European Journal of Human Genetics (2018) 26:1582–1587
https://doi.org/10.1038/s41431-018-0209-0
ARTICLE
Genetic diversity of NDUFV1-dependent mitochondrial complex I
deficiency
Anshika Srivastava1 Kinshuk Raj Srivastava2 Malavika Hebbar3 Chelna Galada3 Rajagopal Kadavigrere4
Fengyun Su5,6 Xuhong Cao5,6 Arul M. Chinnaiyan5,6 Katta M. Girisha3 Anju Shukla3 Stephanie L. Bielas
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Received: 16 January 2018 / Revised: 7 May 2018 / Accepted: 12 June 2018 / Published online: 5 July 2018
© European Society of Human Genetics 2018
Abstract
Medical genomics research performed in diverse population facilitates a better understanding of the genetic basis of
developmental disorders, with regional implications for community genetics. Autosomal recessive mitochondrial complex I
deficiency (MCID) accounts for a constellation of clinical features, including encephalopathies, myopathies, and Leigh
Syndrome. Using whole-exome sequencing, we identified biallelic missense variants in NDUFV1 that encodes the 51-kD
subunit of complex I (NADH dehydrogenase) NDUFV1. Mapping the variants on published crystal structures of
mitochondrial complex I demonstrate that the novel c.1118T > C (p.(Phe373Ser)) variant is predicted to diminish the affinity
of the active pocket of NDUFV1 for FMN that correlates to an early onset of debilitating MCID symptoms. The c.1156C > T
(p.(Arg386Cys)) variant is predicted to alter electron shuttling required for energy production and correlate to a disease onset
in childhood. NDUFV1 c.1156C > T (p.(Arg386Cys)) represents a founder variant in South Asian populations that have
value in prioritizing this variant in a population-specific manner for genetic diagnostic evaluation. In conclusion, our results
demonstrate the advantage of analyzing population-specific sequences to understand the disease pathophysiology and
prevalence of inherited risk variants in the underrepresented populations.
Introduction
Electronic supplementary material The online version of this article
(https://doi.org/10.1038/s41431-018-0209-0) contains supplementary
material, which is available to authorized users.
* Stephanie L. Bielas
sbielas@umich.edu
1
Department of Human Genetics, University of Michigan Medical
School, Ann Arbor, Michigan, USA
2
Life Sciences Institute, University of Michigan, Ann Arbor,
Michigan, USA
3
Department of Medical Genetics, Kasturba Medical College,
Manipal University, Manipal, India
4
Department of Radiodiagnosis, Kasturba Medical College,
Manipal University, Manipal, India
5
Howard Hughes Medical Institute, Michigan Center for
Translational Pathology, University of Michigan, Ann Arbor,
Michigan, USA
6
Department of Pathology, University of Michigan Medical School,
Ann Arbor, Michigan, USA
Aggregation of large-scale sequencing data and increasing
clinical use of whole-exome sequencing (WES) for genetic
diagnosis, is uncovering genetic variation that aids in
annotation and interpretation of disease alleles. Increased
inclusion of WES data from diverse populations, underrepresented in genomic databases uncovers clinically significant genetic variation that advances our understanding of
disease alleles and underlying biology. Elucidating rare
genetic diversity and how it contributes to Mendelian
inherited disorders has implications for community genetics, where a better understanding of the ancestry-specific
genomics can direct carrier testing, presymptomatic diagnosis, and potential interventions to delay the onset of
symptoms in a community-specific manner.
