Reassessment of causality of ABCC6 missense variants associated with pseudoxanthoma elasticum based on Sherloc

PurposePseudoxanthoma elasticum (PXE) is a heritable disorder affecting elastic fibers in the skin, eyes, and cardiovascular system. It is caused by biallelic pathogenic variants in the ABCC6 gene. To date, over 300 ABCC6 variants are associated with PXE, more than half being missense variants. Correct variant interpretation is essential for establishing a direct link between the variant and the patient’s phenotype and has important implications for diagnosis and treatment.MethodsWe used a systematic approach for interpretation of 271 previously reported and 15 novel ABCC6 missense variants, based on the semiquantitative classification system Sherloc.ResultsOnly 35% of variants were very likely to contribute directly to disease, in contrast to reported interpretations in ClinVar, while 59% of variants are currently of uncertain significance (VUS). Subclasses were created to distinguish VUS that are leaning toward likely benign or pathogenic, increasing the number of (likely) pathogenic ABCC6 missense variants to 47%.ConclusionBesides highlighting discrepancies between the Sherloc, American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP), ClinVar, and Leiden Open Variation Database (LOVD) classification, our results emphasize the need for segregation analysis, functional assays, and detailed evidence sharing in variant databases to reach a confident interpretation of ABCC6 missense variants and subsequent appropriate genetic and preconceptual counseling.     

Genome sequencing as a first-line genetic test in familial dilated cardiomyopathy

PurposeWe evaluated genome sequencing (GS) as an alternative to multigene panel sequencing (PS) for genetic testing in dilated cardiomyopathy (DCM).MethodsForty-two patients with familial DCM underwent PS and GS, and detection rates of rare single-nucleotide variants and small insertions/deletions in panel genes were compared. Loss-of-function variants in 406 cardiac-enriched genes were evaluated, and an assessment of structural variation was performed.ResultsGS provided broader and more uniform coverage than PS, with high concordance for rare variant detection in panel genes. GS identified all PS-identified pathogenic or likely pathogenic variants as well as two additional likely pathogenic variants: one was missed by PS due to low coverage, the other was a known disease-causing variant in a gene not included on the panel. No loss-of-function variants in the extended gene set met clinical criteria for pathogenicity. One BAG3 structural variant was classified as pathogenic.ConclusionOur data support the use of GS for genetic testing in DCM, with high variant detection accuracy and a capacity to identify structural variants. GS provides an opportunity to go beyond suites of established disease genes, but the incremental yield of clinically actionable variants is limited by a paucity of genetic and functional evidence for DCM association.     

eyeVarP: A computational framework for the identification of pathogenic variants specific to eye disease

PurposeDisease-specific pathogenic variant prediction tools that differentiate pathogenic variants from benign have been improved through disease specificity recently. However, they have not been evaluated on disease-specific pathogenic variants compared with other diseases, which would help to prioritize disease-specific variants from several genes or novel genes. Thus, we hypothesize that features of pathogenic variants alone would provide a better model.MethodsWe developed an eye disease–specific variant prioritization tool (eyeVarP), which applied the random forest algorithm to the data set of pathogenic variants of eye diseases and other diseases. We also developed the VarP tool and generalized pipeline to filter missense and insertion-deletion variants and predict their pathogenicity from exome or genome sequencing data, thus we provide a complete computational procedure.ResultseyeVarP outperformed pan disease–specific tools in identifying eye disease–specific pathogenic variants under the top 10. VarP outperformed 12 pathogenicity prediction tools with an accuracy of 95% in correctly identifying the pathogenicity of missense and insertion-deletion variants. The complete pipeline would help to develop disease-specific tools for other genetic disorders.ConclusioneyeVarP performs better in identifying eye disease–specific pathogenic variants using pathogenic variant features and gene features. Implementing such complete computational procedure would significantly improve the clinical variant interpretation for specific diseases.     

Accurate assignment of disease liability to genetic variants using only population data

PurposeThe growing size of public variant repositories prompted us to test the accuracy of pathogenicity prediction of DNA variants using population data alone.MethodsUnder the a priori assumption that the ratio of the prevalence of variants in healthy population vs that in affected populations form 2 distinct distributions (pathogenic and benign), we used a Bayesian method to assign probability to a variant belonging to either distribution.ResultsThe approach, termed Bayesian prevalence ratio (BayPR), accurately parsed 300 of 313 expertly curated CFTR variants: 284 of 296 pathogenic/likely pathogenic variants in 1 distribution and 16 of 17 benign/likely benign variants in another. BayPR produced an area under the receiver operating characteristic curve of 0.99 for 103 functionally confirmed missense CFTR variants, which is equal to or exceeds 10 commonly used algorithms (area under the receiver operating characteristic curve range = 0.54-0.99). Application of BayPR to expertly curated variants in 8 genes associated with 7 Mendelian conditions led to the assignment of a disease-causing probability of ≥80% to 1350 of 1374 (98.3%) pathogenic/likely pathogenic variants and of ≤20% to 22 of 23 (95.7%) benign/likely benign variants.ConclusionIrrespective of the variant type or functional effect, the BayPR approach provides probabilities of pathogenicity for DNA variants responsible for Mendelian disorders using only the variant counts in affected and unaffected population samples.     

