Dilated cardiomyopathy (DCM) is characterized by left ventricular dilatation and systolic dysfunction in the absence of significant coronary artery disease or volume overload. Although relatively rare in children, with an annual incidence estimated at 0.57 per 100,000, DCM is a major cause of heart failure and cardiac transplantation. ¹

In pediatric forms, the aetiology is often difficult to establish. Approximately 40% of childhood DCM cases remain idiopathic after standard investigations, but a growing number of studies have highlighted the significant role of genetic causes accounting for about 35% of cases. ^{2, 3} Over 50 genes have been implicated in the pathogenesis of dilated cardiomyopathy (DCM) to date. ^{4, 5} A number of these genes encode proteins essential for mitochondrial function and the preservation of cellular integrity, such as those coding for sarcomeric, cytoskeletal, nuclear envelope and Z-disk-associated proteins proteins. ^{6, 7}

Among the most frequently implicated genes in familial DCM, the Tittin gene ( TTN), encoding the sarcomeric protein Titin, is considered the main susceptibility gene in adults. It was reported that truncating variants in TTN (TTNtv) gene account for about 25% of non-ischemic DCM cases in adults. ^{8, 9} However, the contribution of TTN in pediatric cases is less defined, with a lower mutation rate and significant phenotypic variability. ¹⁰

CTNNA3 gene, encoding alpha-T catenin, has been associated with inherited forms of arrhythmogenic right ventricular cardiomyopathy (ARVC). In a murine model, van Hengel et al. showed that CTNNA3 inactivation disrupted intercellular junction architecture and led to ventricular dilation. ¹¹

Whole-exome sequencing (WES) is increasingly emerging as a diagnostic tool, allowing the identification of rare and potentially pathogenic mutations even in the absence of a clear family history. The relevance of WES in clarifying complex cardiomyopathies and guiding familial screening has been reported. ^{12, 13}

This study reports the clinical features of a severe DCM in a 2-year-old boy and links this pathogenesis with the co-existence of two pathogenic genetic variants in TTN and CTNNA3 genes. Overall, the identification of these mutations broadens the base of pathogenic variants linked to DCM.

Dilated cardiomyopathy (DCM) is a clinically and genetically heterogeneous disorder characterized by dilation and impaired contraction of the left or both ventricles, leading to systolic dysfunction. ^{1, 14, 15} Mutations in genes encoding sarcomeric and cytoskeletal proteins are major contributors to both familial and sporadic forms of DCM. ^{5, 16, 17} Among these genes, TTN is the most frequently mutated in DCM. ^{8, 9}

The TTN gene, encoding the sarcomeric protein titin, plays a critical role in maintaining myocardial elasticity and mechanical stability. Truncating variants in TTN (TTNtv) including nonsense mutations, frameshift mutations, and out-of-frame splice site variants, represent the most prevalent genetic cause of dilated cardiomyopathy (DCM). ⁹ Studies on large cohorts reported that TTNtv were identified in 11-15% of adult cases of sporadic DCM and 23-27% of familial DCM. ^{18– 23}

The pathogenic effect of TTNtv is position-dependent, with variants located in the A-band region of the gene being particularly deleterious, given their substantial disruption of sarcomeric structure and function. ²⁴

In this study, we identified a heterozygous nonsense variant, c.95008C>T (p.Arg31670*), in exon 342 of the TTN gene, which introduces a premature stop codon. This variant has been reported previously in familial forms of DCM. ²⁵ Familial segregation analysis confirmed that the variant co-segregated with the disease phenotype, being present in all affected individuals, while unaffected family members carried the wild-type allele.

The CTNNA3 gene encodes alpha-T catenin, a key component of the intercalated disc that plays a crucial role in myocardial intercellular adhesion. ¹¹ Although CTNNA3 has been less extensively studied in the context of DCM, it has emerged as a potential candidate gene. ^{26, 27} Notably, Janssen et al. proposed CTNNA3 as a candidate disease gene in a family with DCM linked to the 10q21–q23 locus, where this gene is located. However, no pathogenic variants were identified in that study, and the gene’s involvement in DCM remained inconclusive. ²⁸ Subsequent research demonstrated that alpha-T catenin protein levels are significantly reduced in DCM, and that decreased CTNNA3 expression correlates with left ventricular ejection fraction (LVEF) and ventricular dimensions, suggesting a potential role in disease progression. ²⁹ Recently, proteomic and phosphoproteomic approaches have provided evidence supporting a role for CTNNA3 phosphorylation in the pathophysiology of DCM. ³⁰

In this study, we identified a novel heterozygous nonsense variant in the CTNNA3 gene, c.2023G>T (p.Glu675*), in the proband. This variant introduces a premature stop codon and is classified as pathogenic based on ClinVar annotations and ACMG criteria.

Interestingly, both germline CTNNA 3 and TTN variants coexist in the proband and other affected family members, suggesting a potential digenic contribution to the disease phenotype. Furthermore, two younger family members, who are currently asymptomatic, carried only one of the two identified variants either the TTN or the CTNNA3 nonsense pathogenic/likely pathogenic variant. This finding suggests the possibility of incomplete penetrance, variable expressivity, or age-dependent onset of the disease. Clinical evaluation, including transthoracic echocardiography, revealed no significant abnormalities in those individuals. The absence of a detectable phenotype in carriers of a single variant supports the hypothesis that co-inheritance of both TTN and CTNNA3 variants may be required for disease expression, consistent with a potential digenic inheritance model contributing to the pathogenesis of dilated cardiomyopathy in this pedigree. In our study, the early and severe disease onset at the age of 2 may reflect an additive or synergistic effect of the TTN and CTNNA3 variants, supporting the hypothesis that combined genetic hits may increase disease severity and accelerate its clinical manifestation.

The co-occurrence of pathogenic variants in cardiomyopathy-associated genes is relatively rare but has been increasingly recognized as a potential contributor to more severe or early-onset phenotypes. Recently, Han et al. report a case of DCM with a novel TTN variant, as well as two rare variants in the SCN5A and LDLR genes. ³¹ It is interesting to mention that the TTN/CTNNA3 combination has not been reported previously in DCM.

This finding further underscores once again the genetic complexity of cardiomyopathy and highlights the utility of whole-exome sequencing (WES) in identifying novel candidate genes to improve patient management. In our previous work, we performed WES on a Tunisian family presenting with complex hypertrophic cardiomyopathy and identified a variant in the MRPL44 gene that segregates with the disease phenotype, suggesting a potential role for MRPL44 in the genetic etiology of hypertrophic cardiomyopathy. ³²

In conclusion, this case highlights the critical importance of comprehensive genetic evaluation in pediatric DCM, particularly when familial inheritance is suspected. The co-occurrence of two pathogenic variants in the TTN and CTNNA3 genes in a symptomatic family member underscores the genetic complexity of cardiomyopathies and supports the hypothesis of a multigenic model contributing to severe, early-onset phenotypes. These findings directly informed a targeted family screening strategy and prompted close cardiologic monitoring for at-risk relatives, enabling early identification and intervention.

The Institutional human ethics committee of the Faculty of Medicine of Sfax-Tunisia., approved this work (N ^o65/24). Written informed consent to participate in this study was provided by all participants and, for children, by their legal guardian/close relative. All data were fully anonymized prior to analysis to ensure confidentiality, in compliance with ethical standards, and in adherence to the Declaration of Helsinki.