Identification and functional characterization of LDB3 gene variants in DCM patients from Indian Population

Abstract

The LIM domain-binding protein 3 (LDB3), also known as Z-band alternatively spliced protein (ZASP), is a key component of the Z-disc, essential for maintaining sarcomeric integrity and transmitting contractile force in cardiomyocytes. Genetic variants in LDB3 have been implicated in cardiomyopathies, including dilated cardiomyopathy (DCM). In this study, we have screened 100 idiopathic DCM cases along with controls by Sanger sequencing and identified nine non-synonymous (p.S184I, p.D193N, p.K204R, p.R229C, p.P295T, p.Q402P, p.F465I, p.F496L, p.P606S), seven synonymous (p.T91=, p.A152=, p.D168=, p.S182=, p.A279=, p.S347=, p.A358=), and seven intronic (c.322-50G>C, c.548+53A>C, c.718+47G>C, c.718+81G>A, c.755+11G>A, c.755+86G>A, c.*30C>G) variants, among which six (p.S184I, p.D193N, p.P295T, p.F465I, p.F496L, p.P606S) missense variants were novel. In-vitro studies demonstrated that p.K204R increased LDB3 expression whereas p.S184I, p.F465I, p.F496L, p.P606S reduced LDB3 expression. Immunostainning revealed cytoplasmic aggregation of the p.K204R-LDB3 protein. The variant p.R229C also over-expressed the mutant protein. In case of variant, p.P295T, Z-disc was severely disrupted indicating impaired Z-disc integrity. Furthermore, synonymous variants showed altered mRNA folding, stability, and codon usage bias, potentially affecting translation efficiency. In-silico analyses predicted p.F465I, p.F496L, p.P606S, and p.S184I variants to be deleterious, significantly altering protein structure. Structural modeling using AlphaFold2 showed high RMSD values, suggesting conformational destabilization. Collectively, these findings highlight LDB3 as a hypermutable candidate gene for DCM in our cohort that may contribute to DCM pathogenesis by perturbing protein structure, expression, and cellular localization, underscoring the critical role of LDB3 in cardiac muscle function and disease.