Arrhythmia Variant Associations and Reclassifications in the eMERGE-III Sequencing Study

Background: Sequencing Mendelian arrhythmia genes in individuals without an indication for arrhythmia genetic testing can identify carriers of pathogenic or likely pathogenic (P/LP) variants. However, the extent to which these variants are associated with clinically meaningful phenotypes before or after return of variant results (RoR) is unclear. In addition, the majority of discovered variants are currently classified as Variants of Uncertain Significance (VUS), limiting clinical actionability. Methods: The eMERGE-III study is a multi-center prospective cohort which included 21,846 participants without prior indication for cardiac genetic testing. Participants were sequenced for 109 Mendelian disease genes, including 10 linked to arrhythmia syndromes. Variant carriers were assessed with Electronic Health Record (EHR)-derived phenotypes and follow-up clinical examination. Selected VUS (n=50) were characterized in vitro with automated electrophysiology experiments in HEK293 cells. Results: As previously reported, 3.0% of participants had pathogenic or likely pathogenic (P/LP) variants in the 109 genes. Herein, we report 120 participants (0.6%) with P/LP arrhythmia variants. Compared to non-carriers, arrhythmia P/LP carriers had a significantly higher burden of arrhythmia phenotypes in their EHRs. Fifty four participants had variant results returned. Nineteen of these 54 participants had inherited arrhythmia syndrome diagnoses (primarily long QT syndrome), and 12/19 of these diagnoses were made only after variant results were returned (0.05%). After in vitro functional evaluation of 50 variants of uncertain significance (VUS), we reclassified 11 variants: 3 to likely benign and 8 to P/LP. Conclusions: Genome sequencing in a large population without indication for arrhythmia genetic testing identified phenotype-positive carriers of variants in congenital arrhythmia syndrome disease genes. As large numbers of people are sequenced, the disease risk from rare variants in arrhythmia genes can be assessed by integrating genomic screening, EHR phenotypes, and in vitro functional studies.

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Arrhythmia variant associations and reclassifications in the eMERGE-III sequencing study

10.1101/2021.03.30.21254549 ◽

2021 ◽

Author(s):

Andrew M Glazer ◽

Giovanni Davogustto ◽

Christian M Shaffer ◽

Carlos G Vanoye ◽

Reshma R Desai ◽

...

Keyword(s):

Rare Variants ◽

Disease Risk ◽

Disease Genes ◽

Functional Evaluation ◽

Functional Studies ◽

Large Numbers ◽

Inherited Arrhythmia ◽

Genomic Screening ◽

Uncertain Significance

In 21,846 eMERGE-III participants, sequencing 10 arrhythmia syndrome disease genes identified 123 individuals with pathogenic or likely pathogenic (P/LP) variants. Compared to non-carriers, P/LP carriers had a significantly higher burden of arrhythmia phenotypes in their electronic health records (EHRs). Fifty one participants had variant results returned. Eighteen of these 51 participants had inherited arrhythmia syndrome diagnoses (primarily long QT syndrome), and 11/18 of these diagnoses were made only after variant results were returned. After in vitro functional evaluation of 50 variants of uncertain significance (VUS), we reclassified 11 variants: 3 to likely benign and 8 to P/LP. As large numbers of people are sequenced, the disease risk from rare variants in arrhythmia genes can be assessed by integrating genomic screening, EHR phenotypes, and in vitro functional studies.

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A Bayesian method using sparse data to estimate penetrance of disease-associated genetic variants

10.1101/571158 ◽

2019 ◽

Cited By ~ 1

Author(s):

Brett M. Kroncke ◽

Derek K. Smith ◽

Andrew M. Glazer ◽

Dan M. Roden ◽

Jeffrey D. Blume

Keyword(s):

