The SMYD1 p.Asn101Ser is a partial loss-of-function variant that impairs mitochondrial function and leads to...

Infantile cardiomyopathies are rare, life-threatening disorders for which genetic diagnosis has been accelerated by next-generation sequencing approaches, including gene panel, exome, and genome sequencing. However, determining the functional consequences of identified variants remains a major challenge. Variants in SMYD1, a striated muscle-specific lysine methyltransferase critical for cardiac development and mitochondrial function, have only recently been linked to human cardiomyopathy. Here, we functionally characterize a homozygous SMYD1 variant (c.302A>G; p.Asn101Ser) identified in a patient with severe early-onset cardiomyopathy requiring cardiac transplantation. Structural modeling predicts that the N101S substitution perturbs a highly conserved residue near the cofactor binding pocket within SMYD1's catalytic domain, disrupting local interactions and modestly destabilizing the protein. Consistent with these predictions, in vitro studies demonstrate that the N101S variant impairs mitochondrial respiratory capacity in myocytes. Quantification of SMYD1 protein levels in patient cardiac tissue revealed increased SMYD1 abundance, suggesting that the N101S variant results in functional impairment rather than protein instability and may trigger compensatory upregulation of SMYD1 expression. Together, these findings support a hypomorphic mechanism in which the N101S variant disrupts SMYD1 activity, leading to mitochondrial dysfunction and cardiomyopathy. This study provides mechanistic insight into SMYD1-associated cardiomyopathy and highlights the importance of integrating genetic, structural, and functional analyses to establish the pathogenicity of rare variants.