In a total of 1,364 HCM probands who presented at the cardiogenetics clinic of the Antwerp University Hospital for genetic testing, from January 2015 until October 2023, a recurrent MYBPC3 (ENST00000545968; NM_000256.3) variant, a large heterozygous deletion of exon 23 to 26 (del23-26), was identified in 24 distinct families (1.8% of probands). Probands were defined as patients with a new diagnosis of HCM, presenting for cardiogenetic diagnosis, without prior knowledge of a genetic variant within their family. For each proband a family tree was drawn up to four generations. Through comparison of family trees, possible closely related probands could be identified, This was the case for two separately presenting individuals of family three, which were subsequently merged in 1 single family with the first identified patient serving as proband.

This cardiogenetic screening was performed using a cardiomyopathy (CM) next-generation sequencing (NGS) gene panel encompassing 59 genes associated with CMs. In short, genomic DNA of HCM patients was extracted from EDTA blood using standard procedures (Chemagic DNA bloodkit special, Perkin Elmer, Waltham, MA, United States). Genetic analysis was performed using either custom HaloPlex target enrichment followed by NGS (Illumina) as previously described (Proost et al., 2015) (before June 2021) or whole exome sequencing after enrichment of the exome (Twist Human Core Exome kit, Twist Bioscience with additional custom probes) (starting from June 2021). The deletion was identified through CNV analysis using a read-depth strategy (SeqPilot, JSI) and confirmed with a multiplex amplicon quantification (MAQ) assay. When applying a read-depth strategy for CNV analysis, the raw number of reads for each region of interest (ROI) is normalized against the number of reads of selected control regions within the sample and compared between all samples within the same sequencing run. A deletion is characterized by approximately 50% of the reads found when compared to controls, while a duplication will show approximately 150% of the reads.

Cascade-screening of first-degree relatives was performed. Family members were invited to participate by family letter and genetic counselling by a multidisciplinary team was offered before and after variant testing. After family members underwent genetic testing (using MAQ) for the MYPBC3 del23-26 variant, genotype positive (G+) relatives were subsequently offered clinical evaluation. If the proband carried an additional variant, identified with the 59 CM-panel, this additional variant was sequenced in the family members as well, using sanger sequencing.

Demographic characteristics, cardiac assessment and outcome were collected from probands and relatives’ patient files. Composite cardiac outcome included death, heart transplantation (HTx), life-threatening arrhythmia, heart failure hospitalization or septal reduction therapy.

To assess the exact localization of the breakpoints of the deletion a touchdown PCR was performed using a forward primer located at the end of exon 22 and a reverse primer at the beginning of exon 27 of the MYBPC3 gene (95°C 5′, 20× (95°C 30″, 65 (−0.5×)°C 30″, 72°C 1′), 15× (95°C 30″, 55°C 30″, 72°C 1′), 72°C 10′, 15°C 10′).

MYBPC3-E22F-M13F: GTT​TTC​CCA​GTC​ACG​ACG​AAT​AAG​GCC​CCA​GCC​AGG​CCA

MYBPC3-E27R-M13R: CAG​GAA​ACA​GCT​ATG​ACA​TGT​GTG​CTC​TGT​CAG​CCC​CTG​CA

The presence of a PCR product within the expected range was confirmed on Qiaxcel^® capillary electrophoresis (Qiagen). For the non-affected allele, we did not expect a PCR product as this would be too big (>4,000 bp) for amplification. Sanger sequencing was used to sequence the resulting PCR product (3500 Dx Genetic Analyzer, Applied Biosystems) and sequence was compared to the reference sequence ENST00000545968.6 retrieved from Ensembl (Martin et al., 2022) using CLC DNA workbench software version 5.7.1 (CLC Bio, Aarhus, Denmark).

First, a shared genetic background was identified in all index patients, based on intragenic SNPs within the MYBPC3 gene (Table 2). Next, haplotype analysis, using microsatellite markers surrounding MYBPC3, was performed to confirm the presence and assess the size of the common haplotype in all probands (Figure 1).

Thirteen microsatellite markers surrounding MYBPC3 were analyzed in all selected probands as has been described before (Bergman et al., 2001) (D11S1785; D11S1993; D11S986; D11S4174; D11S1344; D11S2016; D11S1326; D11S1395; D11S1920; D11S1313; D11S4191; D11S913; D11S4113). Forward primers were labeled with 6-fluorescein amidite (FAM), and PCR products were analyzed in the presence of an internal sizing standard (ROX) using ABI3130XL (Applied Biosystems). The sizes of the amplicons were determined using ABI GeneMapper software v3.7 (Applied Biosystems).

