Discussion

This study utilised whole-exome sequencing on eight Egyptian patients with hypertrophic cardiomyopathy to identify and report prioritised variants, focusing on rare variants in hypertrophic cardiomyopathy-associated genes. This investigation represents the first exploratory genetic characterisation of hypertrophic cardiomyopathy in an Egyptian cohort using whole-exome sequencing, addressing a significant gap in the cardiovascular genomics literature, which has historically underrepresented populations from North Africa and the Middle East. By identifying 21 rare variants across 10 hypertrophic cardiomyopathy-associated genes in this small cohort, these preliminary findings offer an initial contribution to a more global understanding of hypertrophic cardiomyopathy’s genetic architecture. They highlight the importance of diversifying genomic datasets; however, larger studies are essential to determine the true prevalence and spectrum of these variants in the Egyptian population. Our sample size (n = 8) restricts the generalizability of our observations. Larger, population-based cohorts with appropriately ethnically matched controls are required to confirm these preliminary findings and to determine the true prevalence and spectrum of these variants in the Egyptian population.

According to the American Heart Association (AHA), cardiomyopathies are classified into three categories: genetic, non-genetic (acquired), and mixed (genetic and non-genetic).^{Reference Corrado, Graziano and Bauce21} In hereditary hypertrophic cardiomyopathy, a complex genetic architecture is connected to approximately 290 genes (https://hpo.jax.org/). After stringent filtering for rarity and predicted pathogenicity, we identified 21 rare variants in 10 genes with definitive or moderate evidence for hypertrophic cardiomyopathy causation, as defined by the ClinGen Hereditary Cardiovascular Disease Gene Curation Expert Panel. These variants were classified as pathogenic, likely pathogenic, or of uncertain significance (VUS) according to ACMG/AMP guidelines, with at least three in silico prediction tools consistently indicating a deleterious effect and an elevated CADD score.

A methodological constraint of this study is the exclusive reliance on in silico predictive algorithms (SIFT, PolyPhen‑2, MutationTaster, and CADD) and ACMG/AMP classification criteria, without the corroborative support of functional assays or familial segregation analyses. This is particularly critical for the numerous variants classified as VUS (e.g., in TTN, ACTN2, ALPK3, and others), which constitute the majority of our findings. The true contribution of these VUS to hypertrophic cardiomyopathy pathogenesis remains speculative, and we urge caution in their interpretation. We have therefore revised our discussion to explicitly emphasise this uncertainty, especially for non‑sarcomeric genes and VUS findings. Furthermore, we did not have access to an ethnically matched control population from Egypt, which would have strengthened the interpretation of variant frequencies and pathogenicity.

Crucially, these acknowledged constraints do not diminish the substantive contribution of our research. Rather, they serve as a framework for responsible data interpretation and highlight the inherent complexities of genotypic–phenotypic correlation in hypertrophic cardiomyopathy. Our findings represent a vital foundational genomic survey of an understudied population, providing a critical baseline of rare variant prevalence that will inform and necessitate future prospective investigations. The identification of these VUS, particularly within non-sarcomeric genes, offers compelling candidate targets for downstream mechanistic studies and underscores the urgent need for population-specific reference datasets. Consequently, we frame these limitations not as weaknesses, but as essential directives for the next phase of validation and discovery within this demographic.

Patients with hypertrophic cardiomyopathy most frequently have mutations in the genes that code for sarcomeric proteins. Early guidelines caused the classification of this cardiomyopathy as a disease affecting the sarcomere. Most sarcomeric diseases follow an autosomal dominant pattern of inheritance, with variations in the age of onset and the level of penetration.^{Reference Lorenzini, Norrish and Field22} Sarcomeric gene mutations are linked to a range of clinical manifestations (genetic pleiotropy), including cardiomyopathies such as restrictive, non-compaction, and dilated cardiomyopathies, as well as, infrequently, structural cardiomyopathies and congenital cardiac conditions such as Ebstein anomaly or septal defects.^{Reference García-Hernández, De la Higuera and Ochoa23}

The MYBPC3 (myosin-binding protein C3) gene was found to have variants in two patients in our study. The leading cause of hypertrophic cardiomyopathy is mutations in MYBPC3. One of the primary mechanisms driving hypertrophic cardiomyopathy associated with MYBPC3 is haploinsufficiency.^{Reference Josephs, Roberts and Theotokis24} Consequently, whether they are evaluated with high or low confidence, non-truncating variants (i.e., stop-loss variants, missense variants, a protein-truncating variant anticipated to evade nonsense-mediated decay, and in-frame indels) are given precedence, irrespective of their pathogenic predictions. Two rare MYBPC3 variants were found in our data; they were an intron variant, a missense variant, and, in one patient (EGP7), a heterozygous missense variant (p.Gly1206Asp), which is classified as a likely pathogenic variant in the MYBPC3 gene.

