infantile Refsum • Know the disorders for which molecular genetic stud-
disease, the least severe. Both ALD and infantile Refsum ies are clinically indicated, such as cystic fibrosis, and
disease have no renal cortical cysts and chondrodysplasia how to interpret test results.
punctata. As a group, ZSD shows elevated plasma con-
centrations of C26:0 and C26:1; elevated ratios of C24/
C22 and C26/C22; elevated phytanic acid and/or pris-
tanic acid; raised pipecolic acid in both plasma and urine; Acknowledgments
and reduced C16 and C18 plasmalogens. However, in The authors thank Dr S. Chatterji, CEO, Dr LH Hiran-
dani Hospital, Powai, Mumbai, Maharashtra, India, for
RCDP, levels of plasma very-long-chain fatty acids are
approving publication of this article.
normal, but plasma phytanic acid levels are increased
and erythrocyte plasmalogen levels are decreased. (6)(7)
Two major exceptions in neonatal presentation of ZSDs References
are normal serum phytanic and pristanic acid levels 1. De Domenico R, Faraci M, Hyseni E, et al. Increased nuchal traslucency
in normal karyotype fetuses. J Prenat Med. 2011;5(2):23–26
(minimal dietary intake of phytanic acid in formula and
2. Abi Berger. What is fetal nuchal translucency? BMJ. 1999;318:81
breast-fed infants) and high urinary excretion of pipecolic
3. Vieira LA, Silva SV, de Faria RB, Lippi UG, Lopes RG. Perinatal and
acid in the neonatal period, leading to the suggestion of
pediatric follow up of children with increased nuchal translucency
urine testing of neonates and plasma testing in older and normal karyotype [article in Portuguese]. Rev Bras Ginecol Obstet.
children. (6) 2013;35(6):274–280

Vol. 23, No. 12 D E C E M B E R 2 0 2 2 e843

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 4. Socolov D, Socolov R, Gorduza VE, et al. Increased nuchal 6. Steinberg SJ, Raymond GV, Braverman NE, Moser AB. Peroxisome
translucency in fetuses with a normal karyotype-diagnosis and biogenesis disorders, Zellweger syndrome spectrum. In:
management: An observational study. Medicine (Baltimore). Pagon RA, Adam MP, Ardinger HH, et al, eds.
2017;96(29):e7521 GeneReviewsVR . Seattle, WA: University of Washington,

5. Irving MD, Chitty LS, Mansour S, Hall CM. Chondrodysplasia 1993–2018.

punctata: a clinical diagnostic and radiological review. Clin 7. Lee PR, Raymond GV. Child neurology: Zellweger syndrome.
Dysmorphol. 2008;17(4):229–241 Neurology. 2013;80(20):e207–e210

e844 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 INDEX OF SUSPICION IN THE NURSERY

Jaundice in a 10-hour-old Infant

Elizabeth A. Hagan, MD,* Abdul-Azeez Abdullahi, MD,† Issam Halabi, MD,‡ Akshaya Vachharajani, MD§
*Department of Pediatrics, University of Missouri Health Care, Columbia, MO
†
Department of Internal Medicine, University of Missouri Health Care, Columbia, MO
‡
Division of Neonatal Medicine, Department of Pediatrics, University of Missouri School of Medicine, Columbia, MO
§
Division of Gastroenterology, Department of Pediatrics, University of Missouri School of Medicine, Columbia, MO

CLINICAL COURSE
A 2,733-g male infant is born at 39 1/7 weeks gestational age to a 29-year-old primi-
gravida. The mother has had routine prenatal care. Cesarean delivery is precipitated
by non-reassuring fetal heart sounds. Amniotic fluid is meconium stained, and the
infant requires supplemental oxygen for 3 minutes after birth. His Apgar scores are 8
and 9 at 1 and 5 minutes, respectively. At 9 hours of age, he is noted to have a jaun-
diced appearance and at 11 hours of age, total bilirubin is 10.15 mg/dL (173.6 mmol/L)
and direct bilirubin is 3.7 mg/dL (63.3 mmol/L). Initial hematocrit is 48.8% with a re-
ticulocyte count of 9.3%. Both the infant and mother have blood type O1 and are
Coombs negative. He is transferred to the NICU for further evaluation.
On arrival in the NICU, he is given nothing by mouth and evaluated for neona-
tal cholestasis. Phototherapy is unnecessary, per American Academy of Pediatrics
(AAP) guidelines, because total bilirubin never rises above the minimum thresh-
old for phototherapy for even high-risk patients. He is started on empiric antibiot-
ics. Antibiotics are discontinued after negative sepsis evaluation. At 24 hours of
age, findings of a comprehensive metabolic panel, thyroid studies, and coagula-
tion studies are all within normal limits. Additional laboratory tests for urine cyto-
megalovirus polymerase chain reaction, g-glutamyl transferase, Epstein-Barr virus,
and a1-antitrypsin also have unremarkable findings. Abdominal ultrasonography
findings are normal and feedings are restarted with a lactose-free formula. He is
started on phenobarbital in preparation for hepatobiliary iminodiacetic acid
(HIDA) scan which shows no excretion of radiotracer in bowel or gallbladder sug-
gestive of biliary atresia (BA). His first newborn screening results show borderline
elevated galactose-1-phosphate uridyltransferase (GALT) at 3.4 U/dL, concerning
for galactosemia. Tests for galactosemia-1-phosphate (G1P) level and glucose-6-
phosphate dehydrogenase (G6PD) level; bile acid spectrophotometry on urine
sample; and genetic analysis for “cholestasis panel: sequencing and CNV analysis
and Alagille syndrome panel” are all performed.

AUTHOR DISCLOSURES Drs Hagan,

DISCUSSION
Abdullahi, Halabi, and Vachharajani have
Jaundice in the neonate is commonly associated with the relatively benign, tran- disclosed no financial relationships
relevant to this article. This commentary
sient buildup of unconjugated bilirubin due to immature hepatic conjugation of
does not contain a discussion of an
bilirubin. As elevated levels of both unconjugated and conjugated bilirubin can unapproved/investigative use of a
cause clinical disease requiring early intervention, the AAP’s 1994 guidelines on commercial product/device.

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e845

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 the management of hyperbilirubinemia recommends that and portal stromal edema, interlobular bile duct injury and
all infants should be routinely screened between 24 and fibrosis, and bile duct proliferation with bile duct plugs. A
48 hours of age. (1) Jaundice that appears before 24 hours 2016 analysis of the Childhood Liver Disease Research and
of age, with high blood bilirubin levels, or persists beyond Education Network (ChiLDReN) data found that ductal plugs
14 days of age requires prompt investigation to potentially and portal stroma edema on biopsy, along with no decrease
prevent severe liver disease. in bile duct count and rare to no hepatocellular giant cell
Neonatal cholestasis refers to elevated conjugated bilirubin transformation or lobular extramedullary hematopoiesis, have
during the first 3 months of age. If the total bilirubin is at or a concordance rate of 0.89 for BA diagnosis. (5) Many of
below 5 mg/dL (85 mmol/L), direct bilirubin of more than 1.0 these features are also seen on liver biopsy of non-BA causes
mg/dL (17.1 mmol/L) is generally considered abnormal. For of neonatal cholestasis including Alagille syndrome, neonatal
total bilirubin values higher than 5 mg/dL (85 mmol/L), direct hepatitis, and viral hepatitis. Immunohistologic staining of
bilirubin of more than 20% of the total bilirubin is consid-
biopsies can help diagnose or rule out other causes such as
ered abnormal (Fig 1). (2) Conjugated hyperbilirubinemia can
various forms of a1-antitrypsin disease, cystic fibrosis, mito-
be caused by anatomic obstruction to flow of bile, infection
chondrial disorders, panhypopituitarism, bile acid synthesis
or hepatitis, thyroid disorders, genetic or metabolic disorders,
disorders, Niemann-Pick disease type C, progressive familial
gestational alloimmune liver disease, and toxins.
intrahepatic cholestasis, or nonsyndromic bile duct paucity.
The most common cause of neonatal cholestasis, BA
The infant in the current case underwent exploratory
(25%–40%), requires surgical intervention to reestablish
laparoscopy, cholangiography, and liver wedge biopsy at
bile flow. Uncorrected, average survival is 24 to 36 months
16 days of age. Biopsies showed portal fibrosis, prominent
without a liver transplant, but surgical intervention can
ductular reaction, and ductular bile plugs. Lobular inflam-
improve the 10-year transplant-free survival rate 75% to
mation was mild and nonspecific. No steatosis was seen.
90%. (3) Other anatomic conditions such as biliary cysts,
inspissated bile/plug syndrome (eg, patients with cystic The portal veins and hepatic arteries were histologically unre-
fibrosis), gallstones, tumors, or neonatal sclerosing cholan- markable. Trichrome stain showed portal/periportal and
gitis also require surgical evaluation, making early imaging perisinusoidal fibrosis. Staining with periodic acid–Schiff
essential. If BA is suspected, hepatobiliary scintigraphy or (PAS) and PAS with diastase was negative for a1-antitrypsin
HIDA scan should be performed to rule out biliary obstruc- inclusion in hepatocytes. Prussian blue stain revealed severe
tion, though specificity ranges from 69% to 72%. (4) Ultra- iron deposition in hepatocytes. Rhodamine stain revealed
sound findings of abnormal gallbladder has a diagnostic focal copper deposition in periportal hepatocytes. Intraopera-
accuracy of 71% and triangular cord sign of 95%. (5) tive findings included an enlarged, non-nodular liver that
The gold standard for diagnosing BA is intraoperative chol- was dark/black. The gallbladder was small and pale. Cholan-
angiography with liver biopsy. Intraoperative cholangiography giography showed appropriate transit of contrast from the
will confirm bile duct obstruction. Biopsy will show bile duct common bile duct to the duodenum, ruling out BA.

Figure 1. Total and direct bilirubin levels. G6PD=glucose-6-phosphate dehydrogenase, TSB=total serum bilirubin.

e846 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 6-phosphogluconate dehydrogenase, and G6PD to avoid
false positives (Fig 2). (10)(11) The assay measures the fluo-
rescence of the reduced form of nicotinamide adenine di-
nucleotide phosphate in the last step of the conversion
from galactose-1-phosphate to ribulose-5-P, in which GALT
is only the first step. The patient was switched back to
breast milk after G1P results ruled out galactosemia, and
the mother has started a G6PD diet.
Genetic analysis eventually revealed that this patient had
class 1 variant of the G6PD gene, c.1178 G>A p.Arg393His,
or G6PDNashville, observed to have activity at only 16% of nor-
mal. (12) The patient was discharged at 16 days of age with
follow-up appointments with gastroenterology, genetics, and
hematology/oncology, and routine newborn appointments.
Total bilirubin and direct bilirubin continued their downward
Figure 2. Beutler spot assay pathway. GALT = galactose-1-phosphate uridyl- trend over the next 2 months and ursodeoxycholic acid was
transferase, G6PD = glucose-6-phosphate dehydrogenase, NADP = nicotin-
amide adenine dinucleotide phosphate, 6PGDH=6-phosphogluconate stopped, with parents continuing to care for their child with
dehydrogenase, NADPH=reduced form of nicotinamide adenine dinucleo- regular G6PD precautions.
tide phosphate.

