Biallelic variants of ATP13A3 cause dose-dependent childhood-onset pulmonary arterial hypertension characterised by extreme morbidity and mortality.

BACKGROUND: The molecular genetic basis of pulmonary arterial hypertension (PAH) is heterogeneous, with at least 26 genes displaying putative evidence for disease causality. Heterozygous variants in the ATP13A3 gene were recently identified as a new cause of adult-onset PAH. However, the contribution of ATP13A3 risk alleles to child-onset PAH remains largely unexplored. METHODS AND RESULTS: We report three families with a novel, autosomal recessive form of childhood-onset PAH due to biallelic ATP13A3 variants. Disease onset ranged from birth to 2.5 years and was characterised by high mortality. Using genome sequencing of parent-offspring trios, we identified a homozygous missense variant in one case, which was subsequently confirmed to cosegregate with disease in an affected sibling. Independently, compound heterozygous variants in ATP13A3 were identified in two affected siblings and in an unrelated third family. The variants included three loss of function variants (two frameshift, one nonsense) and two highly conserved missense substitutions located in the catalytic phosphorylation domain. The children were largely refractory to treatment and four died in early childhood. All parents were heterozygous for the variants and asymptomatic. CONCLUSION: Our findings support biallelic predicted deleterious ATP13A3 variants in autosomal recessive, childhood-onset PAH, indicating likely semidominant dose-dependent inheritance for this gene.

Biallelic Loss-of-Function Variants in BICD1 Are Associated with Peripheral Neuropathy and Hearing Loss.

Hearing loss and peripheral neuropathy are two clinical entities that are genetically and phenotypically heterogeneous and sometimes co-occurring. Using exome sequencing and targeted segregation analysis, we investigated the genetic etiology of peripheral neuropathy and hearing loss in a large Ashkenazi Jewish family. Moreover, we assessed the production of the candidate protein via western blotting of lysates from fibroblasts from an affected individual and an unaffected control. Pathogenic variants in known disease genes associated with hearing loss and peripheral neuropathy were excluded. A homozygous frameshift variant in the BICD1 gene, c.1683dup (p.(Arg562Thrfs*18)), was identified in the proband and segregated with hearing loss and peripheral neuropathy in the family. The BIDC1 RNA analysis from patient fibroblasts showed a modest reduction in gene transcripts compared to the controls. In contrast, protein could not be detected in fibroblasts from a homozygous c.1683dup individual, whereas BICD1 was detected in an unaffected individual. Our findings indicate that bi-allelic loss-of-function variants in BICD1 are associated with hearing loss and peripheral neuropathy. Definitive evidence that bi-allelic loss-of-function variants in BICD1 cause peripheral neuropathy and hearing loss will require the identification of other families and individuals with similar variants with the same phenotype.

Expanding the phenotypic spectrum of COLEC10-Related 3MC syndrome: A glimpse into COLEC10-Related 3MC syndrome in the Ashkenazi Jewish population.

Bi-allelic variants in COLEC11 and MASP1 have been associated with 3MC syndrome, a clinical entity made of up four rare autosomal recessive disorders: Carnevale, Mingarelli, Malpuech, and Michels syndromes, characterized by variable expression of facial dysmorphia, cleft lip/palate, postnatal growth deficiency, hearing loss, cognitive impairment, craniosynostosis, radioulnar synostosis, and genital and vesicorenal anomalies. More recently, bi-allelic variants in COLEC10 have been described to be associated with 3MC syndrome. Syndromic features seen in 3MC syndrome are thought to be due to disruption of the chemoattractant properties that influence neural crest cell migration. We identified nine individuals from five families of Ashkenazi Jewish descent with homozygosity of the c.311G > T (p.Gly104Val) variant in COLEC10 and phenotype consistent with 3MC syndrome. Carrier frequency was calculated among 52,278 individuals of Jewish descent. Testing revealed 400 carriers out of 39,750 individuals of Ashkenazi Jewish descent, giving a carrier frequency of 1 in 99 or 1.01%. Molecular protein modeling suggested that the p.Gly104Val substitution alters local conformation. The c.311G > T (p.Gly104Val) variant likely represents a founder variant, and homozygosity is associated with features of 3MC syndrome. 3MC syndrome should be in the differential diagnosis for individuals with short stature, radioulnar synostosis, cleft lip and cleft palate.