Autosomal recessive mitochondrial complex I deficiency
(MCID) accounts for approximately 23% of childhood
respiratory chain deficiency cases [1]. Mitochondrial complex I is composed of 44 structural subunits and over 10
assembly factors, which underscores its diverse clinical
manifestations that diverge based on severity and age of
onset [2]. The clinical presentations range from infant onset
Genetic diversity of NDUFV1-dependent mitochondrial complex I deficiency
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Fig. 1 Brain imaging and
NDUFV1 variants in Proband 1
and 2. a T1-weighted axial brain
MRI of proband 1 at 6 months
demonstrates pachy diffusion
involving centrum semiovale,
corona radiate (arrows), and
periventricular external capsule
(stars). Proband 2 T2 weighted
axial brain MRI at 6 years of age
shows diffuse white matter
demyelination with gliotic areas
and atrophy. b Pedigrees for
family 1 and family 2, both of
South Asian population. Filled
symbols denote individuals
affected with the MCID. Double
lines denote consanguinity. c
Sequence chromatograms
showing the biallelic inheritance
of NDUFV1 missense variants
(black arrows) in proband 1 and
2 (lower panel, proband)
consistent with consanguinity in
the family
subacute necrotizing encephalomyelopathy (Leigh syndrome [MIM: 256000]) to adult-onset exercise-induced
myopathy. Genetic studies have identified disease-causing
variants in 11 nuclear-encoded complex I genes [MIM:
252010], including NDUFV1 [3–13].
In the present study, we report genetic findings from two
unrelated probands from the South Asian population
(Country of origin: India, region: Southern India) that presented with divergent features of MCID. Proband 1 exhibited symptoms of MCID at 6 months of age and presented
with a novel homozygous c.1118T > C (p.(Phe373Ser))
missense variant in exon 8 of NDUFV1. Proband 2 was
homozygous for the previously reported c.1156C > T (p.
(Arg386Cys)) variant, also in exon 8 of NDUFV1 (Figs. 1c,
2a), but did not exhibit symptoms until 6 years of age [14–
16]. The discrepancy in clinical presentation and age of
onset between these cases is supported by the molecular
impact as modeled on published crystal structures of
mitochondrial complex I. The NDUFV1 p.(Arg386Cys)
variant exhibits higher frequency in South Asian population
suggesting a founder effect with implication for community
genetics [14].
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A. Srivastava et al.
Fig. 2 a Schematic showing the intron-exon structure of human
NDUFV1. The cDNA sequence from position 1111 (NM_007103.3)
and the corresponding peptide sequence from Ile371 to Glu387 are
shown. Genetic variants that affect function or amino acid substitutions identified in mitochondrial complex I deficiency patient are
marked in red. b Structure of mammalian mitochondria and close-up
depicting membrane embedded respiratory chain complexes. The circled region shows the N module of complex I, composed of NDUFV1,
NDUFV2, and NDUFS1 proteins. Crystal structure of N module
derived from T. thermophilus (3IAM), where colors denote distinct
proteins (blue = NDUFV1, pink = NDUFS, and green = NDUFV2).
Colored elements: Fe–S cluster = red and orange, FMN binding
pocket = blue and green, and missense variants identified in proband 1
and 2 are encircled with bold circle. Dashed red lines denote the
trajectory of shuttled electrons. c Magnified image of substituted
amino acid close to FMN binding pocket in proband 1. The amino acid
in black protruding out from alpha helix is the phenylalanine at
position 373. The variation p.(Phe373Ser) disrupts the FMN binding to
the NDUFV1. (d) Magnification of amino acids close to Fe–S cluster
disrupted by proband 2 substitution. Arginine at position 386 is shown
in red. The p.(Arg386Cys) substitution will alter buffering of Fe–S
clusters and electrons transfer across NDUFV1
Clinical reports
objects after the use of spectacles. On clinical examination
at the age of 1 year, height was 78.5 cm (−0.9 SD), head
circumference was 49 cm (+1 SD), and weight was 9.8 kg
(−1 SD). He was observed to have bilateral lower set ears,
nystagmus, mosaic pigmentary anomalies, hepatomegaly,
and spasticity in lower limbs, extreme plantar responses and
brisk deep tendon reflexes. Electroencephalogram and
fundus examinations were found to be normal. Brain imaging sequences show diffuse hyperintensity in the cerebral
white matter, cerebellar white matter and brainstem white
matter, and small cystic areas in the periventricular white
matter (Fig. 1a). Magnetic resonance spectrometry showed
Proband 1
One-year-old proband 1, the first born of third-degree
consanguineous parents (Fig. 1b), was referred for developmental delay. He was born at full term by normal vaginal
delivery. He weighed 2.5 kg at birth. An excessive cry was
noted. Seizures were first observed at the age of 6 months.