The patient’s G6PD level was found to be 2.2 units/g Lessons for the Clinician
• Consider hemolysis including G6PD deficiency Coombs-
of hemoglobin, or 20% of mean normal, though reference
negative nonspherocytic hemolytic anemia in neonates with
values (8.8–13.4 units/g of hemoglobin) are only estab-
conjugated hyperbilirubinemia.
lished for patients older than 12 months. (6) While it is
• Beware of false-positive results for galactosemia on new-
reasonable to assume that hemolysis was triggered by peri-
born screening that may be due to impaired functionality
natal oxidative stress and resulted in elevated bilirubin,
of G6PD, 6PGDH, or PGM1.
only some infants with G6PD deficiencies present with
neonatal jaundice. A study of G6PD-deficient neonates in
1996 found no correlation between increased hemolysis
and jaundice. (7) However, a later study of black neo- American Board of Pediatrics
nates with G6PD deficiency described a subset of these Neonatal-Perinatal Content
neonates with elevated rates of hemolysis. (8) Neonates
have 2 types of unconjugated hyperbilirubinemia: exag-
Specifications
• Know the factors, including red cell life span, enzyme
gerated physiologic jaundice or jaundice related to he-
defects, and red cell structural abnormalities, associ-
molysis. (9) In our case, total bilirubin peaked on the ated with an increase in bilirubin production.
3rd day of age, while direct bilirubin peaked on the 11th • Know the factors associated with a decrease in neona-
day of age, suggesting that in addition to elevated levels tal serum bilirubin excretion, including those that af-
of unconjugated bilirubin released from red blood cells, fect the enterohepatic circulation of bilirubin.

the ability to conjugate and subsequently excrete biliru- • Know the disorders for which molecular genetic stud-
ies are clinically indicated, such as cystic fibrosis, and
bin was decreased, leading to cholestasis. The infant how to interpret test results.
was switched to non-soy formula to halt hemolysis due • Know the range of normal serum bilirubin concentra-
to G6PD deficiency. He was started on ursodeoxycholic tion and the effects of an infant’s age, race, and feed-
acid to facilitate excretion of conjugated bile. ing circumstances on serum bilirubin.
Repeat newborn screening showed elevated GALT level • Know the differences between physiologic and non-
physiologic jaundice.
of 3.2 U/dL; however, G1P level was at 27.8 nmoL/h per
• Know the clinical and laboratory features of neonatal
milligram of hemoglobin (reference value, >24.5 nmol/h
hemoglobinopathies, including the thalassemias.
per milligram of hemoglobin). The mechanism of false-
• Know the indications for and approaches to screening
positive newborn screening results is intriguing. The new- for hemoglobinopathies in the newborn population.
born screen uses the Beutler spot assay, which requires a
normal level and function of GALT, phosphoglucomutase-1,

Vol. 23, No. 12 D E C E M B E R 2 0 2 2 e847

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 at: https://www.mayocliniclabs.com/test-catalog/Overview/
References 64567
1. American Academy of Pediatrics, Provisional Committee for Quality 7. Kaplan M, Vreman HJ, Hammerman C, Leiter C, Abramov A,
Improvement and Subcommittee on Hyperbilirubinemia. Practice Stevenson DK. Contribution of haemolysis to jaundice in Sephardic
parameter: management of hyperbilirubinemia in the healthy term Jewish glucose-6-phosphate dehydrogenase deficient neonates. Br J
newborn. [published correction appears in Pediatrics 1995 Mar;95(3): Haematol. 1996;93(4):822–827
458-61] Pediatrics. 1994;94(4 pt 1):558–565
8. Kaplan M, Herschel M, Hammerman C, Hoyer JD, Stevenson
2. American Academy of Pediatrics Subcommittee on Hyperbilirubinemia. DK. Hyperbilirubinemia among African American, glucose-6-
Management of hyperbilirubinemia in the newborn infant 35 or more phosphate dehydrogenase-deficient neonates. Pediatrics. 2004;
weeks of gestation. [published correction appears in Pediatrics. 2004 114(2):e213–e219
Oct;114(4):1138] Pediatrics. 2004;114(1):297–316 9. Luzzatto L, Poggi V. Glucose-6-phosphate dehydrogenase
3. Sundaram SS, Mack CL, Feldman AG, Sokol RJ. Biliary atresia: deficiency. In: Orkin SH, Fisher DE, Ginsburg D, Look AT, Lux
indications and timing of liver transplantation and optimization of SE, Nathan DG, eds. Nathan and Oski’s Hematology and Oncology
pretransplant care. Liver Transpl. 2017;23(1):96–109 of Infancy and Childhood. Philadelphia, PA: Elsevier/Saunders;
4. Kianifar HR, Tehranian S, Shojaei P, et al. Accuracy of hepatobiliary 2015:609–629.
scintigraphy for differentiation of neonatal hepatitis from biliary 10. Beutler E. G6PD deficiency. Blood. 1994;84(11):3613–3636
atresia: systematic review and meta-analysis of the literature. Pediatr 11. Stuhrman G, Perez Juanazo SJ, Crivelly K, Smith J,
Radiol. 2013;43(8):905–919 Andersson H, Morava E. False-positive newborn screen
5. Kanegawa K, Akasaka Y, Kitamura E, et al. Sonographic diagnosis of using the Beutler spot assay for galactosemia in
biliary atresia in pediatric patients using the “triangular cord” sign glucose-6-phosphate dehydrogenase deficiency. JIMD Rep.
versus gallbladder length and contraction. AJR Am J Roentgenol. 2017;36:1–5
2003;181(5):1387–1390 12. Beutler E, Kuhl W, Gelbart T, Forman L. DNA sequence
6. Mayo Clinic Laboratories. Test ID: G6PD Glucose-6-Phosphate abnormalities of human glucose-6-phosphate dehydrogenase
Dehydrogenase (G-6-PD), Quantitative, Erythrocytes. Available variants. J Biol Chem. 1991;266(7):4145–4150

e848 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 INDEX OF SUSPICION IN THE NURSERY

Term Neonate with Hematochezia

Mariha Khan, MD,* Thomas G. Boyce, MD, MPH,*,† Aditya Joshi, MD, MPH*,‡
*Department of Pediatrics,
†
Division of Pediatric Infectious Diseases, and
‡
Division of Neonatology, Marshfield Clinic Health System, Marshfield, WI

CASE PRESENTATION
A 3,910-gram female infant is born at 39 weeks, 3 days’ gestation via spontaneous
vaginal delivery to a 28-year-old gravida 2, para 2 woman. Testing for maternal
group B Streptococcus, hepatitis B surface antigen, human immunodeficiency virus,
gonorrhea, and chlamydia is negative, whereas testing for rubella antibodies dem-
onstrates immunity. The mother had genital herpes 3 years before this delivery, but
she does not have visible lesions currently. She has been taking prophylactic acyclo-
vir since 36 weeks’ gestation to prevent recurrence. She had gastroenteritis 3 days
before the delivery, which resolved by the time of delivery. Spontaneous rupture of
membranes occurs, with clear amniotic fluid 1 hour before delivery. The infant’s
Apgar scores are 8 and 9 at 1 and 5 minutes, respectively. The infant receives an
intramuscular injection of vitamin K shortly after birth.
The infant develops multiple episodes of bright red blood in the stool and fever
(rectal temperature of 102.3 F [39.1 C]) on the first day after birth. The rest of her
vital signs are normal. She is well appearing with a nontender, nondistended abdo-
men. No anal tears or fissures are noted, and no petechiae are observed on the
skin. The infant is breastfeeding every 2 hours, and there is no evidence of cracked
or bleeding nipples in the mother. An abdominal radiograph is obtained, which re-
veals a normal bowel gas pattern. A test for occult blood in the stool is positive. An
Apt-Downey test cannot be performed due to nonavailability of the reagent. Com-
plete blood cell count (CBC) reveals normal platelet and white blood cell counts.
C-reactive protein is mildly elevated at 2.9 mg/dL (29 mg/L). Prothrombin time
and partial thromboplastin time are normal. The patient is started on empiric genta-
micin, ceftazidime, and acyclovir after obtaining blood and cerebrospinal fluid
(CSF) cultures. Stool testing reveals the cause of the infant’s symptoms.

DISCUSSION
Hematochezia is bright red blood in the stool and is different from melena,
which is tarry black stool with a pungent odor. Hematochezia is bleeding from
AUTHOR DISCLOSURE Drs Khan, Boyce,
the lower gastrointestinal tract, mostly below the ligament of Trietz. Melena is and Joshi have disclosed no financial
related to upper gastrointestinal bleeding that results from the action of gastric relationships relevant to this article.
acid and intestinal bacteria on the iron of the hemoglobin. This commentary does not contain a
discussion of an unapproved/
In a retrospective study by Maayan-Metzger et al, approximately 0.4% of neo- investigative use of a commercial
nates presented with hematochezia. (1) Causes of hematochezia are numerous, product/device.

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e849

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 ranging from isolated bleeding to life-threatening condi- fissure was observed during the examination. Abdominal
tions, such as malrotation with volvulus, necrotizing en- radiographs and CBC were normal. Culture and polymerase
terocolitis, and hemorrhagic disease of the newborn. chain reaction (PCR) of the infant’s stool were both positive
Identifying the cause of the bleeding is essential for the for Salmonella, whereas blood and CSF cultures were nega-
timely management of infants. tive. Because the amniotic fluid was clear upon rupture of
Hematochezia that presents immediately after birth could membranes, and there were no cracks in the nipples, we
be due to swallowed maternal blood during delivery or due ruled out swallowed maternal blood. The infant was started
to swallowed maternal blood from cracked nipples in the on broad-spectrum antibiotics, which were narrowed to cef-
breastfeeding infant. It is usually isolated, and no systemic tazidime monotherapy after the sensitivity report was avail-
clinical signs are present. Swallowed maternal blood could able. Lumbar puncture was performed because of neonatal
be tested for with the alkali denaturation test (Apt test). fever and to exclude Salmonella meningitis, which can be
Other isolated causes of hematochezia are allergic proctocoli- severe. Once blood in the stool resolved by day 4 after birth,
tis (which usually presents at 4–6 weeks of age) and anal fis- she was started on oral feeds. The infant tolerated
sure. Systemic diseases that can present with hematochezia enteral feeds, and antibiotics were discontinued on day
are necrotizing enterocolitis (NEC), malrotation with or with- 10 after birth.
out volvulus, Hirschsprung disease, infectious colitis, and The rate of salmonellosis in pregnant women is the
hemorrhagic disease of the newborn (Table). same as in nonpregnant women (ie, 0.2%). (5)(6) Poultry
Term infants account for less than 10% of all NEC diag- represents the main source of nontyphoidal Salmonella in-
noses. (2)(3)(4) NEC usually presents with other clinical signs fection. (7) The incubation period ranges between 6 and
such as increasing gastric residual feedings, abdominal disten- 48 hours but may be up to 1 week. Salmonella infection
tion, bilious aspirates, increasing apnea, lethargy, metabolic during pregnancy can be associated with septicemia, cho-
acidosis, thrombocytopenia, and hyponatremia. Full-term rioamnionitis, preterm labor, fetal growth restrictions, and
infants present with NEC earlier compared with preterm in- abortion. (5)(6)(7)(8) Routine use of antibiotics is not indi-
fants (within the first week after birth compared with 2 to 3 cated in healthy adults (including pregnant women) for
weeks after birth, but rarely in the first 24 hours). In term Salmonella gastroenteritis because treatment does not
infants, there are fewer systemic manifestations, and intes- shorten the duration of illness but can prolong fecal shed-
tinal perforation is more common. (2)(3)(4) Malrotation pre- ding. Treatment should focus on rehydration, and antibiot-
sents with frequent, bilious emesis. Hematologic disorders ics are usually given only if invasive disease is suspected.
will present with a history of maternal medication that Salmonella infections are a major cause of acute gastro-
could impair vitamin K metabolism in infants or bleeding enteritis worldwide. (8) Children under 5 years of age have
disorders with abnormal prothrombin time or partial throm- the highest incidence rate. (8) Neonatal Salmonella infec-
boplastin time. It is important to confirm the administration tion usually presents during the first week after birth.
of intramuscular vitamin K on the first day after birth. Bloody stools within the first 24 hours after birth due to
Our infant presented with rectal bleeding and fever Salmonella infection have been reported in isolated case
within the first 6 hours after birth. Maternal history was reports. Neonates are particularly vulnerable to Salmonella
positive for Salmonella gastroenteritis 3 days before the infection because of hypochlorhydria and rapid gastric
delivery, but her symptoms had resolved. The infant was emptying time. The colonization and initial invasion prob-
delivered vaginally. Although the mother was asymptom- ably occur at the distal ileum, whereas mucosal edema
atic, she was still likely colonized with Salmonella. No anal and cryptic abscesses are frequently noted in the colon.