Rare Variants ◽

Disease Risk ◽

Genomic Medicine ◽

Disease Genes ◽

Mendelian Disease ◽

Phenotypic Data ◽

Prior Probabilities ◽

Cardiac Gene ◽

Risk Of Disease

AbstractPurposeA major challenge in genomic medicine is how to best predict risk of disease from rare variants discovered in Mendelian disease genes but with limited phenotypic data. We have recently used Bayesian methods to show that in vitro functional measurements and computational pathogenicity classification of variants in the cardiac gene SCN5A correlate with rare arrhythmia penetrance. We hypothesized that similar predictors could be used to impute variant-specific penetrance prior probabilities.MethodsFrom a review of 756 publications, we developed a pattern mixture algorithm, based on a Bayesian Beta-Binomial model, to generate SCN5A variant-specific penetrance priors for the heart arrhythmia Brugada syndrome (BrS).ResultsThe resulting priors correlate with mean BrS penetrance posteriors (cross validated R2= 0.41). SCN5A variant function and structural context provide the most information predictive of BrS penetrance. The resulting priors are interpretable as equivalent to the observation of affected and unaffected carriers.ConclusionsBayesian estimates of penetrance can efficiently integrate variant-specific data (e.g. functional, structural, and sequence) to accurately estimate disease risk attributable to individual variants. We suggest this formulation of penetrance is quantitative, probabilistic, and more precise than, but consistent with, discrete pathogenicity classification approaches.

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Functional evaluation of gene mutations in Long QT Syndrome: strength of evidence from in vitro assays for deciphering variants of uncertain significance

Journal of Congenital Cardiology ◽

10.1186/s40949-020-00037-9 ◽

2020 ◽

Vol 4 (1) ◽

Author(s):

Jules C. Hancox ◽

Alan G. Stuart ◽

Stephen C. Harmer

Keyword(s):

Patch Clamp ◽

High Throughput ◽

Long Qt Syndrome ◽

Patient Specific ◽

Functional Evaluation ◽

Long Qt ◽

Variants Of Uncertain Significance ◽

Uncertain Significance ◽

Qt Syndrome

Abstract Background Genetic screening is now commonplace for patients suspected of having inherited cardiac conditions. Variants of uncertain significance (VUS) in disease-associated genes pose problems for the diagnostician and reliable methods for evaluating VUS function are required. Although function is difficult to interrogate for some genes, heritable channelopathies have established mechanisms that should be amenable to well-validated evaluation techniques. The cellular electrophysiology techniques of ‘voltage-’ and ‘patch-’ clamp have a long history of successful use and have been central to identifying both the roles of genes involved in different forms of congenital Long QT Syndrome (LQTS) and the mechanisms by which mutations lead to aberrant ion channel function underlying clinical phenotypes. This is particularly evident for KCNQ1, KCNH2 and SCN5A, mutations in which underlie > 90% of genotyped LQTS cases (the LQT1-LQT3 subtypes). Recent studies utilizing high throughput (HT) planar patch-clamp recording have shown it to discriminate effectively between rare benign and pathological variants, studied through heterologous expression of recombinant channels. In combination with biochemical methods for evaluating channel trafficking and supported by biophysical modelling, patch clamp also provides detailed mechanistic insight into the functional consequences of identified mutations. Whilst potentially powerful, patient-specific stem-cell derived cardiomyocytes and genetically modified animal models are currently not well-suited to high throughput VUS study. Conclusion The widely adopted 2015 American College of Medical Genetics (ACMG) and Association for Molecular Pathology (AMP) guidelines for the interpretation of sequence variants include the PS3 criterion for consideration of evidence from well-established in vitro or in vivo assays. The wealth of information on underlying mechanisms of LQT1-LQT3 and recent HT patch clamp data support consideration of patch clamp data together (for LQT1 and LQT2) with information from biochemical trafficking assays as meeting the PS3 criterion of well established assays, able to provide ‘strong’ evidence for functional pathogenicity of identified VUS.

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Genetic Studies of Hypertrophic Cardiomyopathy in Singaporeans Identify Variants in TNNI3 and TNNT2 That Are Common in Chinese Patients

Circulation Genomic and Precision Medicine ◽

10.1161/circgen.119.002823 ◽

2020 ◽

Vol 13 (5) ◽

pp. 424-434

Author(s):

