Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disease, defined by left ventricular (LV) wall thickness greater than 15 mm, in the absence of other loading conditions that could explain the hypertrophy (1). The degree, localization, and distribution of the hypertrophy are variable (2). The LV systolic function can be preserved, increased, or reduced (2). Consequently, HCM is characterized by a phenotypic heterogeneity that could be partly explained by the heterogeneity of the genetic underlying etiology.

The estimated prevalence of HCM is 1:500 in the general population based on the recognition of the disease phenotype (3–5). However, considering familial transmission, genotype-positive cases for sarcomeric genes, and subclinical cases, a higher prevalence (1:200) is reported (6).

The clinical manifestations of HCM typically occur between the ages of 20 and 40, although they can develop at any age (7). It is noteworthy that 50–60% of cases are diagnosed after the age of 30, reflecting the variable penetrance and expressivity of the disease (3). The average life expectancy for HCM patients is typically favorable, approaching 70 years in effectively managed cases (6), although outcomes are greatly dependent on the presence of risk factors for complications such as early onset of the disease, arrhythmias, and heart failure. The annual risk of sudden cardiac death (SCD) in high-risk patients is estimated to range from 0.5 to 1%, with the highest risk observed in young cases under 30 years of age (8).

HCM is widely recognized as a sarcomeric disease since the vast majority of patients carry variants within the eight core genes encoding sarcomeric proteins (9). Indeed, the myosin-binding protein C (MYBPC3) and the β-myosin heavy chain (MYH7) genes account for 50–70% of HCM cases that undergo genetic testing (1–3, 10, 11). The remaining patients harbor variants in other sarcomeric genes, such as myosin light chain (MLC) genes (MYL2 and MYL3), troponin encoding genes (TNNT2 and TNNI3), tropomyosin 1 (TPM1), and actin α-cardiac muscle 1 (ACTC1) (4, 11, 12). Collectively, actionable variants in sarcomeric-positive (sarc+) patients account for >90% of the totality of pathogenic (P) variants in HCM patients (12). Of note, these percentages vary widely depending on the studied populations, the clinical profiling, the sequencing method used (panel, whole-exome sequencing [WES], etc.), and variant prioritization criteria.

Several additional genes have been linked to HCM encoding non-sarcomeric proteins, including, but not restricted to, proteins of the Z-disk (ACTN2, TCAP, and VCL), calcium handling genes (TNNC1, RYR2, JPH2, PLN), and the proteasome (TRIM63) (4). Similarly to the sarcomeric genes, both inheritance models are reported in these genes with a predominance of the autosomal dominant pattern (12, 13). However, the contribution of these genes is minor, ranging from 0.06 to 8.7% based on combined published data involving genes with strong, moderate, and weak evidence of causality and HCM-associated genes supported only by functional data (13). These findings reinforce the fact that sarcomeric genes predominantly cause HCM and emphasize the significant proportion of the missing heritability in HCM (13). Therefore, up to 50% of HCM patients do not have an identifiable pathogenic disease-causing variant (14, 15). So far, this missing heritability is increasingly explained by the fact that sarcomeric-negative (Sarc-) and genotype-negative patients have a non-Mendelian or near-Mendelian disease caused by a joint effect of genetic and non-genetic factors (13, 16). Thus, the additive effect of allelic heterogeneity and additional genetic variants, along with variants in cis causing allelic imbalance, could also partially explain the missing heritability, the phenotypic variability, and the incomplete penetrance. In this line, many studies have suggested the oligogenic inheritance model in HCM patients lacking an identifiable highly penetrant variant (16–20). Furthermore, rare variants are broadly acknowledged among the plausible sources of missing heritability and the likely contributors to the oligogenic inheritance rather than common variants. Indeed, variants with strong effects are expected to be kept under selective pressure and remain at low to extremely low frequencies in the population (21, 22). Altogether, incomplete penetrance, variable expressivity, and missing heritability in HCM make the genetic diagnosis and clinical prognosis challenging.

