全外显子测序在新生儿糖尿病研究中的应用

发布时间：2018-07-24 10:04

【摘要】：目的新生儿糖尿病(Neonatal Diabetes Mellitus, NDM)通常是指出生后半年内发生的糖尿病,是一种罕见疾病,常以单基因模式遗传。迄今报导的致病基因已将近20种,均与胰岛B细胞相关,如KCNJ11、ABCC8、INS、GCK等,但仍有30%左右的患者未找到相应的致病基因。NDM患者通常需要终身胰岛素治疗,但近期研究显示钾通道(KATP)基因ABCC8和KCNJ11突变的患者大多可以早期用口服磺脲类药物成功替代胰岛素治疗。因此基因诊断对于NDM的治疗也有一定的指导意义。全外显子测序(Whole exome sequencing)技术的引入使得单基因疾病的研究有了极大的突破。外显子区域起着调控和编码蛋白质的重要功能,与遗传表型相关的功能性变异也大多位于外显子区,因此自2009年此技术开展以来,已经成功找到了数百种孟德尔疾病的未知致病基因,目前它也逐渐被用于复杂疾病易感基因的筛选研究。本研究旨在利用全外显子测序探索NDM的未知致病基因对象和方法 1.挑选病例我们首先用普通测试法对本中心的33名NDM患者进行了最常见致病基因(KCNJ11/ABCC8/INS/GCK)的测序分析,排除了已知致病基因者(共8名)。在剩余25名患者中挑选了具有遗传规律且症状典型的姐弟两名患者,但父母血糖水平正常。对此小家系共四人进行了全外显子测序。数据分析采用了两条思路。 2.数据分析：思路一：来自同一家系的两名患儿在相似的极低年龄发展为同一种疾病,最有可能的致病原因是遗传(称为猜想1),由于父母均不患病,考虑为常染色体隐性遗传模式,继而按照姐弟由同一种致病基因(猜想1.1)和两种不同致病基因(猜想1.2)突变所致两种情况来统计数据。另外也不能完全排除两名患儿分别新发突变的情况(猜想2),但其发生率极低,暂不予考虑。思路二：NDM的已知致病基因均与胰岛B细胞相关,按照功能分为与胰腺发育相关,与胰岛B细胞团数量相关及与胰岛素分泌相关三类。推测未知致病基因也极有可能与B细胞相关。我们进行了文献检索列出所有可能的B细胞相关基因,逐个分析,列出每个成员的突变基因及位点,再分析得出可能的致病基因。 3.验证在NCBI上对此候选基因编码的蛋白进行了功能和结构的检索,并完善了家系成员与此基因相关的临床资料和生化检查。结果：思路一结果：猜想1.1：没有一个共同基因符合常染色体隐性遗传模式。猜想1.2：由于致病基因不同,将四人家系分成两个小家系分析,即弟弟家系(包含弟弟和父母)及姐姐家系(包含姐姐及父母)。在两家系中分别统计得到10个(SIPA1L1、SIPA1L3、OBSCN、KIAA0430、SERINC2、MS4A14、P2RX5、 TRIM52、MAMDC2、PCSK1)和4个(KRTAP5-10、DCP1B、MAGI1、SLC28A1)候选基因,综合基因功能和SIFT预测结果,得出PCSK1可能是NDM新的责任基因,但此基因不能解释姐姐的发病情况,需要进一步的验证。思路二结果：检索并逐个分析所有B细胞相关基因,得出家系中有三个基因(CDKAL1, THADA, PCSK1)存在突变,只有PCSK1符合孟德尔疾病遗传模式。总结：PCSK1可能是NDM的一种新的责任基因。该基因编码一种胰岛素原加工酶,调节胰岛素的合成,PCSK1突变与肥胖及高胰岛素原血症相关。结论：本研究旨在通过全外显子测序寻找NDM的未知致病基因,通过分析一个有两名NDM患者的4人小家系找到了候选基因PCSK1,它与胰岛素原的加工有关,但仍需进一步验证。本研究初步探索了全外显子测序在单基因疾病中的应用及数据统计方法,同时也证实了其在孟德尔疾病研究中的重要性。
[Abstract]:objective
Neonatal Diabetes Mellitus (NDM) usually refers to diabetes occurring half a year after birth. It is a rare disease and is often inherited in a single gene mode. So far, nearly 20 kinds of pathogenic genes have been reported. All of them are related to the B cells of the islet, such as KCNJ11, ABCC8, INS, and GCK, but there are still about 30% of the patients who have not found the corresponding pathogeny. .NDM patients usually need life-long insulin therapy, but recent studies have shown that most patients with ABCC8 and KCNJ11 mutations in the potassium channel (KATP) gene can successfully replace insulin therapy with oral sulfonylureas early. Therefore, the gene diagnosis also has some guidance for the treatment of NDM.
The introduction of total exon sequencing (Whole exome sequencing) technology has made a great breakthrough in the study of single gene disease. The exons region plays an important role in regulating and encoding proteins, and most of the functional variations associated with genetic phenotype are in the exons. Since this technology was carried out in 2009, the number of functional variants has been successfully found. The unknown pathogenic gene of hundreds of Mendel diseases is currently being used in screening for the susceptibility genes of complex diseases. This study aims to explore the unknown pathogenic genes of NDM by exon sequencing.
Objects and methods
1. selection of cases
