外显子测序探寻双侧纹状体坏死家系致病基因的研究

Bilateral striatal necrosis (BSN) is a group of nervous system functional syndromes. Hereditary BSN is a clinical and genetic heterogeneous neurological disease, characterized mainly by generalized dystonia. Our group has collected a pedigree with 3 affected children whose clinial symptoms are inconsistent with those caused by the genes currently known. Genetic variants in genes NUP62、SLC19A3、SLC25A19、ATPase6,mitochondrial ND1，3，4，6 appeared not to be co-segregated with BSN in this family. As such, we speculate that this might be a new type of disease. To identify the causal genetic mutations and better understand the pathogenesis of these clinical symptoms, we conducted whole genome exome sequencing in two affected individuals and one unaffected individual from this BSN family and identified ten genes including point mutation and one gene including large deletions using bioinformatic analysis. Our next step aims to identify the real causal genetic mutations among these eleven candidate genes, and to explore the potential underlying mechanisms. First, we will predict the function of these candidate genes using ANNOVAR and GERPscore software, and then genotype the mutations that might have a severe impact on the corresponding molecule in all of the affected and unaffected individuals of this family with available DNA sample, using Sanger sequencing to identify complete co-segregation between the mutations and BSN phenotype. In addition, we will sequence all coding exons and flanking introns of the gene co-segregated with the disease in another BSN family and 100 normal controls to identify the real causative gene. Finally, further functional analysis will be conducted to explore the relationship between genotype and phenotype according to the structural and functional characteristics of the causative gene, which will provide a theoretical basis in order to reveal the BSN pathogenesis and gene therapy.

遗传性双侧纹状体坏死（BSN）是一类具有临床和遗传异质性神经遗传病，主要表现为全身肌张力障碍。本课题组已收集一个家系，其表现与已知基因引起的临床表型不一致，有可能为新的疾病类型，并已通过扩增测序已知基因得以验证。前期工作采用全外显子测序方法，测定两名患者和家系中一名正常对照的外显子突变图谱，利用生物学分析手段，确定了10个含有点突变或者缺失和1个含有大片段缺失的候选基因。下一步将对这些候选基因进行功能预测，筛选出可能与疾病相关的突变位点，并针对这些位点在家系其他成员中进行进一步的验证和排除，以确定家系中与疾病共分离的基因突变。对与疾病共分离的突变所在基因在其他BSN家系以及正常对照中进行该基因全外显子和侧翼区测序，筛选出真正的致病基因。并根据致病基因的结构和功能特点，进一步进行功能分析，研究突变型与表型的关系，探讨基因突变致BSN的机理，为揭示BSN的发病机制及基因治疗提供理论依据。

遗传性双侧纹状体坏死（BSN）是一类罕见的具有临床和遗传异质性神经系统遗传病，主要表现为全身肌张力障碍。由于本病致残致死率高，确定其致病基因对于产前诊断、优生优育至关重要。通过三年的课题研究，我们发现了该双侧纹状体坏死家系的致病基因，并发现了新的致病突变，丰富了遗传突变谱，为将来的遗传咨询、产前诊断及基因治疗打下理论基础。.我们课题组对该家系患者及正常对照进行了两次全基因外显子测序，第一次外显子测序未找到候选基因，分析原因可能与个别样本外显子测序覆盖度、深度不足有关。再次的外显子测序覆盖度和深度足够支持分析，得到候选基因6个。通过检索文献，并结合该家系特点和疾病特点找到BSN(代码)基因。针对该基因在家系内通过PCR sanger测序的方法进行验证，验证结果为三个患者均为复合杂合突变，发现的两个突变均为未报道过的新突变。父母亲分别携带其中一个突变，未患病的两个正常同胞携带其中一个突变。故该基因在家系内验证符合疾病共分离、表型共传递的特点。该基因功能与线粒体复合物活性有关，在家系成员中进行外周血线粒体复合物I活性的检查发现先证者明显低于其正常母亲的水平，但是仍属于正常水平,正常低值。候选致病基因BSN功能验证通过体外过表达检测其对相关指标的影响，构建BSN基因野生型和突变型的质粒载体，导入HEK293和Hela细胞模型。从3个方面来观察相关指标的变化：（1）检测蛋白降解量；（2）检测蛋白在细胞中的定位，以及野生型和突变型在细胞定位中有无区别；（3）对线粒体的影响，利用试剂盒检测线粒体复合物I的活性，如果有明显影响检测ATP的释放，电镜观察线粒体形态是否发生改变。此部分目前试验结果不理想，仍在进一步探索中。