目标区域测序在新生儿和不典型1型糖尿病遗传基础研究中的应用

发布时间：2019-03-05 13:28

【摘要】：目的新生儿糖尿病(Neonatal DiabetesMellitus, NDM)是一种罕见的单基因遗传疾病,通常指出生后6个月内发生的糖尿病。也有少数出生6个月之后发生的糖尿病经基因检测后被确诊为单基因糖尿病。迄今为止,已确证的致病基因超过20种,以编码钾离子通道(ATP sensitive Potassium Channel, KATP)基因ABCC8、KCNJ11发生率最高。携带KATP突变患者大部分可从胰岛素注射成功转换为口服磺脲类药物治疗。明确基因诊断同时有助于患者家系进行遗传咨询。新生儿糖尿病的遗传基础具有高度异质性。目前,仍有部分患者病因未明。此外,不典型1型糖尿病患者临床表现和疾病进展迥异于典型患者,其背后的病因学机制也可能不同。尽管经典的Sanger测序技术至今仍是单基因疾病诊断领域的金标准,探索常规基因筛查阴性的新生儿糖尿病患者以及不典型1型糖尿病患者潜在的遗传基础,传统测序技术己不再适用。2009年第二代高通量测序技术(High-throughput sequencing technology)的引入使单基因疾病的研究进展迅速。本研究旨在结合传统测序与第二代测序技术(目标区域测序),进一步研究新生儿糖尿病和不典型1型糖尿病的分子遗传学基础,着重于未知致病基因的探索。对象和方法 1.病例入组本中心共收集30例符合入组要求患者,经充分知情同意后,采集患者临床病史,包括出生体重、起病年龄、起病时症状、其他糖尿病综合征相关异常表现、实验室检查(空腹血糖、空腹胰岛素及C肽、糖化血红蛋白、1型糖尿病相关抗体等)和家族史；留取患者、患者同胞及父母外周血2-4ml。 2.基因检测流程入组患者按起病年龄分为两组。一组：6个月以内起病患者,采用Sanger测序法对常见致病基因KCNJ11、INS和ABCC8进行筛查,筛查阴性患者进行目标区域外显子捕获及高通量测序。另一组：6个月以后起病患者,其中1岁以内起病患者常规筛查KCNJ11和INS基因,阴性患者和1岁以后起病患者一起进行高通量测序。其中,如患者表现为糖尿病综合征,采用Sanger测序法筛查相关致病基因,阴性患者进入高通量测序。 3.数据分析高通量测序数据产出后,根据测序深度和覆盖度对测序质量进行评价。去除读取次数小于20次的低质量数据,余下的数据按照下面的标准进行功能性变异的筛检：1)变异位点位于功能区：编码区和剪切区；2)单碱基变异为无义突变、错义突变,或致剪切位点改变；插入/缺失变异导致移码突变、无义突变或剪切位点改变；3)突变位点MAF0.01,或为非SNP位点。 4.验证对筛选出的功能性变异设计引物,进行Sanger测序验证。验证阳性的突变位点,在家系内再次验证。符合孟德尔疾病遗传模式的变异位点,在表型正常的样本中进一步扩大验证。结果 6例新生儿糖尿病患者常规筛查检出4例阳性患者,2例为KCNJ11突变,2例为INS突变；24例不典型1型糖尿病患者中,1例表现为Wolfram综合征,检出WFS1基因纯合突变。常规筛查阴性的2例新生儿糖尿病患者和22/23例不典型1型糖尿病患者进行目标区域测序,结果未发现可以致病突变。结论常规Sanger测序仍然是新生儿糖尿病基因诊断的首选方式。扩大范围的目标区域测序对病因未明的新生儿糖尿病患者是必要的。对不典型1型糖尿病患者进行常规基因筛查是有意义的,但现有临床筛选标准有待改进。扩大范围的目标区域测序对不典型1型糖尿病患者是否必要有待进一步探索研究。
[Abstract]:Purpose Neonate Diabetes Mellitus (NDM) is a rare single-gene genetic disease, which usually indicates the glycosuria occurring within 6 months of life. A few six-month-old diabetes is diagnosed as a single-gene glycosuria after gene detection. To date, more than 20 pathogenic genes have been confirmed to encode the ATP sensitive Potassic Channel (KATP) gene ABCC8 and the KCNJ11 incidence is the most High. The majority of patients with KATP mutant can be successfully converted from insulin injection into oral sulfas. Therapy. It is clear that the diagnosis of the gene can also help the family of the patient to carry on the genetic analysis. Inquire. The genetic basis of neonatal diabetes is highly heterogeneous Sex. Currently, some of the patient's causes are not in addition, that clinical and disease progression of the non-typical type 1 diabetic patient is significantly different from that of a typical patient, and the underlying etiology mechanism may not In the same way, while the classical Sanger sequencing technology is still a gold standard in the field of single-gene disease diagnosis, it is possible to explore the potential of a conventional gene screening-negative neonatal diabetic patient and a non-typical type 1 diabetic patient. The traditional sequencing technology is no longer applicable. The introduction of the second-generation high-throughput sequencing technology in 2009 leads to the research of single-gene disease. The aim of this study was to further study the molecular genetic basis of neonatal and non-typical type 1 diabetes, focusing on unknown pathogenic genes, in combination with traditional sequencing and second generation sequencing technology (target area sequencing). Exploration. Object