Interpreting Incidentally Identified Variants in Genes Associated with Catecholaminergic Polymorphic Ventricular Tachycardia in a Large Cohort of Clinical Whole Exome Genetic Test Referrals

Authors, Journal, Affiliations, Type, DOI

• Authors: Andrew P. Landstrom, Andrew L. Dailey-Schwartz (co-first authors), Jill A. Rosenfeld, Yaping Yang, Margaret J. McLean, Christina Y. Miyake, Santiago O. Valdes, Yuxin Fan, Hugh D. Allen, Daniel J. Penny, Jeffrey J. Kim
• Journal: Circulation: Arrhythmia and Electrophysiology 2017 April;10(4):e004742
• Affiliations: Department of Pediatrics (Section of Pediatric Cardiology) and Cardiovascular Research Institute, Baylor College of Medicine; Baylor Miraca Genetics Laboratories, Houston, Texas
• Type: Original article — retrospective genetic cohort study
• DOI: 10.1161/CIRCEP.116.004742

Overview

This study asks whether incidentally identified RYR2 and CASQ2 variants found on clinical whole-exome sequencing (WES) are markers of CPVT. It compares 6,517 WES referrals from Baylor Miraca Genetics Laboratories with 60,706 ostensibly healthy ExAC controls and a 155-patient CPVT case cohort, and adds retrospective clinical review of 140 variant-positive referrals from Texas Children's Hospital. Rare CPVT-gene variants were present in 8.8% of WES referrals versus 6.0% of controls and 60.1% of clinically robust CPVT cases; 97.7% of the WES variants were VUS, they co-localised with control rather than case residues, and almost none fell in canonical RyR2 hotspots. Among referrals with low pre-test suspicion, no individual had clinical CPVT. The authors conclude that incidental CPVT-gene VUSs are unlikely to represent disease and that a targeted CPVT panel, not WES, remains the preferred diagnostic test.

Keywords

Catecholaminergic polymorphic ventricular tachycardia; genetic testing; genomics; RYR2 gene; whole exome sequencing

Key Takeaways

Cohorts and variant selection

• Clinical WES cohort: 6,517 individuals from 6,477 families (54.2% male, median age 6.1 years) referred to Baylor Miraca Genetics Laboratories between October 2011 and October 2015, independent of referral diagnosis.
• Variants were included if they were in coding or predicted splice sequence of RYR2 or CASQ2 and were classified as likely pathogenic or VUS at the time of testing; synonymous and "not pathogenic" variants were excluded. TRDN and CALM1 were not analysed.
• CPVT case cohort: 155 clinically diagnosed patients, of whom 33 were "clinically robust" (exertional syncope plus documented bidirectional or polymorphic VT).
• Control cohort: 60,706 ostensibly healthy ExAC individuals, including 33,370 non-Finnish Europeans, with variants filtered at minor allele frequency <0.01.
• Texas Children's Hospital subgroup: 140 variant-positive probands from 136 families with clinical records available for retrospective review.

Prevalence of CPVT-gene variants in unselected WES referrals

• 8.8% (573/6,517) carried a rare RYR2 or CASQ2 variant; the rate was unchanged when only unrelated probands were analysed.
• 547 carriers had one variant, 25 had two and one had three; there were 599 variants in total.
• 97.7% (585/599) were VUS and only 2.3% (14) were classified likely pathogenic.
• RYR2 accounted for 84.5% of variants (506) and CASQ2 for 15.5% (93). Likely pathogenic variants were proportionally more frequent in CASQ2 (8.6%) than RYR2 (1.2%; P = 0.0005).
• 16.9% of variants were radical (nonsense, insertion/deletion or canonical splice) and 83.1% were missense; all likely pathogenic variants were heterozygous, and four variant-positive individuals carried homozygous VUSs.

Comparison with CPVT cases and healthy controls

• 47.1% (73/155) of CPVT cases were mutation-positive, rising to 60.1% (20/33) of clinically robust cases — more than six-fold the 8.8% WES rate (P < 0.0001).
• Controls had a rare-variant rate of 6.0%, significantly lower than the WES cohort (P < 0.0001).
• The case excess was driven by missense variants (57.4% of cases vs 5.2% of controls and 7.6% of WES referrals); radical-variant rates were similar across groups (2.6%, 0.9% and 1.6%).
• The resulting signal-to-noise ratio for CPVT genetic testing was approximately 10:1, comparable to LQTS (~15:1) and HCM (~12:1).

