Current affiliations:
Center for the Study of Rare Hereditary Diseases, Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy
Istitute of Clinical Biochemistry, Faculty of Medicine and Surgery, Catholic University of the Sacred Heart, Rome, Italy
Previous affiliations:
Department of Biomedical Sciencies, Faculty of Medicine and Surgery, University of Milan, Milan, Italy
IRCCS San Raffaele Scientific Institute, Milan, Italy
Department of Pharmacology, Faculty of Medicine and Surgery, University of Milan, Italy
Center for the Study of Rare Hereditary Diseases, Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy
Istitute of Clinical Biochemistry, Faculty of Medicine and Surgery, Catholic University of the Sacred Heart, Rome, Italy
Previous affiliations:
Department of Biomedical Sciencies, Faculty of Medicine and Surgery, University of Milan, Milan, Italy
IRCCS San Raffaele Scientific Institute, Milan, Italy
Department of Pharmacology, Faculty of Medicine and Surgery, University of Milan, Italy
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CLINICAL GENETICS of RARE HEREDITARY DISEASES by Roberto Colombo
Methods: DNA from three stone-formers in a Southampton family (UK) and two from an Italian family were analyzed independently by whole exome sequencing and selected variants were genotyped across all available members of both pedigrees. A disease variant of SLC25A25 (OMIM 608745), encoding the mitochondrial ATP-Mg/Pi carrier 3 (APC3) was identified, and analyzed structurally and functionally with respect to its calcium-regulated transport activity.
Results: All five patients had a heterozygous dominant SLC25A25 variant (rs140777921; GRCh37.p13: chr 9 130868670 G>C; p.Gln349His; Reference Sequence NM_001006641.3). Non-stone formers also carried the variant indicating incomplete penetrance. Modeling suggests that the variant lacks a conserved polar interaction, which may cause structural instability. Calcium-regulated ATP transport was reduced to ~20% of the wild type, showing a large reduction in function.
Conclusion: The transporter is important in regulating mitochondrial ATP production. This rare variant may increase urine lithogenicity through impaired provision of ATP for solute transport processes in the kidney, and/or for purinergic signaling. Variants found in other genes may compound this abnormality.
Methods: Here we describe a series of de novo missense variants in LMBRD2 in 10 unrelated individuals with overlapping features. Exome sequencing or genome sequencing was performed on all individuals, and the cohort was assembled through GeneMatcher.
Results: LMBRD2 encodes an evolutionary ancient and widely expressed transmembrane protein with no known disease association, although two paralogues are involved in developmental and metabolic disorders. Exome or genome sequencing revealed rare de novo LMBRD2 missense variants in 10 individuals with developmental delay, intellectual disability, thin corpus callosum, microcephaly and seizures. We identified five unique variants and two recurrent variants, c.1448G>A (p.Arg483His) in three cases and c.367T>C (p.Trp123Arg) in two cases. All variants are absent from population allele frequency databases, and most are predicted to be deleterious by multiple in silico damage-prediction algorithms.
Conclusion: These findings indicate that rare de novo variants in LMBRD2 can lead to a previously unrecognised early-onset neurodevelopmental disorder. Further investigation of individuals harbouring LMBRD2 variants may lead to a better understanding of the function of this ubiquitously expressed gene.
Methods: For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow.
Findings: We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222; excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism.
Interpretation: In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations.
METHODS: We performed exome, targeted capture, and Sanger sequencing of patients with undiagnosed developmental disorders, in multiple independent diagnostic or research centers. Phenotypic and mutational comparisons were facilitated through data exchange platforms. Whole-transcriptome sequencing was performed on RNA from patient- and control-derived fibroblasts.
RESULTS: We identified heterozygous missense variants in TRAF7 as the cause of a developmental delay-malformation syndrome in 45 patients. Major features include a recognizable facial gestalt (characterized in particular by blepharophimosis), short neck, pectus carinatum, digital deviations, and patent ductus arteriosus. Almost all variants occur in the WD40 repeats and most are recurrent. Several differentially expressed genes were identified in patient fibroblasts.
CONCLUSION: We provide the first large-scale analysis of the clinical and mutational spectrum associated with the TRAF7 developmental syndrome, and we shed light on its molecular etiology through transcriptome studies.
Here we report three novel families, compound heterozygous for missense variants in HARS2 identified by next-generation sequencing, namely c.172A>G (p.Lys58Glu) and c.448C>T (p.Arg150Cys) identified in two sisters aged 13 and 16 years and their older brother, c.448C>T (p.Arg150Cys) and c.980G>A (p.Arg327Gln) identified in a seven year old girl, and finally c.137T>A (p.Leu46Gln) and c.259C>T (p.Arg87Cys) identified in a 32 year old woman.
Clinically, all five individuals presented with early onset, rapidly progressive hearing impairment. Whereas the oldest female fulfilled the criteria of Perrault syndrome, the three younger females, aged 7, 13 and 16, all had apparently normal ovarian function, apart from irregular menstrual periods in the oldest female at age 16.
The present report expands the list of HARS2 variants and helps gain further knowledge to the phenotype.
Familial hypercholesterolemia (FH) is a primary hyperlipemia. It is an autosomal dominant genetic disorder of lipoproteins metabolism mainly caused by mutations in the low density lipoprotein receptor gene (LDLR). We aimed to investigate the functional impact on the low density lipoprotein receptor (LDLR) activity of six uncharacterised variants located in the coding region of the LDLR gene, namely c.428G > T, c.640T > C, c.1708C > T, c.1736A > T, c.1981C > G and c.2114C > G (NM_000527.4) and to attempt to define their clinical status.