Table. Differential Diagnosis of Hematochezia in Term Neonates

Isolated rectal bleeding
 Swallowed maternal blood during delivery or from cracked nipples
 Milk protein allergy, which presents mostly at 4–6 weeks of age
 Anal fissure
Rectal bleeding with other systemic diseases
 Gastrointestinal: Hirschsprung disease with enterocolitis, malrotation with volvulus, necrotizing enterocolitis
 Hematological: Thrombocytopenia, vitamin K deficiency, clotting factor deficiency, von Willebrand disease
 Infectious: Bacterial infection (gastroenteritis)

e850 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 The diarrhea is due to secretion of fluid and electrolytes
by the small and large intestines. In a term neonate with American Board of Pediatrics
hematochezia and suspected infection, CBC, blood, and Neonatal-Perinatal Content
stool culture or stool PCR testing should be obtained. Ab- Specification
dominal radiography would be helpful in assessing for • Know the clinical manifestations and differential diagno-
NEC and midgut volvulus. sis of GI bleeding in newborn infants, including the vari-
Treatment with antibiotics is recommended for infants ous coagulation disorders that cause GI hemorrhage.
with Salmonella gastroenteritis who are younger than 3 months
due to a higher incidence of bacteremia and the risk of seed-
ing the meninges. (8) Empiric antimicrobial therapy should be
given with a parenteral third-generation cephalosporin. If test- References
ing shows that the strain is sensitive, ampicillin can be used. 1. Maayan-Metzger A, Ghanem N, Mazkereth R, Kuint J. Characteristics
In infants older than 3 months, antibiotics are not recom- of neonates with isolated rectal bleeding. Arch Dis Child Fetal Neonatal
Ed. 2004;89(1):F68–F70
mended due to the risk of prolonging fecal excretion and con-
2. O’Neil AM, Homme JL. Evaluation of hematochezia in a two-day-old
cerns for antibiotic resistance. (8) However, empiric therapy is
infant. J Emerg Med. 2016;50(1):41–43
indicated in immunocompromised patients and those with in- 3. Wen Q, Liu K, Yue W, et al. Clinical significance of positive fecal
vasive disease. occult blood test in neonates. Sci Rep. 2019;9(1):17898
4. Bray-Aschenbrenner A, Feldenberg LR, Kirby A, Fitzpatrick CM,
Josephsen JB. Bloody stools in a 3-day-old term infant. Pediatrics.
2017;140(3):e20170073
Lessons for the Clinician
5. Delcourt C, Yombi JC, Vo B, Yildiz H. Salmonella enteritidis during
• Salmonella infections are common; if they occur during pregnancy, a rare cause of septic abortion: case report and review of
pregnancy, they can lead to fetal complications. the literature. J Obstet Gynaecol. 2019;39(4):554–555
• Transplacental transmission of Salmonella infection is 6. S
anchez-Vargas FM, Abu-El-Haija MA, G
omez-Duarte OG. Salmonella
rare; however, mothers can transmit the disease to neo- infections: an update on epidemiology, management, and prevention.
Travel Med Infect Dis. 2011;9(6):263–277
nates during and shortly after delivery.
7. Coughlin LB, McGuigan J, Haddad NG, Mannion P. Salmonella
• In a neonate with hematochezia, it is important to take sepsis and miscarriage. Clin Microbiol Infect. 2003;9(8):866–868
a history of recent maternal illness and obtain stool and 8. Bula-Rudas FJ, Rathore MH, Maraqa NF. Salmonella infections in
blood cultures from the infant. childhood. Adv Pediatr. 2015;62(1):29–58

Vol. 23, No. 12 D E C E M B E R 2 0 2 2 e851

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 MATERNAL-FETAL CASE STUDIES

Neonatal Implications of Maternal

Thrombocytopenia during Pregnancy
Arlin Delgado, MD, Stephanie Ros, MD, MSCI
Department of Obstetrics and Gynecology, University of South Florida, Tampa, FL

CASE PRESENTATION
A 32-year-old gravida 6, para 3-0-2-3 woman presented at 39 weeks’ 2 days’ ges-
tation for scheduled repeat cesarean section. Her history was notable for 2 spon-
taneous vaginal deliveries, 1 cesarean section, and macrosomia affecting 1 of her
pregnancies. She had developed gestational diabetes, which was diet-controlled,
in the current pregnancy. She had no history of anemia, and results of routine
screening via hemoglobin solubility testing were within normal limits. She also
had no known history of thrombocytopenia until the current pregnancy when
her platelet count at 32 weeks’ gestation showed a downward trend, at 98,000/mL
(98 × 109/L). The differential diagnosis included gestational thrombocytopenia,
viral illness, hypertensive disorders in pregnancy, and immune thrombocytope-
nia (ITP). Serial blood pressure evaluations to monitor for hypertensive disor-
ders in pregnancy (ie, preeclampsia with severe features, and hemolysis,
elevated liver enzymes, and low platelets [HELLP] syndrome) were performed.
She remained asymptomatic and normotensive, and other findings of her labora-
tory evaluation were otherwise unremarkable with a hemoglobin level of 12.1 g/dL
(121 g/L), and no evidence of hemolysis, normal creatinine, and normal liver enzymes.
There was no evidence of preeclampsia or HELLP syndrome. She was found to have
a platelet count of 85,000/mL (85 × 109/L) at her preoperative visit approximately
1 week before surgery. She was not taking any medications except for prenatal
vitamins, and reported no sick contacts or known exposures.
On presentation 3 days before delivery, she continued to be asymptomatic
and normotensive. Routine admission laboratory testing showed severe throm-
bocytopenia, with a platelet count of 24,000/mL (24 × 109/L). Repeat testing to
rule out laboratory error as a cause for the abnormal value confirmed the low
platelet count. The patient was also noted to have worsening anemia with a
hemoglobin level of 8.3 g/dL (83 g/L). Testing for anemia revealed elevated lac-
tate dehydrogenase and low haptoglobin, which were concerning for hemolysis;
Author Disclosures Dr Ros receives however, her total bilirubin was normal and her reticulocyte count was low. Iron
royalty payments from UptoDate. Dr
Delgado has disclosed no financial studies were consistent with iron-deficiency anemia, and coagulation and fibrin-
relationships relevant to this article. This ogen studies were within normal limits. A peripheral blood Wright smear
commentary does not contain a showed no evidence of schistocytes but did show small spherocytes and
discussion of an unapproved/
investigative use of a commercial decreased platelets. Due to severe thrombocytopenia, the hematology team was
product/device. consulted and her scheduled surgery delayed. The leading differential diagnosis

e852 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Figure 1. Reassuring fetal testing on hospital day 1.

was ITP. She was administered a dose of intravenous im- secondary to general anesthesia; however, ongoing hemor-
munoglobulin (IVIG) and started on oral dexamethasone rhage was then thought to be exacerbated by her thrombocy-
daily. Achieving a platelet count of at least 50,000/mL topenia after her uterine atony was treated. After stabilization,
(50 × 109/L) was the goal before surgery to reduce risk of she was immediately transferred to the intensive care unit.
hemorrhage. The planned mode of delivery was repeat ce- She continued to receive IVIG for 2 days and oral dexametha-
sarean per patient preference. sone for 4 days. On postoperative day 5, her platelets normal-
Fetal testing was reassuring on day 1 (Fig 1). ized to 154,000/mL (154 × 109/L).
On hospital day 2, her thrombocytopenia had not im-
proved significantly. Delivery was deferred again to con- Neonatal Outcome
tinue to optimize her platelet count before surgery. The neonate was delivered at 39 weeks, 3 days of gestation,
On hospital day 3, her platelet count was noted to rise to and had an Apgar score of 7 and 9 at 1 and 5 minutes, re-
50,000/mL (50 × 109 /L). During daily fetal surveillance, spectively, with a birthweight of 3,790 g (77th percentile). The
recurrent decelerations were noted, and a repeat low
transverse cesarean section via Pfannenstiel incision
was performed under general anesthesia given the con-
traindication for spinal anesthesia secondary to mater-
nal thrombocytopenia.
She was administered 1 unit of platelets and 1 unit of
packed red blood cells before proceeding to the operating
room. Additional blood products were on hold in anticipa-
tion of a potential hemorrhage.

Maternal Outcome
During and immediately after the cesarean section, the Figure 2. Blood smear demonstrating maternal thrombocytopenia with
patient experienced a hemorrhage, with a total blood loss fewer platelets than expected, with this slide demonstrating a platelet
count of approximately 80,000/mL (80 × 109/L). The platelets are circled in
of 4,500 mL. The hemorrhage was believed to be due in red. (Photo courtesy of Patrick Malafronte, MD, Anatomic/Clinical Pathology
part to uterine atony at the time of the cesarean section and Neuropathology.)

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e853

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Table. Thrombocytopenia in Pregnancy: Risk of Neonatal Thrombocytopenia
Maternal Diagnosis Risk of Severe Neonatal Thrombocytopenia
Immune thrombocytopenic purpura Low
Gestational thrombocytopenia Not expected
Preeclampsia with severe features/HELLP syndrome Low
Acute HIV infection Low
Lupus Low
Medications associated with immune-mediated platelet Low
destruction (such as carbamazepine, phenytoin, valproate)

HELLP = hemolysis, elevated liver enzymes, and low platelets, HIV = human immunodeficiency virus.

