新一代测序在中国青少年起病的成人型糖尿病致病基因检测中的应用

发布时间：2018-04-30 19:03

本文选题：单基因糖尿病 + 青少年起病的成人型糖尿病　；参考：《北京协和医学院》2014年博士论文

【摘要】：目的单基因糖尿病(monogenetic diabetes)是指由单个基因突变导致的糖尿病,以单基因模式遗传,约占全部糖尿病患者的1%-2%,其中最常见的类型为青少年起病的成人型糖尿病(maturity onset diabetes of the young, MODY)。MODY目前已知13种致病基因,分为13种亚型,功能均与胰岛B细胞发育、凋亡、功能、葡萄糖代谢和胰岛素相关,如INS、HNF1A、GCK等。MODY的临床表型、预后、治疗方案和胰腺外表型与其致病基因相关,因此基因诊断对于MODY的治疗、预后及家系中患病成员的诊治具有非常重要的指导意义。至今仍有10%-20%的临床表现典型的MODY患者未能检测到致病基因。因此,利用新的技术手段探索新发致病基因具有重要意义。目标区域测序技术使基因检测不再局限于单个片段的局部检测,它可以针对众多已知的或研究者感兴趣的目的基因进行大范围的检测,与全外显子测序技术相比,它价格低廉,产生的数据量少,目的性明确,已经成为单基因疾病,甚至复杂多基因疾病研究领域的一种成熟的基因检测手段。本研究旨在利用目标区域测序,针对胰岛B细胞功能及能量代谢通路中的重要基因进行单基因糖尿病未知致病基因的探索。对象和方法 1.挑选病例我们首先用Sanger测序法根据本中心45例单基因糖尿病家系的临床表型进行相应致病基因(GCK/HNF1A/HNF4A/KCNJ11)的测序分析,排除了已知致病基因家系12例。从剩余家系中选择遗传规律明显且症状典型的32例患者进行目标区域测序。 2.数据分析单基因糖尿病的己知致病基因均与胰岛B细胞功能及能量代谢相关,推测未知致病基因也具有相似的基因功能。针对检测到的、符合家系遗传规律的少见突变的基因及位点逐个进行分析,搜集文献,再进一步整合得出可能的致病基因。 3.验证在NCBI上进行候选基因的功能和蛋白结构的检索,且完善家系成员与此基因的相关临床资料和生化检查。结果检索并逐个分析32例家系患者所得的少见基因突变,根据位点突变蛋白功能预测和基因功能等相关文献报导进行排除,最终确定UCP3可能是MODY的一种新的致病基因。该基因编码线粒体内膜上的解偶联蛋白,控制离子转运,维持能量梯度和质子浓度差,与脂肪酸在线粒体中的氧化调节相关。有关联性分析发现其突变与儿童早发性肥胖和成人肥胖、高脂血症、2型糖尿病相关。结论本研究旨在通过目标区域测序寻找单基因糖尿病的未知致病基因,通过分析45例糖尿病家系找到了候选基因UCP3,它与能量代谢相关,但仍需进一步验证。本研究初步探索了目标区域测序在单基因疾病中的应用和数据统计方法,证实了其在单基因疾病领域的研究重要性。
[Abstract]:Purpose Monogenic diabetes mellitus refers to diabetes caused by a single gene mutation, which is inherited in a single gene pattern. About 1- 2% of all patients with diabetes, the most common type of disease is adult diabetes maturity onset diabetes of the young, MODY).MODY, the most common type is the adult diabetes maturity onset diabetes of the young, MODY).MODY, which is known to cause 13 kinds of genes, including 13 subtypes, all of which are related to the development, apoptosis and function of islet B cells. Glucose metabolism is associated with insulin, such as the clinical phenotype, prognosis, therapeutic regimen and appearance of pancreas of INSN HNF1A, GCK, et al., so gene diagnosis is associated with the treatment of MODY. The prognosis and the diagnosis and treatment of the family members are of great significance. So far, 10-20% of MODY patients with typical clinical manifestations have failed to detect pathogenic genes. Therefore, it is of great significance to explore new pathogenic genes by means of new techniques. The target region sequencing technique does not limit the gene detection to the local detection of a single fragment. It can be used to detect a large range of known or interesting target genes. Compared with total exon sequencing technology, it is cheaper than the whole exon sequencing technology. Because of its small amount of data and clear purpose, it has become a mature method for gene detection in the field of single gene diseases and even complex polygenic diseases. The purpose of this study was to explore the unknown genes involved in single gene diabetes mellitus (MDM) by using target region sequencing to identify the important genes in the islet B cell function and energy metabolism pathway. Objects and methods 1. Selection of cases We first analyzed the corresponding pathogenic gene (GCK / HNF1A / HNF4A / KCNJ11) by Sanger sequencing according to the clinical phenotype of 45 monogenic diabetic families in our center, and excluded 12 families with known pathogenetic genes (GCK / HNF1A / HNF4A / KCNJ11). The target regions were sequenced from 32 patients with obvious inheritance and typical symptoms from the remaining families. 2. Data analysis The known pathogenetic genes of single gene diabetes are related to the function of islet B cells and energy metabolism. It is inferred that unknown pathogenetic genes have similar gene functions. The rare mutation genes and loci were analyzed one by one and the literature was collected and the possible pathogenic genes were further integrated. 3. Verification The function and protein structure of candidate gene were searched on NCBI, and the clinical data and biochemical examination of family members were improved. Result The rare gene mutations obtained from 32 pedigrees were retrieved and analyzed one by one. According to the functional prediction of locus mutant protein and gene function, UCP3 may be a new pathogenetic gene of MODY. The gene encodes uncoupling proteins in mitochondrial intima, controls ion transport, maintains low energy gradient and proton concentration, and is related to the oxidation regulation of fatty acids in mitochondria. The mutation was associated with early childhood obesity, adult obesity, hyperlipidemia and type 2 diabetes. Conclusion The purpose of this study was to search for unknown genes of single gene diabetes by sequencing the target region. The candidate gene UCP3 was found by analyzing 45 diabetic families. UCP3 is related to energy metabolism, but it still needs to be further verified. In this study, the application and statistical method of target region sequencing in single gene diseases were preliminarily explored, and its importance in the field of single gene diseases was confirmed.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.1;R440

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