METHODS:
Functional studies were carried out using site-directed mutagenesis techniques and expression of LDLR protein in vitro. Results were correlated with clinical data and in silico analyses in order to assess the physiopathological role of these variants.
RESULTS:
This work provides functional information about 6 uncharacterised mutations in LDLR.
CONCLUSIONS:
The six variants studied here appeared to affect the LDLR function in vitro to different degrees, ranging from receptors with normal to slightly reduced activity to receptors exhibiting less than 10% of the wild-type activity. According to these studies and The American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines, two variants could be classified as "Likely Benign" (p.(Ala705Gly) and p.(Leu570Phe)), three variants as "Pathogenic" (p.(Asp579Val), p.(Cys143Phe) and p.(Trp214Arg)) and one variant as "Likely Pathogenic" (p.(Pro661Ala)).
Methods: We performed whole-exome or targeted next-generation sequencing on autosomal dominant NAFLD patients.
Results: We report a heritable form of NAFLD and/or dyslipidemia due to monoallelic ABHD5 mutations, with complete clinical expression after the fourth decade of life, in seven unrelated multiplex families encompassing 39 affected subjects.
Conclusion: We associate a mendelian form of NAFLD and/or dyslipidemia with monoallelic ABHD5 mutations.
METHODS: We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype-phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs.
RESULTS: The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001).
CONCLUSION: We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients.
METHODS AND RESULTS: We present genetic, clinical and functional data in five individuals from four different families with homozygous or compound heterozygous BCAT2 mutations which were all detected following abnormal biochemical profile results or familial mutation segregation studies. We demonstrate that BCAT2 deficiency has a recognizable biochemical profile with raised plasma BCAAs and, in contrast with MSUD, low-normal branched-chain keto acids (BCKAs) with undetectable L-allo-isoleucine. Interestingly, unlike in MSUD, none of the individuals with BCAT2 deficiency developed acute encephalopathy even with exceptionally high BCAA levels. We observed wide-ranging clinical phenotypes in individuals with BCAT2 deficiency. While one adult was apparently asymptomatic, three individuals had presented with developmental delay and autistic features. We show that the biochemical characteristics of BCAT2 deficiency may be amenable to protein-restricted diet and that early treatment may improve outcome in affected individuals.
CONCLUSIONS: BCAT2 deficiency is an inborn error of BCAA catabolism. At present, it is unclear whether developmental delay and autism are parts of the variable phenotypic spectrum of this condition or coincidental. Further studies will be required to explore this.
Methods: Patients underwent a detailed ophthalmic evaluation, including funduscopic examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potentials (VEP). Genetic analysis was performed with a combination of whole exome sequencing (WES) and Sanger sequencing. Bioinformatic analysis of the WES results was performed via a customized pipeline. Pathogenicity of the identified intronic variant was evaluated in silico using the web tool Human Splicing Finder, and in vitro, using a minigene-based splicing assay. Linkage disequilibrium (LD) analysis was used to demonstrate a founder effect, and the decay of LD over generations around the mutation in Caucasus Jewish chromosomes was modeled to estimate the age of the most recent common ancestor.
Results: In eight patients with RP from six unrelated families, all of Caucasus Jewish ancestry, we identified a novel homozygous intronic variant, located at position −9 of PDE6B intron 15. The c.1921–9C>G variant was predicted to generate a novel acceptor splice site, nine bases upstream of the original splice site of intron 15. In vitro splicing assay demonstrated that this novel acceptor splice site is used instead of the wild-type site, leading to an 8-bp insertion into exon 16, which is predicted to cause a frameshift. The presence of a common ancestral haplotype in mutation-bearing chromosomes was compatible with a founder effect.
Conclusions: The PDE6B c.1921–9C>G intronic mutation is a founder mutation that accounts for at least 40% (6/15 families) of autosomal recessive RP among Caucasus Jews. This result is highly important for molecular diagnosis, carrier screening, and genetic counseling in this population.
METHODS: Clinical data from 60 cases with MYLK pathogenic variants were analyzed (five null and two missense variants), and the effect of missense variants on kinase activity was assessed.
RESULTS: Twenty-three individuals (39%) experienced an aortic event (defined as aneurysm repair or dissection); the majority of these events (87%) were aortic dissections. Aortic diameters were minimally enlarged at the time of dissection in many cases. Time-to-aortic-event curves showed that missense pathogenic variant (PV) carriers have earlier-onset aortic events than null PV carriers. An MYLK missense variant segregated with aortic disease over five generations but decreases MYLK kinase acitivity marginally. Functional Assays fail to identify all pathogenic variants in MYLK.
CONCLUSION: These data further define the aortic phenotype associated with MYLK pathogenic variants. Given minimal aortic enlargement before dissection, an alternative approach to guide the timing of aortic repair is proposed based on the probability of a dissection at a given age.
Multi centre study concerning the course of disease for each organ system, together with metabolic, neuroradiological and genetic findings.
