Enhancing rare variant interpretation in inherited arrhythmias through quantitative analysis of consortium disease cohorts and population controls

Abstract

Purpose: Stringent variant interpretation guidelines can lead to high rates of variants of uncertain significance (VUS) for genetically heterogeneous disease like long QT syndrome (LQTS) and Brugada syndrome (BrS). Quantitative and disease-specific customization of American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines can address this false negative rate.

Methods: We compared rare variant frequencies from 1847 LQTS (KCNQ1/KCNH2/SCN5A) and 3335 BrS (SCN5A) cases from the International LQTS/BrS Genetics Consortia to population-specific gnomAD data and developed disease-specific criteria for ACMG/AMP evidence classes-rarity (PM2/BS1 rules) and case enrichment of individual (PS4) and domain-specific (PM1) variants.

Results: Rare SCN5A variant prevalence differed between European (20.8%) and Japanese (8.9%) BrS patients (p = 5.7 × 10^-18) and diagnosis with spontaneous (28.7%) versus induced (15.8%) Brugada type 1 electrocardiogram (ECG) (p = 1.3 × 10^-13). Ion channel transmembrane regions and specific N-terminus (KCNH2) and C-terminus (KCNQ1/KCNH2) domains were characterized by high enrichment of case variants and >95% probability of pathogenicity. Applying the customized rules, 17.4% of European BrS and 74.8% of European LQTS cases had (likely) pathogenic variants, compared with estimated diagnostic yields (case excess over gnomAD) of 19.2%/82.1%, reducing VUS prevalence to close to background rare variant frequency.

Conclusion: Large case-control data sets enable quantitative implementation of ACMG/AMP guidelines and increased sensitivity for inherited arrhythmia genetic testing.

Keywords: ACMG/AMP guidelines; Brugada; LQTS; variant interpretation.

Fig. 1. For rare nontruncating variants in autosomal dominant disease, evidence classes and American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) rules (rule codes from Richards et al.) can be broadly grouped by their power to distinguish between pathogenic and benign variants (y-axis) and the likelihood that such evidence will be available (x-axis).

Variant-specific evidence (such as cosegregation in family pedigrees) is powerful but often unavailable for genetically heterogeneous diseases. Supporting evidence (such as population frequency) can be applied to most variants but is rarely sufficient for definitive classification. If available, data from case–control studies, relating to enrichment of specific variants or classes of variants, provide powerful gene/disease-specific evidence and help to address the high false negative rate associated with stringent contemporary guidelines.

Fig. 2. Frequency of rare variants in KCNQ1, KCNH2 and SCN5A in inherited arrhythmia cohorts and gnomAD population controls.

(a) The odds ratio for disease association for long QT syndrome (LQTS) (KCNQ1, KCNH2, SCN5A) and Brugada syndrome (BrS) (SCN5A) stratified by filtering allele frequency, based on the prevalence of rare variants in the European arrhythmia cohorts and gnomAD exomes. Data for each bin are plotted at the upper frequency cutoff. Error bars represent 95% confidence intervals (CIs). The dashed gray line indicates an odds ratio (OR) of 1. (b) Proportion of cases in the BrS and LQTS European and Japanese cohorts with rare nontruncating (missense and inframe insertions/deletions) and truncating (frameshift, nonsense, splice) variants (blue) and comparison with the frequency of such variants in population-specific gnomAD data sets (gray). The darker shades indicate the rarest variants corresponding to an estimated penetrance of ≥50% filtering allele frequencies (FAF < 1.0 × 10⁻⁵ and 1.1 × 10⁻⁵ for BrS and LQTS respectively), while the lighter shades represent variants in the 10–50% penetrance range.

Fig. 3. Identification of ion channel gene domains enriched in case variants and effect of case-control analyses on variant classification in inherited arrhythmia patients.

(a) Distribution of rare, nontruncating variants in the primary Brugada syndrome (BrS) and long QT syndrome (LQTS) cohorts across the domains of KCNQ1, KCNH2, and SCN5A and equivalent variant classes in gnomAD (full data set). Domain coordinates are derived from UniProt entries, with the exception of the KCNQ1 C-terminus highly conserved regions (from Kapplinger et al.) and the KCNH2 N-terminus cluster (based on variant distribution observed in this cohort and published referral cohort). Regions with poor coverage in gnomAD exome sequencing, and therefore excluded from etiological fraction (EF) calculations, are in white. Darker gray indicates higher variant density (overlapping variants not plotted separately). The coordinates describe amino acid position. (b–d) Effect of case–control evidence (PM1/PS4 rules) on American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) classification of rare nontruncating variants. For BrS, the proportion of cases with pathogenic, likely pathogenic, and variants of uncertain significance (VUS) are displayed before and after use of these evidence classes, for European (b) and Japanese (c) cases. Classification using case–control evidence for both European and Japanese LQTS cases is shown in (d). The sensitivity of variant classification methods can be measured by comparison with the rate of rare benign variation in gnomAD (gray); any excess beyond this is expected to reflect pathogenic variation in cases and therefore represents the target cohort yield of pathogenic variants.