Utilizing ethnic-specific differences in minor allele frequency to recategorize reported pathogenic deafness variants.

Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) > 0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness.

Splice site, frameshift, and chimeric GFAP mutations in Alexander disease.

Alexander disease (AxD) is a usually fatal astrogliopathy primarily caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed in astrocytes. We describe three patients with unique characteristics, and whose mutations have implications for AxD diagnosis and studies of intermediate filaments. Patient 1 is the first reported case with a noncoding mutation. The patient has a splice site change producing an in-frame deletion of exon 4 in about 10% of the transcripts. Patient 2 has an insertion and deletion at the extreme end of the coding region, resulting in a short frameshift. In addition, the mutation was found in buccal DNA but not in blood DNA, making this patient the first reported chimera. Patient 3 has a single-base deletion near the C-terminal end of the protein, producing a short frameshift. These findings recommend inclusion of intronic splice site regions in genetic testing for AxD, indicate that alteration of only a small fraction of GFAP can produce disease, and provide caution against tagging intermediate filaments at their C-terminal end for cell biological investigations.

A founder mutation in the MPL gene causes congenital amegakaryocytic thrombocytopenia (CAMT) in the Ashkenazi Jewish population.

Congenital amegakaryocytic thrombocytopenia (MIM #604498) (CAMT) is a rare inherited disease presenting as severe thrombocytopenia in infancy. Untreated, many CAMT patients develop aplastic anemia within the first decade of life; the only effective treatment of CAMT is bone marrow transplantation. CAMT is the result of the presence of homozygous or compound heterozygous mutations in the thrombopoietin receptor-encoding gene, MPL. We report here the identification and characterization of a founder mutation in MPL in the Ashkenazi Jewish (AJ) population. This mutation, termed c.79+2T>A, is a T to A transversion in the invariant second base of the intron 1 donor splice site. Analysis of a random sample of 2018 individuals of AJ descent revealed a carrier frequency of approximately 1 in 75. Genotyping of six loci adjacent to the MPL gene in the proband and in the 27 individuals identified as carriers of the c.79+2T>A mutation revealed that the presence of this mutation in the AJ population is due to a single founder. The observed carrier frequency predicts an incidence of CAMT in the AJ population of approximately 1 in 22,500 pregnancies. The identification of this mutation will enable population carrier testing and will facilitate the identification and treatment of individuals homozygous for this mutation.

ABCC8 mutation allele frequency in the Ashkenazi Jewish population and risk of focal hyperinsulinemic hypoglycemia.

PURPOSE: Congenital hyperinsulinism of infancy (OMIM# 256450) is a devastating disease most commonly caused by dominant or recessive mutations in either ABCC8 or KCNJ11, the genes that encode for the ß-cell adenosine triphosphate-regulated potassium channel. A unique combination of a paternally inherited germline mutation and somatic loss-of-heterozygosity causes the focal form of the disease (Focal-congenital hyperinsulinism of infancy [Focal-CHI]), the incidence of which in genetically susceptible individuals is not known. METHODS: We genotyped 21,122 Ashkenazi Jewish individuals for two previously identified ABCC8 founder mutations and utilized a clinical database of 61 unrelated Ashkenazi patients with congenital hyperinsulinism of infancy to obtain an estimate of the risk of Focal-CHI in a genetically susceptible fetus. RESULTS: The combined mutation carrier rate in Ashkenazi Jews was 1:52, giving an estimated frequency of homozygosity or compound heterozygosity of 1:10,816 in this population. The risk of Focal-CHI is 1:540 per pregnancy in offspring of carrier fathers. CONCLUSION: We recommend that these mutations be included in the genetic screening program for the Ashkenazi Jewish population. As the risk of Focal-CHI is not expected to be mutation specific, the data reported in this study are useful for counseling all families in which the father was found to carry a recessive ABCC8 or KCNJ11 mutation.

Identification of Novel and Recurrent Variants in MYO15A in Ashkenazi Jewish Patients With Autosomal Recessive Nonsyndromic Hearing Loss.