67 individuals (39 previously unreported) from 59 families were included (age range 5 days - 33.4 years, median age 9 years). A total of 41 different SERAC1 variants were identified, including 20 that have not been reported before. With exception of two families with a milder phenotype, all affected individuals show a strikingly homogeneous phenotype and time course. Severe, reversible neonatal liver dysfunction and hypoglycemia was seen in more than 40% of all cases. Starting at a median age of six months muscular hypotonia (91%) was seen, followed by progressive spasticity (82%, median onset 15 months) and dystonia (82%, 18 months). The majority of affected individuals never learnt to walk (68%). 79% suffered hearing loss, 58% never learnt to speak, nearly all had significant intellectual disability (88%). MRI features were accordingly homogenous with bilateral basal ganglia involvement (98%), the characteristic "putaminal eye" was seen in 53%. The urinary marker 3-methylglutaconic aciduria is virtually present in all patients (98%). Supportive treatment focused on spasticity and drooling, was effective in individuals treated, hearing aids or cochlear implants did not improve communication skills.
MEGDHEL syndrome is as a progressive deafness-dystonia syndrome with frequent and reversible neonatal liver involvement and a strikingly homogenous course of disease.
Methods: DNA from three stone-formers in a Southampton family (UK) and two from an Italian family were analyzed independently by whole exome sequencing and selected variants were genotyped across all available members of both pedigrees. A disease variant of SLC25A25 (OMIM 608745), encoding the mitochondrial ATP-Mg/Pi carrier 3 (APC3) was identified, and analyzed structurally and functionally with respect to its calcium-regulated transport activity.
Results: All five patients had a heterozygous dominant SLC25A25 variant (rs140777921; GRCh37.p13: chr 9 130868670 G>C; p.Gln349His; Reference Sequence NM_001006641.3). Non-stone formers also carried the variant indicating incomplete penetrance. Modeling suggests that the variant lacks a conserved polar interaction, which may cause structural instability. Calcium-regulated ATP transport was reduced to ~20% of the wild type, showing a large reduction in function.
Conclusion: The transporter is important in regulating mitochondrial ATP production. This rare variant may increase urine lithogenicity through impaired provision of ATP for solute transport processes in the kidney, and/or for purinergic signaling. Variants found in other genes may compound this abnormality.
Methods: Here we describe a series of de novo missense variants in LMBRD2 in 10 unrelated individuals with overlapping features. Exome sequencing or genome sequencing was performed on all individuals, and the cohort was assembled through GeneMatcher.
Results: LMBRD2 encodes an evolutionary ancient and widely expressed transmembrane protein with no known disease association, although two paralogues are involved in developmental and metabolic disorders. Exome or genome sequencing revealed rare de novo LMBRD2 missense variants in 10 individuals with developmental delay, intellectual disability, thin corpus callosum, microcephaly and seizures. We identified five unique variants and two recurrent variants, c.1448G>A (p.Arg483His) in three cases and c.367T>C (p.Trp123Arg) in two cases. All variants are absent from population allele frequency databases, and most are predicted to be deleterious by multiple in silico damage-prediction algorithms.
Conclusion: These findings indicate that rare de novo variants in LMBRD2 can lead to a previously unrecognised early-onset neurodevelopmental disorder. Further investigation of individuals harbouring LMBRD2 variants may lead to a better understanding of the function of this ubiquitously expressed gene.
Methods: For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow.
Findings: We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222; excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism.
Interpretation: In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations.
METHODS: We performed exome, targeted capture, and Sanger sequencing of patients with undiagnosed developmental disorders, in multiple independent diagnostic or research centers. Phenotypic and mutational comparisons were facilitated through data exchange platforms. Whole-transcriptome sequencing was performed on RNA from patient- and control-derived fibroblasts.
RESULTS: We identified heterozygous missense variants in TRAF7 as the cause of a developmental delay-malformation syndrome in 45 patients. Major features include a recognizable facial gestalt (characterized in particular by blepharophimosis), short neck, pectus carinatum, digital deviations, and patent ductus arteriosus. Almost all variants occur in the WD40 repeats and most are recurrent. Several differentially expressed genes were identified in patient fibroblasts.
CONCLUSION: We provide the first large-scale analysis of the clinical and mutational spectrum associated with the TRAF7 developmental syndrome, and we shed light on its molecular etiology through transcriptome studies.
Here we report three novel families, compound heterozygous for missense variants in HARS2 identified by next-generation sequencing, namely c.172A>G (p.Lys58Glu) and c.448C>T (p.Arg150Cys) identified in two sisters aged 13 and 16 years and their older brother, c.448C>T (p.Arg150Cys) and c.980G>A (p.Arg327Gln) identified in a seven year old girl, and finally c.137T>A (p.Leu46Gln) and c.259C>T (p.Arg87Cys) identified in a 32 year old woman.
Clinically, all five individuals presented with early onset, rapidly progressive hearing impairment. Whereas the oldest female fulfilled the criteria of Perrault syndrome, the three younger females, aged 7, 13 and 16, all had apparently normal ovarian function, apart from irregular menstrual periods in the oldest female at age 16.
The present report expands the list of HARS2 variants and helps gain further knowledge to the phenotype.
Familial hypercholesterolemia (FH) is a primary hyperlipemia. It is an autosomal dominant genetic disorder of lipoproteins metabolism mainly caused by mutations in the low density lipoprotein receptor gene (LDLR). We aimed to investigate the functional impact on the low density lipoprotein receptor (LDLR) activity of six uncharacterised variants located in the coding region of the LDLR gene, namely c.428G > T, c.640T > C, c.1708C > T, c.1736A > T, c.1981C > G and c.2114C > G (NM_000527.4) and to attempt to define their clinical status.