His development was delayed mildly, with momentary loss
of head control, and roll over at 7 months of age. He was
diagnosed with myopia. He started smiling and reaching for
Genetic diversity of NDUFV1-dependent mitochondrial complex I deficiency
reduced N-acetyl aspartate and elevated choline levels and
an inverted double peak of lactate.
Proband 2
Proband 2, second born of third-degree consanguineous
parents (Fig. 1b), developed normally until the 6 years of
age at which time he presented with neuroregression, mild
cognitive decline with regressive speech deficiencies,
bilateral optic atrophy, and marked motor decline. Proband
2 was of normal height and weight at 137 cm and 25 kg.
History of seizures was noted. Sibling with similar clinical
features died at the age of 15 years. Physical examination
showed a height of 137 cm (−1 SD), head circumference of
46 cm (−4 SD), and weight 25 kg (−3 SD) with spasticity
in all four limbs, clonus, and nystagmus. Optic atrophy was
reported on ophthalmology evaluation. However, blood
lactate and pyruvate levels were within normal range. Brain
imaging displayed a diffuse white matter demyelination
with cystic areas consistent with neurodegeneration
(Fig. 1a). Based on these features, proband 2 was provided
with a diagnosis of leukodystrophy.
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genetic variants [20]. Variants were further filtered against
public databases such as 1000 Genomes Project phase 3,
ExAC, National Heart, Lung, and Blood Institute and
Exome Sequencing Project Exome Variant Server
(ESP6500SI-V2). Variants flagged as low quality or putative false positives (Phred quality score < 20 and low quality
by depth < 20) and minor allele frequency > 1% was
excluded from the analysis.
Protein modeling
Mitochondrial complex I crystal structure of bacterial [T.
thermophilus (PDB:3IAM)] and bovine [Bos Taurus
(PDB:5lc5)] was obtained from Protein Data Bank
(http://www.rcsb.org/pdb/). Sequences were mapped to
Homo sapiens using ClustalW. Pymol software was used to
model the structure of N-modules of bacterial and bovine
mitochondrial complex I.
Results
Variant prioritization
Material and methods
Ethics statement and clinical sample collection
The present study is a part of an ongoing combined clinical/
research project (Ethical approval number: Indo-Foreign/
Neuro/154/2015) that started in the year 2013 to recruit
individuals with inherited neurodevelopmental disorders. A
total of 450 consanguineous families from Southern India
have been recruited to date. The parents provided written
informed consent for WES. DNA was isolated from peripheral blood by standard procedures [17].
Whole-exome sequencing
Exome libraries of affected and unaffected genomic DNA
were generated using the Illumina TruSeq DNA Sample
Prep kit, following the manufacturer’s instructions. Parentsproband WES was performed on family 1. Coding
sequences were prepared and captured with the Agilent
SureSelect All Exon kit-v4 and sequenced on an Illumina
HiSeq 2500 instrument as described previously [18]. Only
proband 2 underwent WES in family 2, as previously
described [19].
Variant calling and filtering
WES data was processed using GATK callers and SeqMule.
ANNOVAR was used to functionally annotate the detected
In Proband 1, seven rare variants, including those in
NDUFV1 (NM_007103.3) were prioritized as candidates
for the clinical presentation. Among these, only one was
responsible for neurodevelopmental disorders; homozygous
missense variant in NRXN2 was ruled out because of its
prevalence in public databases and inconsistencies in
associated phenotypic outcomes (Supplemental Table 1).
Alternatively, the novel homozygous missense c.1118T > C
(p.(Phe373Ser)) NDUFV1 variant accounts for the MCID
phenotype that overlaps with the clinical features of proband 1. The NDUFV1 c.1118T > C (p.(Phe373Ser)) variant
was not detected in ExAC browser. The variant was submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/va
riation/431452/; Submission Accession: SCV000588197.1).