In contrast to the MYBPC3 gene, which primarily causes disease through allelic imbalance and haploinsufficiency, the missense variations of the MYH7 (myosin heavy chain 7) gene function via a negative dominant effect, indicating that MYH7 is tolerant to loss-of-function mutations.^{Reference Park, Packard and Levin19} Giving priority to the MYH7 missense variant, which is predicted to have a deleterious effect by various in silico algorithms, could enhance the detection of actionable variants. Since rare and likely causal variants are often clustered in specific gene regions to enhance the outcomes of genetic testing in hypertrophic cardiomyopathy, a novel ACMG rule based on the aetiological fraction for rare missense MYH7 variants has been proposed.^{Reference Kim, Kim and Kim25}

Two rare MYH7 variants found in this analysis were missense variants, one of them a pathogenic variant (p.Phe252Ser) and the other a VUS (p.Glu1708Lys) with a predicted deleterious effect, and with a gnomAD frequency of 0.000003983. Mutations in the MYH7 gene were related to considerable left ventricular hypertrophy, a hallmark of hypertrophic cardiomyopathy, as well as an adverse prognosis, compared with patients with mutations in other genes associated with hypertrophic cardiomyopathy.^{Reference Mathew, Zahavich and Lafreniere-Roula26}

Our analysis reflects the distinct pathological mechanisms employed by different genes. The MYBPC3 variants suggest haploinsufficiency as a primary driver, whereas the MYH7 missense variants are indicative of a dominant-negative effect. This mechanistic distinction is crucial for understanding genotype–phenotype correlations.

Three variants of the sarcomeric gene TNNT2, which encodes for cardiac troponin T2, a protein that regulates the complex of thin-filament troponin in the sarcomere and is essential for contractile function, have been identified.^{Reference Pham, Giudicessi, Tweet, Boucher, Newman and Geske27} Notably, all three patients with TNNT2 variants (EGP1, EGP5, and EGP6) exhibited arrhythmic manifestations, including atrial fibrillation and non-sustained ventricular tachycardia, as documented in Table 1. This consistent association supports the established role of TNNT2 as a marker for increased arrhythmic risk and sudden cardiac death, reinforcing the clinical utility of identifying such variants for arrhythmic risk stratification.

The identification of rare, prioritised variants in core sarcomeric genes such as MYBPC3, MYH7, and TNNT2 aligns with the well-characterised molecular basis of hypertrophic cardiomyopathy. The presence of both pathogenic and VUS missense variants in MYH7, a gene associated with a severe phenotypic prognosis, underscores its critical role and the need for careful variant interpretation.

The cardiac sarcomere functions as part of a complex network within the myocyte, interconnected with the cell membrane and other sarcomeres. Structural and Z-disk proteins, and intermediate filaments that link the Z-disk to the cell membrane via costameres, are important components of its function. Additionally, several of the genes in this network are involved in calcium management, sarcomeric protein turnover, and transcription regulation. While a lot of genes have been studied as potential candidates in hypertrophic cardiomyopathy, only a few have a confirmed association with the phenotype.^{Reference Walsh, Offerhaus, Tadros and Bezzina5}

According to the ACMG/AMP categorisation, we found a single patient to have a rare missense mutation in the PLN gene (p.Arg25Cys), which is likely pathogenic and has an expected deleterious impact. The PLN gene encodes phospholamban, which is essential for controlling calcium reuptake in the sarcoplasmic reticulum. A particular kind of arrhythmogenic/dilated cardiomyopathy and, less frequently, variants of the null type in simple heterozygosity and incomplete penetrance to hypertrophic cardiomyopathy have been associated with mutations in this protein.^{Reference Vafiadaki, Chaudhari and Soliman28} The main causes of PLN-induced cardiomyopathy are abnormal Ca2+ handling and SERCA2 dysfunction, which significantly affect the heart’s contraction and relaxation processes.^{Reference Brand, Baumgarten and Denzinger29} There are just a few known variants of PLN since it is a diminutive gene with only one coding exon, with an expected contribution to hypertrophic cardiomyopathy that is small—less than 1%, to be exact.

