Redefining the Genetic Architecture of Hypertrophic Cardiomyopathy: Role of Intermediate-Effect Variants

Abstract

Background: Hypertrophic cardiomyopathy (HCM) is a genetically heterogeneous disorder linked primarily to rare variants in sarcomeric genes, although recently certain nonsarcomeric genes have emerged as important contributors. Nonmendelian genetic variants with reproducible moderate-effect sizes and low penetrance, intermediate-effect variants (IEVs), can play a crucial role in modulating disease expression. Understanding the clinical impact of IEVs is crucial to unravel the complex genetic architecture of HCM.

Methods: We conducted an ancestry-based enrichment analysis of 14 validated HCM genes, including the 9 core sarcomeric and 5 nonsarcomeric genes (ALPK3, CSRP3, FHOD3, FLNC, and TRIM63). Enrichment of intermediate frequency missense variants was evaluated in 10 981 patients with HCM, 4030 internal controls of European-ancestry, and 590 000 external controls from gnomAD non-Finnish Europeans. The population-attributable fraction was calculated to assess contribution of IEVs to HCM. Age-related disease penetrance, phenotypic severity (left ventricular maximum wall thickness), and major adverse cardiac events were analyzed in 11 991 HCM cases of the whole cohort according to 5 genetic groups: genotype negative, isolated IEV, monogenic, monogenic+IEV, and double monogenic.

Results: Fourteen IEVs in 8 genes were identified in 731 individuals (6.1% of the cohort), of whom 570 patients (4.8%) had IEVs in isolation: 198 (34.7%) in sarcomeric genes and 372 (65.3%) in nonsarcomeric genes. The contribution of IEVs to HCM genetics according to population-attributable fraction was estimated to be 4.9% (95% CI, 3.2-6.7). A significant gradient in penetrance, phenotypic severity, and major adverse cardiac events was observed across genetic groups. Compared with genotype-negative patients, IEV carriers displayed a younger median age at diagnosis (59 years of age [95% CI, 46-69] versus 61 years [95% CI, 49-70]; P=0.0073) and a higher mean left ventricular maximum wall thickness (18.1±3.7 versus 19.0±4.3; P=0.0043). IEVs also modified disease expression in individuals with monogenic variants, causing a more aggressive phenotype than in individuals from the monogenic-only group with HCM onset at younger age and a higher left ventricular maximum wall thickness (all P<0.0001), with major adverse cardiac event-free survival being significantly lower (93.3% versus 69.3% at 70 years of age; P<0.0001).

Conclusions: IEVs are present in 6.1% of HCM cases and account for 4.8% of HCM genetic burden. IEVs also influence disease severity and outcomes, particularly when combined with monogenic disease-causing variants. Evaluation of IEVs should be considered when HCM genetic testing is performed.

Keywords: cardiomyopathy, hypertrophic; genetic predisposition to disease; genetic testing; genetic variation; inheritance patterns; penetrence; risk factors.

Figure 1.

Study IEVs selection flow chart and methodology for filtering and selection of IEVs. A, Methodology for variant selection and cohort composition. Global numbers for cases and controls, both before and after selection of the non-Finnish European (NFE) with principal component analysis (PCA), the genes targeted for exploration, and the criteria applied for variant filtering. B, Selection and validation of intermediate-effect variants (IEVs). Comparison of strategies and enrichment analysis with internal and external controls, global and PCA analysis. Venn diagram illustrates the overlap in variants identified by the global and PCA-adjusted strategies. Numbers represent the count of variants meeting specified criteria for each strategy and their overlap. C, Simplified IEV filtering and selection. FAF indicates filtered allele frequency; HCM, hypertrophic cardiomyopathy; and OR, odds ratio.

Figure 2.

