Impact of cardiac myosin light chain kinase gene mutation on development of dilated cardiomyopathy
Impact of cardiac myosin light chain kinase gene mutation on development of dilated cardiomyopathy
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DOI：
10.1002/ehf2.12410
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发表时间：
2019-04-01
影响因子：
3.8
通讯作者：
Yamagishi, Masakazu
中科院分区：
文献类型：
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作者：
Hodatsu, Akihiko;Fujino, Noboru;Yamagishi, Masakazu
目的心肌肌球蛋白轻链激酶(CMLCK)磷酸化心肌肌球蛋白调节轻链2(MLC2v)，调节肌节和心肌细胞结构。然而，关于cMLCK突变和MLC2v磷酸化之间的关系，特别是在发现cMLCK基因突变的家族性扩张型心肌病(DCM)方面，几乎没有数据。本研究的目的是研究cMLCK突变对扩张型心肌病患者的功能影响。方法和结果扩张型心肌病的诊断基于患者的病史和超声心动图。我们用高分辨熔融法对DCM先证者进行cMLCK基因突变筛查。已知的引起DCM的基因突变通过家族成员的外显子组测序排除。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析MLC2v的磷酸化。我们还进行了ADP-Glo分析，以确定激酶反应中使用的三磷酸腺苷的总量。研究对象为无血缘关系的DCM先证者(男109例，女40例)，其中家族性16例，散发性133例。通过突变筛选，发现了一个c1915-1g>；t(p.Pro639Valfs*15)的截短突变，该突变在2 0 0名健康志愿者的4 0 0条染色体中未检测到，该突变被列入人类遗传变异数据库，等位基因频率为lt；0.001。在先证者中，通过外显子组分析排除了已知的引起DCM的基因突变的存在。家族性分析证实一位19岁男性携带者，表现为轻微的左心室扩张，但收缩功能未受影响。用PHOS-Tag SDS-PAGE进行的磷酸化分析表明，已鉴定的p.Pro639Valfs*15突变导致完全缺乏激酶活性，尽管它不影响野生型cMLCK的活性。ADP-Glo分析证实突变型cMLCK没有任何激酶活性，而野生型cMLCK的Km值为5.93+/-1.47muM，V-max为1.28+/-0.03moL/min/molkins.结论cMLCK基因p Pro639Valfs*15的截短突变可能与MLC2v磷酸化的显著损害有关，并可能与DCM的发生有关，但需要对DCM患者进行更大规模的研究来确定该突变的发生率，并进一步加强其与疾病发展的相关性。
Aims Cardiac 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 results The 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 mu M and a V-max of 1.28 +/- 0.03 mol/min/mol kinase.Conclusions These 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.