After confirming a shared haplotype, the age of the variant was estimated based on the common microsatellite markers using a linkage-disequilibrium based approach (Bergman et al., 2001). The age of the variant (t, in number of generations) was estimated using the following formula: t = ln P d 1 − P n 2 1 − P n 2 ln 1 − r

Where Pd1 is the frequency of the founder marker allele in variant-carrying haplotypes and Pn2 is the frequency of the founder marker allele in non-variant carrying haplotypes. The founder marker allele was assumed to be the most frequent allele among the variant-carrying haplotypes. When the founder marker allele is present in all variant-carrying haplotypes (Pd1 = 1), but absent from all non-variant carrying haplotypes (Pn2 = 0), this formula can no longer be used as the estimated mutation age would equal 0. r is the recombination fraction between a marker and the variant and is calculated as: r = 1 − e − 2 d 2 where d is the distance of the marker to the variant in cM/100. Distance of the shared haplotype in cM was based on the distance in Mb, assuming that 1 Mb ≈ 1 cM. Each generation was assumed to be 25 years when calculating the age of the variant. An estimation of mutation age for each tested marker was calculated, after which the average of estimations was used to derive the final estimation as has been reported before (Bergman et al., 2001).

Frequencies are expressed as numbers and percentages. Continuous variables are expressed as mean ± SD if normally or median ± interquartile range if non-normally distributed. Unpaired T-test or Mann-Whitney U test was used for comparisons of continuous variables. Chi-square test was used for comparisons of categorical variables. All analyses were performed using SPSS Statistics version 28 (IBM Corporation). A p-value of <0.05 was considered statistically significant.

From January 2015 till October 2023, we identified 24 probands with HCM carrying the MYBPC3 exon 23-26 deletion (Table 1), with a mean age 51 ± 16 years at time of clinical HCM diagnosis and 62 ± 10 years at time of genetic diagnosis. A positive family history of HCM was present in ten (41.7%) probands, sudden cardiac death (SCD) had occurred in nine families (37.5%) of which in two (8.3%) families before the age of 35.

Using a nested-PCR approach we delineated the exact breakpoints of the MYBPC3 deletion of exon 23 to 26. The complete deletion spans 3546 base pairs from intron 22 to intron 26: Chr11:g.47360721 – 47357175del (GRCh37/hg19). Exon 23 and part of exon 24 encode the last part of the C5 immunoglobulin-I domain whereas the remainder of exon 24, exon 25 and exon 26 encode the C6 fibronectin-3 domain of the cMyBP-C protein (Carrier et al., 1997).

The variant was classified as pathogenic (class 5) according to ACMG guidelines (Richards et al., 2015; Riggs et al., 2020) with the following arguments: 1. Copy number loss content 1A (contains protein-coding functionally important element; 0p); 2. Overlap with established haplo-insufficient gene 2E [both breakpoints are within the same gene; classification according to sequence variant interpretation criteria: PVS1 (null variant in a gene where loss of function is a known mechanism), PS4/PM2 (increased prevalence in affected individuals compared to controls/absent from controls); PP1 (co-segregation with disease in multiple affected family members); 0.9 p]; and 3. Evaluation of number of genes in the CNV: 3A (0-24 genes; 0p); 4. Detailed evaluation of genomic content 4F and 5D (7 or more observed segregations; 0.45 p). 66 informative meioses were present over all families. Of a total of 20 relatives with HCM, 19 carried the MYBPC3 variant. An overview of the pedigrees of all 24 families is available in the Supplementary Material.

A common genetic background, defined by intragenic SNPs within the MYBPC3 gene was identified in all 24 probands (Table 2).

The length of the common haplotype was assessed in all probands by 13 microsatellite markers spanning 26.4 Mb. A common haplotype of 1.19 Mb was identified in all 24 probands, with 19 of the probands sharing a 13.8 Mb haplotype (Figure 1). The founder event was estimated to have happened five generations ago. Taking into account that probands were on average 63 years old (ranging 50–87 years) and assuming that each generation spans circa 25 years (5*25 + 63), the founder event presumably took place 175–200 years ago (around the year 1830). All probands now live in a well-defined region within central Flanders, presumably the founder lived close to the region of Aalst (Figure 2).