and method 1. A total of 30 patients enrolled in the study were enrolled in the study, and after full and informed consent, the patient's clinical history was collected, including the weight of the birth, the age of the onset of the disease, the symptoms of the onset of the disease, and other diabetes. Syndrome-related abnormal performance, laboratory test (fasting blood glucose, fasting insulin and C-peptide, glycosylated hemoglobin, type 1 diabetes-related antibody, etc.) and family history; retention of the patient, the patient's compatriots and the father maternal peripheral blood 2-4 m l.2. Gene detection process The patients enrolled were divided into two groups according to the age of onset. One group:6 months from the patient, the common pathogenic gene KCNJ11, INS and ABCC8 were screened by the Sanger sequencing method, and the negative patients were screened for the target. Regional exon capture and high-throughput sequencing. The other group:6 months later, patients with 1 year of age were routinely screened for KCNJ11 and INS genes, negative patients and 1 year old High-throughput sequencing with a patient, where, for example, the patient appears to be a diabetic syndrome, the relevant pathogenic base is screened using the Sanger sequencing method As a result, the negative patient high-throughput sequencing.3. after data analysis of high-throughput sequencing data output, The sequencing quality was evaluated by sequencing depth and coverage. The lower quality data of less than 20 times of reading was removed, and the remaining data were screened for functional variations according to the following criteria:1) The mutation site was located on the functional area: the coding region and the shear region;2) the single base variation was not A change in a sense mutation, a missense mutation, or a shear site; insertion/ deletion variation results in a change in the transcode mutation, a nonsense mutation, or a shear site; and 3) a mutation position. point MAF 0.01, or non-SNP site.4. Verify the work to be screened Functional variation design primer for Sanger sequencing Verification. Verify the positive mutation site and re-verify in the home system. It is in accordance with the Mendelian disease genetic model. variation In 6 cases of neonatal diabetes,4 positive patients were detected in routine screening,2 were KCNJ11 and 2 were INS, and 24 were not typical of type 1 diabetes. Now, Wolfram syndrome, a homozygous mutation of the WFS1 gene was detected.2 cases of neonatal diabetes and 22/23 of the conventional screening-negative cases were not typical of type 1 glycosuria The patient was sequenced with the target area and the result was not found to be able to cause a mutation. Conventional Sanger sequencing is still the first choice for the diagnosis of neonatal diabetic genes. The expanded range of target area sequencing is necessary for neonatal diabetic patients with unknown etiology. It is not typical of type 1 diabetes The routine gene screening of the patient is significant, but the existing clinical screening criteria need to be improved. The scope of the extension
【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】：R722.1

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