METHODS:
Functional studies were carried out using site-directed mutagenesis techniques and expression of LDLR protein in vitro. Results were correlated with clinical data and in silico analyses in order to assess the physiopathological role of these variants.
RESULTS:
This work provides functional information about 6 uncharacterised mutations in LDLR.
CONCLUSIONS:
The six variants studied here appeared to affect the LDLR function in vitro to different degrees, ranging from receptors with normal to slightly reduced activity to receptors exhibiting less than 10% of the wild-type activity. According to these studies and The American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines, two variants could be classified as "Likely Benign" (p.(Ala705Gly) and p.(Leu570Phe)), three variants as "Pathogenic" (p.(Asp579Val), p.(Cys143Phe) and p.(Trp214Arg)) and one variant as "Likely Pathogenic" (p.(Pro661Ala)).
Methods: We performed whole-exome or targeted next-generation sequencing on autosomal dominant NAFLD patients.
Results: We report a heritable form of NAFLD and/or dyslipidemia due to monoallelic ABHD5 mutations, with complete clinical expression after the fourth decade of life, in seven unrelated multiplex families encompassing 39 affected subjects.
Conclusion: We associate a mendelian form of NAFLD and/or dyslipidemia with monoallelic ABHD5 mutations.
METHODS: We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype-phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs.
RESULTS: The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001).
CONCLUSION: We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients.
METHODS AND RESULTS: We present genetic, clinical and functional data in five individuals from four different families with homozygous or compound heterozygous BCAT2 mutations which were all detected following abnormal biochemical profile results or familial mutation segregation studies. We demonstrate that BCAT2 deficiency has a recognizable biochemical profile with raised plasma BCAAs and, in contrast with MSUD, low-normal branched-chain keto acids (BCKAs) with undetectable L-allo-isoleucine. Interestingly, unlike in MSUD, none of the individuals with BCAT2 deficiency developed acute encephalopathy even with exceptionally high BCAA levels. We observed wide-ranging clinical phenotypes in individuals with BCAT2 deficiency. While one adult was apparently asymptomatic, three individuals had presented with developmental delay and autistic features. We show that the biochemical characteristics of BCAT2 deficiency may be amenable to protein-restricted diet and that early treatment may improve outcome in affected individuals.
CONCLUSIONS: BCAT2 deficiency is an inborn error of BCAA catabolism. At present, it is unclear whether developmental delay and autism are parts of the variable phenotypic spectrum of this condition or coincidental. Further studies will be required to explore this.
Methods: Patients underwent a detailed ophthalmic evaluation, including funduscopic examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potentials (VEP). Genetic analysis was performed with a combination of whole exome sequencing (WES) and Sanger sequencing. Bioinformatic analysis of the WES results was performed via a customized pipeline. Pathogenicity of the identified intronic variant was evaluated in silico using the web tool Human Splicing Finder, and in vitro, using a minigene-based splicing assay. Linkage disequilibrium (LD) analysis was used to demonstrate a founder effect, and the decay of LD over generations around the mutation in Caucasus Jewish chromosomes was modeled to estimate the age of the most recent common ancestor.
Results: In eight patients with RP from six unrelated families, all of Caucasus Jewish ancestry, we identified a novel homozygous intronic variant, located at position −9 of PDE6B intron 15. The c.1921–9C>G variant was predicted to generate a novel acceptor splice site, nine bases upstream of the original splice site of intron 15. In vitro splicing assay demonstrated that this novel acceptor splice site is used instead of the wild-type site, leading to an 8-bp insertion into exon 16, which is predicted to cause a frameshift. The presence of a common ancestral haplotype in mutation-bearing chromosomes was compatible with a founder effect.
Conclusions: The PDE6B c.1921–9C>G intronic mutation is a founder mutation that accounts for at least 40% (6/15 families) of autosomal recessive RP among Caucasus Jews. This result is highly important for molecular diagnosis, carrier screening, and genetic counseling in this population.
METHODS: Clinical data from 60 cases with MYLK pathogenic variants were analyzed (five null and two missense variants), and the effect of missense variants on kinase activity was assessed.
RESULTS: Twenty-three individuals (39%) experienced an aortic event (defined as aneurysm repair or dissection); the majority of these events (87%) were aortic dissections. Aortic diameters were minimally enlarged at the time of dissection in many cases. Time-to-aortic-event curves showed that missense pathogenic variant (PV) carriers have earlier-onset aortic events than null PV carriers. An MYLK missense variant segregated with aortic disease over five generations but decreases MYLK kinase acitivity marginally. Functional Assays fail to identify all pathogenic variants in MYLK.
CONCLUSION: These data further define the aortic phenotype associated with MYLK pathogenic variants. Given minimal aortic enlargement before dissection, an alternative approach to guide the timing of aortic repair is proposed based on the probability of a dissection at a given age.
Multi centre study concerning the course of disease for each organ system, together with metabolic, neuroradiological and genetic findings.
