AbstractAimsCardiac myosin light chain kinase (cMLCK) phosphorylates ventricular myosin regulatory light chain 2 (MLC2v) and regulates sarcomere and cardiomyocyte organization. However, few data exist regarding the relationship between cMLCK mutations and MLC2v phosphorylation, particularly in terms of developing familial dilated cardiomyopathy (DCM) in whom cMLCK gene mutations were identified. The purpose of the present study was to investigate functional consequences of cMLCK mutations in DCM patients.Methods and resultsThe diagnosis of DCM was based on the patients' history and on echocardiography. We screened cMLCK gene mutations in DCM probands with high resolution melting analysis. Known DCM‐causing genes mutations were excluded by exome sequencing of family members. MLC2v phosphorylation was analysed by Phos‐tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis assays. We also performed ADP‐Glo assays for determining the total amount of adenosine triphosphate used in the kinase reaction. Unrelated DCM probands (109 males and 40 females) were enrolled in this study, of which 16 were familial and 133 sporadic. By mutation screening, a truncation variant of c1915‐1 g>t (p.Pro639Valfs*15) was identified, which was not detected in 400 chromosomes of 200 healthy volunteers; it is listed in the Human Genetic Variation Database with an allele frequency < 0.001. In the proband, the presence of mutations in known DCM‐causing genes was excluded with exome analysis. Familial analysis identified a 19‐year‐old male carrier who manifested slight left ventricular dilation with preserved systolic function. Phosphorylation assays analysed by Phos‐tag SDS‐PAGE revealed that the identified p.Pro639Valfs*15 mutation results in a complete lack of kinase activity, although it did not affect wild‐type cMLCK activity. ADP‐Glo assays confirmed that the mutant cMLCK had no kinase activity, whereas wild‐type cMLCK had a Km value of 5.93 ± 1.47 μM and a Vmax of 1.28 ± 0.03 mol/min/mol kinase.ConclusionsThese results demonstrate that a truncation mutation in the cMLCK gene p.Pro639Valfs*15 can be associated with significant impairment of MLC2v phosphorylation and possibly with development of DCM, although a larger study of DCM patients is required to determine the prevalence of this mutation and further strengthen its association with disease development.