Chee Jian Pua ◽

Nevin Tham ◽

Calvin W.L. Chin ◽

Roddy Walsh ◽

Chiea Chuen Khor ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

East Asian ◽

Left Ventricular ◽

Chinese Patients ◽

Disease Genes ◽

Variant Of Uncertain Significance ◽

Variants Of Uncertain Significance ◽

Functional Studies ◽

Uncertain Significance

Background: To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry. Methods: We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3634), compared findings with additional populations and White HCM cohorts (n=6179), and performed in vitro functional studies. Results: Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (pathogenic/likely pathogenic: 18%, P <0.0001) but an excess of variants of uncertain significance (24%, P <0.0001), as compared to Whites (pathogenic/likely pathogenic: 31%, excess of variants of uncertain significance: 7%). Two missense variants in thin filament encoding genes were commonly seen in Singaporean HCM (TNNI3:p.R79C, disease allele frequency [AF]=0.018; TNNT2:p.R286H, disease AF=0.022) and are enriched in Singaporean HCM when compared with Asian controls (TNNI3:p.R79C, Singaporean controls AF=0.0055, P =0.0057, genome aggregation database-East Asian AF=0.0062, P =0.0086; TNNT2:p.R286H, Singaporean controls AF=0.0017, P <0.0001, genome aggregation database-East Asian AF=0.0009, P <0.0001). Both these variants have conflicting annotations in ClinVar and are of low penetrance (TNNI3:p.R79C, 0.7%; TNNT2:p.R286H, 2.7%) but are predicted to be deleterious by computational tools. In population controls, TNNI3:p.R79C carriers had significantly thicker left ventricular walls compared with noncarriers while its etiological fraction is limited (0.70 [95% CI, 0.35–0.86]) and thus TNNI3:p.R79C is considered variant of uncertain significance. Mutant TNNT2:p.R286H iPSC-CMs (induced pluripotent stem cells derived cardiomyocytes) show hypercontractility, increased metabolic requirements, and cellular hypertrophy and the etiological fraction (0.93 [95% CI, 0.83–0.97]) support the likely pathogenicity of TNNT2:p.R286H. Conclusions: As compared with Whites, Chinese HCM patients commonly have low penetrance risk alleles in TNNT2 or TNNI3 but exhibit few clinically actionable HCM variants overall. This highlights the need for greater study of HCM genetics in non-White populations.

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Novel MSX1 variants identified in families with nonsyndromic oligodontia

International Journal of Oral Science ◽

10.1038/s41368-020-00106-0 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Jinglei Zheng ◽

Miao Yu ◽

Haochen Liu ◽

Tao Cai ◽

Hailan Feng ◽

...

Keyword(s):

Current Data ◽

Phenotypic Characterization ◽

Functional Evaluation ◽

Gene Variants ◽

Chinese Families ◽

Whole Exome ◽

Bioinformatics Databases ◽

Uncertain Significance ◽

Vitro Analysis

AbstractThe goal of this study was to identify MSX1 gene variants in multiple Chinese families with nonsyndromic oligodontia and analyse the functional influence of these variants. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify the causal gene variants in five families with nonsyndromic oligodontia, and a series of bioinformatics databases were used for variant confirmation and functional prediction. Phenotypic characterization of the members of these families was described, and an in vitro analysis was performed for functional evaluation. Five novel MSX1 heterozygous variants were identified: three missense variants [c.662A>C (p.Q221P), c.670C>T (p.R224C), and c.809C>T (p.S270L)], one nonsense variant [c.364G>T (p.G122*)], and one frameshift variant [c.277delG (p.A93Rfs*67)]. Preliminary in vitro studies demonstrated that the subcellular localization of MSX1 was abnormal with the p.Q221P, p.R224C, p.G122*, and p.A93Rfs*67 variants compared to the wild type. Three variants (p.Q221P, p.G122*, and p.A93Rfs*67) were classified as pathogenic or likely pathogenic, while p.S270L and p.R224C were of uncertain significance in the current data. Moreover, we summarized and analysed the MSX1-related tooth agenesis positions and found that the type and variant locus were not related to the severity of tooth loss. Our results expand the variant spectrum of nonsyndromic oligodontia and provide valuable information for genetic counselling.

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Most myopathic lamin variants aggregate: a functional genomics approach for assessing variants of uncertain significance

npj Genomic Medicine ◽

10.1038/s41525-021-00265-x ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Corey L. Anderson ◽

Emma R. Langer ◽

Timothy C. Routes ◽

Seamus F. McWilliams ◽

Igor Bereslavskyy ◽

...