Hearing loss is a genetically and phenotypically heterogeneous disorder. The purpose of this study was to determine the genetic cause underlying hearing loss in four Ashkenazi Jewish families. We screened probands from each family using a combination of targeted mutation screening and exome sequencing to identifiy the genetic cause of hearing loss in each family. We identified four variants in MYO15A, two novel variants never previously linked to deafness (c.7212+5G>A and p.Leu2532ArgfsTer37) and two recurrent variants (p.Tyr2684His and p.Gly3287Gly). One family showed locus heterogeneity, segregrating two genetic forms of hearing loss. Mini-gene assays revealed the c.7212+5G>A variant results in abnormal splicing and is most likely a null allele. We show that families segregrating the p.Gly3287Gly variant show both inter and intra-familial phenotypic differences. These results add to the list of MYO15A deafness-causing variants, further confirm the pathogenicity of the p.Gly3287Gly variant and shed further light on the genetic etiology of hearing loss in the Ashkenazi Jewish population.

Recommendation of premarital genetic screening in the Syrian Jewish community based on mutation carrier frequencies within Syrian Jewish cohorts.

BACKGROUND: There is a paucity of information available regarding the carrier frequency for autosomal recessive pathogenic variants among Syrian Jews. This report provides data to support carrier screening for a group of autosomal recessive conditions among Syrian Jews based on the population frequency of 40 different pathogenic variants in a cohort of over 3800 individuals with Syrian Jewish ancestry. METHODS: High throughput PCR amplicon sequencing was used to genotype 40 disease-causing variants in 3840 and 5279 individuals of Syrian and Iranian Jewish ancestry, respectively. These data were compared with Ashkenazi Jewish carrier frequencies for the same variants, based on roughly 370,000 Ashkenazi Jewish individuals in the Dor Yeshorim database. RESULTS: Carrier screening identified pathogenic variants shared among Syrian, Iranian, and Ashkenazi Jewish groups. In addition, alleles unique to each group were identified. Importantly, 8.2% of 3401 individuals of mixed Syrian Jewish ancestry were carriers for at least one pathogenic variant. CONCLUSION: The findings of this study support the clinical usefulness of premarital genetic screening for individuals with Syrian Jewish ancestry to reduce the incidence of autosomal recessive disease among persons with Syrian Jewish heritage.

Further Delineation of the Clinical and Pathologic Features of HIKESHI-Related Hypomyelinating Leukodystrophy.

BACKGROUND: A recurrent homozygous missense variant, c.160G>C;p.(Val54Leu) in HIKESHI, was found to cause a hypomyelinating leukodystrophy with high frequency in the Ashkenazi Jewish population. We provide extended phenotypic classification of this disorder based on clinical history of a further seven affected individuals, assess carrier frequency in the Ashkenazi Jewish population, and provide a neuropathological study. METHODS: Clinical information, neuroimaging, and biosamples were collected. Brain autopsy was performed for one case. RESULTS: Individuals with HIKESHI-related disease share common clinical features: early axial hypotonia evolving to dystonia or with progressive spasticity, hyperreflexia and clonus, feeding difficulties with poor growth, and nystagmus. Severe morbidity or death during febrile illness occurred in five of the nine affected individuals. Magnetic resonance images of seven patients were analyzed and demonstrated diffuse hypomyelination and thin corpus callosum. Genotyping data of more than 125,000 Ashkenazi Jewish individuals revealed a carrier frequency of 1 in 216. Gross pathology examination in one case revealed abnormal white matter. Microscopically, there was a near-total absence of myelin with a relative preservation of axons. The cerebral white matter showed several reactive astrocytes and microglia. CONCLUSIONS: We provide pathologic evidence for a primary disorder of the myelin in HIKESHI-related leukodystrophy. These findings are consistent with the hypomyelination seen in brain magnetic resonance imaging and with the clinical features of early-onset spastic/dystonic quadriplegia and nystagmus. The high carrier rate of the recurrent variant seen in the Ashkenazi Jewish population requires increased attention to screening and diagnosis of this condition, particularly in this population.

A synonymous variant in MYO15A enriched in the Ashkenazi Jewish population causes autosomal recessive hearing loss due to abnormal splicing.