67 individuals (39 previously unreported) from 59 families were included (age range 5 days - 33.4 years, median age 9 years). A total of 41 different SERAC1 variants were identified, including 20 that have not been reported before. With exception of two families with a milder phenotype, all affected individuals show a strikingly homogeneous phenotype and time course. Severe, reversible neonatal liver dysfunction and hypoglycemia was seen in more than 40% of all cases. Starting at a median age of six months muscular hypotonia (91%) was seen, followed by progressive spasticity (82%, median onset 15 months) and dystonia (82%, 18 months). The majority of affected individuals never learnt to walk (68%). 79% suffered hearing loss, 58% never learnt to speak, nearly all had significant intellectual disability (88%). MRI features were accordingly homogenous with bilateral basal ganglia involvement (98%), the characteristic "putaminal eye" was seen in 53%. The urinary marker 3-methylglutaconic aciduria is virtually present in all patients (98%). Supportive treatment focused on spasticity and drooling, was effective in individuals treated, hearing aids or cochlear implants did not improve communication skills.
MEGDHEL syndrome is as a progressive deafness-dystonia syndrome with frequent and reversible neonatal liver involvement and a strikingly homogenous course of disease.
OBJECTIVE: To characterize the contribution of known disease genes to POI and identify molecular etiologies and biological underpinnings of POI.
DESIGN, SETTING, AND PARTICIPANTS: We applied exome sequencing (ES) and family-based genomics to 42 affected female individuals from 36 unrelated Turkish families, including 31 with reported parental consanguinity.
RESULTS: This analysis identified likely damaging, potentially contributing variants and molecular diagnoses in 16 families (44%), including 11 families with likely damaging variants in known genes and five families with predicted deleterious variants in disease genes (IGSF10, MND1, MRPS22, and SOHLH1) not previously associated with POI. Of the 16 families, 2 (13%) had evidence for potentially pathogenic variants at more than one locus. Absence of heterozygosity consistent with identity-by-descent mediated recessive disease burden contributes to molecular diagnosis in 15 of 16 (94%) families. GeneMatcher allowed identification of additional families from diverse genetic backgrounds.
CONCLUSIONS: ES analysis of a POI cohort further characterized locus heterogeneity, reaffirmed the association of genes integral to meiotic recombination, demonstrated the likely contribution of genes involved in hypothalamic development, and documented multilocus pathogenic variation suggesting the potential for oligogenic inheritance contributing to the development of POI.
AIM: To investigate the role of the ABCB4 gene in Italian women with intrahepatic cholestasis of pregnancy and raised gamma-glutamyl transpeptidase by, analyzing the complete coding sequence and mRNA splicing products.
METHODS: Among 299 women with intrahepatic cholestasis of pregnancy, 10 showing raised gamma-glutamyl transpeptidase were enrolled in this study. DNA and RNA were extracted from peripheral blood mononuclear cells using standard procedures. The 27 coding exons and the promoter region were amplified by polymerase chain reaction and analyzed by sequencing. Reverse transcript-polymerase chain reaction analysis of ABCB4 mRNA and cDNA analysis were also performed.
RESULTS: A novel splicing mutation that causes a truncated protein of 249 amino acid was identified in a woman who had the highest serum levels of gamma-glutamyl transpeptidase, alkaline phosphatase, bile acids, and the highest pruritus score. We identified also one already described p.R590Q mutation in a woman who had significantly higher serum levels of alkaline phosphatase, aspartate, and alanine aminotransferase.
CONCLUSIONS: Our study demonstrates that splicing mutations in the ABCB4 gene can cause ICP in women with high gamma-glutamyl transpeptidase and thus a complete analysis of coding sequence and cDNA products is required.
The 1278insTATC is the most prevalent beta-hexosaminidase A ( HEXA) gene mutation causing Tay-Sachs disease (TSD), one of the four lysosomal storage diseases (LSDs) occurring at elevated frequencies among Ashkenazi Jews (AJs). To investigate the genetic history of this mutation in the AJ population, a conserved haplotype (D15S981:175-D15S131:240-D15S1050:284-D15S197:144-D15S188:418) was identified in 1278insTATC chromosomes from 55 unrelated AJ individuals (15 homozygotes and 40 heterozygotes for the TSD mutation), suggesting the occurrence of a common founder. When two methods were used for analysis of linkage disequilibrium (LD) between flanking polymorphic markers and the disease locus and for the study of the decay of LD over time, the estimated age of the insertion was found to be 40+/-12 generations (95% confidence interval: 30-50 generations), so that the most recent common ancestor of the mutation-bearing chromosomes would date to the 8th-9th century. This corresponds with the demographic expansion of AJs in central Europe, following the founding of the Ashkenaz settlement in the early Middle Ages. The results are consistent with the geographic distribution of the main TSD mutation, 1278insTATC being more common in central Europe, and with the coalescent times of mutations causing two other LSDs, Gaucher disease and mucolipidosis type IV. Evidence for the absence of a determinant positive selection (heterozygote advantage) over the mutation is provided by a comparison between the estimated age of 1278insTATC and the probability of the current AJ frequency of the mutant allele as a function of its age, calculated by use of a branching-process model. Therefore, the founder effect in a rapidly expanding population arising from a bottleneck provides a robust parsimonious hypothesis explaining the spread of 1278insTATC-linked TSD in AJ individuals.
Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive disorder with a high prevalence in the Japanese population, is characterised by severe muscular dystrophy associated with brain malformation (cortical dysgenesis) and mental retardation. In Japan, 87% of FCMD-bearing chromosomes carry a 3-kb retrotransposal insertion of tandemly repeated sequences within the disease gene recently identified on chromosome 9q31, and most of them share a common founder haplotype. FCMD is the first human disease known to be caused primarily by an ancient retrotransposal integration. By applying two methods for the study of linkage disequilibrium between flanking polymorphic markers and the disease locus, and of its decay over time, the age of the insertion mutation causing FCMD in Japanese patients is calculated to be approximately 102 generations (95% confidence interval: 86-117 g), or slightly less. The estimated age dates the most recent common ancestor of the mutation-bearing chromosomes back to the time (or a few centuries before) the Yayoi people started migrating to Japan from the Korean peninsula. This finding makes the molecular population genetics of FCMD understandable in the context of Japan's history and the founder effect consistent with the prevalent theory on the origins of the modern Japanese population.