Keyword(s):

Protein Misfolding ◽

Striated Muscle ◽

Induced Pluripotent Stem Cell ◽

Functional Assay ◽

Human Cardiomyocytes ◽

Variants Of Uncertain Significance ◽

Uncertain Significance ◽

Data Driven Approach ◽

The Impact

AbstractHundreds of LMNA variants have been associated with several distinct disease phenotypes. However, genotype–phenotype relationships remain largely undefined and the impact for most variants remains unknown. We performed a functional analysis for 178 variants across five structural domains using two different overexpression models. We found that lamin A aggregation is a major determinant for skeletal and cardiac laminopathies. An in vitro solubility assay shows that aggregation-prone variants in the immunoglobulin-like domain correlate with domain destabilization. Finally, we demonstrate that myopathic-associated LMNA variants show aggregation patterns in induced pluripotent stem cell derived-cardiomyocytes (iPSC-CMs) in contrast to non-myopathic LMNA variants. Our data-driven approach (1) reveals that striated muscle laminopathies are predominantly protein misfolding diseases, (2) demonstrates an iPSC-CM experimental platform for characterizing laminopathic variants in human cardiomyocytes, and (3) supports a functional assay to aid in assessing pathogenicity for myopathic variants of uncertain significance.

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Genetic Testing in Patients with Hypertrophic Cardiomyopathy

International Journal of Molecular Sciences ◽

10.3390/ijms221910401 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10401

Author(s):

Jiri Bonaventura ◽

Eva Polakova ◽

Veronika Vejtasova ◽

Josef Veselka

Keyword(s):

Genetic Testing ◽

Hypertrophic Cardiomyopathy ◽

Genetic Basis ◽

Molecular Genetic ◽

Mendelian Disease ◽

Sarcomeric Proteins ◽

Pathogenic Variants ◽

Large Numbers ◽

Uncertain Significance ◽

Common Clinical Practice

Hypertrophic cardiomyopathy (HCM) is a common inherited heart disease with an estimated prevalence of up to 1 in 200 individuals. In the majority of cases, HCM is considered a Mendelian disease, with mainly autosomal dominant inheritance. Most pathogenic variants are usually detected in genes for sarcomeric proteins. Nowadays, the genetic basis of HCM is believed to be rather complex. Thousands of mutations in more than 60 genes have been described in association with HCM. Nevertheless, screening large numbers of genes results in the identification of many genetic variants of uncertain significance and makes the interpretation of the results difficult. Patients lacking a pathogenic variant are now believed to have non-Mendelian HCM and probably have a better prognosis than patients with sarcomeric pathogenic mutations. Identifying the genetic basis of HCM creates remarkable opportunities to understand how the disease develops, and by extension, how to disrupt the disease progression in the future. The aim of this review is to discuss the brief history and recent advances in the genetics of HCM and the application of molecular genetic testing into common clinical practice.

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Thermodynamic destabilization informs pathogenicity assessment of a variant of uncertain significance in cardiac myosin binding protein C

10.1101/789081 ◽

2019 ◽

Author(s):

Maria Rosaria Pricolo ◽

Elías Herrero-Galán ◽

Cristina Mazzaccara ◽

Maria Angela Losi ◽

Jorge Alegre-Cebollada ◽

...

Keyword(s):

Genetic Testing ◽

Rna Splicing ◽

Protein C ◽

Binding Protein ◽

Cardiac Myosin ◽

Variants Of Uncertain Significance ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Uncertain Significance ◽