umbilical cord platelet count was 220,000/L (220 × 109/L). cord venipuncture or directly into a collection tube with freely
Despite exposure to general anesthesia, the neonate did not flowing cord blood (Fig 3).
experience any respiratory effects from the anesthetic agents. Management of ITP in pregnancy includes serial platelet
Serial neonatal platelet counts were obtained due to maternal count assessment and ongoing evaluation for other obstetrical
ITP and were within normal limits for 2 days. The neonate etiologies for thrombocytopenia. Expert opinion recommends
had an uncomplicated course. keeping maternal platelet count above 30,000/mL (30 ×109/L)
to reduce the risk of spontaneous bleeding until the late third
trimester when higher thresholds are advised closer to delivery
DISCUSSION
(at least 50,000/mL [50 × 109/L] for cesarean delivery). A pa-
ITP occurs in 1 in 1,000 to 10,000 patients in pregnancy tient’s platelet count should be optimized before delivery to
and is the most common reason for platelet counts less minimize the risk of hemorrhage. (8) First-line treatment for
than 50,000/mL (50 × 109/L) in pregnant patients during
the second trimester. (1) Thrombocytopenia in pregnancy is
defined as platelet counts less than 150,000/mL (150 × 109/L)
in pregnancy. Suspicion for ITP is high when an otherwise
healthy patient has newly found platelet levels of less than
70,000/mL (70 × 109/L) with peripheral smear most com-
monly showing no platelet abnormalities. (1)(2)(3) However,
large platelets may be sometimes seen (Fig 2). (4) ITP is
caused by T cell–mediated platelet destruction and is consid-
ered a diagnosis of exclusion.
When thrombocytopenia is noted in pregnancy, it is
important to maintain a broad differential diagnosis.
Etiologies of maternal thrombocytopenia in the third
trimester include gestational thrombocytopenia, ITP, pre-
eclampsia with severe features, HELLP syndrome, a new diag-
nosis of HIV, viral illness or lupus, and thrombocytopenia due
to medication effects (Table). (5) Common presenting symp-
toms of ITP include petechiae, purpura, nosebleeds, or easy
bruising; however, most women will be asymptomatic. ITP in-
creases the maternal risk for hemorrhage particularly at the
time of delivery if thrombocytopenia is severe, defined fre-
quently as a platelet count of less than 50,000/mL (50 × 109/L).
(6) There is additionally a potential for maternal platelet anti-
bodies to cross the placenta and result in fetal and neonatal
thrombocytopenia with a rare risk of intracerebral hemorrhage.
(7) An umbilical cord platelet count can provide an initial plate- Figure 3. Umbilical cord blood collection for a neonatal platelet count as
the maternal patient has thrombocytopenia. (Photo courtesy of Natasha
let count for the neonate, which may be collected via umbilical Rich, MD, and Melissa Chan, MD.)

e854 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 ITP includes steroid administration and IVIG. Subsequent
treatment for ITP that is refractory to steroids or IVIG may in- American Board of Pediatrics
clude chemotherapeutic agents or, rarely, splenectomy. Neonatal-Perinatal Content
Neonatal risks for maternal ITP are relatively low. Ap-
Specifications
proximately 1% to 5% of neonates born to women with
• Know the causes and pathophysiology of neonatal
ITP have a platelet count below 20,000/mL (20 × 109/L) thrombocytopenia and thrombocytosis.
at birth, with 5% to 15% of these neonates requiring • Know the clinical and laboratory manifestations and man-
treatment, (1) and approximately 1% can have neonatal agement of neonatal thrombocytopenia and thrombo-
intracranial hemorrhage. (9) Studies show that there is cytosis.
no clear correlation between maternal platelet count and • Know the effects on the fetus and/or newborn infant of
neonatal platelet count at birth. Risk factors for neonatal analgesics and anesthetics administered to the mother
during labor.
thrombocytopenia include maternal splenectomy and a
prior sibling with thrombocytopenia at birth. (3) At birth,
umbilical cord platelet count is assessed, and when
platelet counts are less than 50,000/mL (50 × 109/L),
References
head imaging may be considered in addition to the ad-
1. Cines DB, Levine LD. Thrombocytopenia in pregnancy. Hematology
ministration of steroids or IVIG. (10) If neonatal throm-
Am Soc Hematol Educ Program. 2017;2017(1):144–151
bocytopenia occurs, the duration is most often short-
2. Provan D, Stasi R, Newland AC, et al. International consensus
lived and resolves when maternal immunoglobulin G
report on the investigation and management of primary immune
antibodies leave the neonatal circulation. Of note, neona- thrombocytopenia. Blood. 2010;115(2):168–186
tal thrombocytopenia as a result of neonatal alloimmune
3. ACOG practice bulletin no. 207: thrombocytopenia in pregnancy.
thrombocytopenia is associated with a normal platelet in Obstet Gynecol. 2019;133(3):e181–e193
the mother. 4. McCrae K. Immune thrombocytopenia: no longer ‘idiopathic’. Cleve
If a cesarean is necessary, typically a minimum platelet Clin J Med. 2011;78(6):358–373
count needs to be stable and over 70,000/mL (70 × 109/L) 5. Chaiworapongsa T, Chaemsaithong P, Yeo L, Romero R. Pre-eclampsia
for regional anesthesia. (11) As this patient’s platelet count part 1: current understanding of its pathophysiology. Nat Rev Nephrol.
was not above this minimum count, the anesthesiologists 2014;10(8):466–480

recommended general anesthesia. General anesthesia in 6. American College of Obstetricians and Gynecologists’ Committee on
pregnancy has small but significant maternal and neona- Practice Bulletins—Obstetrics. ACOG practice bulletin no. 203:
chronic hypertension in pregnancy. Obstet Gynecol. 2019;133(1):e26–e50
tal risks. General anesthesia is systemic, and anesthetic
agents will cross the placenta. Maternal risks include an 7. Hwa HL, Chen RJ, Chen YC, Wang TR, Huang SC, Chow SN.
Maternal and fetal outcome of pregnant women with idiopathic
increased likelihood of uterine atony or difficult intuba- thrombocytopenic purpura: retrospective analysis of 25 pregnancies.
tion. Neonatal risks include respiratory depression due J Formos Med Assoc. 1993;92(11):957–961
to anesthetic crossing the placenta, and, therefore, expe- 8. Li J, Gao YH, Su J, Zhang L, Sun Y, Li ZY. Diagnostic ideas and
dited delivery is advised when cesarean delivery is per- management strategies for thrombocytopenia of unknown causes in
formed under general anesthesia. pregnancy. Front Surg. 2022;9:799826

It is unclear if the heavy bleeding this patient experi- 9. Neunert C, Lim W, Crowther M, Cohen A, Solberg L Jr, Crowther
enced at delivery was due to atony from her general anes- MA; American Society of Hematology. The American Society of
Hematology 2011 evidence-based practice guideline for immune
thetic, ITP, or a combination of the 2. Fortunately, this thrombocytopenia. Blood. 2011;117(16):4190–4207
neonate did not experience thrombocytopenia secondary
10. Al-Jama FE, Rahman J, Al-Suleiman SA, Rahman MS. Outcome of
to a maternal diagnosis of ITP. This case demonstrates pregnancy in women with idiopathic thrombocytopenic purpura.
the importance of obstetrical and pediatric collaboration Aust N Z J Obstet Gynaecol. 1998;38(4):410–413
in the setting of maternal comorbidities such as throm- 11. Hess AS, Ramamoorthy J, Hess JR. Perioperative platelet
bocytopenia from ITP. transfusions. Anesthesiology. 2021;134(3):471–479

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e855

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 VISUAL DIAGNOSIS

Early Term Infant with Prenatal Brain

Abnormalities and Decreased Oral Intake
Parvathi Nataraj, MD,* Dhanashree Rajderkar, MD,† Diomel de la Cruz, MD,* Michael D. Weiss, MD*
Departments of *Pediatrics and
†
Radiology, University of Florida, Gainesville, FL

THE CASE
A fetus with abnormal findings on brain imaging at 33 weeks’ 5 days’ gestation
that persisted on postnatal imaging.

Prenatal and Birth Histories

• 30-year-old gravida 1, para 1 woman.
• Maternal history significant for maternal type 2 diabetes mellitus controlled
with insulin during the pregnancy and preeclampsia. Prenatal testing included
normal findings on noninvasive prenatal screening.
• Prenatal ultrasonography at 33 weeks, 5 days’ gestation showed abnormalities
and referral to our center showed persistence of these abnormalities.
• Fetal magnetic resonance imaging (MRI) was performed at 35 weeks’ gestation
(Fig 1A, B). Neurosurgery was consulted.
• Estimated gestational age: 37 weeks.
• Cesarean section for arrest of descent during induction of labor for preeclamp-
sia. Fetal bradycardia occurred for 6 to 7 minutes before delivery.
• The female infant required bag-mask ventilation in the delivery room followed
by continuous positive airway pressure and was subsequently transferred to
the NICU.
• Apgar scores: 4, 6, and 7 at 1, 5, and 10 minutes, respectively.
• Cord arterial blood gas: pH 7.0, PCO2 91.3 mm Hg (12.1 kPa), base deficit 12.9.
• Cord venous blood gas: pH 7.07, PCO2 79.5 mm Hg (10.6 kPa), base deficit
10.7.

Postnatal Presentation (Newborn Day)

Upon admission to the NICU, the infant underwent serial neurologic examinations
AUTHOR DISCLOSURES Drs Nataraj, due to the low arterial cord pH and fetal bradycardia. Initially, the infant had mild
Rajderkar, de la Cruz, and Weiss have hypotonia, mild weakness of the palmar grasp, uncoordinated suck, and delayed
disclosed no financial relationships
relevant to this article. Dr Nataraj’s
gag reflex. She had normal pupillary reflexes, deep tendon reflexes, and plantar
current affiliation is Pediatrix Medical grasps. The infant’s first arterial blood gas at 2 hours after birth had a pH of 7.29
Group, Sunrise, FL. This commentary does with PCO2 of 46 mm Hg (6.1 kPa), bicarbonate of 22 mEq/L (22 mmol/L), and
not contain a discussion of an
unapproved/investigative use of a base excess of 4 mEq/L ( 4 mmol/L). A decision was made to not proceed with
commercial product/device. therapeutic hypothermia because the infant’s neurologic findings normalized

e856 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 • Oxygen saturation: 99% (in room air)
• Temperature: 98.2 F (36.8 C)

Physical Examination (Day 5)

• Birthweight = 3,820 g (88.8th percentile), length = 51.4 cm
(88.7th percentile), head circumference = 33 cm (23rd
percentile)
• Current weight = 3,860 g
• General: Infant awake, no apparent distress
Figure 1. A. Fetal magnetic resonance imaging at 35 weeks’ gestation, • Head: Anterior fontanelle soft, open, and flat
coronal T2 weighted image. B. Fetal magnetic resonance imaging at
35 weeks’ gestation, sagittal T2 weighted image. • Lungs: Clear to auscultation bilaterally, no retractions
• Cardiovascular: Regular rate and rhythm, normal S1 and
over the first few hours after birth and an amplitude elec- S2, no murmurs noted
troencephalogram was normal. • Abdomen: Soft, nontender, nondistended, bowel sounds
The patient was weaned off respiratory support within present, no organomegaly
12 hours after birth. She had severe and persistent hypo- • Genitourinary: Normal term female genitalia
glycemia that did not respond to intravenous glucose sup- • Skin: No rashes, slight jaundice present
plementation. Additional testing revealed that she had • Neurologic: Tone and reflexes normal for age, suck and
hyperinsulinism (insulin level of 14.86 mIU/mL when her gag reflexes present
glucose was 45 mg/dL [2.5 mmol/L]) for which she was
treated with diazoxide. There was no concern for clinical Laboratory Studies
seizures related to the hypoglycemia. • Complete blood cell count on newborn day
Due to the prenatal ultrasound and MRI findings, pedi- • White blood cells: 14,300/mL (14.3 × 109/L)
atric neurosurgery was consulted after delivery and a post- • Hemoglobin: 14.6 g/dL (146 g/L)
natal head ultrasound scan was obtained (Fig 2A, B). The • Hematocrit: 45.9%
infant was noted to have poor oral intake requiring naso- • Platelet count: 250 × 103/mL (250 × 109/L)
gastric feedings. This, along with the findings on the pre- • Nucleated RBC: 127%
natal MRI, prompted the medical team to obtain a brain
MRI on day 5 (Fig 3 A,B,C). DIFFERENTIAL DIAGNOSIS
This is a 5-day old infant born at 37 weeks’ gestation with
PROGRESSION brain abnormalities identified prenatally that persisted post-
Vital Signs (Day 5) natally, a low arterial cord pH, and poor oral feeding.
• Heart rate: 167 beats/min
• Respiratory rate: 60 breaths/min • Fetal stroke
• Blood pressure: 64/29 mm Hg • Hydrocephalus
• Hypoxic-ischemic encephalopathy
• Isolated intracranial hemorrhage
• Prader-Willi syndrome
• Sepsis

ACTUAL DIAGNOSIS
Fetal stroke.