The efficiency of WES reanalysis has been demonstrated to improve genetic testing yield, especially by adding the newly associated genes previously unrecognized as clinically relevant. It is estimated that the genetic diagnostic yield could be enhanced by approximately 15% when including new disease-gene associations, up-to-date software, variant frequency databases, and text mining for genetic and clinical re-evaluation (23). Moreover, it is recommended that the dataset initially analyzed over 2 years ago should be prioritized for reanalysis (24).

In this study, we sought to refine the initial Hypertension Genetic Epidemiology Network (HYPERGEN) analysis by focusing on HCM-causing genes (that are known and recently associated). Additionally, we assessed the usefulness of our reanalysis regarding the clinical actionability of the identified variants by applying the American College of Medical Genetics and Genomics/Association of Molecular Pathologists (ACMG/AMP) criteria.

This study was approved by an institutional review committee. All subjects gave informed consent for genetic studies. WES was performed on the NGS Illumina HiSeq2500 platform using Agilent SureSelect V6 technology (Genwiz, United States) (25).

Despite the advent of next-generation sequencing, approximately 50% of cases remain genotype-elusive. In this study, a reanalysis of exome data from 200 HCM patients was carried out 5 years after the initial analysis with the goal of refining the initial analysis, reporting all the relevant, prioritized variants, and particularly identifying rare variants within the novel HCM-associated genes.

In the present reanalysis, nearly 34% of patients were found to carry variants in MYBPC3 and MYH7 genes. This yield of sarc+ patients reached 37% when including patients with TNNT2, MYL2, and MYL3 variants.

Haploinsufficiency is the primary mechanism driving HCM linked to MYBPC3 (myosin binding protein C3) gene (38, 48). Thus, non-truncating variants (missense, in-frame indels, PTV predicted to escape non-sense mediated decay [NMD], and stop-loss) are prioritized whether they are predicted as pathogenic with high or low confidence. Our reanalysis yielded a total of 40 MYBPC3 variants, including 24 missense, 4 splice-site, 6 stopgain, and 6 frameshift variants. The most recurrent and common HCM causal variant MYBPC3, p.Arg502Trp, was identified in only three patients (1.5%). This variant was found in 2.4% of the European-descent patients (15, 49–51). However, the pathogenic MYBPC3: c1928-2A > G variant was the most commonly identified variant in the HYPERGEN cohort (n = 7; 3.5%). Notably, these variants are unique except for one patient (HCM-5) harboring a second MYBPC3 variant (p.Leu183Ile) (Table 1). Contrary to the MYBPC3 gene, mostly known with haploinsufficiency and allelic imbalance as the underlying mechanism of the disease, the pathophysiology mechanism of MYH7 (myosin heavy chain 7) missense variants is the dominant negative effect, which implies that the MYH7 gene is tolerant to loss of function (LoF) variants (10, 38, 39, 52, 53). Thus, prioritizing the MYH7 missense variant with a predicted deleterious impact according to different in silico algorithms may increase the identification rate of actionable variants. Moreover, gene regions in which rare and likely causal variants are significantly clustered; therefore, a new etiological fraction-based ACMG rule on rare missense MYH7 variants was proposed to improve genetic testing yield in HCM (30). All the identified MYH7 variants in this study are missense (n = 16) with 8 P/LP variants. Of note, variants in the MYH7 gene were associated with significant LV hypertrophy, which is the hallmark of HCM and an unfavorable prognosis compared to patients carrying variants in the other HCM genes (54).

We identified a small proportion of patients (3%) carrying variants in TNNT2, MYL2, and MYL3 genes. Only two variants in the TNNT2 gene were identified. The TNNT2 gene encodes the cardiac troponin T2 which is a regulatory protein of the thin filament troponin complex in the sarcomere playing a crucial role in contractility function (39, 55). More than 30 TNNT2 P/LP variants have been linked to HCM, with individual variants being unique and private to distinct families (55). Moreover, phenotypic variability was reported among patients carrying the same TNNT2 variant (55). The MLCs are composed of a regulatory light chain (MYL2) and an essential light chain (MYL3). These genes contribute to the stability of myosin head and the regulation of cardiac contraction by phosphorylation and Ca²⁺ binding (52). Despite these genes being considered well-established HCM genes, their contribution to HCM etiology is limited. A study by Borrelli et al. estimated that the contribution of genes encoding sarcomere thin filaments does not exceed 5% (11). In our cohort, no high or moderate confidence variants were found in ACTC1, TPM1, TNNC1, or TNNI3 genes. A recent study by Allouba et al. (56) showed that homozygous variants are more prevalent within MYL2 and MYL3 genes than within major sarcomeric HCM genes. In the HYPERGEN cohort, only one homozygous variant was identified (MYL3: p.Ala57Asp) in a patient with a family history of HCM.