Nonsyndromic hearing loss is genetically heterogeneous. Despite comprehensive genetic testing, many cases remain unsolved because the clinical significance of identified variants is uncertain or because biallelic pathogenic variants are not identified for presumed autosomal recessive cases. Common synonymous variants are often disregarded. Determining the pathogenicity of synonymous variants may improve genetic diagnosis. We report a synonymous variant c.9861 C > T/p.(Gly3287=) in MYO15A in homozygosity or compound heterozygosity with another pathogenic or likely pathogenic MYO15A variant in 10 unrelated families with nonsyndromic sensorineural hearing loss. Biallelic variants in MYO15A were identified in 21 affected and were absent in 22 unaffected siblings. A mini-gene assay confirms that the synonymous variant leads to abnormal splicing. The variant is enriched in the Ashkenazi Jewish population. Individuals carrying biallelic variants involving c.9861 C > T often exhibit progressive post-lingual hearing loss distinct from the congenital profound deafness typically associated with biallelic loss-of-function MYO15A variants. This study establishes the pathogenicity of the c.9861 C > T variant in MYO15A and expands the phenotypic spectrum of MYO15A-related hearing loss. Our work also highlights the importance of multicenter collaboration and data sharing to establish the pathogenicity of a relatively common synonymous variant for improved diagnosis and management of hearing loss.

A founder deletion in the TRPM1 gene associated with congenital stationary night blindness and myopia is highly prevalent in Ashkenazi Jews.

Congenital stationary night blindness (CSNB) is a disease affecting the night vision of individuals. Previous studies identified TRPM1 as a gene involved in reduced night vision. Homozygous deletion of TRPM1 was the cause of CSNB in several children in 6 Ashkenazi Jewish families, thereby prompting further investigation of the carrier status within the families as well as in large cohorts of unrelated Ashkenazi and Sephardi individuals. Affected children were tested with a CSNB next-generation (NextGen) sequencing panel. A deletion of TRPM1 exons 2 through 7 was detected and confirmed by PCR and sequence analysis. A TaqMan-based assay was used to assess the frequency of this deletion in 18266 individuals of Jewish descent. High-throughput amplicon sequencing was performed on 380 samples to determine the putative deletion-flanking founder haplotype. Heterozygous TRPM1 deletions were found in 2.75% (1/36) of Ashkenazi subjects and in 1.22% (1/82) individuals of mixed Ashkenazi/Sephardic origin. The homozygous deletion frequency in our data was 0.03% (1/4025) and was only found in Ashkenazi Jewish individuals. Homozygous deletion of exons 2-7 in TRPM1 is a common cause of CSNB and myopia in many Ashkenazi Jewish patients. This deletion is a founder Ashkenazi Jewish deletion.

High-throughput carrier screening using TaqMan allelic discrimination.

Members of the Ashkenazi Jewish community are at an increased risk for inheritance of numerous genetic diseases such that carrier screening is medically recommended. This paper describes the development and evaluation of 30 TaqMan allelic discrimination qPCR assays for 29 mutations on 2 different high-throughput platforms. Four of these mutations are in the GBA gene and are successfully examined using short amplicons due to the qualitative nature of TaqMan allelic discrimination. Two systems were tested for their reliability (call rate) and consistency with previous diagnoses (diagnostic accuracy) indicating a call rate of 99.04% and a diagnostic accuracy of 100% (+/-0.00%) from one platform, and a call rate of 94.66% and a diagnostic accuracy of 93.35% (+/-0.29%) from a second for 9,216 genotypes. Results for mutations tested at the expected carrier frequency indicated a call rate of 97.87% and a diagnostic accuracy of 99.96% (+/-0.05%). This study demonstrated the ability of a high throughput qPCR methodology to accurately and reliably genotype 29 mutations in parallel. The universally applicable nature of this technology provides an opportunity to increase the number of mutations that can be screened simultaneously, and reduce the cost and turnaround time for accommodating newly identified and clinically relevant mutations.

Premarital and prenatal screening for cystic fibrosis: experience in the Ashkenazi Jewish population.