DISCUSSION
The fetal MRI was concerning for an area of gliosis, and
the postnatal MRI showed periventricular cystic encepha-
Figure 2. A. Postnatal head ultrasound scan obtained on the first day after
lomalacia and ependymal blood products, which are con-
birth, coronal view. B. Postnatal head ultrasound scan obtained on the first
day after birth, sagittal view. sistent with a diagnosis of fetal stroke.

Vol. 23, No. 12 D E C E M B E R 2 0 2 2 e857

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Patient Follow-up
Occupational therapy worked closely with the infant for
the impaired oral feeding with improvement. She did not
require supplemental nasogastric feedings after 9 days of
age. Her hypoglycemia also improved and she was weaned
off diazoxide before discharge. She was discharged from
the NICU at 15 days of age.
The infant has continued to be followed by pediatric
neurosurgery after her discharge from the NICU. At her
15-month follow-up, she had no signs of elevated intracra-
nial pressure. She had an MRI at that visit, which showed
stable size of ventricles. No further follow-up with pediatric
neurosurgery was scheduled. The patient did have some mild
left-sided weakness, for which she was being seen by physical
therapy and occupational therapy. The infant had not had any
hematologic evaluation at this point.

What the Experts Say

In our patient, the findings of diffuse white matter gliosis with
Figure 3. A. Postnatal T2 sequence on magnetic resonance imaging at ventricular enlargement, watershed periventricular infarct, and
5 days of age. B. Postnatal magnetic resonance imaging, T1 weighted,
5 days of age. C. Postnatal magnetic resonance imaging at 5 days of age,
intracranial hemorrhage seen on the fetal MRI at 35 weeks’
susceptibility weighted imaging. gestation points to a hypoxic event in utero. This is a unique
case in that no intracranial hemorrhage was detected on prena-
tal ultrasonography, which identified bilateral ventriculomegaly,
leading to a fetal MRI, which showed blood products in the
Prenatal ultrasonography performed at 33 weeks, 5 days’
infarcted area on the fetal MRI. The postnatal MRI findings
gestation at the outside hospital was concerning for bilat-
5 days after birth further supported the prenatal diagnosis of a
eral ventriculomegaly. After referral to our center, repeat
watershed periventricular stroke, ventricular enlargement, and
ultrasonography at 34 weeks, 5 days’ gestation showed
diffuse white matter volume loss consistent with a diagnosis of
severe ventriculomegaly (images from the outside hospital
perinatal stroke with global hypoxia-ischemia. The patient had
were not available for comparison). The fetal MRI
a watershed infarct next to the lateral ventricle that contained
(Fig 1A and 1B) showed significant enlargement of the fetal
temporal horns with prominence of the third and fourth hemosiderin on susceptibility weighted imaging on postnatal
ventricles, periventricular cystic areas above the right day 5. Therefore, the focal watershed infarct had bleeding into
superior lateral ventricle (white arrows), and blood prod- the infarcted region, confirming the prenatal MRI findings.
ucts (yellow arrows) (Fig 1A, B). Postnatal head The lateral ventricles were bilaterally enlarged on MRI on post-
ultrasonography showed ventriculomegaly of the lateral natal day 5, with a head circumference at birth of 33 cm (50%
ventricles (R>L) as well as right-sided periventricular for 37-week estimated gestational age neonate). Based on the
cystic changes (Fig 2A, B). postnatal imaging, the most likely explanation for the ventricu-
The postnatal MRI (Fig 3A) performed at 5 days of age lomegaly was diffuse injury to the white matter with diffuse
showed periventricular cystic encephalomalacia (white ar- white matter gliosis, again confirming the prenatal suspicion
row), ependymal blood products (yellow arrow) and bilat- of white matter gliosis and volume loss. (1) A small amount of
eral ventriculomegaly of the lateral ventricles (R>L). The blood in the lateral ventricles was noted on MRI but this small
T1 sequence (Fig 3B) showed periventricular multicystic amount of blood products, even if it produced arachnoiditis,
encephalomalacia (white arrow) and hemosiderin lining would be unlikely to account for the degree of ventriculome-
(yellow arrow) surrounding the gliotic region. Figure 3C galy and diffuse loss of white matter. Further, the presence of
shows subependymal blood products (yellow arrow) and intraventricular blood fits with a diffuse in utero hypoxic-ische-
evidence of fetal germinal matrix hemorrhage seen on this mic event. An association has been suggested between the
susceptibility weighted imaging. amount of nucleated red blood cells at birth and perinatal

e858 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 hypoxia. (2) Interestingly, our patient’s initial nucleated RBC In our patient’s case, because the arterial cord pH was
count was 127%, suggestive of in utero hypoxia. (3)(4) 7.00, an evaluation was conducted due to concern for hyp-
Perinatal/neonatal strokes, as seen in our case, have sig- oxic-ischemic encephalopathy. The infant’s serial neuro-
nificant clinical implications. It has been shown that up to logic examinations after delivery quickly showed normal
57% of patients with perinatal strokes had neurologic/cogni- findings within the first 6 hours after birth, and so her
tive abnormalities. (5) Fetal strokes occur between 14 weeks clinical picture was not consistent with an encephalopathy
of gestation and delivery. (6) Following an ischemic injury, due to acute or subacute hypoxia-ischemia. The infant’s
hemorrhagic transformation may occur at the area of ische- oral feeding also improved before discharge, which made
mia such as seen in our case. The hemorrhagic transforma- a genetic syndrome such as Prader-Willi syndrome un-
tion at the area of ischemia in a fetal stroke can be identified likely. Prader-Willi syndrome was included in the initial
as a hemorrhage on intracranial imaging. Our patient had differential diagnosis due to the poor feeding and mild
mild left-sided weakness for which she continues to receive hypotonia noted on initial examination; however, the im-
physical and occupational therapy. aging findings, improvement in both enteral feedings and
Although prenatal ultrasonography is a good tool to iden- tone over time, and absence of specific facial features
tify abnormalities in fetal anatomy, fetal MRI has been shown ruled out a diagnosis of Prader-Willi syndrome. A blood
to be of benefit for prenatal diagnoses, especially central ner- culture conducted on admission was sterile, making sepsis
vous system abnormalities. (7) A fetal MRI may not always be an unlikely cause of the infant’s symptoms.
helpful because of the possibility of significant motion artifact.
(8) In our case, the fetus was found to have ventriculomegaly
and evidence of focal cystic changes in the periventricular
white matter consistent with an in utero stroke. Salomon et al
American Board of Pediatrics
reported the mean ventricular width at 35 weeks’ gestation to Neonatal-Perinatal Content
be 6.84 mm, with approximately 1% of all fetuses having ven- Specifications
tricular width measuring greater than 10 mm. (9) • Know the role and risks of magnetic resonance imaging,
Based on the prenatal imaging that revealed blood prod- as well as other non-ultrasonographic imaging techniques
ucts in the cystic space next to the lateral ventricles, the differ- in assessing fetal anatomy.
ential diagnosis of the fetus in our case includes other causes • Know the indications for and limitations of various neuro-
imaging studies and be able to recognize normal and ab-
of intracranial bleeding. Fetal intracranial hemorrhage itself
normal structures and changes during development and
is a rare phenomenon. A previous report demonstrated that growth.
there were 6 cases of fetal intracranial bleeding from 1992 to
1994 out of a total of 6,641 patients (however, the authors
stated that this may have been an overestimation as the hos-
pital was a large referral center). (10) Determining an exact
References
cause of fetal intracranial hemorrhage is still not always possi-
1. Khwaja O, Volpe JJ. Pathogenesis of cerebral white matter injury
ble. In a series of 5 cases of fetal intracranial hemorrhage
of prematurity. Arch Dis Child Fetal Neonatal Ed. 2008;93(2):
published in 2001, all of the women had sustained trauma F153–F161
during their pregnancy. (11) In this case series, hydrocephalus 2. Buonocore G, Perrone S, Gioia D, et al. Nucleated red blood cell
was found in 3 of the 5 patients and 3 patients required a ven- count at birth as an index of perinatal brain damage. Am J Obstet
triculoperitoneal shunt within the first 2 months of age. One Gynecol. 1999;181(6):1500–1505
3. Ghosh B, Mittal S, Kumar S, Dadhwal V. Prediction of perinatal
fetus died in utero and in another case, the parents elected to
asphyxia with nucleated red blood cells in cord blood of newborns.
terminate. In contrast to other case studies, no known mater- Int J Gynaecol Obstet. 2003;81(3):267–271
nal trauma was sustained in our case, which may explain 4. Walsh BH, Boylan GB, Murray DM. Nucleated red blood cells and
the fetal intracranial hemorrhage. However, trauma is not early EEG: predicting Sarnat stage and two year outcome. Early
the only postulated mechanism causing in utero intracranial Hum Dev. 2011;87(5):335–339

hemorrhages. A 2013 case series of 6 patients with fetal intra- 5. Lynch JK, Nelson KB. Epidemiology of perinatal stroke. Curr Opin
Pediatr. 2001;13(6):499–505
cranial hemorrhage found that 2 cases were associated with
6. Ozduman K, Pober BR, Barnes P, et al. Fetal stroke. Pediatr Neurol.
intrauterine growth restriction. (12) Maternal vitamin K defi- 2004;30(3):151–162
ciency has also been proposed as being associated with fetal 7. Weisstanner C, Kasprian G, Gruber GM, Brugger PC, Prayer D.
intracranial hemorrhage. (13) MRI of the fetal brain. Clin Neuroradiol. 2015;25(Suppl 2):189–196

Vol. 23, No. 12 D E C E M B E R 2 0 2 2 e859

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 8. Putbrese B, Kennedy A. Findings and differential diagnosis of 11. Strigini FA, Cioni G, Canapicchi R, Nardini V, Capriello P,
fetal intracranial haemorrhage and fetal ischaemic brain injury: Carmignani A. Fetal intracranial hemorrhage: is minor maternal
what is the role of fetal MRI? Br J Radiol. 2017;90(1070): trauma a possible pathogenetic factor? Ultrasound Obstet Gynecol.
20160253 2001;18(4):335–342
9. Salomon LJ, Bernard JP, Ville Y. Reference ranges for fetal 12. Kutuk MS, Yikilmaz A, Ozgun MT, et al. Prenatal diagnosis and
ventricular width: a non-normal approach. Ultrasound Obstet Gynecol. postnatal outcome of fetal intracranial hemorrhage. Childs Nerv Syst.
2007;30(1):61–66 2014;30(3):411–418
10. Vergani P, Strobelt N, Locatelli A, et al. Clinical significance of fetal 13. Eventov-Friedman S, Klinger G, Shinwell ES. Third trimester fetal
intracranial hemorrhage. Am J Obstet Gynecol. 1996;175(3 Pt intracranial hemorrhage owing to vitamin K deficiency associated with
1):536–543 hyperemesis gravidarum. J Pediatr Hematol Oncol. 2009;31(12):985–988