Informing variant assessment using structured evidence from prior classifications (PS1, PM5, and PVS1 sequence variant interpretation criteria)

PurposeThis study aimed to explore whether evidence of pathogenicity from prior variant classifications in ClinVar could be used to inform variant interpretation using the American College of Medical Genetics and Genomics/Association for Molecular Pathology clinical guidelines.MethodsWe identified distinct single-nucleotide variants (SNVs) that are either similar in location or in functional consequence to pathogenic variants in ClinVar and analyzed evidence in support of pathogenicity using 3 interpretation criteria.ResultsThousands of variants, including many in clinically actionable disease genes (American College of Medical Genetics and Genomics secondary findings v3.0), have evidence of pathogenicity from existing variant classifications, accounting for 2.5% of nonsynonymous SNVs within ClinVar. Notably, there are many variants with uncertain or conflicting classifications that cause the same amino acid substitution as other pathogenic variants (PS1, N = 323), variants that are predicted to cause different amino acid substitutions in the same codon as pathogenic variants (PM5, N = 7692), and loss-of-function variants that are present in genes in which many loss-of-function variants are classified as pathogenic (PVS1, N = 3635). Most of these variants have similar computational predictions of pathogenicity and splicing effect as their associated pathogenic variants.ConclusionBroadly, for >1.4 million SNVs exome wide, information from previously classified variants could be used to provide evidence of pathogenicity. We have developed a pipeline to identify variants meeting these criteria that may inform interpretation efforts.     

Integration of functional assay data results provides strong evidence for classification of hundreds of BRCA1 variants of uncertain significance

PurposeBRCA1 pathogenic variant heterozygotes are at a substantially increased risk for breast and ovarian cancer. The widespread uptake of testing has led to a significant increase in the detection of missense variants in BRCA1, the vast majority of which are variants of uncertain clinical significance (VUS), posing a challenge to genetic counseling. Here, we harness a wealth of functional data for thousands of variants to aid in variant classification.MethodsWe have collected, curated, and harmonized functional data for 2701 missense variants representing 24.5% of possible missense variants in BRCA1. Results were harmonized across studies by converting data into binary categorical variables (functional impact versus no functional impact). Using a panel of reference variants we identified a subset of assays with high sensitivity and specificity (≥80%) and apply the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant interpretation guidelines to assign evidence criteria for classification.ResultsIntegration of data from validated assays provided ACMG/AMP evidence criteria in favor of pathogenicity for 297 variants or against pathogenicity for 2058 representing 96.2% of current VUS functionally assessed. We also explore discordant results and identify limitations in the approach.ConclusionHigh quality functional data are available for BRCA1 missense variants and provide evidence for classification of 2355 VUS according to their pathogenicity.     

Suspected clonal hematopoiesis as a natural functional assay of TP53 germline variant pathogenicity

PurposeSome variants identified by multigene panel testing of DNA from blood present with low variant allele fraction (VAF), often a manifestation of clonal hematopoiesis. Research has shown that the proportion of variants with low VAF is especially high in TP53, the Li-Fraumeni syndrome gene. Based on the hypothesis that variants with low VAF are positively selected as drivers of clonal hematopoiesis, we investigated the use of VAF as a predictor of TP53 germline variant pathogenicity.MethodsWe used data from 260,681 TP53 variants identified at 2 laboratories to compare the distribution of pathogenic and benign variants at different VAF intervals.ResultsLikelihood ratios toward pathogenicity associated with a VAF < 26% equated to the American College of Medical Genetics/Association of Molecular Pathology strong strength level and were applicable for 1 in 5 variants of unknown significance.ConclusionIn conclusion, detection of variants with low VAF in blood can be considered an in vivo functional assay to aid assessment of TP53 variant pathogenicity.     

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.     