PURPOSE: Since the early 1990s, Dor Yeshorim (DY) and the Mount Sinai School of Medicine (MSSM) have conducted premarital and prenatal carrier screening for cystic fibrosis (CF) in the Ashkenazi Jewish (AJ) population as part of their genetic testing programs, respectively. Together, over 170,000 screenees have been tested. In this study, we report the CF mutation frequencies in over 110,000 screenees who reportedly were of 100% AJ descent from the DY program and MSSM. In addition, the CF mutation frequencies in a group of > 7,000 screenees for AJ diseases who were of < 100% AJ descent are reported. METHODS: Testing for CF mutations was performed by either PCR and restriction digestion or ASO hybridization analyses at MSSM or sent to various academic and commercial laboratories by DY. RESULTS: The overall (and individual) carrier frequency for the five common AJ mutations, W1282X (0.020), DeltaF508 (0.012), G542X (0.0024), 3849+10kb C>T (0.0020), and N1303K (0.0016), among screenees who were 100% AJ was 1 in 26; when D1152H and the rare 1717-1G>A were included, the overall carrier frequency increased to approximately 1 in 23. In four families with D1152H, five compound heterozygotes for D1152H and W1282X (n = 2), DeltaF508 (1) or 3849+10kb C>T (1) were identified. In contrast, the carrier frequency for screenees reporting < 100% AJ descent was approximately 1 in 30 for the seven mutations. CONCLUSIONS: The carrier frequency for five common CF mutations in a large 100% AJ sample increased from 1 in 26 to 1 in 23 when D1152H was included in the panel. Addition of D1152H to mutation panels when screening the AJ population should be considered because compound heterozygosity is associated with a variable disease phenotype. Further studies to delineate the phenotype of CF patients with this mutation are needed.

Geographic distribution of disease mutations in the Ashkenazi Jewish population supports genetic drift over selection.

The presence of four lysosomal storage diseases (LSDs) at increased frequency in the Ashkenazi Jewish population has suggested to many the operation of natural selection (carrier advantage) as the driving force. We compare LSDs and nonlysosomal storage diseases (NLSDs) in terms of the number of mutations, allele-frequency distributions, and estimated coalescence dates of mutations. We also provide new data on the European geographic distribution, in the Ashkenazi population, of seven LSD and seven NLSD mutations. No differences in any of the distributions were observed between LSDs and NLSDs. Furthermore, no regular pattern of geographic distribution was observed for LSD versus NLSD mutations-with some being more common in central Europe and others being more common in eastern Europe, within each group. The most striking disparate pattern was the geographic distribution of the two primary Tay-Sachs disease mutations, with the first being more common in central Europe (and likely older) and the second being exclusive to eastern Europe (primarily Lithuania and Russia) (and likely much younger). The latter demonstrates a pattern similar to two other recently arisen Lithuanian mutations, those for torsion dystonia and familial hypercholesterolemia. These observations provide compelling support for random genetic drift (chance founder effects, one approximately 11 centuries ago that affected all Ashkenazim and another approximately 5 centuries ago that affected Lithuanians), rather than selection, as the primary determinant of disease mutations in the Ashkenazi population.

Mutation frequencies for glycogen storage disease Ia in the Ashkenazi Jewish population.

Glycogen storage disease type Ia (GSDIa) is a severe autosomal recessive disorder caused by deficiency of the enzyme D-glucose-6-phosphatase (G6Pase). While numerous mutations have been found in cosmopolitan European populations, Ashkenazi Jewish (AJ) patients appear to primarily carry the R83C mutation, but possibly also the Q347X mutation found generally in Caucasians. To determine the frequency for both these mutations in the AJ population, we tested 20,719 AJ subjects for the R83C mutation and 4,290 subjects for the Q347X mutation. We also evaluated the mutation status of 30 AJ GSDIa affected subjects. From the carrier screening, we found 290 subjects with R83C, for a carrier frequency for this mutation of 1.4%. This carrier frequency translates into a predicted disease prevalence of 1 in 20,000, five times higher than for the general Caucasian population, confirming a founder effect and elevated frequency of GSDIa in the AJ population. We observed no carriers of the Q347X mutation. Among the 30 GSDIa affected AJ subjects, all were homozygous for R83C. These results indicate that R83C is the only prevalent mutation for GSDIa in the Ashkenazi population.

Nemaline myopathy in the Ashkenazi Jewish population is caused by a deletion in the nebulin gene.

Nemaline myopathy (NM) is a neuromuscular disorder that is clinically diverse and can be attributed to mutations in any of several genes. The Ashkenazi Jewish population, which represents a relatively genetically homogeneous group, has an increased frequency of several genetic disorders and has been the beneficiary of genetic screening programs that have reduced the incidence of these diseases. The identification of individuals with NM in this population has prompted a study of its cause. Our study has revealed that five NM patients from five families bear an identical 2,502-bp deletion that lies in the nebulin gene and that includes exon 55 and parts of introns 54 and 55. The absence of this exon results in the generation of a transcript that encodes 35 fewer amino acids. An analysis of the gene frequency of this mutation in a random sample of 4,090 Ashkenazi Jewish individuals has revealed a carrier frequency of one in 108.