中国青少年成人起病型糖尿病（MODY）患者筛查及GCK基因突变功能学研究

【作者】 王志新；

【导师】 肖新华；

【作者基本信息】 北京协和医学院 ， 内科学内分泌（专业学位）， 2014， 博士

【摘要】 第一部分中国青少年的成人起病型糖尿病（MODY）患者筛查[目的]探讨中国青少年的成人起病型糖尿病患者（MODY）的临床特点和分子遗传学特征。[方法]设定血糖异常患者进行MODY致病基因检测的入组标准。按照该标准收集2010年至2014年在北京协和医院疑诊为MODY的糖尿病家系患者,系统性分析其临床特点及实验室检查资料。根据患者的临床特点判断其可能的MODY类型,抽提相关家系成员的基因组DNA,聚合酶链式反应（PCR）扩增后进行MODY致病基因的直接测序。[结果]1.共入组33例家系,基因检测发现11例MODY2家系和1例MODY3家系,共发现和诊断MODY2患者32例,MODY3患者2例,由GCK基因复合杂和突变引起的新生儿糖尿病患者1例。2.发现不同的GCK基因突变位点11个（R43C、T168A、K169N、R191W、Y215X、 E221K、R25OH, G261R、M235T、W257X、A379E）,其中K169N （c.507G>C）、 Y215X（c.645C>A）、R250H（c.749G>A）、W257X（c.771G>A）、G261R（c.781G>C）为新报道突变。发现HNF1A基因突变位点1个（P519L）。3.对11例MODY2家系的研究显示,起病时年龄跨度大、缺乏糖尿病典型症状以及较低的血甘油三酯水平是MODY2患者的临床特征。大多数MODY2患者OGTT试验有正常的胰岛素分泌曲线。4.中国MODY2患者和致病基因检测阴性的早发糖尿病患者均具有较低的hsCRP水平,可能和其较高的rs1169288和/或rs2464196携带率有关。[结论]MODY2和MODY3分别占中国MODY家系的33%和3%,大部分MODY患者其致病基因未明。第二部分中国妊娠期糖代谢异常人群GCK基因突变初步筛查[目的]初步明确中国妊娠期血糖异常人群中葡萄糖激酶（GCK）基因突变情况。[方法]回顾性研究,选择2005年7月至2008年5月在北京协和医院进行妊娠期血糖筛查并进行了口服葡萄糖耐量（OGTT）试验和糖化血红蛋白（HbAlc）检测的妊娠妇女。以空腹血糖在5.5～10.0mmol/L、OGTT试验2h与Oh血糖差值小于4.6mmol/L且HbAlc值小于8.0%作为筛选条件,对满足所有条件者进行GCK基因外显子区和启动子区-71G>C的突变筛查。[结果]共纳入577例受试者,符合GCK基因检测条件者30例,可获得标本数17例,发现1例GCK基因突变致青少年的成人起病型糖尿病2型（MODY2）患者和1处非编码区新变异。该MODY2患者6号外显子区c.626C>T（NM₀00162.3）突变导致第209位编码氨基酸从苏氨酸变为甲硫氨酸（p. T209M, NP₀00153.1）.推测中国妊娠期血糖异常人群GCK最小突变率为0.27%,估测中国总人群中MODY2的最小患病率为21/10万。[结论]中国妊娠期血糖异常的人群中GCK基因突变并不常见。第三部分GCK基因突变功能学研究[目的]探讨不同葡萄糖激酶（GCK）基因突变R43C、K169N、R191W、E221K、R250H、R275H和A379E引起青少年的成人起病型糖尿病2型（MODY2）的分子机制,以及K90R、M197V突变引起先天性高胰岛素血症（CHI）的分子机制。[方法]构建携带有谷胱甘肽S转移酶（GST）标签的野生型和各突变型GCK质粒。体外表达和纯化野生型和各突变型GST-GCK重组蛋白。酶偶联分析法进行动力学和热稳定性分析。[结果]1.与野生型相比,R43C、R191W、E221K、R250H、A379E、K90R突变导致蛋白产量下降,M197V突变导致蛋白产量增加。2.与野生型相比,K169N、R191W、A379E突变导致葡萄糖S0.5值显著升高,催化常数（Kcat）显著下降,R191W, A379E突变还导致ATP-Km值升高。总体相对活性指数（Ia）分别为<0.001,0.037和0.233。3.与野生型相比,E221K突变导致葡萄糖S0.5、ATP-Km值升高,Kcat下降,工。为0.477；R43C突变未导致葡萄糖S0.5、ATP-Km值的显著改变,Kcat的显著降低使其Ia为0.429。4.R250H突变仅导致Kcat轻度下降,Ia与野生型无明显差异；R275H突变导致葡萄糖S0.5和Kcat的轻度下降,Ia与野生型也无明显差异。5.K9OR和M197V突变导致葡萄糖S0.5和希尔系数（h）下降、ATP-Km升高,K90R突变同时导致Kcat轻度下降,总体Ia分别为1.620和4.690。6.热稳定性分析显示,K169N、R191W（?）A379E突变随着孵育温度升高或时间延长酶活性有明显下降。[结论]1.酶动力学异常、催化活性下降和热稳定性下降是K169N、R191W和A379E突变导致高血糖的原因。2.酶动力学异常、催化活性下降参与E221K突变导致的高血糖发病；催化活性下降参与R43C突变导致的高血糖发病。3.酶动力学异常是M197V和K9OR突变导致先天性高胰岛素血症的原因。4.酶动力学和热稳定性研究未能揭示R25OH和R275H突变导致高血糖的原因。更多还原