Cystinuria is an autosomal recessive disorder of the transepithelial transport of amino acids, clinically manifested by the development of kidney stones. Mutations in the gene encoding rBAT (SLC3A1, on chromosome 2p16.3) are linked to type I cystinuria, while the SLC7A9 locus (19q13.1), expressing b0,+ AT protein, is involved in non-type I cystinuria, which is very common among Libyan Jews. Applying two methods for linkage disequilibrium analysis to haplotype data spanning six 19q12-q13.1 polymorphic markers, and relying on the physical distances between the markers and the recently mapped SLC7A9 (CSNU3) locus, the age of the founder missense V170M mutation causing non-type I cystinuria in Jews of Libyan ancestry is calculated to be approximately 14 to 15 generations (g) (95% confidence interval: 9-20 g) or slightly more. The estimated age dates the most recent common ancestor of the mutation-bearing chromosomes back to the time (or some decades before) Jewish families settled in Libya following their expulsion from the Iberian Peninsula. This finding makes the molecular population genetics of cystinuria understandable in the context of the Libyan Jews' history.
Methods: A literature search was performed on PubMed and Google Scholar databases in order to retrieve data regarding Antidepressants effects on specific RDoC constructs. Further, the targets of drugs of interest were identified through Drugbank database, and their possible function within RDoC constructs was discussed.
Discussion: In this review we summarize and discuss the significance of the results of pre-clinical and clinical studies investigating specific RDoC paradigms relevant to depressive phenotypes and antidepressant effects.
Recent evidence suggests that psychiatric symptoms share a common genetic liability across diagnostic categories. The present study investigated the effects of variants within previously identified relevant genes on specific symptom clusters, independently from the diagnosis.
Methods
1550 subjects affected by Schizophrenia (SCZ), Major Depressive Disorder or Bipolar Disorder were included. Symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS) and the Hamilton Depression Rating Scale (HDRS). Principal component analysis and a further clinical refinement were used to define symptom clusters. Clusters scores were tested for association with 46 genetic variants within nine genes previously linked to one or more major psychiatric disorders by large genome wide association studies (ANK3, CACNA1C, CACNB2, FKBP5, FZD3, GRM7, ITIH3, SYNE1, TCF4). Exploratory analyses were performed in each disorder separately to further elucidate the SNPs effects.
Results
Five PANSS clusters (Negative; Impulsiveness; Cognitive; Psychotic; Depressive) and four HDRS clusters (Core Depressive; Somatic; Psychotic-like; Insomnia) were identified. CACNA1C rs11615998 was associated with HDRS Psychotic cluster in the whole sample. In the SCZ sample, CACNA1C rs11062296 was associated with PANSS Impulsiveness cluster and CACNA1C rs2238062 was associated with PANSS negative cluster.
Discussion
CACNA1C rs11615998 was associated with psychotic symptoms (C-allele carriers have decreased psychotic-risk) independently from the diagnosis, in line with the evidence of a cross disorder effect of many risk variants. This gene was previously associated with SCZ and cross-disorder liability to psychiatric disorders. Our findings confirmed that deep phenotyping is pivotal to clarify the role of genetic variants on symptoms patterns.
Methods: We used two samples of different ethnic origin: a Korean sample, composed by 242 patients diagnosed with Major Depression and 132 patients diagnosed with Bipolar Disorder and 326 healthy controls; an Italian sample composed 151 Major Depression subjects, 189 Bipolar Disorder subjects and 38 outpatients diagnosed for a primary Anxiety disorder.
Results: Our analyses reported an association of rs1344706 with psychotic phenotype in the Cross-diagnostic pooled sample (geno p=4.15x10-4, allelic p=1.06x10-4). In the cross-diagnosis Italian sample but not in the Korean one, rs7597593 was involved with depressive symptoms improvement after treatment (geno p=0.025, allelic p=0.007).
Conclusion: The present study evidenced the role of ZNF804A alterations in symptoms improvement after treatment. Both manic and depressive symptoms seem to be modulated by ZNF804A, though the latter was observed in the Bipolar pooled sample only. The role of this factor is likely related to synaptic development and maintenance, however further analyses will be needed to better understand the molecular mechanics involved with ZNF804A.
OBJECTIVE: Given the need of refinement of the findings obtained in large but poorly phenotyped samples, this study investigated the possible role of variants within FKBP5 in a small but deeply phenotyped BPD sample.
METHODS: A sample (N = 131) of bipolar patients were investigated with 10 polymorphisms within the FKBP5 gene. A control sample (N = 65) was also used for the analyses. Treatment response and remission of symptoms were evaluated using of the Hamilton Depression Rating Scale (HDRS), Hamilton Anxiety Rating Scale (HARS), and Young Mania Rating Scale (YMRS). The same analyses were also performed on the depressive subsample of BPD (D.BPD).
RESULTS: rs3800373 was associated with disorder risk in the depressive BPD subsample with the G allele being more frequent in subjects with a D.BPD phenotype. This was the only association that survived statistical correction.