Myosin Binding

ABSTRACTIn the era of Next Generation Sequencing (NGS), genetic testing for inherited disorders identifies an ever-increasing number of variants whose pathogenicity remains unclear. These variants of uncertain significance (VUS) limit the reach of genetic testing in clinical practice. The VUS for Hypertrophic Cardiomyopathy (HCM), the most common familial heart disease, constitute over 60% of entries for missense variants shown in ClinVar database. We have studied a novel VUS (c.1809T>G-p.I603M) in the most frequently mutated gene in HCM, MYBPC3, which codes for cardiac myosin-binding protein C (cMyBPC). Our determinations of pathogenicity integrate bioinformatics evaluation and functional studies of RNA splicing and protein thermodynamic stability. In silico prediction and mRNA analysis indicated no alteration of RNA splicing induced by the variant. At the protein level, the p.I603M mutation maps to the C4 domain of cMyBPC. Although the mutation does not perturb much the overall structure of the C4 domain, the stability of C4 I603M is severely compromised as detected by circular dichroism and differential scanning calorimetry experiments. Taking into account the highly destabilizing effect of the mutation in the structure of C4, we propose reclassification of variant p.I603M as likely pathogenic. Looking into the future, the workflow described here can be used to refine the assignment of pathogenicity of variants of uncertain significance in MYBPC3.

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Interpreting TMEM67 missense variants of uncertain significance (VUS) in an animal model

10.1101/2021.06.17.448799 ◽

2021 ◽

Author(s):

Karen I Lange ◽

Sunayna Best ◽

Sofia Tsiropoulou ◽

Ian Berry ◽

Colin A Johnson ◽

...

Keyword(s):

Animal Model ◽

Molecular Genetic ◽

Genetic Diagnosis ◽

Knock Out ◽

Variants Of Uncertain Significance ◽

C Elegans ◽

In Vivo Animal Model ◽

Uncertain Significance

Purpose: A molecular genetic diagnosis is essential for accurate counselling and management of patients with ciliopathies. Uncharacterized missense alleles are often classified as variants of uncertain significance (VUS) and are not clinically useful. In this study, we explore the use of a tractable animal model (C. elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a gene frequently mutated as a cause of ciliopathies. Methods: CRISPR/Cas9 gene editing was used to generate homozygous worm strains carrying TMEM67 patient variants. Quantitative phenotypic assays (dye filling, roaming, chemotaxis) assessed cilia structure and function. Results were validated by genetic complementation assays in a human TMEM67 knock-out hTERT-RPE1 cell line. Results: Quantitative assays in C. elegans distinguished between known benign (Asp359Glu, Thr360Ala) and pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of seven missense VUS alleles predicted two benign (Cys173Arg, Thr176Ile) and four pathogenic variants (Cys170Tyr, His782Arg, Gly786Glu, His790Arg). Results from one VUS (Gly979Arg) were inconclusive in worms, but additional in vitro validation suggested it was likely benign. Conclusion: Efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model that allows stratification and rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.

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Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance

eLife ◽

10.7554/elife.71137 ◽

2022 ◽

Vol 11 ◽

Author(s):

Hirokazu Kimura ◽

Raymond M Paranal ◽

Neha Nanda ◽

Laura D Wood ◽

James R Eshleman ◽

...

Keyword(s):

Functional Characterization ◽

Ductal Adenocarcinoma ◽

Proliferation Assay ◽

Variants Of Uncertain Significance ◽

Pathogenic Variants ◽

Deleterious Alleles ◽

Increased Risk ◽

Uncertain Significance

Pathogenic germline CDKN2A variants are associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC). CDKN2A variants of uncertain significance (VUSs) are reported in up to 4.3% of patients with PDAC and result in significant uncertainty for patients and their family members as an unknown fraction are functionally deleterious, and therefore, likely pathogenic. Functional characterization of CDKN2A VUSs is needed to reclassify variants and inform clinical management. 29 germline CDKN2A VUSs previously reported in patients with PDAC or in ClinVar were evaluated using a validated in vitro cell proliferation assay. 12 of the 29 CDKN2A VUSs were functionally deleterious (11 VUSs) or potentially functionally deleterious (1 VUS) and were reclassified as likely pathogenic variants. Thus, over 40% of CDKN2A VUSs identified in patients with PDAC are functionally deleterious and likely pathogenic. When incorporating VUSs found to be functionally deleterious, and reclassified as likely pathogenic, the prevalence of pathogenic/likely pathogenic CDKN2A in patients with PDAC reported in the published literature is increased to up to 4.1% of patients, depending on family history. Therefore, CDKN2A VUSs may play a significant, unappreciated role in risk of pancreatic cancer. These findings have significant implications for the counselling and care of patients and their relatives.

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