利用全外显子组和转录组高通量测序探索Lynch综合征的致病基因

The incidence rate of colorectal cancer is rapidly increasing and ranks the fifth most common cancer in China. Around 3%-5% of colorectal cancer belongs to the Lynch syndrome (hereditary nonpolyposis colorectal cancer [HNPCC]), which shows an autosomal dominant inheritance. Previous studies have showed that the Lynch syndrome is mainly caused by mutations in five DNA-mismatch repair genes (MLH1, MSH2, MSH6, PMS2 and EPCAM). However, in Chinese patients with Lynch syndrome, only 50% of them had mutations in the above genes, suggesting that there are unknown pathogenic genes for this syndrome. The aim of this study is to comprehensively screen the pathogenic genes / mutations in patients with Lynch syndrom by using the high-throughput sequencing technologies. We have collected more than 50 families showing the Lynch syndrome and 300 patients with other types of colorectal cancer. We will perform whole exome sequencing for these probands with no reported mutations of the five DNA mismatch repair genes, then validate the mutations / mutated genes in 300 patients and 300 healthy controls. Meanwhile, we will carry out RNA-seq analysis for the cancerous tissues and paracancerous normal tissues in these patients with newly identified pathogenic mutations, to reconstruct the profiles of the altered genes and pathways that are affected by the mutation(s). Our results will help to create a quick diagnosis chip with the reported and newly identified pathogenic genes based on the next-generation sequencing for Lynch syndrome. We will further characterize the potential change of the candidate rare variants at the cellular level. We believe that this will provide a theoretical and workable basis for genetic diagnosis of family members in hereditary colorectal cancer and facilitate with the early diagnosis and treatment.

我国结直肠癌的发病率逐年增高，现已位居恶性肿瘤发病率第五位。Lynch综合征是一类遗传性结直肠癌，占结直肠癌的3%～5%，该病的发生与DNA错配修复基因（MMR）的种系突变相关。然而，我国患者MMR基因突变检出率不足50%，提示我国患者人群中存在新的未知致病基因。本项目拟对我们前期收集到的50个Lynch综合征家系，在排除具有已报道的MMR基因突变的家系外，深入开展全外显子组分析，确定与该病潜在相关的基因突变，并在患者和对照人群各300例中开展验证。同时，对于含有新突变的相应个体的肿瘤组织和正常组织开展转录组学分析，构建出基因差异表达谱和潜在的调控网络。潜在的致病变异也将进行相应的体外功能分析。研究结果将为后续探索和定制基于下一代测序技术的Lynch综合征患者早期诊断基因Panel提供理论基础，实现Lynch综合征患者早期筛查，达到早诊早治，降低遗传性结直肠癌死亡率的目的，具有重要的意义。

结直肠癌（CRC）是全球高发癌症，在所有CRC病例中，至少20%是由于遗传因素引起。目前的研究结果仅能解释5-10%的遗传性结直肠癌。多数患病家系的遗传原因仍是未知的。为此，这项研究的目的是采用二代测序（NGS）的方法对符合遗传性结直肠癌诊断家系（包括MSS与MSI-H家系），在排除具有已报道的基因突变家系后，探索发现与该病潜在相关的基因突变。经过筛选，目前共入组12个遗传性结直肠癌家系，其中4个家系经检测为已知致病基因—DNA错配修复基因(MMR)的微卫星不稳定(MSI)，其他家系微卫星稳定(MSS)。我们对余8个家系进行了全外显子组测序，发现在家系7中基因X的杂合无义突变与家系成员疾病表型存在完全共分离，提示该位点是潜在遗传性结直肠癌致病位点。针对该基因突变位点，我们在内部对已收集的家系以及外部纳入300例散发结直肠癌对照人群进行基因X目标区域Sanger测序，均未发现突变。在COSMIC数据库中，提示该基因位点突变被发现分别存在于1例散发IV期脑星型细胞瘤及1例散发结直肠癌中，提示该无义突变可能与肿瘤发生有关。我们在接下来的独立家系验证中，进一步收集了8个符合入选条件家系，并对其核心成员进行该位点Sanger测序，发现家系13也存在该位点突变并实现共分离。这一发现为遗传性结直肠癌致病基因的列表增加了新成员，目前针对该致病位点作用已进行部分功能实验验证，也提示了可能的新机制有待研究确定。