Paralogue annotation identifies novel pathogenic variants in patients with Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia

Abstract

Background: Distinguishing genetic variants that cause disease from variants that are rare but benign is one of the principal challenges in contemporary clinical genetics, particularly as variants are identified at a pace exceeding the capacity of researchers to characterise them functionally.

Methods: We previously developed a novel method, called paralogue annotation, which accurately and specifically identifies disease-causing missense variants by transferring disease-causing annotations across families of related proteins. Here we refine our approach, and apply it to novel variants found in 2266 patients across two large cohorts with inherited sudden death syndromes, namely catecholaminergic polymorphic ventricular tachycardia (CPVT) or Brugada syndrome (BrS).

Results: Over one third of the novel non-synonymous variants found in these studies, which would otherwise be reported in a clinical diagnostics setting as 'variants of unknown significance', are categorised by our method as likely disease causing (positive predictive value 98.7%). This identified more than 500 new disease loci for BrS and CPVT.

Conclusions: Our methodology is widely transferable across all human disease genes, with an estimated 150 000 potentially informative annotations in more than 1800 genes. We have developed a web resource that allows researchers and clinicians to annotate variants found in individuals with inherited arrhythmias, comprising a referenced compendium of known missense variants in these genes together with a user-friendly implementation of our approach. This tool will facilitate the interpretation of many novel variants that might otherwise remain unclassified.

Keywords: Cardiovascular Medicine; Clinical Genetics; Congenital Heart Disease; Genetics.

Figure 1

An overview of paralogue annotation. (1) Paralogues (evolutionarily related genes, with homologous sequence and protein domain structures) are identified for a gene of interest. A subset of paralogues for SCN5A is shown here for illustration. (2) Protein sequences of paralogues are aligned, identifying functionally equivalent amino acids across the protein family. (3) Disease-causing variants in paralogues are identified from previous literature reports, and their locations are mapped to the gene of interest. Variants at these sites have a high probability of pathogenicity.

Figure 2

Genetic variation in cohorts with inherited arrhythmia syndromes. RYR2 or SCN5A were sequenced in 2266 patients with an inherited arrhythmia syndrome, as shown. Three hundred and fifty-seven variants were identified in 511 patients, of which 153 were novel missense variants. In the absence of segregation or functional data, these would typically be reported as variants of unknown significance. However, paralogue annotation provided additional information for 65 (36%) variants, that would further inform a clinical genetic report. BrS, Brugada syndrome; CPVT, catecholaminergic polymorphic ventricular tachycardia.

Figure 3

Disease-causing variation in human ryanodine receptors, RYR1 and RYR2. An alignment of RYR1 and RYR2 reveals the structural similarity of the proteins and homologous clustering of pathogenic variation in these related proteins. The protein is represented in light grey, with reported ‘mutation hotspots’ marked in dark grey, and exon boundaries highlighted. The locations of missense variants previously reported to be pathogenic are shown with black lines above and below the protein graphic—longer lines indicate more than one pathogenic DNA variant affecting the same protein residue.

Figure 4

A web-based application makes paralogue annotation easily accessible for genes causing inherited arrhythmia syndromes. A web-based application is available at http://cardiodb.org/Paralogue_Annotation/. Users enter the position of a novel variant using complementary DNA or protein coordinates: in this example a substitution has been found in RYR2, affecting glycine residue 357. This residue maps to RYR1 residue 341, and two cDNA variants at that location (c.1021G>A and c.1021G>C) that each cause substitution of Arg for Gly at that position have been reported to cause malignant hyperthermia. Users should check the alignment quality—here the mapping quality is high: the surrounding region is highly homologous, the reference amino acid is the same in both proteins, and the alignment has a high consensus score. Pubmed links give access to the reports relating to the paralogue mutation(s), allowing users to assess the quality of evidence for pathogenicity. Here functional characterisation has been performed on this variant in the highlighted publication, adding confidence that the variant is disease causing and the residue is intolerant of variation in both RYR1 and its paralogue RYR2.