Splice-altering variant in COL11A1 as a cause of nonsyndromic hearing loss DFNA37

PurposeThe aim of this study was to determine the genetic cause of autosomal dominant nonsyndromic hearing loss segregating in a multigenerational family.MethodsClinical examination, genome-wide linkage analysis, and exome sequencing were carried out on the family.ResultsAffected individuals presented with early-onset progressive mild hearing impairment with a fairly flat, gently downsloping or U-shaped audiogram configuration. Detailed clinical examination excluded any additional symptoms. Linkage analysis detected an interval on chromosome 1p21 with a logarithm of the odds (LOD) score of 8.29: designated locus DFNA37. Exome sequencing identified a novel canonical acceptor splice-site variant c.652-2A>C in the COL11A1 gene within the DFNA37 locus. Genotyping of all 48 family members confirmed segregation of this variant with the deafness phenotype in the extended family. The c.652-2A>C variant is novel, highly conserved, and confirmed in vitro to alter RNA splicing.ConclusionWe have identified COL11A1 as the gene responsible for deafness at the DFNA37 locus. Previously, COL11A1 was solely associated with Marshall and Stickler syndromes. This study expands its phenotypic spectrum to include nonsyndromic deafness. The implications of this discovery are valuable in the clinical diagnosis, prognosis, and treatment of patients with COL11A1 pathogenic variants.     

Extrusion pump ABCC1 was first linked with nonsyndromic hearing loss in humans by stepwise genetic analysis

PurposeTo determine the genetic etiology of deafness in a family (HN-SD01) with autosomal dominant nonsyndromic hearing loss (NSHL).MethodsStepwise genetic analysis was performed on family HN-SD01, including hotspot variant screening, exome sequencing, virtual hearing loss gene panel, and genome-wide linkage analysis. Targeted region sequencing was used to screen ABCC1 in additional cases. Cochlear expression of Abcc1 was evaluated by messenger RNA (mRNA) and protein levels. Computational prediction, immunofluorescence, real-time quantitative polymerase chain reaction, and flow cytometry were conducted to uncover functional consequences of candidate variants.ResultsStepwise genetic analysis identified a heterozygous missense variant, ABCC1:c.1769A>G (p.Asn590Ser), cosegregating with phenotype in HN-SD01. Screening of ABCC1 in an additional 217 cases identified candidate pathogenic variants c.692G>A (p.Gly231Asp) in a sporadic case and c.887A>T (p.Glu296Val) in a familial proband. Abcc1 expressed in stria vascularis and auditory nerve of mouse cochlea. Immunofluorescence showed p.Asn590Ser distributed in cytomembrane and cytoplasm, while wild type was shown only in cytomembrane. Besides, it generated unstable mRNA and decreased efflux capacity of ABCC1.ConclusionStepwise genetic analysis is efficient to analyze the genetic etiology of NSHL. Variants in ABCC1 are linked with NSHL and suggest an important role of extruding pumps in maintaining cochlea function.     

Genotype–phenotype correlation and mutation spectrum in a large cohort of patients with inherited retinal dystrophy revealed by next-generation sequencing

PurposeInherited retinal dystrophy (IRD) is a leading cause of blindness worldwide. Because of extreme genetic heterogeneity, the etiology and genotypic spectrum of IRD have not been clearly defined, and there is limited information on genotype–phenotype correlations. The purpose of this study was to elucidate the mutational spectrum and genotype–phenotype correlations of IRD.MethodsWe developed a targeted panel of 164 known retinal disease genes, 88 candidate genes, and 32 retina-abundant microRNAs, used for exome sequencing. A total of 179 Chinese families with IRD were recruited.ResultsIn 99 unrelated patients, a total of 124 mutations in known retinal disease genes were identified, including 79 novel mutations (detection rate, 55.3%). Moreover, novel genotype–phenotype correlations were discovered, and phenotypic trends noted. Three cases are reported, including the identification of AHI1 as a novel candidate gene for nonsyndromic retinitis pigmentosa.ConclusionThis study revealed novel genotype–phenotype correlations, including a novel candidate gene, and identified 124 genetic defects within a cohort with IRD. The identification of novel genotype–phenotype correlations and the spectrum of mutations greatly enhance the current knowledge of IRD phenotypic and genotypic heterogeneity, which will assist both clinical diagnoses and personalized treatments of IRD patients.Genet Med17 4, 271–278.     

Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides

PurposeUsing exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability.MethodsSequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects.ResultsIn 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects.ConclusionOur data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.     

Exome sequencing revealed Notch ligand JAG1 as a novel candidate gene for familial exudative vitreoretinopathy

PurposeFamilial exudative vitreoretinopathy (FEVR) is a blindness-causing retinal vascular disease characterized by incomplete vascularization of the peripheral retina and by the absence or abnormality of the second/tertiary capillary layers in the deep retina. Variants in known FEVR disease genes can only explain about 50% of FEVR-affected cases. We aim to identify additional disease genes in patients with FEVR.MethodsWe applied exome sequencing analysis in a cohort of 49 FEVR families without pathogenic variants in known FEVR genes. Functions of the affected proteins were evaluated by reporter assay. Knockout mouse models were generated by endothelial-specific Cre line.ResultsThree novel rare heterozygous variants in Notch ligand JAG1 were identified in FEVR families—c.413C>T p. (A138V), c.1415G>A p. (R472H), and c.2884A>G p. (T962A)—and verified by Sanger sequencing analysis. Notch reporter assay revealed that mutant JAG1 proteins JAG1-A138V and JAG1-T962A lost almost all of their activities, and JAG1-R472H lost approximately 50% of its activity. Deletion of Jag1 in mouse endothelial cells resulted in reduced tip cells at the angiogenic front and retarded vessel growth, reproducing FEVR-like phenotypes.ConclusionOur data suggest that JAG1 is a novel candidate gene for FEVR and pinpoints a potential target for therapeutic intervention.     

Copy-number variation contributes 9% of pathogenicity in the inherited retinal degenerations

PurposeCurrent sequencing strategies can genetically solve 55–60% of inherited retinal degeneration (IRD) cases, despite recent progress in sequencing. This can partially be attributed to elusive pathogenic variants (PVs) in known IRD genes, including copy-number variations (CNVs), which have been shown as major contributors to unsolved IRD cases.MethodsFive hundred IRD patients were analyzed with targeted next-generation sequencing (NGS). The NGS data were used to detect CNVs with ExomeDepth and gCNV and the results were compared with CNV detection with a single-nucleotide polymorphism (SNP) array. Likely causal CNV predictions were validated by quantitative polymerase chain reaction (qPCR).ResultsLikely disease-causing single-nucleotide variants (SNVs) and small indels were found in 55.6% of subjects. PVs in USH2A (11.6%), RPGR (4%), and EYS (4%) were the most common. Likely causal CNVs were found in an additional 8.8% of patients. Of the three CNV detection methods, gCNV showed the highest accuracy. Approximately 30% of unsolved subjects had a single likely PV in a recessive IRD gene.ConclusionCNV detection using NGS-based algorithms is a reliable method that greatly increases the genetic diagnostic rate of IRDs. Experimentally validating CNVs helps estimate the rate at which IRDs might be solved by a CNV plus a more elusive variant.     

Risks of breast and ovarian cancer for women harboring pathogenic missense variants in BRCA1 and BRCA2 compared with those harboring protein truncating variants

PurposeGermline genetic testing for BRCA1 and BRCA2 variants has been a part of clinical practice for >2 decades. However, no studies have compared the cancer risks associated with missense pathogenic variants (PVs) with those associated with protein truncating (PTC) variants.MethodsWe collected 582 informative pedigrees segregating 1 of 28 missense PVs in BRCA1 and 153 pedigrees segregating 1 of 12 missense PVs in BRCA2. We analyzed 324 pedigrees with PTC variants in BRCA1 and 214 pedigrees with PTC variants in BRCA2. Cancer risks were estimated using modified segregation analysis.ResultsEstimated breast cancer risks were markedly lower for women aged >50 years carrying BRCA1 missense PVs than for the women carrying BRCA1 PTC variants (hazard ratio [HR] = 3.9 [2.4-6.2] for PVs vs 12.8 [5.7-28.7] for PTC variants; P = .01), particularly for missense PVs in the BRCA1 C-terminal domain (HR = 2.8 [1.4-5.6]; P = .005). In case of BRCA2, for women aged >50 years, the HR was 3.9 (2.0-7.2) for those heterozygous for missense PVs compared with 7.0 (3.3-14.7) for those harboring PTC variants. BRCA1 p.[Cys64Arg] and BRCA2 p.[Trp2626Cys] were associated with particularly low risks of breast cancer compared with other PVs.ConclusionThese results have important implications for the counseling of at-risk women who harbor missense PVs in the BRCA1/2 genes.     