Although HCM has been recognized as a monogenic disease for a long time, the wide utilization of high-throughput sequencing has demonstrated that it may be caused by the occurrence of more than one variant, particularly for sarc−patients. In this reanalysis, considering sarc+patients, we identified three patients with two variants within the MYBPC3 gene, seven cases carrying digenic variants, and five patients carrying more than two variants (Tables 1, 2). In some sarc−cases, unique variants were found in genes encoding proteins located in the Z-disc, namely, FLNC, ACTN2, CSRP3, TRIM63, and SVIL (Tables 5–8). Of note, variants within CAV3, VCL, and JPH2 genes were found along with additional sarcomeric and non-sarcomeric variants.

We identified three CSRP3 variants, including the known p.Trp4Arg variant. The CSRP3 gene, encoding cysteine and glycine-rich protein 3, is one of the non-sarcomeric HCM-associated genes with strong evidence for a primary pathogenic role in HCM (13). It plays different roles in mechanosensory functions and actin cytoskeleton assembly (12, 57). Functional analysis of Csrp3 knock-in animals (Csrp3^Trp4Arg/+) and (Csrp3^{Trp4Arg/Trp4Arg}) showed an age-and gene dosage-dependent HCM and heart failure, characterized by a nearly complete loss of contractile function under catecholamine-induced stress. Moreover, Cspr3 mRNA and protein levels were significantly decreased in the hearts of heterozygous and homozygous Cspr3^Trp4Arg knock-in animals (57).

We identified five variants in the ACTN2 gene, three of which were unique, and two patients were digenic and were carrying additional variants in MYBPC3 and TRIM63 genes. The ACTN2 gene (major Z-disc cross-linking protein) is of particular interest as variants within this gene have been linked to diverse cardiac phenotypes such as HCM, dilated cardiomyopathy (DCM), LV non-compaction (LVNC), and SCD (Supplementary File 3) (58–61). Patients with ACTN2 pathogenic variants showed no specific hypertrophy pattern, as septal, apical, concentric, and biventricular hypertrophy were reported. More importantly, patients with mild hypertrophy had severe complications such as resuscitated cardiac arrest and heart failure (12, 61–64).

A small number of variants was identified within the ALPK3 gene (n = 4), 3 out of the 4 variants were unique. Of note, the ALPK3 gene (α-protein kinase 3) was initially linked to an autosomal recessive form of severe pediatric mixed cardiomyopathy (HCM/DCM phenotype) (65–67). In 2020, the ALPK3 gene reached a definitive classification of strong evidence for an autosomal recessive form of HCM with an infant onset (46). However, cases harboring heterozygous LoF variants were reported with mild-to-moderate phenotypes, and an autosomal dominant pattern of inheritance was subsequently associated with an adult-onset HCM (12, 46, 68, 69). Recently, rare missense ALPK3 variants have been identified in Asian HCM patients (46, 70).