CONCLUSIONS: rs3800373 FKBP5 may increase the risk of developing predominantly depressed BPD, probably through the creation of an enhancer consensus sequence in the 3'UTR of the gene, thus potentially increasing its expression. This finding seems to be partially supported by literature data, which evidenced increased levels of FKBP5 in psychiatric subjects.
METHODS: A total of 114 depressed outpatients (HDRS > 13) were included. "Pure" depressed (D, n = 76) were divided from "mixed" depressed (Mx, n = 38) by the number of concomitant manic symptoms. All patients were evaluated by the Hamilton Depression Rating Scale (HDRS), the Hamilton Anxiety Rating Scale (HARS), the Young Mania Rating Scale (YMRS), the Global Assessment of Functioning (GAF), the Social Adjustment Self-reported Scale (SASS) and the Quality of Life Scale (QoL), at baseline and after 1, 3, 6 and 12 months of treatment.
RESULTS: Mx patients were significantly younger at the onset of BD. Manic features persisted significantly higher in Mx than in D patients all over the follow-up period. Axis I comorbidities had a negative impact on the course of social functioning over the medium term period, while Mx patients showed a faster improvement in social adjustment than "pure" depressed patients.
CONCLUSIONS: Mixed features may persist relatively stable throughout a depressive episode, having a negative impact over clinical and functional outcome, but not on social adjustment.
Methods - 90 depressed patients (Group 1, HDRS: 18.2±4.6, YMRS: 1.4±2.9) and 41 patients presenting a mixed subthreshold symptomathology (Group 2, HDRS: 9.1±3.2, YMRS: 9.6±6.4) were included in the study. All patients underwent structured diagnostic interviews for axis I and axis II disorders (SCID-I, SCID-II) and have been submitted to psychometric assessment with Hamilton Depression Rating Scale (HDRS), Hamilton Anxiety Rating Scale (HARS), Young Mania Rating Scale (YMRS), Global Assessment Scale (GAS), Social Adjustment Self-reported Scale (SASS), Quality of Life Scale (QoL), at baseline and repeated follow-up (1, 3, 6, 12 months). Personality dimensions were evaluated by Temperament and Character Inventory (TCI-R).
Results - The most important result is the slight increase in depressive symptoms in subthreshold mixed patients after 3 months of treatment. These patients were more likely diagnosed as BP-I, were non-SUD patients and/or non-comorbid for axis II personality disorders and had a shorter duration of current episode prior intake. Measures of social and functional adjustment were not different in the two groups, though a slight trend for depressed patients to report a poorer quality of life. No difference in personality traits were observed among the two samples.
Conclusions - Our study confirms the importance of considering subthreshold symptoms in the evaluation and treatment of patients with Bipolar Disorder.
BACKGROUND: Mixed mood states, even in their sub-threshold forms, may significantly affect the course and outcome of bipolar disorder (BD).
AIM: To compare two samples of BD patients presenting a major depressive episode and a sub-threshold mixed state in terms of global functioning, clinical outcome, social adjustment and quality of life during a 1-year follow-up.
METHODS: The sample was composed by 90 subjects (Group 1, D) clinically diagnosed with a major depressive episode and 41 patients (Group 2, Mx) for a sub-threshold mixed state. All patients were administered with a pharmacological treatment and evaluated for depressive, anxious and manic symptoms by common rating scales. Further evaluations included a global assessment of severity and functioning, social adjustment and quality of life. All evaluations were performed at baseline and after 1, 3, 6 and 12 months of treatment.
RESULTS: The two groups were no different for baseline as well as improvement in global severity and functioning. Though clearly different for symptoms severity, the amount of change of depressive and anxiety symptoms was also no different. Manic symptoms showed instead a trend to persist over time in group 2, whereas a slight increase of manic symptoms was observed in group 1, especially after 6 months of treatment. Moreover, in group 1, some manic symptoms were also detected at the Young Mania Rating Scale (n = 24, 26.6%). Finally, improvement in quality of life and social adjustment was similar in the two groups, though a small trend toward a faster improvement in social adjustment in group 1.
CONCLUSIONS: Sub-threshold mixed states have a substantial impact on global functioning, social adjustment and subjective well-being, similarly to that of acute phases, or at least major depression. In particular, mixed features, even in their sub-threshold forms, tend to be persistent over time. Finally, manic symptoms may be still often underestimated in depressive episodes, even in patients for BD.
INTRODUCTION: Bipolar disorder patients (BP) with comorbid Substance Use Disorder (SUD) may present clinical features that could compromise adherence and response to pharmacological treatment. The purpose of this study was to examine clinical and psychopathological features of BP with and without comorbid SUD in a real-world setting.
METHODS: The sample was composed by 131 affective patients. Sixty-five patients were affected by Bipolar Disorder I (BP-I, 49.2%), 29 by Bipolar Disorder II (BP-II, 22.3%) and 37 by Cyclothymic Disorder (CtD, 28.5%), according to DSM-IV. Sixty-six patients were diagnosed for a comorbid SUD. All patients have been submitted to psychometric assessment with Hamilton Depression Rating Scale (HDRS), Hamilton Anxiety Rating Scale (HARS), Young Mania Rating Scale (YMRS), Global Assessment Scale (GAS), Social Adjustment Self-reported Scale (SASS), Quality of Life Scale (QoL), at baseline and repeated follow-up periods (1, 3, 6, 12 months).