We first analyzed the most common pathogenicity gene (KCNJ11/ABCC8/INS/GCK) of 33 NDM patients in the center with the common test method, excluding the known pathogenicity (8). Among the remaining 25 patients, two patients with genetic regularity and typical symptoms were selected, but their parents' blood glucose level was normal. Total exon sequencing was carried out in four persons. Two ideas were used in the data analysis.
2. data analysis:
Idea one: two children from the same family developed the same disease at a similar very low age. The most likely cause of disease is heredity (called guess 1). Because parents are not sick, they are considered autosomal recessive genetic model, and then according to the same pathogenicity gene (guess 1.1) and two different pathogenic genes (guessing 1) .2) two cases of mutation were caused by statistical data. In addition, the new mutation of two children could not be completely eliminated (guess 2), but the incidence was very low and was not considered for the time being.
Idea two: the known pathogeny genes of NDM are all related to the islet B cells. According to their function, they are related to the development of the pancreas, the number of the islet B cell clusters and the three classes of insulin secretion. It is speculated that the unknown pathogenic gene may also be related to the B cells. We have conducted a literature search to list all the possible B cell related genes, one by one. Analysis, listing each member of the mutation genes and sites, and then analyze the possible pathogenic genes.
3. verification
The function and structure of the protein encoded by the candidate gene were retrieved on NCBI, and the clinical data and biochemical examination related to the gene were improved.
Result:
One result: conjecture 1.1: no common gene conforms to the autosomal recessive pattern. Conjecture 1.2: the four family lines are divided into two small family analyses due to different pathogenic genes, that is, the brother family (including younger brother and parent) and sister family (including sister and parent). 10 (SIPA1L1, SIPA) are obtained in the two families respectively. 1L3, OBSCN, KIAA0430, SERINC2, MS4A14, P2RX5, TRIM52, MAMDC2, PCSK1) and the 4 (KRTAP5-10, DCP1B, MAGI1) genes, combined with the gene function and the prediction results, may be a new responsible gene, but this gene does not explain the incidence of sister sister, and needs further validation.
Two results: all B cell related genes were retrieved and analyzed one by one, and three genes (CDKAL1, THADA, PCSK1) in the family were mutated, only PCSK1 conformed to the genetic model of Mendel disease.
Summary: PCSK1 may be a new responsible gene for NDM, which encodes a primary insulin processing enzyme that regulates the synthesis of insulin, and the PCSK1 mutation is associated with obesity and hyperinsulinemia.
Conclusion:
The purpose of this study is to search for the unknown pathogenic gene of NDM by full exon sequencing, and to find the candidate gene PCSK1 by analyzing a small family of 4 people with two NDM patients. It is related to the processing of proinsulin, but it still needs to be further verified. This study explored the application of all exons in the single gene disease and the data statistics. It also confirms its importance in the study of Mendelian diseases.
【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】：2012
【分类号】：R725.8

【相似文献】