Through cascade screening 59 first-degree relatives were genetically tested, of whom 37 (62.7%) were genotype positive (G+) and 22 (37.3%) genotype negative (G-). Screened family members were on average 38 ± 19 years old at the time of genetic testing. Differences between G+ and G- family members are displayed in Table 3.

Subsequent clinical assessment revealed an HCM phenotype in 19 (51.4%) G+ relatives, of whom only 5 (26.3% of newly identified HCM phenotypes) were symptomatic (NYHA class >1, angina, or syncope) at the time of genetic diagnosis (Table 1). G+ relatives with an HCM phenotype were older than those without an HCM phenotype (52.7 ± 21.3 vs. 30.6 ± 15.3; p < 0.001).

Characteristics of probands and G+ relatives are displayed in Table 1. Probands were older than G+ relatives (current age 63 ± 10 years vs. 42 ± 21 years; p < 0.001) and had more severe phenotypes with more clinical symptoms, non-sustained ventricular tachycardia (nsVT), more overt hypertrophy and left ventricular outflow tract obstruction (LVOTO) on echocardiography. The average interventricular septum thickness (IVS) was significantly higher in probands than G+/phenotype positive family members (20.6 ± 3.8 vs. 15.3 ± 3.5 mm; p < 0.001). Probands also presented with worse outcomes: more life-threatening arrhythmias, more heart failure hospitalizations, and a higher need for device therapy.

One might hypothesize that the differences between probands and G+ relatives is related to the age difference but when probands were compared to an age matched subgroup of G+ relatives (n = 16) we could still observe significant differences in hypertrophy (IVS 20.6 ± 3.8 vs. 15.2 ± 3.4; p ≤ 0.001) prevalence of device implantation (25.0% vs. 0%; p = 0.035) and prevalence of LVOTO (43.5% vs. 6.7%; p = 0.014) (Figure 3). Gender distribution was similar between probands and matched relatives. At the age of 50, a penetrance of 78.6% was observed, defined as the presence of HCM in 11 of 14 G+ relatives with age ≥50 years.

A large phenotypic variability has been reported (Page et al., 2012; Helms et al., 2020) in MYPBC3-related HCM and this hinders the assignment of prognostic significance to specific disease-associated mutations or groups of mutations, complicating (genetic) counselling of family members.

Based on literature, the disease penetrance of MYBPC3 variants was 58%–83% at age 50 years (Charron et al., 1998; Niimura et al., 1998; Maron et al., 2001), which resembles our founder population’s disease penetrance of 78.6% at age 50 (in family members). Average age of clinical onset for MYBPC3-harbouring individuals is around 40 years (Charron et al., 1998; Niimura et al., 1998; Van Driest et al., 2004; Sedaghat-Hamedani et al., 2018; Lorenzini et al., 2020b). In our MYBPC3 founder population, age of clinical diagnosis was considerably later, with an average of 51 ± 17 years (probands and family members). Considering that the presence of HCM leads to a loss in quality of life, and HCM is often progressive, an earlier onset can be seen as a more severe phenotypic presentation when compared to late onset.

After the initial discovery of MYBPC3, several studies reported on a less severe phenotype in MYBPC3 variants compared to other known HCM-related genes, especially MYH7. The degree of hypertrophy in our probands (IVS 20.6 mm) was similar to the described MYBPC3 literature [20.6 (19-22)mm, current age not known (Sedaghat-Hamedani et al., 2018)]. However, when all G+ individuals were considered, IVS was lower (16.3 ± 5.5 mm). In a large meta-analysis of 31 studies with 6123 HCM patients, MYBPC3 variant carriers (20% of all HCM patients) had less cardiac conduction disease, less ventricular arrythmia and less HTx (present in 15%, 20% and 0.6% of all MYBPC3 variant carriers respectively) compared to MYH7 variant carriers (Sedaghat-Hamedani et al., 2018). We observed intraventricular conduction disturbances in 25.5% and ventricular arrhythmia in 25.5% of the G+ individuals in our cohort which was not significantly higher than the observation in the MYBPC3 group described above (p = 0.1 for conduction disease and p = 0.47 for ventricular arrythmia). None of the patients who were followed in our cohort died during follow-up (5.5 years [1-22]), but three (5.2%) did experience a life-threatening arrhythmia.

A large study stratified MYBPC3 families according to prognosis, from benign (no death or HTx before the age of 60 years in the family, including the proband) to intermediate (1 event in the family) and malignant (2 or more events in the family). Overall, 90% of families with a MYBPC3 variant were stratified as having a benign or intermediate prognosis (Richard et al., 2003). According to this classification three families with the founder variant can be classified as malignant (12.5%), one family as intermediate (4.2%) and the other 20 families as benign (83.3%). This inter-familial variability indicates that even a single specific variant can be identified with a range of disease severities.