Optical genome mapping and revisiting short-read genome sequencing data reveal previously overlooked structural variants disrupting retinal disease−associated genes

PurposeStructural variants (SVs) play an important role in inherited retinal diseases (IRD). Although the identification of SVs significantly improved upon the availability of genome sequencing, it is expected that involvement of SVs in IRDs is higher than anticipated. We revisited short-read genome sequencing data to enhance the identification of gene-disruptive SVs.MethodsOptical genome mapping was performed to improve SV detection in short-read genome sequencing?negative cases. In addition, reanalysis of short-read genome sequencing data was performed to improve the interpretation of SVs and to re-establish SV prioritization criteria.ResultsIn a monoallelic USH2A case, optical genome mapping identified a pericentric inversion (173 megabase), with 1 breakpoint disrupting USH2A. Retrospectively, the variant could be observed in genome sequencing data but was previously deemed false positive. Reanalysis of short-read genome sequencing data (427 IRD cases) was performed which yielded 30 pathogenic SVs affecting, among other genes, USH2A (n = 15), PRPF31 (n = 3), and EYS (n = 2). Eight of these (>25%) were overlooked during previous analyses.ConclusionCritical evaluation of our findings allowed us to re-establish and improve our SV prioritization and interpretation guidelines, which will prevent missing pathogenic events in future analyses. Our data suggest that more attention should be paid to SV interpretation and the current contribution of SVs in IRDs is still underestimated.     

Systematic misclassification of missense variants in BRCA1 and BRCA2 “coldspots”

PurposeGuidelines for variant interpretation incorporate variant hotspots in critical functional domains as evidence for pathogenicity (e.g., PM1 and PP2), but do not use “coldspots,” that is, regions without essential functions that tolerate variation, as evidence a variant is benign. To improve variant classification we evaluated BRCA1 and BRCA2 missense variants reported in ClinVar to identify regions where pathogenic missenses are extremely infrequent, defined as coldspots.MethodsWe used Bayesian approaches to model variant classification in these regions.ResultsBRCA1 exon 11 (~60% of the coding sequence), and BRCA2 exons 10 and 11 (~65% of the coding sequence), are coldspots. Of 89 pathogenic (P) or likely pathogenic (LP) missense variants in BRCA1, none are in exon 11 (odds <0.01, 95% confidence interval [CI] 0.0–0.01). Of 34 P or LP missense variants in BRCA2, none are in exons 10–11 (odds <0.01, 95% CI 0.0–0.01). More than half of reported missense variants of uncertain significance (VUS) in BRCA1 and BRCA2 are in coldspots (3115/5301 = 58.8%). Reclassifying these 3115 VUS as likely benign would substantially improve variant classification.ConclusionIn BRCA1 and BRCA2 coldspots, missense variants are very unlikely to be pathogenic. Classification schemes that incorporate coldspots can reduce the number of VUS and mitigate risks from reporting benign variation as VUS.     

Investigation of TREM2, PLD3, and UNC5C variants in patients with Alzheimer's disease from mainland China

Recently, 3 rare coding variants significantly associated with Alzheimer's disease (AD) risk have been identified in western populations using whole exome sequencing method, including p.R47H in TREM2, p.V232M in PLD3, and p.T835M in UNC5C. To examine whether these variants are genetic risk factors in patients with AD from mainland China, we sequenced exon 2 of TREM2, exon 9 of PLD3, and exon 15 of UNC5C in Chinese Han population including 360 patients with AD and 400 control individuals. As a result, none of these 3 variants were identified in all subjects, however, 1 novel variant (p.A130V) in TREM2 and 4 novel variants (p.Q860H, p.T837K, p.S843G, and p.V836V) in UNC5C were detected in unrelated patients with late-onset AD. These findings suggest the 3 rare coding variants might not play an important role in AD risk in mainland China.     