However, in proband 2, three rare variants were prioritized
in the genes NDUFV1, KIRREL3, and TFG. Among these,
the biallelic NDUFV1 variant c.1156C > T (p.(Arg386Cys))
located in exon 8 (numbered as in NG_013353.1;
NM_007103.3) was deemed to affect function in silico by
PolyPhen-2 http://genetics.bwh.harvard.edu/pph2/) supported
by a NDUFV1 c.1157G > A (p.(Arg386His)) substitution
previously associated with severe MCID and onset of
symptoms in infancy (Fig. 2a) [21]. Variant p.(Arg386Cys) is
a previously published variant in additional unrelated individuals from the South Asian population and its submission
accession is SCV000566902.2. The parents were heterozygous for both the variants identified in Proband 1 and 2, as
confirmed by Sanger sequencing and consistent with consanguinity (Fig. 1c)
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Protein modeling analysis
We modeled the functional impact of proband 2 homozygous NDUFV1 p.(Arg386Cys) variant relative to the
previously characterized p.(Arg386His) variant that alters
the same amino acid Arg386, on the bacterial and bovine
crystal structure of mitochondrial complex I [22–25]
(Fig. 2b). The amino acid substitution p.(Arg386Cys) is
predicted to disrupt the protein–protein interactions that
facilitate Fe–S cluster buffering. In the three-dimensional
conformation of complex I, Arg386 is in close proximity to
Cys385, which participates in Fe–S cluster stabilization
within the complex and buffering (Fig. 2d). The volume of
the Cys side chain is small compared to that of His as
observed in amino acid substitution p.(Arg386His).
Increased bulkiness of His may perturb local interactions
between Fe–S binding motifs of NDUFV1. However, the
functional consequence of having two consecutive Cys at
the Fe–S binding motif, as would be the case with the p.
(Arg386Cys) substitution is predicted to be less disruptive
to the Fe–S binding motif, and further compensate for this
substitution by facilitating Fe–S cluster buffering.
Protein modeling and crystal structure analysis of p.
(Phe373Ser) suggests that the variant is located in FMN/
NADH binding site of complex I and is involved in FMN
binding. Substitution of the highly hydrophobic Phe to a
small and polar Ser is predicted to diminish the affinity for
FMN to the active pocket of NDUFV1, altering the first step
of electron transfer that promotes the redox activity of
complex I. We surveyed the published variants in NDUFV1
relative to the functions ascribed by yeast genetic screens to
demonstrate the predictive value of evaluating pathogenicity relative to the available mammalian crystal structure
(Fig. 2c). These findings are summarized in supplemental
Table 2.
Variant interpretation: ACMG guidelines
According to the variant classification guidelines of ACMG,
genetic variant p.(Phe373Ser) is categorized as “likely
pathogenic” whereas variant p.(Arg386Cys) is categorized
as “pathogenic” (Supplemental Table 3).
Discussion
Mitochondrial complex I of the respiratory chain functions
to liberate and transfer electrons from NADH to ubiquinone
for ATP production. Missense variants in NDUFV1 can
disrupt three important functions of complex I: (1) binding
of FMN and NADH, (2) transfer of electrons between
iron–sulfur clusters, and (3) structural integrity required to
maintain the interactions between complex I subunits. Yeast
A. Srivastava et al.
functional studies have proven useful for assaying deleterious alleles in NDUFV1, but do not differentiate between
alleles corresponding to the clinical outcome of differing
severity. Modeling the impact of missense substitutions in
NDUFV1 relative to these functions, using the bacterial and
bovine complex I crystal structures, provide evidence to
support the molecular impact of deleterious amino acid
substitutions. Using this approach, we provide a rationale
for how substitutions of the same amino acid residue, i.e., p.