Three genes were recently associated with HCM and identified in this reanalysis: TRIM63, FHOD3, and SVIL. TRIM63 is one of the rare genes recently described as a cause of HCM with autosomal-recessive inheritance. TRIM63 encodes muscle-specific RING-finger protein 1 (MuRF1), a member of the ubiquitin ligases subfamily, such as MuRF-2 and MuRF-3 (71, 72). It is an E3 ubiquitin−protein that regulates the degradation of sarcomeric proteins such as Mybpc3 and Myh6 through ubiquitylation (12, 72). Homozygous and compound heterozygous rare variants in the TRIM63 gene were linked to HCM. TRIM63 variant carriers showed concentric LV hypertrophy, significant cardiac fibrosis, LV systolic dysfunction, and arrhythmias (12, 40, 41). Furthermore, systolic dysfunction and late gadolinium enhancement have been reported as characteristic features of TRIM63-associated cardiomyopathies (41). Rare missense variants in TRIM63 at the heterozygous state were reported as genetic modifiers of HCM. Although the TRIM63 missense variants had limited evidence of disease causality, TRIM63 knockouts are likely to be associated with HCM, given their enrichment in HCM patients and their absence in gnomAD (12, 41). In our reanalysis, the two LP missense variants (p.Cys23Tyr and p.Cys75Tyr) were identified in 5 patients of the HYPERGEN cohort. The TRIM63: (p.Gln247*) stop variant was identified in three patients, one at the homozygous state and two patients were heterozygous. The p.Gln247* variant is reported in the ClinVar database with a conflicting interpretation of pathogenicity. Nevertheless, in vitro and in vivo functional studies showed near complete loss of auto-ubiquitination in cells transduced with the TRIM63Q247* lentiviral construct (12, 41). Several other TRIM63 variants impair the ubiquitination of Trim63 substrates in adult cardiomyocytes. These findings implicate the impaired protein degradation as a pathophysiology mechanism of HCM (40, 41).

Of note, in the HYPERGEN cohort, five patients (2.5%) carried homozygous variants in MYBPC3 (n = 1), MYL3 (n = 1), and TRIM63 (n = 3) genes.

The second recent gene identified in this reanalysis is the FHOD3 gene. FHOD3 encodes the cardiac formin homology 2 domain containing three proteins that localize in the thin filament of the sarcomere and promote actin filament polymerization in cardiomyocytes (44).

Rare pathogenic FHOD3 variants were linked to HCM etiology through association analysis of 3,189 HCM patients and familial segregation studies (43). FHOD3 variants associated with HCM are mostly non-truncating and disturb the diaphanous inhibitory domain of the protein. Patients carrying likely causative FHOD3 variants showed mild-to-moderate LV hypertrophy and a 1% annual incidence of cardiovascular mortality (43). Additionally, FHOD3-HCM patients were diagnosed in adulthood (mean age 46.1 years), and two-thirds (66%) were men. The majority of patients (82%) had asymmetric septal hypertrophy (mean 18.8 ± 5 mm). LV ejection fraction <50% was present in 14% of the cohort and hypertrabeculation in 16% (43). Moreover, the cardiomyopathic phenotype of cMyBP-C null mice was aggravated by Fhod3 overexpression with a sarcomere integrity disruption. This phenotype was partially improved by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a damaging impact on cardiac function under cMyBP-C null conditions where Fhod3 is mis-localized (44). These findings suggest the likely contribution of Fhod3 to the pathogenesis of cMyBP-C-related cardiomyopathy and that Fhod3 is implicated in cardiac cMyBP-C-mediated regulation via direct interaction (44). In this study, rare missense FHOD3 variants were identified in the hotspot exons (12 and 15), and two patients with the recurrent FHOD3 p.Arg637Gln variant were digenic, carrying additional variants in the MYBPC3 gene (p.Cys1202Leufs*35 and p.Arg597Gln).

More recently, the SVIL gene was associated with HCM (46, 47). The SVIL (Supervillin) encodes a multidomain actin and myosin-binding protein in the Z-disc (73, 74). Biallelic LoF SVIL variants were identified in patients with skeletal myopathy, mild LV hypertrophy, and smaller descending aortic diameter (75). Indeed, a 10.5-fold excess burden of SVIL rare PTV variants in HCM cases has been demonstrated in a recent GWAS (47). Patients harboring rare truncating SVIL variants were found to have an increased LV contractility in both obstructive and non-obstructive forms of HCM, as demonstrated by Mendelian randomization analyses (47). In one family, the SVIL: p.(Gln255*) variant was found in two affected cousins, providing some evidence of familial segregation (47). In this reanalysis, clinical data were gathered for 7 out of the 13 patients with SVIL variants (Table 9). Three patients harboring the SVIL: p.(Ser1414Thr), p.(Glu1286Lys), and p.(Lys1960Arg) variants presented an aortic phenotype including bicuspid aortic valve disease, isolated ectasia of Valsalva sinus, and mild aortic insufficiency. Overall, the majority of SVIL patients showed severe intramyocardial late gadolinium enhancement. One patient (HCM-108) experienced SCD, and another patient (HCM-139) had a history of syncopes during physical effort. Of note, the patient (HCM-108) carried an additional variant in the FHOD3 gene (p.Arg638Trp). A severe scoliosis was diagnosed in one patient (HCM-129).

