【摘要】：目的 新生兒糖尿病(Neonatal DiabetesMellitus, NDM)是一種罕見的單基因遺傳疾病,通常指出生后6個月內發(fā)生的糖尿病。也有少數(shù)出生6個月之后發(fā)生的糖尿病經(jīng)基因檢測后被確診為單基因糖尿病。迄今為止,已確證的致病基因超過20種,以編碼鉀離子通道(ATP sensitive Potassium Channel, KATP)基因ABCC8、KCNJ11發(fā)生率最高。攜帶KATP突變患者大部分可從胰島素注射成功轉換為口服磺脲類藥物治療。明確基因診斷同時有助于患者家系進行遺傳咨詢。新生兒糖尿病的遺傳基礎具有高度異質性。目前,仍有部分患者病因未明。此外,不典型1型糖尿病患者臨床表現(xiàn)和疾病進展迥異于典型患者,其背后的病因學機制也可能不同。 盡管經(jīng)典的Sanger測序技術至今仍是單基因疾病診斷領域的金標準,探索常規(guī)基因篩查陰性的新生兒糖尿病患者以及不典型1型糖尿病患者潛在的遺傳基礎,傳統(tǒng)測序技術己不再適用。2009年第二代高通量測序技術(High-throughput sequencing technology)的引入使單基因疾病的研究進展迅速。本研究旨在結合傳統(tǒng)測序與第二代測序技術(目標區(qū)域測序),進一步研究新生兒糖尿病和不典型1型糖尿病的分子遺傳學基礎,著重于未知致病基因的探索。 對象和方法 1.病例入組 本中心共收集30例符合入組要求患者,經(jīng)充分知情同意后,采集患者臨床病史,包括出生體重、起病年齡、起病時癥狀、其他糖尿病綜合征相關異常表現(xiàn)、實驗室檢查(空腹血糖、空腹胰島素及C肽、糖化血紅蛋白、1型糖尿病相關抗體等)和家族史；留取患者、患者同胞及父母外周血2-4ml。 2.基因檢測流程 入組患者按起病年齡分為兩組。一組：6個月以內起病患者,采用Sanger測序法對常見致病基因KCNJ11、INS和ABCC8進行篩查,篩查陰性患者進行目標區(qū)域外顯子捕獲及高通量測序。另一組：6個月以后起病患者,其中1歲以內起病患者常規(guī)篩查KCNJ11和INS基因,陰性患者和1歲以后起病患者一起進行高通量測序。其中,如患者表現(xiàn)為糖尿病綜合征,采用Sanger測序法篩查相關致病基因,陰性患者進入高通量測序。 3.數(shù)據(jù)分析 高通量測序數(shù)據(jù)產(chǎn)出后,根據(jù)測序深度和覆蓋度對測序質量進行評價。去除讀取次數(shù)小于20次的低質量數(shù)據(jù),余下的數(shù)據(jù)按照下面的標準進行功能性變異的篩檢：1)變異位點位于功能區(qū)：編碼區(qū)和剪切區(qū)；2)單堿基變異為無義突變、錯義突變,或致剪切位點改變；插入/缺失變異導致移碼突變、無義突變或剪切位點改變；3)突變位點MAF0.01,或為非SNP位點。 4.驗證 對篩選出的功能性變異設計引物,進行Sanger測序驗證。驗證陽性的突變位點,在家系內再次驗證。符合孟德爾疾病遺傳模式的變異位點,在表型正常的樣本中進一步擴大驗證。 結果 6例新生兒糖尿病患者常規(guī)篩查檢出4例陽性患者,2例為KCNJ11突變,2例為INS突變；24例不典型1型糖尿病患者中,1例表現(xiàn)為Wolfram綜合征,檢出WFS1基因純合突變。常規(guī)篩查陰性的2例新生兒糖尿病患者和22/23例不典型1型糖尿病患者進行目標區(qū)域測序,結果未發(fā)現(xiàn)可以致病突變。 結論 常規(guī)Sanger測序仍然是新生兒糖尿病基因診斷的首選方式。擴大范圍的目標區(qū)域測序對病因未明的新生兒糖尿病患者是必要的。對不典型1型糖尿病患者進行常規(guī)基因篩查是有意義的,但現(xiàn)有臨床篩選標準有待改進。擴大范圍的目標區(qū)域測序對不典型1型糖尿病患者是否必要有待進一步探索研究。
[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
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R722.1

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