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

Abstract

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.

Keywords: cardiomyopathies; hypertrophy; population; troponin I; troponin T.

Figure 1.

Overview of the study design outlining the major components of the study and the various disease and control populations used. ACGV indicates atlas of cardiac genetic variation^,,; AF, allele frequency; CMR, cardiac magnetic resonance; EA, East Asian; exVUS, excess variant of uncertain significance; HCM, hypertrophic cardiomyopathy; LP, likely pathogenic; P, pathogenic; and SG, Singaporean. *https://www.cardioclassifier.org/15

Figure 2.

Pathogenic/likely pathogenic variants and excess variant of uncertain significance (VUS; exVUS = caseVUS%−controlVUS%) in hypertrophic cardiomyopathy (HCM) genes in Singaporean patients with HCM. A, Fifteen genes were assessed including major sarcomeric genes (MYBPC3, MYH7, TNNT2, and TNNI3), other sarcomeric genes (ACTC, MYL2, MYL3, TPM1, and TNNC1), other HCM genes (CSRP3, FHL1, and PLN), and geno/phenocopies (GLA, LAMP, and PRKAG2). The number and percentage refer to the total pathogenic/likely pathogenic (P/LP) case per gene (darker shade) while the number and percentage in parentheses refer to the total case excess of P, LP, and exVUS (lighter shade) as compared to genome aggregation database (gnomAD). B, The secondary pie charts show the proportion of all Singaporean HCM patients with TNNI3:p.R79C or TNNT2:p.R286H VUS as compared to other VUS in these genes, depicted overall in (A) by lighter shading.

Figure 3.

TNNI3:p.R79C and TNNT2:p.R286H: Cardiac indices in the general population. Violin plots comparing (A) indexed left ventricular (LV) mass (LVMi) and (B) left ventricular maximum wall thickness (LVMWT) in population controls with or without TNNI3:p.R79C or TNNT2:p.R286H, derived using cardiac magnetic resonance (CMR). Data were represented as median ± interquartile range (IQR) in a violin box-and-whiskers plot (Tukey rule) with the whiskers representing 1.5× IQR and outliers were plotted as individual dots. P values of regression models were derived using ANOVA where a significance cutoff of P<0.05 was used.

Figure 4.

Contractile characterization, metabolic flux, and cell size analysis of TNNT2:p.R286H iPSC-CMs (induced pluripotent stem cells derived cardiomyocytes). A, Comparison of the percentage sarcomere shortening and (B) relaxation duration for isogenic wild type (WT), TNNT2:p.R286H (R286H/+) and established (+) hypertrophic variant of MYH7:p.R403Q (R403Q/+) iPSC-CMs. C, Measurement of oxygen consumption rate (OCR) and (D) extracellular acidification rate (ECAR) in WT, R286H/+ and R403Q/+ cardiomyocytes using the Seahorse platform and (E) unconstrained cell size in WT (n=586 cells), R286H/+ (n=408 cells) and (+) R403Q/+ (n=488 cells). All iPSC-CMs were generated by mutating an isogenic line, denoted TTN-GFP PGP1.^, Two or more differentiations were studied from 2 independent clones for each genotype. Data, mean ± SEM. Student t test for each mutant compared with WT was used where a significance cutoff of P<0.05 was used.