One patient (EGP2) had two heterozygous variants of the KLHL24 gene: Exon 3: c.307C>T (p.Arg103) and Exon 3: c.604G>A (p.Glu202Lys). The (p.Arg103) variant has a deleterious (fs/PTC) effect and is likely pathogenic according to the ACMG classification. KLHL24 encodes the Kelch-like protein 24, an ubiquitin ligase substrate receptor that contributes to the stability of intermediate filaments. Hypertrophic cardiomyopathy recessive, which presents with early-onset disease, a rising incidence of arrhythmias, and neuromuscular issues, potentially caused by desmin abduction or glycogen accumulation, is associated with pathogenic null mutations in this gene.^{Reference García-Hernández, De la Higuera and Ochoa23}

The ALPK3 gene, which codes for alpha-protein kinase 3, was found to have a heterozygous missense mutation in Exon 14: c.4808T>C (p.Leu1603Pro). This nuclear protein is crucial for the early differentiation of cardiomyocytes, as it is involved in transferase and protein kinase activity and has widespread expression. Pathogenic variants in both alleles are associated with Noonan-like syndromic characteristics and paediatric-onset cardiomyopathy in over 80% of cases, and in more than 50% of cases, a prolonged QT interval is present. While missense variations can arise as compound heterozygotes with one loss-of-function mutation or as homozygotes, most autosomal recessive instances involve a null ALPK3 variant.^{Reference Andrade, Stephan and Furquim30} There is strong evidence that the ALPK3 gene causes an autosomal recessive form of infant-onset hypertrophic cardiomyopathy. Nonetheless, mild-to-moderate symptoms and an autosomal dominant inheritance pattern associated with adult-onset hypertrophic cardiomyopathy have also been connected to heterozygous loss-of-function mutations. Recently, a rare missense variant has been identified in Asian hypertrophic cardiomyopathy patients.^{Reference Chumakova and Baulina31} In our Egyptian study, the (p.Leu1603Pro) variant was classified as a VUS by ACMG, with predictions indicating potential deleterious effects on protein function.

The study successfully implicates genes beyond the canonical sarcomere in the Egyptian hypertrophic cardiomyopathy population. The discovery of a likely pathogenic variant in the PLN gene, a key regulator of calcium cycling, points to dysregulated calcium handling as a contributory pathway to the disease. Furthermore, the identification of variants in the KLHL24 gene and the ALPK3 gene underscores the role of proteins involved in sarcomeric stability, ubiquitination, and cardiomyocyte differentiation, expanding the genetic spectrum of hypertrophic cardiomyopathy to include developmental and proteostatic pathways.

Our analysis expands the known mutational spectrum of hypertrophic cardiomyopathy by identifying variants in both well-established sarcomeric genes and lesser-known or emerging genes, including KLHL24, PLN, ALPK3, and TTN. The detection of a likely pathogenic stop-gained variant in KLHL24 (p.Arg103*) and a likely pathogenic missense variant in PLN (p.Arg25Cys) adds to the growing body of evidence implicating these genes in hypertrophic cardiomyopathy pathogenesis. The submission of these novel variants to ClinVar (accessions SCV004562018, SCV006082319, SCV006082313, and SCV006082320) provides a valuable resource for the international scientific community and aids in future variant classification efforts.