(Arg386Cys) and p.(Arg386His) result in MCID with different age of onset and clinical outcomes. Genetic variants
p.(Arg386Cys) and p.(Arg386His) were both classified as
strongly functionally impacted variants in the yeast genetic
screens [24]. Based on the bacteria and bovine crystal
structure, the p.(Arg386His) substitution results in an amino
acid side chain that is bulkier than the p.(Arg386Cys)
substitution. The bulkier His side chain is predicted to more
severely disrupt protein–protein interaction between
NDUFV1 and NDUFS1 and buffering between the Fe–S
clusters through complex I than the Cys, providing a
molecular rationale for the less severe later onset MCID
associated with the p. (Arg386Cys) variant, as compared
with p. (Arg386His) that is associated with early onset of
severe clinical manifestations followed by death in infancy
[21].
Functional validation of our novel p.(Phe373Ser) variant
has not been performed in yeast. Modeling studies suggest
that p.(Phe373Ser) lies in the close proximity to Arg88,
Lys111, Ala117 and Glu246, NDUFV1 amino acids shown
to be deleterious in functional assays when mutated to
clinically relevant MCID substitutions. These amino acids
maintain the conformation of the FMN/NADH binding
pocket, implicating a role for variant p.(Phe373Ser) in
complex I function. This analysis approach predicts that p.
(Phe373Ser) affects complex I function and the severity of
clinical outcomes can be traced back to the nature of the
amino acid substitution, with those most dissimilar to the
wildtype amino acid negatively impacting FMN binding
kinetics and clinical outcomes.
Heterozygous NDUFV1 c.1156C > T (p.(Arg386Cys))
was detected at a frequency of 0.0001 in the ExAC database. While rare, it is noteworthy that 13 of the 15 c.1156C
> T (p.(Arg386Cys)) alleles described were observed in
South Asian populations and accentuates a pervious report
that associated this allele to late-onset MCID, in unrelated
South Asian families [14]. Additionally, our inherited
neurodevelopmental disorder cohort of 450 consanguineous
families also identified NDUFV1 c.1156C > T (p.(Arg386Cys)) in one of the two families. Altogether, the recessive
inheritance of c.1156C > T, independent of consanguinity,
suggests this allele is a founder variant, yet defining
enrichment in this population is complicated by the underrepresentation of this population in ExAC or related
Genetic diversity of NDUFV1-dependent mitochondrial complex I deficiency
sequence databases [16]. Our results demonstrate the value
of including genetically diverse populations in genomic
medicine research. Consequently, first-tier genetic screening
of c.1156C > T (p.(Arg386Cys)) may prove to have high
molecular diagnostic yield for South Asian children that
present with cardinal features of late-onset MCID.
In conclusion, our results provide a rationale for how
substitutions of the same amino acid residue can be associated with different ages of onset. We also demonstrate the
value of including ancestrally diverse population in genomic medicine and clinical research studies, to improve the
reliability of molecular diagnosis and reduce global health
disparities.
Acknowledgments We thank the patient and all the family members
for their participation. The study was supported by the grant from
National Institute of Health (1R21NS094047-01).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
1. Hoefs SJ, Skjeldal OH, Rodenburg RJ, et al. Novel mutations in
the NDUFS1 gene cause low residual activities in human complex
I deficiencies. Mol Genet Metab. 2010;100:251–6.
2. Alston CL, Compton AG, Formosa LE, et al. Biallelic mutations
in TMEM126B cause severe complex I deficiency with a variable
clinical phenotype. Am J Hum Genet. 2016;99:217–27.
3. Benit P, Beugnot R, Chretien D, et al. Mutant NDUFV2 subunit
of mitochondrial complex I causes early onset hypertrophic cardiomyopathy and encephalopathy. Hum Mutat. 2003;21:582–6.
4. Benit P, Slama A, Cartault F, et al. Mutant NDUFS3 subunit of
mitochondrial complex I causes Leigh syndrome. J Med Genet.
2004;41:14–7.
5. Kirby DM, Salemi R, Sugiana C, et al. NDUFS6 mutations are a
novel cause of lethal neonatal mitochondrial complex I deficiency.
