The present study characterised Slovenian probands with LP/P MYBPC3 variants clinically and echocardiographically and compared characteristics, clinical outcomes and estimated disease penetrance between Slovenian probands with LP/P MYBPC3 variants and Italians with MYBPC3:c.913_914del.

Likely pathogenic and pathogenic variants in MYBPC3 were identified in 41 (60%) of Slovenian probands with an identified genetic cause of HCM. The most frequently identified variant was MYBPC3:c.913_914del, which was present in 19 (46%) individuals with the LP/P MYBPC3 variant. To date, MYBPC3:c.913_914del has been extensively described in Italian patients with HCM. The variant has also been reported in individuals living in the Netherlands, Australia, Canada, USA, Brazil, Belgium, Finland and Germany in ClinVar (variation ID:42,801) [21, 22] and in patients with HCM in the literature [7, 23,24,25,26,27].

The haplotype analysis showed that the probands with MYBPC3:c.913_914del from the Italian and Slovenian cohorts most likely share the same common ancestor. The segregation analysis supports this finding, as in both cohorts the variant was found to be inherited from the tested parents (three segregations in the Slovenian cohort and four in the Italian cohort). Considering that Slovenian and Italian probands with MYBPC3:c.913_914del share not only a pathogenic variant but also a fairly similar environment, we expected that the findings regarding the penetrance and clinical outcome observed in the Italian cohort would be replicated in the Slovenian cohort. However, the results of the present study did not confirm previous findings.

First, the study of Italian patients with HCM found that the variant was significantly associated with an arrhythmic profile leading to more frequent episodes of NSVT, ICD implantation and a higher risk of SCD, and with a milder thickening of the left ventricular wall in probands with MYBPC3:c.913_914del compared to those with other LP/P MYBPC3 variants [10]. We observed that the Slovenian probands with MYBPC3:c.913_914del and those with other LP/P MYBPC3 variants did not differ significantly in any of the above parameters, but both Slovenian groups had a significantly lower incidence of NSVT, ICD implantation, lower calculated risk of SCD and milder wall thickening than the Italian probands with MYBPC3:c.913_914del. Although the results of the present study cannot explain why Italian probands with MYBPC3:c.913_914del differ significantly from the Slovenian probands, the comparison between Slovenian probands with MYBPC3:c.913_914del and those with other LP/P MYBPC3 variants may suggest that the clinical presentation of LP/P MYBPC3 variants is not as different on a larger scale as might be expected. Research to elucidate the effect of specific HCM-causing variants has been ongoing for several decades. It remains unclear why LP/P variants in different genes related to HCM can result in a similar phenotype in unrelated individuals [28] while the same LP/P variant can lead to markedly different phenotypes among family members [8, 29]. To date, there is no evidence that pathogenic MYBPC3 variants lead to variant-specific phenotypes that have been confirmed by functional studies. Currently, only research using a meta-analysis approach can identify clinically important differences at the gene or variant level [3, 8, 30].

Second, Italian probands with HCM and MYBPC3:c.913_914del were found to have a higher incidence of adverse cardiac events compared to those without the pathogenic MYBPC3 variant. The comparison of the frequency of events between Slovenian probands with the LP/P MYBPC3 variant and Italian probands with MYBPC3:c.913_914del showed that the only statistically significant difference was the frequency of adverse cardiac events between Slovenian and Italian probands with MYBPC3:c.913_914del, which was lower in Slovenian probands. However, a recent meta-analysis found no difference in adverse events between patients with HCM with known and unknown genetic background of the disease, suggesting that the effect of the pathogenic variant on the frequency of adverse cardiac events should be studied on a larger scale [8].

Third, the Italian study found that probands with MYBPC3:c.913_914del almost always have a family history of HCM (95%) and that the penetrance of the disease for MYBPC3:c.913_914del is not only age-related but also gender-specific, as men over 20 years of age with MYBPC3:c.913_914del had a significantly higher incidence of HCM than women of the same age. These conclusions were drawn from the segregation analysis, which was performed in 14 out of 19 Italian families. The high proportion of family history for HCM is mainly based on the examination of relatives of the same or younger age as the proband, as only in four families (29%) the inheritance of the variant in the proband was confirmed by the affected parent and in half of the families the clinically unaffected parents were not included in the segregation analysis (5 families, 36%) or information on the clinical status of the parents was not provided (3 families, 21%). This suggests that not only the family history but also the penetrance of the variant was inflared. No significant difference in the overall penetrance of the LP/P MYBPC3 variants was found in either the Italian or the Slovenian cohorts. We did not observe a significant gender difference in the disease penetrance between Slovenian probands with MYBPC3:c.913_914del and those with other L/P MYBPC3 variants employing the same method as in the Italian study. Limited sample size resulting in insufficient statistical power to detect a statistically significant difference is a possible explanation for this finding, given the known male predominance in inherited cardiomyopathy cohorts [3, 8, 30]. Another possible explanation lies in the limitations of the traditional family history-based method for estimating penetrance. This approach may provide information that is valid only for the families studied and may be biased when generalised to the wider population and other world populations. Therefore, especially with the increasing availability of data, population-based studies of unrelated individuals with pathogenic variants provide a more reliable method for estimating disease penetrance [30, 31].

Our findings in the Slovenian cohort with the MYBPC3:c.913_914del variant differed from those reported in the Italian study, suggesting that conclusions about the clinical presentation of a specific pathogenic variant should be drawn with caution, given the incomplete penetrance and heterogeneous clinical presentation of variants associated with hypertrophic cardiomyopathy.

In this retrospective study, participants were recruited on the basis of genetic analysis and cascade screening, with clinical and imaging data collected over 20 years of routine medical care, not as part of a targeted clinical trial. Due to advances in cardiology practice over time, some probands had inadequate initial examination data, which may have biased the assessment of changes in echocardiographic parameters over time. Limited cardiac magnetic resonance imaging data prevented an assessment of differences in cardiac fibrosis between the cohorts. However, all probands were followed up, and data from the last examination are complete. Due to limited imaging data from relatives with HCM, only probands were evaluated for the cardiac phenotype. Extensive segregation analysis was not possible for all probands due to logistical constraints.