Architecture of variants identified in the HCM cohort. A, Genetic variants identified in the study, with the filtered allele frequency in gnomAD v4.1 version on the x axis, and the enrichment (odds ratio [OR]) of the variants in hypertrophic cardiomyopathy (HCM) cases compared with internal controls (using principal component analysis [PCA] analysis) on the y axis. Black dots correspond to the variants that were significantly enriched (OR ≥1) after PCA validation, selecting individuals of European ethnicity in HCM cases and internal controls, and using gnomAD non-Finnish European (NFE) data. Blue line represents the linear regression line for the model of the variants enriched in our study (OR=10^−0.386.FAF^−0.465; R²=0.725, P<0.001), and red line represents the variants not significantly enriched (OR=10^−1.080.FAF^−0.417; R²=0.629, P<0.001). B, Intermediate-effect variants (IEVs), defined as those significantly enriched in HCM cases compared with internal and external controls, with an OR ≥2. Red dots represent variants validated in both global and PCA (NFE) analysis, and gray dots represent variants not significantly enriched in this last analysis. C, Monogenic variants, defined as those with an filtered allele frequency (FAF) <5×10⁻⁵ (0.005%) and significantly enriched in HCM cases compared with internal and external controls, with an OR ≥2. Green dots represent variants validated in both global and PCA (NFE) analysis. D, Near-monogenic variants (green dots), defined as those with an FAF between FAF >5×10⁻⁵ (0.005%) and 0.01 (1%), enriched in HCM cases with an OR ≥2, but an estimated penetrance >15% or an internal OR ≥15 (with a high penetrance to be considered IEV). E, Small-effect variants, defined as those with an FAF >0.01 (1%) and significantly enriched in HCM cases with an OR ≥1. Blue dots represent variants that were validated in both global and PCA (NFE) analysis, and gray dots represent variants not significantly enriched in internal PCA analysis. Tables S4, S5, S7, and S8 provide full details.

Figure 3.

Selected IEVs. Selected intermediate-effect variants (IEVs) with enrichment in hypertrophic cardiomyopathy (HCM) cases (European ancestry) vs internal controls and gnomADv4.1 non-Finnish European (NFE) individuals. Odds ratios (ORs) of the HCM cohort vs internal controls; variant classifications in ClinVar (Feb 2025); minor allele frequencies (MAF) in gnomADv4.1 populations; and additional evidence of pathogenicity. Numbers of biallelic cases and previous functional validation studies in human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) or animal models (Tables S10 and S11 provide details).

Figure 4.

Results of genetic testing. A, Genetic testing results in the whole cohort. B, Contribution of each gene among cases with positive genetic results: sarcomeric (blue bars), primary nonsarcomeric (red bars), and phenocopy genes (yellow bars). C, Proportion of pathogenic/likely pathogenic (P/LP) variants (diagnostic yield) in the whole cohort compared with carriers of intermediate-effect variants (IEVs). D, Co-occurrence rate of a second monogenic variant and IEVs in different genetic subpopulations. HCM indicates hypertrophic cardiomyopathy; and VOUS, variant of uncertain significance.

Figure 5.

Age at diagnosis, survival free of MACEs, and LVMWT according to genetic groups. A, Kaplan-Meier curves showing the age-related penetrance. B, Kaplan-Meier major adverse cardiac event (MACE)–free survival. C, Violin plots of left ventricular maximal wall thickness (LVMWT); black dots represent mean LVMWT. D, Proportion of individuals with severe left ventricular hypertrophy (LVMWT >25; left) and massive left ventricular hypertrophy (LVMWT >30; right). IEV indicates intermediate-effect variant.

Figure 6.

Age at diagnosis according to IEV zygosity (biallelic vs heterozygous carriers). Age at diagnosis in 9 intermediate-effect variants (IEVs) as described in the literature or in our cohort in homozygous/compound-heterozygous carriers compared with heterozygous carriers. Light blue bars represent homozygous/compound heterozygous carriers; gray bars represent heterozygous carriers (Table S10 provides details).