RESULTS: BP comorbid for SUD were more likely diagnosed as BP-II and CtD and were less likely to present a moderate-severe manic symptomatology. Furthermore, personality disorders were more frequent in SUD patients than in non-comorbid BP. BP with SUD were not different for primary outcome measure (HDRS, HARS, YMRS, GAS) from non-comorbid BP; however, BP with SUD were significantly more impaired in social functioning (SASS) at any stage of the follow-up and poor functioning increased the risk of relapse in substance use during treatment. Finally, SUD comorbidity did not represent a risk factor for treatment drop-out, while in our sample young age, low treatment dosage and BP-I diagnosis were significantly associated with drop-out.
DISCUSSION: The primary finding of this work is that BP with comorbid SUD are significantly more compromised in social functioning. Second, these patients were less likely to be diagnosed for BP-I and to present a severe manic symptomatology. Finally, we found that the diagnosis of SUD, but young age, low treatment dosage and BP-I diagnosis to be risk factors for treatment drop-out. Physicians should be alert to these differences in their clinical practice.
Administration of L-kynurenine and some analogous molecules into the right lateral ventricle caused convulsive attacks in rats. These convulsions are tonic-clonic. Various modifications of the structure of L-kynurenine affected the production of convulsions. In particular, methylation of the carboxyl group increased the potency. Methylation of the carboxyl function and hydroxylation of the benzene ring greatly increased the latency and the duration of the convulsions. Removal of the carboxyl function or its reduction resulted in the loss of the epileptogenic effect. Masking of the amino function with formyl or acetyl residues also produced inactive compounds. The results of structural modification of the kynurenine carbon skeleton clearly show that the structure essential for stimulation of convulsive activity includes a free amino group and a free or masked carboxyl function. The molecular structure able to induce convulsive effects appears to be the laevo -isomer of kynurenine, since the dextro-form has been proved to be inactive. The convulsant activity, evaluated as the ED50, for L-kynurenine, is between that of bicuculline and pentamethylenetetrazole . The convulsant mechanism of kynurenine is unknown, but might very possibly involve interference with the activity of certain inhibitory neurotransmitters.
Many compounds deriving from the esterification of p-sulfamoyl benzoic acid, a known inhibitor of carbonic anhydrase, and with structures possessing probable acetylcholinemimetic or cholinelithic effects, have been synthesized and studied. The effects of such compounds on carbonic anhydrase and on gastric acid secretion have been investigated. All the sulfamoyl compounds display a certain degree of carbonic anhydrase inhibition. The type of substituent in the ester group determines whether the compound is inactive, stimulant or inhibiting towards gastric acid secretion. The different way in which the compounds affect the secretion of gastric acid is discussed.
4-(p-sulfamoylbenzoyl)-N-methyl-piperidine and similar molecules, deprived of the sulfonamide function and the tertiary amino group, were prepared. A study was made of the effects that these molecules had on carbonic anhydrase and on basal gastric acid secretion. The compounds with a sulfamoyl group display a certain degree of inhibitory power towards carbonic anhydrase, but only 4-(p-sulfamoylbenzoyl)-N-methyl-piperidine is able to lower basal gastric acid secretion; the similar compounds, deprived of sulfamoyl or tertiary amino groups are ineffective as far as gastric acid secretion is concerned. 4-(p-sulfamoylbenzoyl)-N-methyl-piperidine is capable of lowering gastric acid secretion more than acetazolamide. It is unable to antagonize the contraction of the nictitating membrane and the hypotensive effect of acetylcholine in the cat. The probable mechanism of action of the active compound on gastric acid secretion is discussed.
Two simple methods of differentiating π- and τ-derivatised histidines unambiguously are described. The first involves conversion into one of the two known im-methyl-L-histidines, which, to avoid all possible confusion arising from ambiguous nomenclature and previous work, have been correlated with N(α)-t-butoxycarbonyl-N(π)-benzyloxymethyl-L-histidine, whose structure has been established by X-ray crystallography. The second method, which is appropriate for im-substituents of type RCH2–, involves the measurement of nuclear Overhauser effects. If the substituent is at the π-position, the CH2 signal is enhanced if the low-field adjacent proton between the heterocyclic nitrogens is irradiated, but not if the high-field, more distant, ring proton is irradiated. If the substituent is at the τ-position, the CH, signal is enhanced whichever of the equidistant ring protons is irradiated.
Two functionalised supports for the solid-phase synthesis of peptides under mild reaction conditions were prepared: 4-chloromethylphenoxyacetamidomethyl-copoly (styrene-1%-divinylbenzene) and 4-chloromethylphenoxyacetyl-norleucyl-poly (dimethylacrylamide). They were devised in order to avoid the danger of racemization which exists during base-catalyzed esterification of the first protected amino acid to the 4-alkoxybenzyl alcohol resins formerly employed in combination with N alpha-9-fluorenylmethoxycarbonyl and tert.-butyl side-chain protecting groups. Esterification of N alpha-protected amino acids to the new resins can be achieved easily and without significant levels of racemization by means of their caesium salts, while cleavage from the supports is possible by treatment with trifluoroacetic acid. The 4-chloromethylphenoxyacetyl polystyrene resin was tested by the synthesis of Leu-enkephalin which was cleaved, at the end of the synthesis, from the solid support in 91% yield by 60% trifluoroacetic acid in methylene chloride, and was shown to be more than 99% pure by ion-exchange chromatography and reverse phase high pressure liquid chromatography.