Topology, co-localisation and hotspots

• WES variants mapped to 278 RyR2 residues, case variants to 140 and control variants to 973.
• WES variants overlapped case residues in only 7.9% of instances but control residues in 60.4% — about 7.5-fold more likely to match healthy variation.
• 10.5% of WES-altered residues fell within one of the three canonical RyR2 hotspots, versus 32.2% of control variants and 80.2% of CPVT-case variants (P < 0.0001). WES variants were distributed across the linear sequence without clustering.

Clinical review of variant-positive referrals

• In the 140 Texas Children's probands, 85.7% were referred for neurological disease (66.4% developmental delay), only 12.9% had any cardiac indication and 5% were referred for cardiac disease alone.
• 2/140 (1.4%) had ventricular ectopy; 98.6% had none. Two had syncope (1.4%) and one had a family history of sudden death (0.7%).
• The only individual with a plausible channelopathy was the single cardiac-only referral with a structurally normal heart, syncope and a family history of sudden death; he carried RyR2-G3946S (likely pathogenic) and was classified as "possible CPVT". A 4-year-old with frequent PVCs had a RAB23 variant explaining his syndromic phenotype and a RyR2 VUS.
• No individual with low pre-test suspicion had clinical CPVT, although later manifestation cannot be excluded.

Interpretation and testing strategy

• An incidental CPVT-gene VUS in a patient without clinical suspicion is unlikely to be a marker of CPVT; variant location, class and population context should be weighed, not the label alone.
• Targeted CPVT panel testing remains the optimal diagnostic test for suspected CPVT; WES is unlikely to add diagnostic value as the initial platform.
• The high background rate (6.0%) and ~10:1 signal-to-noise ratio argue for interpreting incidental findings in the context of pre-test probability rather than reporting them as disease.

Limitations of the document

• Retrospective design. Variants were classified by the testing laboratory at the time of WES, not re-adjudicated with current ACMG/AMP criteria or ClinGen gene curation.
• Referral bias. The cohort is a paediatric diagnostic WES population (median age 6.1 years), 85.7% referred for neurological disease; it is not a general population sample, and the 8.8% rate may not generalise to adult or cardiology referrals.
• Gene scope. Only RYR2 and CASQ2 were analysed; TRDN, CALM1/2/3 and TECRL were excluded, so the total CPVT-gene background burden is underestimated.
• No functional or segregation data. Variant pathogenicity was not tested in vitro or by family co-segregation, so the "likely pathogenic" and VUS categories are laboratory classifications rather than experimentally validated calls.
• Control comparison depends on ExAC. The ExAC cohort differs in ancestry composition and sequencing pipeline from the WES referrals, and rare-variant frequencies are sensitive to ancestry and coverage.
• Clinical follow-up is incomplete. The Texas Children's subgroup is small and single-centre, records were retrospective, and late-onset CPVT in currently asymptomatic carriers cannot be excluded.
• Authors' conflicts: two authors receive salary support from Baylor Miraca Genetics Laboratories, the commercial laboratory that generated the WES data.

Key Concepts Mentioned

• entities/CPVT — the disease
• entities/RYR2 — the major CPVT gene and hotspot topology
• entities/CASQ2 — the recessive CPVT2 gene
• concepts/Incidental-Cardiovascular-Variants — the interpretation framework
• concepts/ACMG-Secondary-Findings — reporting policy for incidental CPVT variants
• concepts/Variant-Reclassification — why "VUS" labels are provisional
• concepts/ClinGen-Gene-Disease-Validity — gene-disease validity context

Key Entities Mentioned

• Baylor Miraca Genetics Laboratories — source of the clinical WES cohort
• Texas Children's Hospital — clinical review cohort
• Baylor College of Medicine — academic affiliation
• Exome Aggregation Consortium (ExAC) — control cohort
• Illumina HiSeq and VCRome 2.1 capture — sequencing platform

Wiki Pages Updated

• Created: wiki/sources/cpvt-exome-circep-2017.md
• Updated: wiki/concepts/Incidental-Cardiovascular-Variants.md
• Updated: wiki/entities/CPVT.md
• Updated: wiki/entities/RYR2.md
• Updated: wiki/entities/CASQ2.md
• Updated: wiki/concepts/ACMG-Secondary-Findings.md
• Updated: wiki/sourceindex.md
• Updated: log.md