RNA sequencing solved the most common but unrecognized NEB pathogenic variant in Japanese nemaline myopathy

PurposeThe diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20–40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants.MethodsAberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES.ResultsWe identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases.ConclusionThese results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.     

A pathogenic UFSP2 variant in an autosomal recessive form of pediatric neurodevelopmental anomalies and epilepsy

PurposeNeurodevelopmental disabilities are common and genetically heterogeneous. We identified a homozygous variant in the gene encoding UFM1-specific peptidase 2 (UFSP2), which participates in the UFMylation pathway of protein modification. UFSP2 variants are implicated in autosomal dominant skeletal dysplasias, but not neurodevelopmental disorders. Homozygosity for the variant occurred in eight children from four South Asian families with neurodevelopmental delay and epilepsy. We describe the clinical consequences of this variant and its effect on UFMylation.MethodsExome sequencing was used to detect potentially pathogenic variants and identify shared regions of homozygosity. Immunoblotting assessed protein expression and post-translational modifications in patient-derived fibroblasts.ResultsThe variant (c.344T>A; p.V115E) is rare and alters a conserved residue in UFSP2. Immunoblotting in patient-derived fibroblasts revealed reduced UFSP2 abundance and increased abundance of UFMylated targets, indicating the variant may impair de-UFMylation rather than UFMylation. Reconstituting patient-derived fibroblasts with wild-type UFSP2 reduced UFMylation marks. Analysis of UFSP2’s structure indicated that variants observed in skeletal disorders localize to the catalytic domain, whereas V115 resides in an N-terminal domain possibly involved in substrate binding.ConclusionDifferent UFSP2 variants cause markedly different diseases, with homozygosity for V115E causing a severe syndrome of neurodevelopmental disability and epilepsy.     

ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice,cis-regulatory,structural, and recurrent hypomorphic variants

PurposeABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability.MethodsBy locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4.ResultsWe identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells.ConclusionNoncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.     

Clinical and molecular findings in a cohort of 152 Brazilian severe early onset inherited retinal dystrophy patients

Leber congenital amaurosis (LCA) and early‐onset retinal dystrophy (EORD) are severe inherited retinal dystrophy that can cause deep blindness childhood. They represent 5% of all retinal dystrophies in the world population and about 10% in Brazil. Clinical findings and molecular basis of syndromic and nonsyndromic LCA/EORD in a Brazilian sample (152 patients/137 families) were studied. In this population, 15 genes were found to be related to the phenotype, 38 new variants were detected and four new complex alleles were discovered. Among 123 variants found, the most common were CEP290: c.2991+1655A>G, CRB1: p.Cys948Tyr, and RPGRIP1: exon10‐18 deletion.     

Genetic Epidemiology of Mitochondrial Pathogenic Variants Causing Nonsyndromic Hearing Loss in a Large Cohort of South Indian Hearing Impaired Individuals

Mitochondria play a critical role in the generation of metabolic energy in the form of ATP. Tissues and organs that are highly dependent on aerobic metabolism are involved in mitochondrial disorders including nonsyndromic hearing loss (NSHL). Seven pathogenic variants leading to NSHL have so far been reported on two mitochondrial genes: MT‐RNR1 encoding 12SrRNA and MT‐TS1 encoding tRNA for Ser^(UCN). We screened 729 prelingual NSHL subjects to determine the prevalence of MT‐RNR1 variants at position m.961, m.1555A>G and m.1494C>T, and MT‐TS1 m.7445A>G, m.7472insC m.7510T>C and m.7511T>C variants. Mitochondrial pathogenic variants were found in eight probands (1.1%). Five of them were found to have the m.1555A>G variant, two others had m.7472insC and one proband had m.7444G>A. The extended relatives of these probands showed variable degrees of hearing loss and age at onset. This study shows that mitochondrial pathogenic alleles contribute to about 1% prelingual hearing loss. This study will henceforth provide the reference for the prevalence of mitochondrial pathogenic alleles in the South Indian population, which to date has not been estimated. The m.1555A>G variant is a primary predisposing genetic factor for the development of hearing loss. Our study strongly suggests that mitochondrial genotyping should be considered for all hearing impaired individuals and particularly in families where transmission is compatible with maternal inheritance, after ruling out the most common variants.