ANSWER KEY FOR DECEMBER 2022 NEOREVIEWS

Primary Mitochondrial Disorders in the Neonate 1.E; 2. B; 3.A; 4. C; 5. D
Congenital Disorders of Red Blood Cells 1. D; 2. B; 3. A; 4. E; 5. C

e860 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 COMPLEX FETAL CARE

COMPLEX
FETAL CARE

Fetal Hepatomegaly
Jina Park, MD,* Devlynne Sasha Ondusko, MD,* Bill H. Chang, MD,† Emily A. Edwards, MD,‡ Sylvia Doan, MBBS,§
Ken Gatter, MD,¶ Ibrahim Hajjali, MD,¶ Amanda Kim, MD*
*Division of Neonatology, Oregon Health & Science University, Portland, OR
†
Division of Hematology and Oncology, Oregon Health & Science University, Portland, OR
‡
Division of Diagnostic Radiology, Oregon Health & Science University, Portland, OR
§
Division of Pediatric Gastroenterology, Oregon Health & Science University, Portland, OR
¶
Division of Pathology, Oregon Health & Science University, Portland, OR

CASE REPORT
Prenatal Course
A 29-year-old gravida 3, para 1-0-1-1 woman had an unexpected, short interval
pregnancy detected at 10 weeks’ gestation while taking oral contraceptive pills.
The pregnancy was notable for maternal Crohn’s disease, hypothyroidism, and
gestational diabetes, treated pharmacologically with infliximab infusions every
8 weeks, levothyroxine, and insulin, respectively. At 16 weeks’ gestation, she met
with her gastroenterologist about management of Crohn’s disease during preg-
nancy and was counseled that the literature to date did not show an increased risk
of birth defects or adverse fetal and neonatal outcomes with infliximab use during
pregnancy. Thus, this anti–tumor necrosis factor a (TNF-a) therapy was contin-
ued. Routine prenatal laboratory tests included sequential screening, with the risk
of trisomies 21 and 18 being less than 1 in 10,000; blood type A, Rh positive, anti-
body screen negative; hepatitis B surface antigen negative; human immunodefi-
ciency negative; rapid plasma reagin nonreactive; rubella immune; gonorrhea and
chlamydia negative; and group B Streptococcus negative.
Beginning at 32 weeks’ gestation, the pregnancy was monitored with weekly
fetal nonstress tests (NSTs) given the maternal comorbidities. She reported de-
creased fetal movement for 1 week during her scheduled prenatal care visit at
35 weeks and 3 days’ gestation. Fetal heart tones were easily detected during the
NST but without appropriate accelerations. As a result, she underwent urgent fe-
tal ultrasonography and biophysical profile (BPP) testing that confirmed absent
fetal movement. A fetal ultrasound scan at that time was notable for new polyhy-
dramnios with an amniotic fluid index of 26 cm; a grossly enlarged fetal liver
that filled the upper abdomen and appeared to compress the chest (Fig 1); no ev-
AUTHOR DISCLOSURES Drs Park,
idence of hydrops fetalis or splenomegaly; normal cardiac anatomy and activity;
Ondusko, Chang, Edwards, Doan, Gatter,
and cephalic fetal presentation. Hospital admission was recommended and a re- Hajjali, and Kim have disclosed no
peat fetal BPP showed appropriate fetal activity with a score of 8 out of 8. financial relationships relevant to this
The initial differential diagnosis for polyhydramnios and fetal hepatomegaly at article. This commentary does not
contain a discussion of an unapproved/
the time of maternal admission included infection, transient myeloproliferative investigative use of a commercial
disorder (TMD) associated with trisomy 21, fetal anemia, hepatic tumor, metabolic product/device.

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e861

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Figure 1. Prenatal ultrasound scan at 35 weeks, 4 days’ gestation showing
severe hepatomegaly. On this coronal image, the enlarged hypoechoic
liver (arrows) filled the upper abdomen, with abdominal circumference
approximately 5 weeks larger than expected for gestational age. The heart
(triangle) and lungs (diamond) were mildly cranially displaced but other-
wise normal, and there was no hydrops.

storage disorder, gestational alloimmune liver disease, and Figure 2. Fetal magnetic resonance imaging at 35 weeks, 6 days’ gestation
fetal congestive heart failure. The differential and next steps showed severe hepatomegaly, similar to the prenatal ultrasound findings.
This coronal T2 single shot image shows the massively enlarged liver
for evaluation were presented to the parents by physicians (arrows) that filled the upper abdomen, displacing the thoracic and other
from maternal-fetal medicine and neonatology; in addition, a abdominal contents. Liver signal intensity was uniform without a discrete
mass, and no abnormal iron deposition was noted. A normal gallbladder
pediatric palliative care consultation was sought for family (triangle) and spleen (not pictured) were noted, and there was no hydrops.
support given the diagnostic uncertainty and possible severe
illness. Upon admission, the evaluation for these potential diagnosis, concern of worsening fetal status, and uncer-
etiologies included serology testing for congenital infections, tain benefit to extending gestation, induction of labor was
fetal middle cerebral artery (MCA) Doppler measurements, pursued.
and fetal magnetic resonance imaging (MRI). Daily monitor-
ing with ultrasound imaging and BPPs was performed to as- Initial Postnatal Course
sess for hydrops fetalis or findings of fetal distress. Serology A female infant (Rowan) was delivered vaginally, with
testing for cytomegalovirus (CMV), herpes simplex virus, 65-second abdominal dystocia and a single wrapped nu-
and toxoplasmosis did not show evidence of a recent pri- chal cord. Resuscitation was initiated based on the Neona-
mary infection. The MCA Doppler was 1.04 multiples of the tal Resuscitation Program recommendations, and due to
mean, which is within normal limits for gestational age, persistent apnea, Rowan received positive pressure ventila-
making significant anemia unlikely. tion for 90 seconds followed by continuous positive airway
Fetal MRI at 35 weeks and 6 days’ gestation showed a pressure (CPAP). Her Apgar score was 3, 7, and 8 at 1,
markedly enlarged fetal liver without splenomegaly, abnor- 5, and 10 minutes, respectively. She was brought to the
mal iron deposition, or hydrops (Fig 2). At this point, the dif- NICU for further evaluation and management.
ferential diagnosis included metabolic storage disorders and Rowan’s birthweight was 3.04 kg (77th percentile) and
infiltrative processes such as TMD, leukemia, or lymphoma; the initial physical examination was notable for mild respi-
congenital infection was thought to be less likely given the ratory distress, a distended abdomen, and hepatomegaly
results of the maternal serology tests. At 36 weeks and 1 day with an inferior liver edge that was palpable in the pelvis.
of gestation, repeat ultrasonography showed macrosomia Pertinent negatives on examination included absence of
(estimated fetal weight 3,385 g, >90th percentile), a nor- facial dysmorphisms, normal cardiovascular examination
mal amniotic fluid index (21 cm), and similar hepatomeg- findings without murmur, clear breath sounds bilaterally,
aly. At 36 weeks and 2 days’ gestation, ultrasonography patent normal-appearing anus, normal neurologic tone
revealed new dilation of the rectum of uncertain etiology. and reflexes, no rash, and absence of clinodactyly, sandal
Given the new finding in the setting of unclear underlying gap digits, or single transpalmar creases.

e862 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Her initial laboratory tests showed the following:

• Postnatal arterial blood gas: pH=7.14, PCO2=48 mm Hg

(6.4 kPa), base deficit 12.5, and lactate 74.8 mg/dL (8.3
mmol/L)
• Capillary blood glucose=10 mg/dL (0.56 mmol/L)
• White blood cell count=28,900/mL (28.9 × 109/L), man-
ual differential notable for elevated immature granulo-
cytes (5.7%), atypical cells (3.8%), reactive lymphocytes
(18.9%), and the following cell types within normal lim-
its: neutrophils (14.2%), lymphocytes (42.5%), mono-
cytes (14.2%), eosinophils (0%), and basophils (0.9%).
The laboratory described the atypical cells as having fine
chromatin, high nucleus to cell ratio, prominent nucle-
oli, and dark blue cytoplasm, consistent with blasts.
• Hematocrit=50.5%
• Platelet count=39 × 103/mL (39 × 109/L)
• International normalized ratio=2.86, activated partial
thromboplastin clotting time=55.7 seconds, and fibrino-
gen <35 mg/dL (1.03 g/L) Figure 3. Radiography on the first day after birth showed massive
• Alanine aminotransferase=402 U/L (6.7 mkat/L), aspar- hepatomegaly (arrows), with soft tissue density opacifying much of the
upper and right abdomen. Lung volumes are low but well aerated
tate aminotransferase=55 U/L (0.8 mkat/L) (diamond). Note the bowel gas (triangle) displaced into the left lower
• Indirect bilirubin=2.1 mg/dL (35.9 mmol/L), direct biliru- abdomen by the enlarged liver and the enteric tube within the
displaced stomach.
bin=0.9 mg/dL (15.4 mmol/L), total bilirubin=3.0 mg/dL
(51.3 mmol/L)
• Alkaline phosphatase=200 U/L (3.3 mkat/L) Outcome
• Plasma albumin=2.0 g/dL (20 g/L) Rowan continued to receive 7 cm H2O CPAP to maintain
adequate lung volumes. She continued to require blood
Rowan received multiple normal saline boluses for the lac- products for her coagulopathy. Her hypoglycemia persisted,
tic acidosis and multiple 10% dextrose boluses for hypoglyce- requiring an increased glucose infusion rate to maintain
mia. Her peak lactate level was 80 mg/dL (8.9 mmol/L). To euglycemia. Blood and urine cultures and serum and sur-
treat her thrombocytopenia and coagulopathy, she received face herpes simplex virus polymerase chain reaction (PCR)
transfusions of platelets, cryoprecipitate, and fresh frozen tests were performed, and she was empirically treated with
plasma. Cord blood was collected and banked for future ge- ampicillin, gentamicin, and acyclovir. Abdominal ultraso-
netic studies. nography 1 day after birth showed severe hepatomegaly, dis-
Full body radiography showed low lung volumes, a placing otherwise normal abdominal structures, similar to
normal heart size, and hepatomegaly opacifying most the prenatal imaging (Fig 4).
of the abdomen (Fig 3). No dilated bowel was identified A pediatric infectious diseases specialist was consulted
to correspond with rectal dilation seen on prenatal ultra- to guide further diagnostic testing. Other services consulted
sonography. included hematology/oncology, gastroenterology, molecular
The initial differential diagnosis included the following: and medical genetics, and endocrinology. Additional studies
inflammation (infection, drug injury, autoimmune disorder, conducted based on initial consultation recommendations in-
metabolic disorders), storage disease (glycogen or lipid stor- cluded PCR for parechovirus, enterovirus, and CMV, next-
age disease, a1-antitrypsin deficiency, Wolman disease), con- generation sequencing panel for known mutations in hemato-
gestion and engorgement (congestive heart failure, hepatic logic malignancies, leukemia/lymphoma flow cytometry panel,
vein thrombosis), biliary obstruction (choledochal cyst, bili- peripheral blood morphology, manual differential, urine or-
ary atresia, mass lesion), and infiltrative processes (TMD if ganic acids, serum amino acids, plasma creatinine kinase,
Rowan had trisomy 21 mosaicism without physical features, lipid panel, ammonia, thyroid function studies, g-glutamyl
leukemia, lymphoma, or parasitic). transferase, and urine creatinine.