Interestingly, variants in SVIL and FHOD3 genes accounted for 10 and 7%, respectively, of patients with early HCM onset. Patients with sarcomere mutations have been reported to show earlier adverse complications and a worse prognosis (76). Several published HCM cohorts, studies, and data provided by the Sarcomeric Human Cardiomyopathy Registry (SHaRe) have shown that cases with sarcomeric variants had an early onset of the disease and a more severe phenotype with malignant complications. Thus, patients diagnosed in early adulthood (<40 years) had more severe outcomes with many adverse complications compared to patients with late-onset HCM. Moreover, HCM young patients (20–29 years) had a 4-fold increase in the risk of death compared to the general population (76–78).

All the identified variants in this reanalysis were searched in a French control cohort, gathering 960 healthy individuals. Only seven variants were present (Table 10). Of note, TCAP: p.(Met71Thr), ALPK3: p.(Arg1483Trp), FLNC: p.(Arg1860Cys) and MYPN: p.(Pro1112Leu) variants were unique in HYPERGEN carriers. The VCL: p.(His636Arg) and MYPN: p.P (ro1112Leu) variants were found in Finnish and Ashkenazi Jewish populations, respectively, suggesting that these variants could be more prevalent in these bottleneck populations.

Although the identification of variants in genes lacking strong evidence for disease causality and/or VUS in minor HCM genes does not increase the clinically actionable genes/variants discovery, it sheds light on the possibility of a combinatorial joint effect where VUS variants may act as a small risk increasing genetic factor.

Gathering strong proof of disease causality remains challenging for many reasons, such as the unavailability of family members to undergo co-segregation and funding constraints for functional studies. Indeed, genes such as ALPK3 and FHOD3 have been considered disease-causing by large gene-centric case–control studies. This strategy is an effective approach to reaching the needed statistical power supporting gene–disease association (46). Furthermore, the publication of cases with variants of low to moderate evidence was recommended, as the identical variants may be detected in extended families with similar phenotypes by researchers interested in functional validation (46).

Presently, we aimed to reanalyze a cohort of 200 HCM patients by focusing on genes strongly associated with HCM while also investigating newly linked genes with limited or insufficient evidence of causality to determine their potential involvement in our cohort. Overall, our results strengthen the implication of FHOD3, TRIM63, and SVIL genes in HCM as minor genes. Variants in the SVIL gene, recently linked to HCM, were found in 13 HYPERGEN patients. More importantly, patients harboring SVIL variants presented a similar hypertrophic pattern (significant myocardial fibrosis for six out of the seven patients/apical for three patients), and aortopathy was reported for three patients. The findings from our study extend the clinical and genetic spectrum of SVIL gene carriers. While Tadros et al. (47) identified SVIL variants in HCM patients, our study reveals additional cases, underscoring the need for expanded genetic screening. This broader understanding can facilitate future investigations into the pathophysiological mechanisms associated with SVIL variants, potentially leading to improved risk stratification and management strategies for patients.

The assessing and reporting of the identified variants—particularly within the novel associated genes—may provide additional strength to the previously reported variants and identify novel variants that help an accurate classification and interpretation. This study contributes to defining the genetic landscape of French HCM patients, which facilitates cascade genetic testing in familial cases and ultimately could guide personalized treatment.

The main goal of this study was to assess and report actionable and high-confidence variants in known and emerging HCM genes. However, there are several limitations in our study, making an accurate estimation of the increased diagnostic yield challenging. This is mainly due to the absence of detailed genetic results from the initial analysis. In addition, detailed patient phenotyping and clinical data are lacking. Thus, genotype–phenotype correlations and risk stratification were difficult to achieve.