Delta-Sarcoglycan基因的两个新突变在东亚人遗传性心肌病中的致病作用及其机理

Cardiomyopathy (CM), one of the common cause of heart failure and mortality, is divided into hypertrophic (HCM) and dilated (DCM). The genetic abnormalities would account for about 50-70% of the cases of HCM and 25-30% of the cases of DCM. For HCM, it mainly involves mutational sarcomeric protein genes. For DCM, although the disease owns mutational genes encoding cytoskeleton proteins, only 30~35% mutation has been reported. Dystrophin glycoprotein complex (DGC) is composed of a transmembrane subcomplex of α-, β-, γ- and δ-sarcoglycan (SG), α- and β-dystroglycan (DG), and dystrophin. Dystrophin serves as a link between intracellular F-actin and DGC. In Syrian hamster CM model, a 30 kb deletion located in δ-SG gene promoter area has been reported as the etiology. In human, a few positive reports from Japanese and Amerindian and negative results from Caucasus suggested that the role of the δ-SG gene in inherited CM might be more important in eastern Asians than that in other ethnicities. However, this hypothesis needs to be identified in Chinese population. On the other hand, it is still unclear whether there is any CM-causing abnormality in human δ-SG gene promoter, just like the hamster model. The exact promoter region of the gene has not been even determined. In this study, we aimed to determine the promoter region and then to search for mutations among the promoter, 5'UTR, and exons in Chinese patients with HCM and DCM. In our pilot studies, we found: 1) the promoter region ranging from -338 to +476bp. This region includes 7 E-box and 3 MEF-2 putative motifs. Of them, the 2nd and 6th E-box were proven to be functional; 2) a novel 11bp deletion (Del-100~-110) of a putative MEF2C binding motif located just downstream of the 6th functional E-box; 3) homozygous Del-100~-110 decreased the promoter activity to two third of the control level; 4) the prevalence of homozygous Del-100~-110 is 2.1 times higher in HCM patients; 5) EMSA results showed that the probe without Del-100~-110 lost the binding of an unknown transcriptional factor (TF); 6) a novel heterozygous missence mutation of A848G resulting in Q283R in the highly-conserved C-terminus. 7) The frequency of heterozygous A848G is 4.9 times higher in DCM patients; 8) δ-SG-283R has a significantly decreased binding affinity with b-SG in vitro protein pull-down assay; and 9) both novel mutations were found only in Mongoloid (Chinese, Korean, Japanese, and Thai), but not in Africa and Caucasus population. Next, we plan to: 1) determine which bHLH or MADS TF binds to the WT sequence without Del-100~-110; 2) expand CM patient number and try to link homozygous Del-100~-110 to heterozygous A848G and, if luckily, meet a homozygous 848GG patient to study their cardiac phenotype; and 3) establish a knock-in mice model of homozygous 848GG and to study their cardiac phenotype and try to rescue the cardiac phenotype with virus-guided transfection of normal δ-SG-848AA gene.

遗传性心肌病会发生心衰、猝死、缺乏除心移植之外的有效疗法。其致病基因任然不明，肌细胞膜上dystrophin glycoprotein complex （DGC）中的δ-SG基因的作用有待研究。申请人确定了该基因的启动子部位并找出该部位中东亚人特有的11bp的缺失突变，其在HCM中的发生率比正常人高2.1倍。该缺失突变为TA丰富序列，还可能破坏了前面紧邻的一个E-box，导致启动子活性下降35%，EMSA实验表明该缺失导致一个未知的转录因子的结合力减弱。我们还在该蛋白高度保守的羧基端找出东亚人特有的杂合子Q283R突变，其在DCM中的发生率比正常人高4.9倍。283Q是DGC中其他二成员的羧基端都有的决定DGC稳定性的重要氨基酸。体外证实δ-SG283R与β-SG结合力下降。接下来，要确定与缺失区原本序列结合的转录因子；寻找纯合子848GG病人和制作纯合子848GG小鼠并研究其表型及机理。

心肌病（CM）是心衰，心律失常及心源性死亡的常见原因，主要分为扩张性（DCM）及肥厚性心肌病（HCM）。遗传学异常可以解释70%的HCM和30% 的DCM。与HCM的致病基因已经大致明了不同，DCM的致病基因远远没有清楚。特异性DCM致病基因多数与细胞骨架蛋白组成的DGC糖蛋白复合体有关. 这个复合体主要有α-, β-, γ-, 及δ-SG, α-及β-DG, 以及 dystrophin（肌细胞内F-actin 与DGC复合体之间的桥梁）组成。这个复合体的各个基因突变可以导致肢体肌营养不良，这种病人多伴有心肌病。δ-SG基因的遗传学异常在舒利亚仓鼠中可致心肌病。但在人类DCM及HCM中的发病作用任然不明。我们首先确定了该基因的启动子部位。在104例中国人DCM，145例HCM，以及790正常人的启动子，5’-UTR，以及8个外显子部位找到2种新的SNPs：启动子部位的11bp缺失和在高度保守的羧基端的错义突变（A848Gp.Q283R）。11bp缺失的出现频率在DCM （14.42%）及HCM （14.48%）病人中高于正常对照组（11.01%）。A848G出现频率在DCM 病人（6.73%; OR=9.43; p=0.0002）中明显增高，而在HCM 病人（1.38%; OR=1.37; p=0.62）及正常对照（0.76%）中不高。由基因型11bp缺失和A848G组成的半倍体（-_G）与增高的DCM危险性相关（OR=17.27; 95%CI=3.19-93.56; p=0.001），而与HCM不相关（OR=1.90; 95%CI=0.38-9.55; p=0.44）。基因型-/- 的11bp缺失与 848A/G 组成的纯合体在5名DCM 病人（4.81%; OR=39.85; p=0.0001）及1例正常人（0.13%）中出现，而没有在HCM病人中出现。这二种新的SNPs在日本人群中有发现，而在非洲黑人及高加索白人中没有发现。11bp缺失导致-SG基因启动子活性下降到正常值的64±3%（p<0.01）。免疫共沉淀及体外蛋白质下拉实验都表明 δ-SG-283R与β- 及γ-SG结合正常，但是β/δ/γ-SG复合体的肌膜定位量明显下降。总之, 由11bp缺失及A848G组成的半倍体 （-_G）伴随着我们蒙古黄色人种中人群对DCM的患病易感性。