Alpha-actinin 2, which is encoded by the ACTN2 gene, is an essential structural protein in the Z-disk that senses sarcomeric distention and mechanical traction, transmits force, and anchors the contractile apparatus. With substantial evidence supporting its familial inheritance in an autosomal dominant pattern, the current evidence linking ACTN2 to hypertrophic cardiomyopathy is based on a few missense variants.^{Reference Rodriguez, Schmeckpeper and Landim-Vieira32} Exemplary hypertrophic cardiomyopathy findings, such as enhanced calcium sensitivity in myofilaments, myocyte hypertrophy, hypercontractility, myofibrillar disarray, and extended relaxation, were observed in functional tests conducted using cardiomyocytes derived from human-induced pluripotent stem cells.^{Reference Guo, Wang and Li33} Even within families, disparate patterns of hypertrophy, left ventricular dilatation, obstructive and non-obstructive illness, atrial and ventricular arrhythmias, and conduction disorders make genotype–phenotype associations in hypertrophic cardiomyopathy unconvincing. Since the gene’s worldwide incidence in hypertrophic cardiomyopathy is modest, large case–control studies do not reveal any global enrichment of rare variations, indicating that only certain variants are associated with the disease.^{Reference Santoro, Mango and Mallardi34} In analyses of the ACTN2 gene, one patient was found to be a carrier of a VUS according to the ACMG classification. The variant is found at a low frequency in gnomAD.

The TTN gene encodes the titin protein, which extends from the M to Z lines of the sarcomere and is found only in skeletal muscles and cardiac myocytes. It can tolerate missense variations but is intolerant to loss-of-function variants. Truncating mutations in TTN are the most frequent cause of sporadic and familial dilated cardiomyopathy and are rare in hypertrophic cardiomyopathy.^{Reference Panwar, Singh and Mathur35} In our study, we found seven TTN variants, all of which are missense or intronic variants (i.e., non-truncating) and are categorised as VUS per ACMG guidelines. This aligns with the current understanding that truncating TTN variants are not a major contributor to hypertrophic cardiomyopathy pathogenesis. The role, if any, of these specific non-truncating VUS remains unclear. The high burden of TTN VUS (identified in 7 out of 8 patients) and their variable phenotypic impact—ranging from mild (EGP4) to severe (EGP6 and EGP7)—raises the hypothesis that TTN may act as a disease modifier rather than a primary driver. This is purely speculative at this stage, given the VUS classification of these variants, and warrants further investigation, including family segregation studies and functional assays, to determine if and how they might modify sarcomeric function. With respect to potential oligogenic effects, the co‑occurrence of multiple rare variants (e.g., in EGP6 and EGP7) is intriguing but remains speculative given the VUS status of many of these alleles.

The detection of a variant in ACTN2, encoding essential Z-disk proteins, reinforces the concept of the sarcomere as an integrated mechanosensory unit. Their involvement suggests that defects in force transmission and structural integrity can precipitate the hypertrophic cardiomyopathy phenotype. A significant proportion of the prioritised variants were classified as VUS, particularly in genes like TTN, ACTN2, and ALPK3. This highlights a common challenge in clinical genetics. It is crucial to emphasise that while we discuss the potential biological roles of these genes, the VUS status of these specific variants means their contribution to the patients’ phenotypes remains unconfirmed. This underscores the necessity of functional studies and population-specific data to reclassify these VUS and clarify their true pathogenic potential.

Myosin VI is involved in intracellular vesicular and organelle movement, where it is the unconventional actin-based molecular motor. It is involved in two stages of endocytic trafficking, being recruited to clathrin-coated pits and the subsequent uncoated endocytic vesicles.^{Reference Naccache, Hasson and Horowitz36} One variant was identified in the MYO6 gene, and the MutationTaster tool predicted a deleterious effect for one variant, c.1785G>T (Glu595Asp).

The MYLK2 gene encodes the calcium/calmodulin-dependent enzyme myosin light chain kinase (skMLCK) in fast-twitch fibres in adult skeletal muscle.^{Reference Wiebke, Andreas and Ottmar37} Calmodulin, which binds calcium, undergoes a conformational change upon calcium saturation, allowing it to interact with skMLCK, enhancing calcium affinity and promoting sustained muscle action. The MYLK2 gene variations are associated with mid-ventricular hypertrophic cardiomyopathy.^{Reference Ran, Li and Tan38} In our study, only one 38-year-old patient had a missense variant in the MYLK2 gene, Exon 11: c.1564G>A (p.Val522Ile), which is found at a low frequency in gnomAD (allele frequency = 0.000003977).

The identified genes and their associated OMIM phenotypes are summarised in Table 3, illustrating the broad phenotypic spectrum linked to these loci, including dilated cardiomyopathy, left ventricular non-compaction, and skeletal myopathies.