组蛋白H3K4去甲基化酶KDM5A在先天性心脏病中的作用研究

Congenital heart diseases (CHD) are the most common birth defects and cause significant morbidity and mortality in children worldwide. The incidence of CHD has been estimated to appropriately seven per 1000 newborns which has not changed over the past several decades. The molecular cause of CHD remains largely unknown. Ongoing studies using genomic microarrays and next-generation sequencing have demonstrated that the genetic contributions to cardiovascular diseases have been significantly ignored in the past. We performed high-resolution genome-wide microarrays for chromosomal imbalances in over 200 syndromic CHD patients with an Illumina SNP-array platform, and fortunately, we identified a 417kb duplication at 12p13.33 locus in a child with CHD and polydactyly. This duplicated region contained KDM5A/JARID1, which encodes a histone H3K4 demethylase. The KDM5 subfamily has four members KDM5A, KDM5B, KDM5C and KDM5D, while KDM5B and KDM5C have been linked to heart diseases in conditional knockout mice. Microarray-based gene identification has been demonstrated to be an efficient and reliable method, and zebrafish has also been shown to be a promising model to study cardiac development and human cardiac disease (Cardiovascular Research 2011,91, 279-288). For these reasons, we propose to use the zebrafish to identify KDM5A that underlies CHD. Our study will contribute to the identification of causative gene for CHD, and our understanding of specific human CHDs in the future.

先天性心脏病(CHD)是最常见的出生缺陷。近年来，表观遗传学在CHD发病机制中的研究逐渐兴起。得益于全外显子测序和芯片技术的应用，多个新鉴定的CHD 综合征致病基因涉及组蛋白表观遗传学修饰。本小组前期采用SNP-array芯片技术对综合征型CHD进行研究，在一例CHD合并多指患者中鉴定到包含组蛋白赖氨酸去甲基化酶KDM5A/JARID1的12p13.33微重复，这一发现尚未见文献报道。推测KDM5A过表达可能破坏组蛋白甲基化平衡状态，影响心脏发育关键基因的转录调控，进而影响原始心肌细胞的增殖和分化，导致CHD的发生。为证实这一假说，我们将以斑马鱼为模式生物，利用SNP-array、IP、ChIP-seq以及mRNA显微注射等技术，从群体-个体-分子-模式生物四个层次，探索KDM5A与CHD关系。本课题有可能鉴定新的CHD致病基因并报道一类新综合征，同时从新的视角阐明CHD发生的分子机制。

近年来，表观遗传学在CHD 发病机制中的研究逐渐兴起。得益于全外显子测序和芯片技术的应用，多个新鉴定的CHD 综合征致病基因涉及组蛋白表观遗传学修饰。本小组前期采用SNP-array 芯片技术对综合征型CHD 进行研究，在一例CHD 合并多指患者中鉴定到包含组蛋白赖氨酸去甲基化酶KDM5A/JARID1 的12p13.33 微重复，进一步对搜集的56例综合征型CHD芯片分析没有发现新的与KDM5A相关的微缺失或微重复，但利用高通量测序的靶向测序技术，在排除致病性CNV的CHD患者中鉴定出3例KDM5A变异（p.H808Q,、p.V1047和p.K1680E），突变体的功能及其致病原因尚待新的研究证实。另外，我们报道了4例新的致病性CNVs。这些CNVs分别包含如BMPR1A、SKI、NFIX等基因。通过CRISPR/Cas9技术我们构建了KDMA5A突变体敲入斑马鱼，发现突变成鱼心功能确实降低。同时我们利用质谱技术探索了KDMA5A的相互作用蛋白，从分子水平探索其原始心肌细胞的增殖和分化导致CHD发生的可能性。我们的研究证实过多表达的KDM5A影响成鱼心脏功能。