Next-generation sequencing of 32 genes associated with hereditary aortopathies and related disorders of connective tissue in a cohort of 199 patients

PurposeHeritable factors play an important etiologic role in connective tissue disorders (CTD) with vascular involvement, and a genetic diagnosis is getting increasingly important for gene-tailored, personalized patient management.MethodsWe analyzed 32 disease-associated genes by using targeted next-generation sequencing and exome sequencing in a clinically relevant cohort of 199 individuals. We classified and refined sequence variants according to their likelihood for pathogenicity.ResultsWe identified 1 pathogenic variant (PV; in FBN1 or SMAD3) in 15 patients (7.5%) and ≥1 likely pathogenic variant (LPV; in COL3A1, FBN1, FBN2, LOX, MYH11, SMAD3, TGFBR1, or TGFBR2) in 19 individuals (9.6%), together resulting in 17.1% diagnostic yield. Thirteen PV/LPV were novel. Of PV/LPV-negative patients 47 (23.6%) showed ≥1 variant of uncertain significance (VUS). Twenty-five patients had concomitant variants. In-depth evaluation of reported/calculated variant classes resulted in reclassification of 19.8% of variants.ConclusionVariant classification and refinement are essential for shaping mutational spectra of disease genes, thereby improving clinical sensitivity. Obligate stringent multigene analysis is a powerful tool for identifying genetic causes of clinically related CTDs. Nonetheless, the relatively high rate of PV/LPV/VUS-negative patients underscores the existence of yet unknown disease loci and/or oligogenic/polygenic inheritance.     

Autosomal dominant Zellweger spectrum disorder caused by de novo variants in PEX14 gene

PurposeZellweger spectrum disorders (ZSDs) are known as autosomal recessive disorders caused by defective peroxisome biogenesis due to bi-allelic pathogenic variants in any of at least 13 different PEX genes. Here, we report 2 unrelated patients who present with an autosomal dominant ZSD.MethodsWe performed biochemical and genetic studies in blood and skin fibroblasts of the patients and demonstrated the pathogenicity of the identified PEX14 variants by functional cell studies.ResultsWe identified 2 different single heterozygous de novo variants in the PEX14 genes of 2 patients diagnosed with ZSD. Both variants cause messenger RNA mis-splicing, leading to stable expression of similar C-terminally truncated PEX14 proteins. Functional studies indicated that the truncated PEX14 proteins lost their function in peroxisomal matrix protein import and cause increased degradation of peroxisomes, ie, pexophagy, thus exerting a dominant-negative effect on peroxisome functioning. Inhibition of pexophagy by different autophagy inhibitors or genetic knockdown of the peroxisomal autophagy receptor NBR1 resulted in restoration of peroxisomal functions in the patients’ fibroblasts.ConclusionOur finding of an autosomal dominant ZSD expands the genetic repertoire of ZSDs. Our study underscores that single heterozygous variants should not be ignored as possible genetic cause of diseases with an established autosomal recessive mode of inheritance.     

Variant recurrence in neurodevelopmental disorders: the use of publicly available genomic data identifies clinically relevant pathogenic missense variants

PurposeNext-generation sequencing has revealed the major impact of de novo variants (DNVs) in developmental disorders (DD) such as intellectual disability, autism, and epilepsy. However, a substantial fraction of these predicted pathogenic DNVs remains challenging to distinguish from background DNVs, notably the missense variants acting via nonhaploinsufficient mechanisms on specific amino acid residues. We hypothesized that the detection of the same missense variation in at least two unrelated individuals presenting with a similar phenotype could be a powerful approach to reveal novel pathogenic variants.MethodsWe looked for variations independently present in both our database of >1200 solo exomes and in denovo-db, a large, publicly available collection of de novo variants identified in patients with DD.ResultsThis approach identified 30 variants with strong evidence of pathogenicity, including variants already classified as pathogenic or probably pathogenic by our team, and also several new variants of interest in known OMIM genes or in novel genes. We identified FEM1B and GNAI2 as good candidate genes for syndromic intellectual disability and confirmed the implication of ACTL6B in a neurodevelopmental disorder.ConclusionAnnotation of local variants with denovo-db can highlight missense variants with high potential for pathogenicity, both facilitating the time-consuming reanalysis process and allowing novel DD gene discoveries.     