Most variants identified in MYBPC3 are “private” variants, meaning that they have been identified in a single family. In a large cohort, up to 56% of variants could be considered private (Alfares et al., 2015). When a variant is identified in several families and a shared haplotype is characterized, it represents a founder variant. The proportion of founder variants in HCM patients differs from region to region with higher proportions in isolated populations. Most strikingly, in Iceland up to 58% of the complete HCM population has the same specific MYBPC3 founder variant (MYBPC3 c.927-2A>G), with an overall population prevalence of 0.36% (Adalsteinsdottir et al., 2014). Founder variants are suggested to be associated with a more benign phenotype, as they have withstood negative selection pressure (Jääskeläinen et al., 2004; Thompson and Day, 2017). In several populations, however, no difference in LV hypertrophy severity or prevalence of adverse events between founders and non-founders were observed (Christiaans et al., 2010a; Christiaans et al., 2010b; Ross et al., 2017; Sabater-Molina et al., 2017; van Velzen et al., 2017; Helms et al., 2020).

Our current cohort is characterized by a higher age of onset, but an overall high penetrance at the age of 50 years (78.6% of G+ family members, in comparison to an average penetrance of 50% at this age in genotype positive HCM (Lorenzini et al., 2020b). Despite this late onset, negative outcomes were frequent, suggesting a stronger contribution of this specific founder variant on the HCM phenotype penetrance.

Therefore, the presence of any (MYBPC3) genetic variant seems to be the most important predictor of phenotype and severity, rather than the nature of the exact MYBPC3 variant itself. These MYBPC3 founder variants could withstand negative selection pressure as overall HCM mortality is low and the mean age of HCM related mortality is around 68 years which is long after the age of reproduction (compared to 81 years for non-HCM related mortality, p = 0.02) (Adalsteinsdottir et al., 2014).

In our study cohort, probands were significantly more affected than G+ relatives and some of these differences stayed significant after correction for age by comparison with an age-matched cohort. Probands had thicker left ventricles compared to age- and gender matched G+ family members with a HCM phenotype, had more outflow tract obstruction and worse outcomes. It should be noted however, that overall, probands were more extensively investigated than family members, with a higher proportion of probands undergoing cardiac MRI and longer follow-up periods.

A similar observation was present in other MYBPC3 variant carrying populations, regardless of this being a founder variant or not. Overall, G+ relatives with a clinical HCM phenotype had less hypertrophy, less adverse remodeling and had lower risk of complications and mortality compared to probands (van Velzen et al., 2016; van Velzen et al., 2017). Age at time of genetic diagnosis was lower in G+ family members, but age at time of clinical HCM diagnosis was similar between probands and their family members (van Velzen et al., 2016; van Velzen et al., 2017). Likewise in our cohort, G+ family members were younger at the time of clinical diagnosis, which likely is explained by the active screening of individuals through cascade screening, whereas probands only present when symptoms are obvious, or complications have occurred.

Male sex is a significant predictor for the development of an HCM phenotype in G+ carriers who were phenotype negative at initial investigation. This is true for genotype positive HCM in general, MYBPC3 carriers and MYBPC3 founder carriers, including the founder population presented in the current work (Christiaans et al., 2010a; Page et al., 2012; Terauchi et al., 2015; Sabater-Molina et al., 2017; van Velzen et al., 2017; Lorenzini et al., 2020b). Additionally, female patients with HCM are diagnosed later than men (Adalsteinsdottir et al., 2014; Sabater-Molina et al., 2017; van Velzen et al., 2017; Lakdawala et al., 2021). However, women experience more heart failure symptoms than men, have more LV outflow tract obstruction and more frequently have a need for medical treatment (Adalsteinsdottir et al., 2014; Terauchi et al., 2015; Lakdawala et al., 2021). Several studies, including a large meta-analysis, on a total of 9,427 HCM patients (39.5% women), confirmed that during follow-up, women have a higher risk for the development of heart failure and all-cause mortality even after correction for differences present at baseline (Terauchi et al., 2015; Geske et al., 2017; Lorenzini et al., 2020a; Lakdawala et al., 2021; Trongtorsak et al., 2021).

