目标区域靶向捕获测序及基因功能研究鉴定原发性闭角型青光眼的致病突变

Glaucoma, the leading cause of irreversible blindness worldwide, is classified according to the configuration of the irido-corneal angle into open angle and angle-closure glaucoma. Primary angle closure glaucoma (PACG) is the major form of glaucoma in China and Asia compared to primary open angle glaucoma (POAG), which is more common in Caucasians and Africans. 40% -60% of glaucoma in China is PACG that the prevalence rate of over the age of 50 is 1.2% -2.1%, the blindness rate up to 35%. PACG is a serious hazard to the visual health in our country and is material ophthalmology science problem. The pathogenesis of PACG is thought to occur through the interplay of multiple anatomical and physiological factors, and thus shows many indications of being a complex disease, with both genetic and environmental etiological factors. Our research group conducted a genome-wide association study (GWAS) and found significant associations at three new loci: rs11024102 in PLEKHA7 (per-allele odds ratio (OR) = 1.22; P = 5.33x10−12), rs3753841 in COL11A1 (per-allele OR = 1.20; P = 9.22x10−10), and rs1015213 located between PCMTD1 and ST18 on chromosome 8q (per-allele OR = 1.50; P = 3.29x10−9). Then we conducted another genome-wide association study on anterior chamber depth (ACD) to discover novel genes for PACG on a total of 5,308 population-based individuals of Asian descent. Genome-wide significant association was observed at a sequence variant within ABCC5 (rs1401999; per-allele effect size =20.045 mm, P = 8.17x10-9). We hypothesize that the genetic loci associated with PACG are located in the areas that related with the 4 SNPs. In this study, we plan to focus on the 4 SNPs related areas and identify the pathogenic mutations associated with PACG by targeted capture and high throughput sequencing technique. This will be followed by validation experiments in independent cases and controls; then the study of the genotype—phenotype correlation, and the evaluation of the association with PACG of the significant SNPs/ loci identified in earlier two stages. We may conduct the gene function pilot study based on the sequencing results by setting up gene knock-out and transgenic mice model. We may be successful in unraveling the genetic determinants that confer individual susceptibility to PACG. The research will suggest possible mechanisms explaining the pathogenesis of PACG. An ability to elucidate the spectrum of genetic loci associated with the disease would provide perspectives into the genetic mechanisms responsible for individual susceptibility to the condition. In the future, the information obtained is likely to enhance the efforts to develop a clinically useful genetic profile for early detection or risk stratification for this blinding disorder.

原发性闭角型青光眼是我国首要不可逆性致盲眼病，是复杂性遗传相关疾病，早期诊断及治疗是降低致盲率的关键。临床尚缺乏有效的早期诊断和病因学治疗方法，遗传发病机制研究是解决问题的突破口。课题组前期研究发现了闭青相关的4个新易感位点（rs1401999、rs11024102、rs3753841和rs1015213）。由此提出假说，闭青的致病基因就在这4个SNPs相关范围内。拟将研究范围从全基因组缩小到这些前期发现的目标区域中，采用靶向捕获高通量测序方法鉴定闭青致病基因/位点突变，并在独立样本中进行验证，开展表型与基因型关联分析、基因间相互作用关系分析，建立基因敲除、转基因小鼠及交配后小鼠模型，探索序列变异导致闭青发生的致病机制研究，揭示闭青个体易感性的遗传机制。结果将有助于建立一个整合遗传学易感基因及解剖学危险因素的发病风险预测模型，提高闭青早期诊断率，为后续诊断芯片开发及病因学治疗提供实验依据。

原发性闭角型青光眼是我国首要不可逆性致盲眼病，是复杂性遗传相关疾病，早期诊断及治疗是降低致盲率的关键。临床尚缺乏有效的早期诊断和病因学治疗方法，遗传发病机制研究是解决问题的突破口。本课题主要研究内容包括：1、收集和建立闭青生物样本库、临床资料库。2、采用全外显子测序技术分别对闭青家系和散发病例进行测序，然后通过sequnom MassARRAY技术及Sanger测序进行验证研究；3、对来自全国闭青病例-对照共8000个样本进行GWAS研究，寻找与闭青易感性相关的SNP位点及基因。本研究通过成立“中国青光眼百家研究联盟”收集到来自全国的1万例闭青血样，建立了闭青生物样本库，通过电子化数据病例录入管理EDC系统建立了相应的临床资料库。为后续闭青遗传研究奠定了良好的基础。对1463例散发闭青和1309例正常对照进行全外显子测序，没有发现之前两次GWAS研究发现的SNP位点有显著性差异，但是发现11号染色体突变NUCB2 p.F318C（rs750095360,17336973 T>G）仅出现在闭青患者中，而在正常对照中没有。为进一步明确其与闭青的关系，我们用在“中国青光眼百家联盟”收集到的来自全国各地的散发闭青2053例和正常对照2054例对这个NUCB2 p.F318C位点进行Sanger测序验证。但遗憾的是验证结果为阴性：我们发现病例组和对照组都各有2例有此变异。对散发闭青病例-对照研究没有发现有显著差异的位点。我们选择典型闭青家系进行研究，采用全外显子组测序，然后在散发闭青病例中进行验证，发现HOOK2 rs897804和GTPBP3 rs3745193 的新非同义突变，可能与闭青发病相关。闭青样本量充足是课题组的优势，目前正在进行的是中国人闭青GWAS研究，第一阶段全基因组SNP芯片检测正在进行中。本研究共发表标注基金号的文章10篇，其中一篇发表在Nature Genetics. 2016；48(5): 556-562。课题执行过程中，建立起比较完善的科研团队，核心团队包括医生、护士、统计专家、数据库专家等。同时培养了科研护士和硕士研究生，在全国各地分中心医院中培养了数百名相关研究人员。本研究探究闭青遗传学发病机制，结果将有助于建立一个整合遗传学易感基因及解剖学危险因素的发病风险预测模型，提高闭青早期诊断率，为后续诊断芯片开发及病因学治疗提供实验依据。