J Clin Invest. 2004;114:837–45.
6. Loeffen J, Elpeleg O, Smeitink J, et al. Mutations in the complex I
NDUFS2 gene of patients with cardiomyopathy and encephalomyopathy. Ann Neurol. 2001;49:195–201.
7. Loeffen J, Smeitink J, Triepels R, et al. The first nuclear-encoded
complex I mutation in a patient with Leigh syndrome. Am J Hum
Genet. 1998;63:1598–608.
8. Schuelke M, Smeitink J, Mariman E, et al. Mutant
NDUFV1 subunit of mitochondrial complex I causes leukodystrophy and myoclonic epilepsy. Nat Genet. 1999;21:260–1.
1587
9. Triepels RH, van den Heuvel LP, Loeffen JL, et al. Leigh syndrome associated with a mutation in the NDUFS7 (PSST) nuclearencoded subunit of complex I. Ann Neurol. 1999;45:787–90.
10. van den Heuvel L, Ruitenbeek W, Smeets R, et al. Demonstration
of a new pathogenic mutation in human complex I deficiency: a 5bp duplication in the nuclear gene encoding the 18-kD (AQDQ)
subunit. Am J Hum Genet. 1998;62:262–8.
11. Bénit P, Chretien D, Kadhom N, et al. Large-scale deletion and
point mutations of the nuclear NDUFV1 and NDUFS1 genes in
mitochondrial complex I deficiency. Am J Hum Genet.
2001;68:1344–52.
12. Berger I, Hershkovitz E, Shaag A, Edvardson S, Saada A, Elpeleg
O. Mitochondrial complex I deficiency caused by a deleterious
NDUFA11 mutation. Ann Neurol. 2008;63:405–8.
13. Fernandez-Moreira D, Ugalde C, Smeets R, et al. X-linked
NDUFA1 gene mutations associated with mitochondrial encephalomyopathy. Ann Neurol. 2007;61:73–83.
14. Marin SE, Mesterman R, Robinson B, Rodenburg RJ, Smeitink J,
Tarnopolsky MA. Leigh syndrome associated with mitochondrial
complex I deficiency due to novel mutations In NDUFV1 and
NDUFS2. Gene. 2013;516:162–7.
15. Breningstall GN, Shoffner J, Patterson RJ. Siblings with leukoencephalopathy. Semin Pediatr Neurol. 2008;15:212–5.
16. Lek M, Karczewski KJ, Minikel EV, et al. Analysis of proteincoding genetic variation in 60,706 humans. Nature.
2016;536:285–91.
17. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic
Acids Res. 1988;16:1215.
18. Robinson DR, Wu YM, Vats P, et al. Activating ESR1 mutations
in hormone-resistant metastatic breast cancer. Nat Genet.
2013;45:1446–51.
19. Girisha KM, Shukla A, Trujillano D, et al. A homozygous nonsense variant in IFT52 is associated with a human skeletal ciliopathy. Clin Genet. 2016;90:536–9.
20. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.
Nucleic Acids Res. 2010;38:e164.
21. Vilain C, Rens C, Aeby A, et al. A novel NDUFV1 gene mutation
in complex I deficiency in consanguineous siblings with brainstem
lesions and Leigh syndrome. Clin Genet. 2012;82:264–70.
22. Baradaran R, Berrisford JM, Minhas GS, Sazanov LA. Crystal
structure of the entire respiratory complex I. Nature.
2013;494:443–8.
23. Sazanov LA, Hinchliffe P. Structure of the hydrophilic domain of
respiratory complex I from Thermus thermophilus. Science.
2006;311:1430–6.
24. Vinothkumar KR, Zhu J, Hirst J. Architecture of mammalian
respiratory complex I. Nature. 2014;515:80–4.
25. Varghese F, Atcheson E, Bridges HR, Hirst J. Characterization of
clinically identified mutations in NDUFV1, the flavin-binding
subunit of respiratory complex I, using a yeast model system.
Hum Mol Genet. 2015;24:6350–60.