13q染色体末端先天性心脏病致病基因的鉴定及功能研究

Congenital heart disease (CHD) is the most common birth defect and cause significant morbidity and mortality in infants and children worldwide. The incidence of CHD, which has not changed over the past several decades, has been estimated to appropriately seven per 1000 newborns. The molecular mechanism of CHD remains largely unknown. Ongoing studies of genomic microarrays and next-generation sequencing have demonstrated that the genetic contributions to cardiovascular diseases have been significantly underestimated 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 identified a 13q terminal deletion in a child with CHD and other malformations. By literature review, we proposed a distal 6 Mb region for the causative gene of congenital heart defect. In 2012, we identified a 1.1 Mb deletion at chromosome 13q34 in a new patient with CHD, limbs anomalies and mild mental retardation. Our study narrows the SRO (shortest region of overlap) to a 1.1 Mb region, which encompassing 13 genes. Recent advances in next-generation sequencing and genomic microarrays have greatly facilitated the discovery of causative genes for cardiovascular diseases. Microarray based gene identification has been demonstrated to be an efficient and reliable method. And zebrafish have shown to be a promising model to study cardiac development and human cardiac disease. Here, we propose to use the zebrafish to identify the key gene(s) for CHD in the 1.1 Mb region of 13q terminal deletion region. With the expanding number of distal 13q deletion syndrome patients and accurate genomic microarrays data, as well as the zebrafish MO(Mopholino oligonucleotides) model with development defects, our study may contribute to the identification of causative gene for CHD in distal 13q deletion syndrome and the understanding of specific human CHD.

先天性心脏病(CHD)是最常见的出生缺陷,也是新生儿和婴幼儿主要死亡原因之一。CHD发病机制尚未阐明，最新研究表明遗传因素在CHD发生中的重要作用被低估。我们采用SNP-array技术对综合征型CHD进行全基因组微阵列扫描，鉴定了13q末端 6Mb区间存在与CHD相关核心基因。前期研究中，一例特殊综合征型CHD帮助我们进一步将区间缩小到1.1 Mb，包含13个基因。我们推测其中的候选基因单倍剂量不足可能改变所在信号通路，继而影响原始心肌细胞的增殖、迁移和分化，从而导致CHD的发生。为证实这一假说，我们将以斑马鱼为模式生物，采用反向遗传学研究方法，利用SNP-array、RNA-seq、吗啡啉寡核苷酸法(MO)等技术，从群体-个体-分子-模式生物四个层次，鉴定13q末端CHD核心基因。本研究有望发现新的CHD致病基因,从新的视角阐明CHD发生的分子机制,为CHD遗传咨询和产前诊断提供新依据。

项目背景：先天性心脏病 (CHD)是最常见的出生缺陷,其发病机制尚未完全阐明。全基因组微阵列芯片及新一代测序技术研究表明遗传因素在CHD发生中的重要作用被低估。本课题组采用全基因组微阵列芯片技术和新一代测序技术对综合征型CHD进行全基因组微阵列扫描。前期研究中，我们鉴定一例特殊CHD，进一步将区间缩小，包含13个基因。我们推测候选基因单倍剂量不足可能改变其所在信号通路，影响原始心肌细胞增殖、迁移和分化，从而导致CHD的发生。.研究内容：应用SNP array芯片技术对多种综合型CHD患者进行检测拷贝数变异（CNVs）。使用RT-PCR验证SNP array芯片分析结果。对发现的CNVs区域的基因进行筛选鉴定。运用全外显子测序对相关患者进行分析,并利用基因数据库对测序结果中的已知变异进行筛查；在去除已知变异后选出患者共有而正常人没有的疑似突变；然后运用生信软件对疑似突变进行预测,选出致病性较高的候选突变；最后利用PCR扩增以及Sanger测序法将候选突变在家系内外进行验证。 .主要结果：项目组成功鉴定十余种综合征型CHD患者中的致病性CNVs。项目组诊断并报道了国际首例SHOC2基因的单倍剂量不足可引起RAS相关疾病表型病例，国内首例Emanuel综合征病例、首例1q21.1微重复的病例；首次在CHD患者中报道10p15.1-15.3重复型CNVs，5q13.2缺失型CNVs，5q13.3-14.1重复型CNVs，15q26.3重复型CNVs，16p13.3缺失型CNVs。上述CNVs包含的基因，如SMN1基因等可能是CHD易感基因。首次使用全外显子测序结合CHD相关基因过滤的方法研究小家系遗传致病因素，鉴定出并首次报道全新的家族性房间隔缺损相关性TBX20突变。全外显子测序发现遗传性心律失常致病基因SCN5A上新发现的杂合突变p.Y1495X。.科学意义：上述成果强调了高分辨技术在染色体异常的分子生物学诊断上的必要性与重要性，并为类似患者的致病CNV定位及表型描述奠定基础。新报道的CNVs有助于阐明先心病的关键基因，并为CHD的发病机制探索提供新线索。项目组提出的全外显子测序结合CHD相关基因过滤的方法来研究小家系遗传致病因素既有效又经济。为此类患者的诊断提供新思路。