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e863

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Figure 5. Liver biopsy findings. A. Representative hematoxylin and eosin–
stained fragment of the liver biopsy. B. Higher magnification image of the
liver biopsy. Sinusoidal expansion and extensive parenchymal infiltration
by medium-sized monomorphic lymphoid cells with increased nucleus to
Figure 4. Postnatal abdominal ultrasonography on the first day after birth cell ratios, smooth chromatin, and prominent nucleoli (representative cells
showed severe hepatomegaly (arrows) measuring up to 10.8 cm in cranio- in the yellow circle). Islands of extramedullary hematopoiesis characterized
caudal length. This transverse image of the upper abdomen also shows a by the dark nuclei (black arrow). C. Immunohistochemical staining with
normal spleen (diamond) and a small amount of gas within the com- the B-cell marker PAX5. D. Immunohistochemical staining of the immature
pressed stomach (triangle). marker TdT. Flow cytometry of the peripheral blood confirmed a B-cell
immunophenotype: PAX5, dim CD19, dim CD22, CD38, dim CD45, CD58,
dim cCD79a, and TDT positive; negative for CD10, CD34, and surface light
Given her significant coagulopathy and severe hepato- chains.
megaly, there was concern for intracranial bleeding. Head
ultrasonography showed no hemorrhage but the finding was unsuccessful and her diagnostic lumbar puncture was
was concerning for periventricular white matter disease. traumatic but had some atypical cells. She was diagnosed
Neurology was consulted and recommended a brain MRI with infant B-cell acute lymphocytic leukemia (ALL) with
with and without contrast, which showed small intraven- blood, central nervous system, and liver involvement. She
tricular and subarachnoid hemorrhages, as well as a sub- had no evidence of tumor lysis syndrome based on labora-
galeal hematoma; no white matter abnormalities were tory results, and intravenous fluids were condensed due to
noted. Neurosurgery was consulted and no surgical inter- increased anasarca. On day 6 after birth, she was started
vention was indicated at that time. on small-volume enteral feedings and enrolled in an inves-
On the second day after birth, echocardiography was tigational infant leukemia treatment study.
performed to assess for cardiac anomalies seen with meta- As her hospital stay progressed, results of all of the mater-
bolic storage diseases (including Pompe disease) and to as- nal and infectious disease studies were negative (Table 1).
sess for congenital heart disease or heart failure that may Her blood karyotype was 46,XX and additional genetic and
have led to a congestive hepatomegaly. Echocardiography molecular tests from the blood and the liver biopsy identified
showed an atrial septal defect, a patent ductus arteriosus no known abnormalities found in patients with leukemia.
with left to right flow, elevated estimated right ventricular Whole-genome sequencing was nondiagnostic and no genetic
pressure as expected for age, and normal biventricular variants that could explain the infant’s presentation were de-
function. She was started on total parenteral nutrition tected. Sequence analysis also confirmed that the cells in the
with soybean oil, medium-chain triglycerides, olive oil, liver were of infant and not maternal origin. Thyroid studies
and fish oil (SMOF) lipids, given her direct hyperbilirubi- were within normal limits and additional metabolic findings,
nemia. A percutaneous liver biopsy was performed on the including serum amino acids, ammonia, plasma creatinine
fourth day after birth with expedited pathology; the prelim- kinase, and lipid panel were negative, urine organic acids
inary result was reported as small round cells consistent were normal, and her lactic acidosis resolved. At 10 days of
with leukemia/lymphoma (Fig 5). In addition, her periph- age, with concern for a lymphoproliferative disorder due to
eral blood flow cytometric analysis identified an 11% im- immune dysregulation, her serum infliximab level was mea-
mature atypical B-cell population consistent with B-cell sured, and found to be 7.0 mg/mL, representing transplacen-
lymphoblastic leukemia/lymphoma. The subsequent bone tal transfer of infliximab. Serial weekly head ultrasonography
marrow aspiration to confirm bone marrow involvement demonstrated interval resolution of her periventricular white

e864 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 Table 1. Maternal and Infant Infectious Diseases Results
Laboratory Test Maternal Findings Neonatal Findings
COVID-19 PCR negative PCR negative
Cytomegalovirus IgG, IgM negative PCR negative
Herpes simplex virus 1 and 2 IgG elevated, IgM negative; latent infection PCR negative
Herpes simplex virus 2 IgG, IgM negative
Rapid plasma reagin Nonreactive) Nonreactive
Hepatitis A virus IgG, IgM negative
Hepatits B virus Surface antigen, core antibody negative
Hepatitis C virus Antibody negative
Toxoplasma IgG, IgM negative
Blood culture Negative
Enterovirus PCR negative
Parechovirus PCR negative
Urine culture Negative

COVID-19=coronavirus disease 2019, Ig=immunoglobulin, PCR=polymerase chain reaction.

matter abnormalities by 20 days after birth. Although she of this writing, at 7 months of age, she is doing well, with-
had required intubation for her initial bone marrow biopsy, out evidence of disease, and thriving.
she underwent extubation and was transitioned to high-flow
nasal cannula after the procedure without complications, and DISCUSSION
gradually weaned off respiratory support before being dis- Fetal Hepatomegaly
charged from the hospital. Fetal hepatomegaly can be defined as craniocaudal liver
Due to persistence of elevated transaminases and direct length on ultrasonography greater than the 95th percentile
hyperbilirubinemia despite treatment with ursodiol and for gestational age. Ultrasound findings concerning for fe-
tolerance of full enteral feedings, liver biopsy was repeated tal hepatomegaly include the right hepatic lobe extending
at 2 months of age. Findings were negative for intrahe- below the right kidney and the left hepatic lobe displacing
patic leukemia, but were notable for giant cell hepatitis, the stomach and/or spleen. An abnormal liver surface
periductal cholangitis, bile duct proliferation, and bile duct contour (convex or lobulated) and altered parenchymal
plugs. Giant cell hepatitis was attributed to injury from echogenicity can also be seen with hepatomegaly. Second-
leukemia and chemotherapy. Remaining findings were ary findings that suggest fetal hepatomegaly include en-
concerning for biliary obstruction of unclear etiology. Fur- larged abdominal circumference for gestational age and
ther evaluation with a hepatobiliary iminodiacetic acid enlarged hepatic vein. (1) Once fetal hepatomegaly has
scan and magnetic resonance cholangiopancreatography been identified, further investigation is needed to deter-
did not identify any structural biliary pathology and review mine the etiology. This includes detailed maternal medical
of the whole-genome sequencing identified no mutations and exposure history, family history, additional imaging
to which the cholestasis could be attributed. Her chemo- studies (such as MCA Doppler and fetal MRI), and labora-
therapy regimen was adjusted to account for her hepatitis tory studies (such as amniocentesis for genetic evaluation
and cholestasis. and maternal serologies for an infectious evaluation).
The placental pathology was significant for an extremely Prenatal identification of fetal hepatomegaly is impor-
large placenta for gestational age (834 g; expected range tant because of the potential for in utero treatment for
372–542 g) with villous maturation consistent with stated some conditions (eg, syphilis) and close surveillance and/
gestational age and patchy villous edema. There were also or in utero interventions for others (eg, transfusions for
frequent large, atypical hematopoietic cells in the fetal vas- fetal anemia). Anticipated congenital hepatomegaly also
culature, consistent with blasts, with positive immunohisto- assists with planning for the delivery location, neonatal
chemical staining for CD45, CD20, and TdT, similar to the stabilization, and immediate neonatal management and
liver biopsy. There was no evidence of abruption, and the diagnostic evaluation.
membranes and umbilical cord did not have any significant After birth, a detailed physical examination as well as fur-
abnormality. ther diagnostic evaluation should be completed. Postnatal
Rowan was discharged from the hospital at 82 days of laboratory and imaging studies should be individualized and
age with close outpatient follow-up and ongoing chemo- guided by prenatal evaluations, physical examination find-
therapy. Her cholestasis eventually resolved. At the time ings, and results of initial screening laboratory and imaging

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e865

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 results. The following should be considered: complete blood first year of age. (5) Infant leukemia makes up approxi-
cell count with differential, blood glucose, complete metabolic mately 2.5% to 5% of childhood ALL and 6% to 14% of
panel including transaminases, total and direct bilirubin, co- childhood AML. (6)
agulation studies, evaluation for congenital infections such as Congenital leukemia is a subtype of infant leukemia
CMV, and imaging studies to characterize the liver size and that is defined as leukemia diagnosed within the first 4
appearance, assess for masses and patency of bile ducts, and weeks of age. (7) The incidence of congenital leukemia
evaluate for any additional anatomic abnormalities. Imaging based on surveillance data is between 1 and 5 per million
studies may include abdominal ultrasonography, MRI, nu- live births annually, (8) making up less than 1% of all
clear medicine hepatobiliary scan, and/or echocardiography. childhood leukemia. (9) Two-thirds of cases are AML,
A liver biopsy is typically performed when ultrasound findings with the remainder mostly ALL (predominantly B-cell line-
are nondiagnostic; it can be helpful to definitively evaluate for age) with rare mixed phenotype or blastic plasmacytoid
storage diseases and malignancies. (2) Because solid organ bi- dendritic cell neoplasms. (10) Studies have shown an asso-
opsies are considered high risk for bleeding, laboratory values ciation of fetal and neonatal leukemia with maternal etha-
including platelet count and the international normalized ra- nol consumption during pregnancy. Increased maternal
tio are routinely checked before performing a percutaneous consumption of foods containing DNA topoisomerase II
liver biopsy to assess bleeding risk. In the event that these val- inhibitor has been related to an increased risk of infant
ues are outside the acceptable range (platelets #50,000/mL AML. (11) The dietary source of DNA topoisomerase II
[50 × 109/L], international normalized ratio $1.5), (3) blood inhibitors include catechins in red wine, cocoa, and green
products can be administered as needed during the procedure and black tea, various flavonoids such as quercetin in some
to minimize the risk of clinically significant hemorrhage. vegetables and fruits, and genistein found in soy. (11) Higher
The differential diagnosis of hepatomegaly in a neonate birthweight is also correlated with an increased risk for leu-
is broad and different from that of an older child (Table 2). kemia. (12)
Common features of congenital leukemia include hepa-
Congenital Leukemia tomegaly and splenomegaly. Leukemia cutis (also referred
Infant leukemia is defined as leukemia within the first 12 to as “blueberry spots” or “blueberry muffin rash”) is
months of age. The incidence is estimated to be 41 cases found in approximately 25% to 60% of congenital leuke-
per million in the United States, which equates to approxi- mia cases, (13)(14) and is more common in AML than
mately 160 cases per year with slightly more cases of ALL ALL. (10) The appearance is due to skin infiltration of
(56%) than acute myeloid leukemia (AML), (43%). (4) leukemic cells, and it presents as generalized, firm, mo-
Although girls have a higher risk of developing leukemia bile, purple, blue, brown or red nodules. Approximately
in the first year of age, boys have a higher risk after the 33% to 50% of neonates have central nervous system