Part of the explanation of a smaller portion of women in (MYBPC3-related) HCM could be the routine use of non-indexed measurements of wall thickness for the diagnosis of HCM. As men are on average larger and have thicker ventricle walls, they are more likely to fall above the threshold for hypertrophy (Lang et al., 2015). This is supported by the observation that differences in wall thickness were reversed after indexation for body surface area (van Velzen et al., 2017; Lakdawala et al., 2021). Gender-specific cut-offs, or the routine implementation of BSA indexed cut-offs is therefore warranted. This is further supported by the higher uptake of testing with a genetic panel in female HCM patients compared to male HCM patients (36% vs. 30% p < 0.001 and 51% vs. 43% p < 0.001) (Alfares et al., 2015; Lakdawala et al., 2021). All clinicians should be aware of this gender bias, as the delayed diagnosis in women seems to be related to worse outcomes.

In literature, the presence of an additional (likely) pathogenic variant in MYBPC3 or another HCM-related gene seems to result in worse phenotypes. Homozygosity or compound heterozygosity is present in 0.6%–7.5% of HCM patients and is associated with earlier onset and more severe phenotype (Van Driest et al., 2004; Ingles et al., 2005; Girolami et al., 2010; Marziliano et al., 2012; Page et al., 2012; Teirlinck et al., 2012; Alfares et al., 2015; Lorenzini et al., 2020b). The presence of a VUS next to a (likely) pathogenic variant has been observed in 7.4%–9.8% of HCM patients, but the effect of an additional VUS on the phenotype and disease severity has not been well defined (Alfares et al., 2015; Lakdawala et al., 2021).

In our cohort, 45.8% of probands carried one (or more) additional variants in a gene associated with cardiomyopathy, most of these were VUS, but in one family an additional likely pathogenic/pathogenic MIB1 c.1A>C variant (4.2%) was present. We could not observe any clear differences between probands with or without an additional variant. It should be taken into account that VUS encompasses a broad range of variants, and some VUS might be leaning more to likely pathogenic (hot) whereas others are more likely to be benign after all.

As family members only underwent testing of the MYBPC3 deletion, the presence of additional variants within the 59 CM genes, functioning as genetic modifiers cannot be excluded. As the probands were the most severely affected individuals, we expect genetic modifiers that worsen the phenotype to be, preferentially, identified in these probands. We showed that the G+ family members carrying the additional variant of the proband presented with a more severe phenotype than G+ family members without the additional variant, suggesting that some of the additional variants could act as aggravating modifiers having a synergistic effect on the MYBPC3 founder variant. Identification in more individuals and experiments investigating the actual functional effect of these variants should be performed to prove this hypothesis. Concerning potential protective genetic modifiers that lead to a less severe phenotype, we would expect to detect them in the family members, but we did not perform the full panel screening in these individuals and as such have no data to investigate this. We did not collect genetic data outside of the 59 CM genes, so cannot investigate protective or aggravating modifiers in other parts of the genome. Genome-wide linkage analysis based on short tandem repeat markers or genome-wide associations studies (GWAS) using common SNPs can aid to identify genetic modifiers. However, large families for linkage analysis and large cohorts of patients for GWAS are needed. Additionally, a large number of SNPs or markers need to be assessed, rendering this time- and cost consuming (Ott et al., 2015).

Overall, large differences in penetrance and disease severity are present in patients with the same genotype. Differences in the genetic background can be part of the explanation. Besides compound heterozygosity, more frequent genetic variation based on single nucleotide polymorphisms may play a role. Indeed, it has been shown that a polygenic risk score for HCM could stratify the HCM risk in family members of G+ probands (Zheng et al., 2023). Furthermore, this score aided the prediction of adverse outcomes in HCM patients (Zheng et al., 2023). However, the use of a polygenic risk score requires additional sequencing in all individuals, requiring extra time- and cost investment, which might not be feasible for all patients at the moment. For the current cohort, the data on the SNPs included in the risk score were not available to further stratify HCM risk.

Additionally, environmental factors may play a role. In our cohort G+ relatives with AHT were more likely to present with a HCM phenotype, whereas smoking or BMI did not significantly influenced disease penetrance. Interestingly, a marked difference in disease severity has been observed in three pairs of monogenic twins with a MYBPC3 (c.787G>T; p.Gly263ter) founder variant (Rodríguez Junquera et al., 2022). As they are genetically identical, these differences cannot be explained by genetic background. The authors could not find any clear differences in known environmental risk factors for HCM such as AHT or physical activities (including pregnancy) (Rodríguez Junquera et al., 2022). Other, currently undiscovered, influencing factors (e.g., epigenetics) are therefore likely at play.