Determining pathogenicity of genetic variants in hypertrophic cardiomyopathy: importance of periodic reassessment

PurposeMajor advances have been made in our understanding and clinical application of genetic testing in hypertrophic cardiomyopathy. Determining pathogenicity of a single-nucleotide variant remains a major clinical challenge. This study sought to reassess single-nucleotide variant classification in hypertrophic cardiomyopathy probands.MethodsConsecutive probands with hypertrophic cardiomyopathy with a reported pathogenic mutation or variation of uncertain significance were included. Family and medical history were obtained. Each single-nucleotide variant was reassessed by a panel of four reviewers for pathogenicity based on established criteria together with updated cosegregation data and current population-based allele frequencies.ResultsFrom 2000 to 2012, a total of 136 unrelated hypertrophic cardiomyopathy probands had genetic testing, of which 63 (46%) carried at least one pathogenic mutation. MYBPC3 (n = 34; 47%) and MYH7 (n = 23; 32%) gene variants together accounted for 79%. Five variants in six probands (10%) were reclassified: two variation of uncertain significance were upgraded to pathogenic, one variation of uncertain significance and one pathogenic variant were downgraded to benign, and one pathogenic variant (found in two families) was downgraded to variation of uncertain significance. None of the reclassifications had any adverse clinical consequences.ConclusionGiven the rapid growth of genetic information available in both disease and normal populations, periodic reassessment of single-nucleotide variant data is essential in hypertrophic cardiomyopathy.Genet Med 2014:16(4):286–293.     

Integrated analysis of unclassified variants in mismatch repair genes

PurposeLynch syndrome is a genetic disease that predisposes to colorectal tumors, caused by mutation in mismatch repair genes. The use of genetic tests to identify mutation carriers does not always give perfectly clear results, as happens when an unclassified variant is found. This study aimed to define the pathogenic role of 35 variants present in MSH2, MLH1, MSH6, and PMS2 genes identified in our 15-year case study.MethodsWe collected clinical and molecular data of all carriers, and then we analyzed the variants pathogenic role with web tools and molecular analyses. Using a Bayesian approach, we derived a posterior probability of pathogenicity and classified each variant according to a standardized five-class system.ResultsThe MSH2 p.Pro349Arg, p.Met688Arg, the MLH1 p.Gly67Arg, p.Thr82Ala, p.Lys618Ala, the MSH6 p.Ala1236Pro, and the PMS2 p.Arg20Gln were classified as pathogenic, and the MSH2 p.Cys697Arg and the PMS2 p.Ser46Ile were classified as likely pathogenic. Seven variants were likely nonpathogenic, 3 were nonpathogenic, and 16 remained uncertain.ConclusionQuantitative assessment of several parameters and their integration in a multifactorial likelihood model is the method of choice for classifying the variants. As such classifications can be associated with surveillance and testing recommendations, the results and the method developed in our study can be useful for helping laboratory geneticists in evaluation of genetic tests and clinicians in the management of carriers. Genet Med 2011:13(2):115–124.     

Two integrated and highly predictive functional analysis-based procedures for the classification of MSH6 variants in Lynch syndrome

PurposeVariants in the DNA mismatch repair (MMR) gene MSH6, identified in individuals suspected of Lynch syndrome, are difficult to classify owing to the low cancer penetrance of defects in that gene. This not only obfuscates personalized health care but also the development of a rapid and reliable classification procedure that does not require clinical data.MethodsThe complete in vitro MMR activity (CIMRA) assay was calibrated against clinically classified MSH6 variants and, employing Bayes’ rule, integrated with computational predictions of pathogenicity. To enable the validation of this two-component classification procedure we have employed a genetic screen to generate a large set of inactivating Msh6 variants, as proxies for pathogenic variants.ResultsThe genetic screen-derived variants established that the two-component classification procedure displays high sensitivities and specificities. Moreover, these inactivating variants enabled the direct reclassification of human variants of uncertain significance (VUS) as (likely) pathogenic.ConclusionThe two-component classification procedure and the genetic screens provide complementary approaches to rapidly and cost-effectively classify the large majority of human MSH6 variants. The approach followed here provides a template for the classification of variants in other disease-predisposing genes, facilitating the translation of personalized genomics into personalized health care.     

Mutation spectrum in the ABCC6 gene and genotype–phenotype correlations in a French cohort with pseudoxanthoma elasticum

PurposePseudoxanthoma elasticum (PXE) is an autosomal recessive disorder caused by variants in the ABCC6 gene. Ectopic mineralization of connective tissues leads to skin, eye, and cardiovascular manifestations with considerable phenotypic variability of unknown cause. We aimed to identify genotype–phenotype correlations in PXE.MethodsA molecular analysis was performed on 458 French PXE probands clinically evaluated using the Phenodex score (PS). Variant topographic analysis and genotype–phenotype correlation analysis were performed according to the number and type of identified variants.ResultsComplete molecular analysis of 306 cases allowed the identification of 538 mutational events (88% detection rate) with 142 distinct variants, of which 66 were novel. Missense variant distribution was specific to some regions and residues of ABCC6. For the 220 cases with a complete PS, there was a higher prevalence of eye features in Caucasian patients (P = 0.03) and more severe eye and vascular phenotype in patients with loss-of-function variants (P = 0.02 and 0.05, respectively). Nephrolithiases and strokes, absent from the PS, were prevalent features of the disorder (11 and 10%, respectively).ConclusionWe propose an updated PS including renal and neurological features and adaptation of follow-up according to the genetic and ethnic status of PXE-affected patients.Genet Med advance online publication 19 January 2017     