Table 2. Differential Diagnosis for Neonatal Hepatomegaly

Differential Diagnosis for Neonatal Hepatomegaly Examples
Anemia, immune mediated and nonimmune mediated Rh-isoimmunization, congenital dyserythropoietic anemia
Benign tumors Mesenchymal hamartoma, hepatic hemangioma
Biliary obstruction Biliary atresia, choledochal cyst
Congestive heart failure Severe Ebstein anomaly, ventricular septal defect leading to hepatic
congestion
Exposures Toxins and drugs
Autoimmune hepatitis Congenital hemochromatosis and hydrops fetalis, gestational
alloimmune liver disease
In utero infection TORCH infections: Toxoplasmosis, other (eg, varicella, human
immunodeficiency virus, syphilis, parvovirus B19, hepatitis B virus,
hepatitis C virus), rubella, cytomegalovirus, herpes simplex virus
Malignancies Primary: hepatoblastoma, Secondary or infiltrative: neuroblastoma,
leukemia, lymphoma, transient myeloproliferative disorder
(associated with trisomy 21)
Metabolic/storage disorders Lysosomal storage disorders (eg, Niemann-Pick type C, Gaucher
disease, GM1 gangliosidosis, Wolman disease, sialic acid storage
disease [1])
Overgrowth Overgrowth syndromes (eg, Beckwith-Wiedemann syndrome), maternal
diabetes

e866 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 involvement with leukemic cells in the cerebrospinal fluid, me- Infliximab is a chimeric IgG1 monoclonal antibody against
ninges, and brain parenchyma. (4)(10)(15) The presence of leu- TNF-a used in the treatment of various autoimmune disor-
kocytosis is variable, ranging from none to profound. (9) ders, including Crohn’s disease. Given what is known about
Severe hyperleukocytosis can lead to renal, cardiac, and respira- the transplacental transfer of IgG in pregnancy, infliximab
tory failure and may necessitate an exchange transfusion (10) is thought to have minimal transplacental transfer in the first
or hemodialysis. Other symptoms may include secondary ef- trimester. However, during the late second and third trimes-
fects of leukemia (eg, symptoms of anemia, thrombocytopenia, ters, several studies have demonstrated an efficient placental
and actual or functional neutropenia) and liver dysfunction or transfer, resulting in detectable levels in the neonate such as
failure due to leukemic hepatic infiltration. The neonate may found in this patient, which may remain elevated for several
also have jaundice, ascites, and pleural effusions, though months. (22) Despite this transplacental transfer, current evi-
lymphadenopathy in this population is less common. (10) dence, based on over 300 pregnancy outcomes, suggests that
The prognosis for infant ALL is heavily dependent on the infliximab is safe to use during the first 2 trimesters of preg-
presence of KMT2A rearrangements, in which KMT2A rear- nancy, and various databases, case series, and case reports
ranges and fuses with a number of different partners so that have not revealed an increase in the risk of adverse neonatal
not every KMT2A rearrangement is the same. More than 90 outcomes from in utero exposure to anti-TNF drugs. (22)
different KMT2A partner genes have now been identified. (16) Currently, the US Food and Drug Administration (FDA) clas-
Infants with the type of leukemia that carries a KMT2A rear- sifies infliximab as a category B drug in pregnancy. (23)
rangement have a survival rate of 20% to 40% with intensive There is a theoretical increased risk of infection and
systemic chemotherapy compared to a survival rate of 75% for diminished response to vaccines in infants exposed to in-
patients without a KMT2A rearrangement. (17) In congenital fliximab in utero. However, various case reports and co-
ALL, up to 93% of patients will carry a KMT2A rearrangement hort studies have demonstrated that most infants exposed
(18) with a poor prognosis of 20% survival. The prognosis for to in utero infliximab are able to mount appropriate vac-
congenital ALL without a KMT2A rearrangement is difficult to cine responses and there is no observed increase in the
predict due to the rarity, but may have similar outcomes to their risk of infections during the first year after birth. How-
infant counterparts. Most intriguing are the reports of spontane- ever, given the unknown degree of immunosuppression
ous remission in cases of congenital ALL, suggesting a possible in infants with detectable levels of infliximab, it is recom-
immune response for clearance of the disease. (10)(19) mended that live vaccines be postponed until serum inflix-
The most confounding aspect in the pathologic diagnosis imab levels are undetectable. (22)
of this patient was the absence of a KMT2A rearrangement. Infliximab is thought to be acceptable to use while breast-
Conventional karyotyping, fluorescence in situ hybridization, feeding, with low risk to the infant. Various case studies and
and next-generation sequencing did not identify any known case series have demonstrated that infliximab is usually not
pathologic mutations. As such, the infant was diagnosed with detectable in breast milk, and if detected, the levels are very
congenital ALL without a KMT2A rearrangement, making a low and the drug is minimally absorbed by the infant. (24)
prediction of her prognosis difficult. Furthermore, the infants who breastfed during maternal
infliximab therapy were found to have normal development
Infliximab in Pregnancy and no adverse effects on follow-up. (24)
Immunoglobulin G (IgG) is the only antibody class with What remains unknown is whether infliximab played
significant human transplacental transfer from the preg- any role in the development of this infant’s leukemia. Al-
nant woman to the fetus. (20) This transfer provides the though the FDA maintains a black box warning with anti-
neonate with a layer of protection while the immune sys- TNF therapy and its association with malignancies in the
tem continues to develop postnatally. The active transpla- user, this relationship remains complex. (25) There is cur-
cental transfer of IgG begins around 13 weeks of gestation rently no published information on infant malignancies
with a linear increase throughout the rest of gestation and associated with the maternal use of infliximab. Yet, it is
the majority of IgG being transferred in the last 4 weeks well known that immunodeficiencies as well as long-term
of pregnancy. (21) Therefore, there is a direct relationship immunosuppression increase the risk of developing ma-
between the level of IgG in the neonate and the duration lignancies. (26) One could postulate that in utero exposure
of gestation. (20) By term gestational age, the fetal IgG con- to infliximab may have played a role in allowing immune
centration exceeds maternal IgG concentrations by 20% to escape of this patient’s leukemia. How this may affect her
30%. (21) prognosis remains elusive.

Vol. 23 No. 12 D E C E M B E R 2 0 2 2 e867

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user
 5. Howlader N, Noone AM, Krapcho M, et al. SEER Cancer Statistics
Review, 1975–2010. Bethesda, MD: National Cancer Institute; based
Summary on November 2012 SEER data submission. Available at: http://seer.
cancer.gov/csr/1975_2010. Accessed September 13, 2022
• The differential diagnosis of fetal hepatomegaly is
6. Pui CH, Kane JR, Crist WM. Biology and treatment of infant
broad and warrants a detailed maternal medical and
leukemias. Leukemia. 1995;9(5):762–769
exposure history, family history, and fetal anatomic
7. Kikuchi A, Tamura N, Ishii K, et al. Four cases of fetal hypoechoic
survey to identify the cause of hepatomegaly. hepatomegaly associated with Trisomy 21 and transient abnormal
myelopoiesis. Prenat Diagn. 2007;27(7):665–669
• Detection of fetal hepatomegaly is important, as some
8. Bader JL, Miller RW. US cancer incidence and mortality in the first
causes are treatable during the fetal period and/or may year of life. Am J Dis Child. 1979;133(2):157–159
benefit from close prenatal surveillance and advanced 9. Sande JE, Arceci RJ, Lampkin BC. Congenital and neonatal
planning for evaluation of the neonate at birth. leukemia. Semin Perinatol. 1999;23(4):274–285

• When imaging and standard laboratory studies 10. Roberts I, Fordham NJ, Rao A, Bain BJ. Neonatal leukaemia. Br J
Haematol. 2018;182(2):170–184
are inconclusive, liver biopsy can help diagnose
11. Spector LG, Xie Y, Robison LL, et al. Maternal diet and infant
infiltrative processes in the liver. leukemia: the DNA topoisomerase II inhibitor hypothesis: a report
from the children’s oncology group. Cancer Epidemiol Biomarkers
• Congenital leukemia is a rare entity that may
Prev. 2005;14(3):651–655
present with fetal hepatomegaly.
12. Biondi A, Cimino G, Pieters R, Pui CH. Biological and therapeutic
• Available evidence suggests that infliximab is safe to aspects of infant leukemia. Blood. 2000;96(1):24–33
use during pregnancy, though this medication is 13. Resnik KS, Brod BB. Leukemia cutis in congenital leukemia:
analysis and review of the world literature with report of an
transferred across the placenta in the third trimester,
additional case. Arch Dermatol. 1993;129(10):1301–1306
which may have implications for the neonate.
14. Bresters D, Reus ACW, Veerman AJP, van Wering ER, van der Does-van
• Infants with detectable levels of infliximab should not den Berg A, Kaspers GJL. Congenital leukaemia: the Dutch experience
and review of the literature. Br J Haematol. 2002;117(3):513–524
receive live vaccines until the level is undetectable.
15. Isaacs H Jr. Fetal and neonatal leukemia. J Pediatr Hematol Oncol.
• This is the first report of an infant with an 2003;25(5):348–361
intrauterine exposure to infliximab and a 16. Meyer C, Burmeister T, Gr€oger D, et al. The MLL recombinome of
diagnosis of ALL; further data are needed to acute leukemias in 2017. Leukemia. 2018;32(2):273–284

determine whether there is an association. 17. Pieters R, De Lorenzo P, Ancliffe P, et al. Outcome of infants
younger than 1 year with acute lymphoblastic leukemia treated with
the interfant-06 protocol: Results from an international phase III
randomized study. J Clin Oncol. 2019;37(25):2246–2256
Acknowledgment 18. van der Linden MH, Valsecchi MG, De Lorenzo P, et al. Outcome of
The authors would like to thank Rowan’s parents for congenital acute lymphoblastic leukemia treated on the Interfant-99
sharing their story. To honor Rowan, her parents asked protocol. Blood. 2009;114(18):3764–3768
us to use her name in this publication for which we have 19. Zhang Q, Ren Z, Yang J, Yin A. Analysis of 59 cases of congenital leukemia
received Oregon Health and Science University Institu- reported between 2001 and 2016. J Int Med Res. 2019;47(10):4625–4635
tional Review Board approval. 20. Palmeira P, Quinello C, Silveira-Lessa AL, Zago CA, Carneiro-
Sampaio M. IgG placental transfer in healthy and pathological
pregnancies. Clin Dev Immunol. 2012;2012:985646
References 21. Saji F, Samejima Y, Kamiura S, Koyama M. Dynamics of immunoglobulins
at the feto-maternal interface. Rev Reprod. 1999;4(2):81–89
1. Jensen KK, Oh KY, Patel N, Narasimhan ER, Ku AS, Sohaey R. Fetal
hepatomegaly: causes and associations. Radiographics. 2020;40(2):589–604 22. Djokanovic N, Klieger-Grossmann C, Pupco A, Koren G. Safety of
infliximab use during pregnancy. Reprod Toxicol. 2011;32(1):93–97
2. Wolf AD, Lavine JE. Hepatomegaly in neonates and children. Pediatr
Rev. 2000;21(9):303–310 23. Janssen Biotech, INC. 2013. REMICADE (infliximab); Lyophilized
Concentrate for Injection, for Intravenous Use. Available at: https://
3. Patel IJ, Rahim S, Davidson JC, et al. Society of Interventional
www.accessdata.fda.gov/drugsatfda_docs/label/2013/103772s5359lbl.
Radiology Consensus Guidelines for the periprocedural
pdf. Accessed October 7, 2022
management of thrombotic and bleeding risk in patients
undergoing percutaneous image-guided interventions-part II: 24. Vaughn CJ. Drugs and lactation database: LactMed. J Electron Resour
recommendations endorsed by the Canadian Association for Med Libr. 2012;9(4) doi: https://doi.org/10.1080/15424065.2012.735134
Interventional Radiology and the Cardiovascular and Interventional 25. Dahmus J, Rosario M, Clarke K. Risk of lymphoma associated with
Radiological Society of Europe. J Vasc Interv Radiol. 2019;30(8): anti-TNF therapy in patients with inflammatory bowel disease:
1168–1184.e1 implications for therapy. Clin Exp Gastroenterol. 2020;13:339–350
4. Brown P. Treatment of infant leukemias: challenge and promise. 26. Mortaz E, Tabarsi P, Mansouri D, et al. Cancers related to
Hematology (Am Soc Hematol Educ Program). 2013;2013:596–600 immunodeficiencies: update and perspectives. Front Immunol. 2016;7:365

e868 NeoReviews

Downloaded from /neoreviews/issue/23/12

by Almoosa Hospital user