Genome sequencing identifies multiple deleterious variants in autism patients with more severe phenotypes

PurposeTo maximize the discovery of potentially pathogenic variants to better understand the diagnostic utility of genome sequencing (GS) and to assess how the presence of multiple risk events might affect the phenotypic severity in autism spectrum disorders (ASD).MethodsGS was applied to 180 simplex and multiplex ASD families (578 individuals, 213 patients) with exome sequencing and array comparative genomic hybridization further applied to a subset for validation and cross-platform comparisons.ResultsWe found that 40.8% of patients carried variants with evidence of disease risk, including a de novo frameshift variant in NR4A2 and two de novo missense variants in SYNCRIP, while 21.1% carried clinically relevant pathogenic or likely pathogenic variants. Patients with more than one risk variant (9.9%) were more severely affected with respect to cognitive ability compared with patients with a single or no-risk variant. We observed no instance among the 27 multiplex families where a pathogenic or likely pathogenic variant was transmitted to all affected members in the family.ConclusionThe study demonstrates the diagnostic utility of GS, especially for multiple risk variants that contribute to the phenotypic severity, shows the genetic heterogeneity in multiplex families, and provides evidence for new genes for follow up.     

Genetic diversity and pathogenic variants as possible predictors of severity in a French sample of nonsyndromic heritable thoracic aortic aneurysms and dissections (nshTAAD)

PurposeHeritable thoracic aortic aneurysms and dissections (hTAAD) are life-threatening complications of well-known syndromic diseases or underdiagnosed nonsyndromic heritable forms (nshTAAD). Both have an autosomal dominant transmission and are genetically heterogeneous. Our objective was to describe the relevance of molecular diagnosis in these patients and the contribution of each gene in nshTAAD.MethodsTwo hundred twenty-six consecutive nshTAAD probands, either young (<45 years) sporadic or familial cases were included. A next-generation sequencing capture panel comprising 23 known disease-causing genes was performed.ResultsClass 4 or 5 variants were identified in 18% of the nshTAAD probands, while class 3 variants were found in 10% of them. The yield in familial cases was greater than in sporadic cases. SMAD3 and FBN1 genes were the major disease-causing genes. Unexpectedly, no premature termination codon variant was identified in the FBN1 gene. Furthermore, we report for the first time that aortic dissection or surgery occurred significantly more often and earlier in probands with a class 4 or 5 pathogenic variant.ConclusionThis study indicates that genetic screening using NGS is efficient in young and familial nshTAAD. The presence of a pathogenic variant has a possible predictive value, which needs to be further investigated because it may influence care.     

Revealing hidden genetic diagnoses in the ocular anterior segment disorders

PurposeOcular anterior segment disorders (ASDs) are clinically and genetically heterogeneous, and genetic diagnosis often remains elusive. In this study, we demonstrate the value of a combined analysis protocol using phenotypic, genomic, and pedigree structure data to achieve a genetic conclusion.MethodsWe utilized a combination of chromosome microarray, exome sequencing, and genome sequencing with structural variant and trio analysis to investigate a cohort of 41 predominantly sporadic cases.ResultsWe identified likely causative variants in 54% (22/41) of cases, including 51% (19/37) of sporadic cases and 75% (3/4) of cases initially referred as familial ASD. Two-thirds of sporadic cases were found to have heterozygous variants, which in most cases were de novo. Approximately one-third (7/22) of genetic diagnoses were found in rarely reported or recently identified ASD genes including PXDN, GJA8, COL4A1, ITPR1, CPAMD8, as well as the new phenotypic association of Axenfeld–Rieger anomaly with a homozygous ADAMTS17 variant. The remainder of the variants were in key ASD genes including FOXC1, PITX2, CYP1B1, FOXE3, and PAX6.ConclusionsWe demonstrate the benefit of detailed phenotypic, genomic, variant, and segregation analysis to uncover some of the previously “hidden” heritable answers in several rarely reported and newly identified ocular ASD-related disease genes.