【Abstract】 PART1Screening of maturity-onset diabetes of the young in Chinese diabetic populationObjective To explore the clinical characteristics and molecular genetics of Chinese maturity-onset diabetes of the young.Methods Inclusion criteria for genetic testing in Chinese patients suspected with maturity-onset diabetes of the young（MODY） were developed. According to the criteria, we collected and retrospectively analyzed the clinical characteristcs and features of laboratory data of Chinese MODY pedigrees diagnosed in Peking Union Medical College Hospital （PUMCH） from2010to2014. Genomic DNA of related members in the pedigrees were extracted, followed by PCR amplification. Then direct sequencing were performed to identify mutations in the potential causative gene of MODY.Results1. A total of33pedigrees were collected and analyzed, in which11MODY2pedigrees and1MODY3pedigree were confirmed by genetic testing. In the12pedigrees,32cases of MODY2,2cases of MODY3and one case of permanent neonatal diabetes mellitus （PNDM） caused by compound heterozygous mutation in GCK gene were found.2. Totally11mutations （R43C、T168A、K169N、R191W, Y215X、E221K、R250H、 G261R、M235T、W257X、A379E） were discovered in GCKgene, in which5[K169N (c.507G>C、Y215X (c.645C>A、R250H （c.749G>A）、W257X （c.771G>A）、 G261R （c.781G>C）]were previously unreported. One previously reported mutation （P519L） in HNF1A gene was also detected.3. The study of the11MODY2pedigrees showed that a large span of the age at diagnosis of hyperglycemia, lack of typical clinical manifestations of diabetes at the time of onset and lower levels of plasma triglyceride were clinical characteristics of MODY2patients. The majority of MODY2patients had normal insulin secretion curve in oral glucose tolerance test（OGTT）. 4. Chinese patients with early-onset diabetes, including MODY2and others caused by unknown pathogenic gene, showed low level of hsCRP. This may be associated with the high percentage of carriers of rs1169288and/or rs2464196in both groups.Conclusion MODY2and MODY3account for33%and3%, respectively in Chinese MODY pedigrees. The causative gene are still unknown in majority of MODY cases. PART2Preliminary screening of mutations in the glucokinase gene in Chinese gestational subjects with abnormal glucose metabolismObjective To preliminarily assess the rate of glucokinase（GCK） gene mutation in Chinese gestational subjects with abnormal glucose metabolism.Methods We retrospectively analyzed Chinese gestational subjects who received oral glucose tolerance test and glycosylated hemoglobin （HbA1c） detection in Peking Union Medical College Hospital （PUMCH） from July2005to May2008. Subjects were selected for direct sequencing of GCK gene if they met the following three criteria:（1） fasting plasma glucose was between5.5and10.Ommol/L,（2） a small increase （<4.6mmol/L） in plasma glucose2hours after an oral glucose load,（3） HbAlc below8.0%.Results A total of577subjects were collected in our study, in which30subjects met the criteria for GCK gene mutation testing. Of the17subjects whose DNA samples were obtainable, one case of maturity-onset diabetes of the young type2（MODY2） with GCK gene mutation c.626C>T（NM₀00162.3） in exon6and one case with previously unreported variation in noncoding region had been found. The mutation c.626C>T（NM₀00162.3） resulted in the substitution of methionine for threonine in amino acid209（p. T209M, NP₀00153.1）. According to our finding, we estimated the minimum GCK gene mutation rate was0.27%in Chinese gestational subjects with abnormal glucose metabolism, and the minimum prevalence of MODY2was21/100,000in Chinese population. Conclusion The GCK gene mutations are not common in Chinese gestational subjects with abnormal glucose metabolism. PART3Functional studies of GCK gene mutationsObjective To explore the molecular mechanisms of missense mutations in the GCK gene resulted in hyperglycemia in patients of maturity-onset diabetes of the young type2（MODY2）（R43C, K169N, R191W, E221K, R250H, R275H and A379E） and hypoglycaemia in patients of congenital hyperinsulinism（CHI）（K90R and M197V）.Methods We constructed plasmids of wild type of human islet glucokinase （GCK） recombined with glutathione S-transferase（GST） and mutants carrying the respective mutations by site-directed mutagenesis. Wild type and mutants were bacterially expressed as fusion proteins and affinity-purified. Enzymatic kinetics and thermostability were evaluated for wild type and every mutants by enzyme-coupled analysis.Results1. Compared with wild type, mutants R43C, R191W, E221K, R250H, A379E and K90R resulted in lower protein yield, while mutants M197V had higer protein yield.2. Compared with wild type, mutants K169N, R191W and A379E had significant increased S0.5for glucose and decreased catalytic constant （Kcat）. Mutants R191W and A379E also had increased Km for ATP(ATP-Km. The relative activity index （Ia） for these mutants were<0.001,0.037和0.233, respectively.3. Compared with wild type, mutant E221K resulted in increased So.5and ATP-Km and dereased Kcat. Mutant R43C resulted in significant decreased in Kcat without changes in S0.5and ATP-Km. The Ia for both mutants were0.477and0.429, respectively.4. Though mutation of R250H caused mild decrease in Kcat, R275H caused mild decrease in both S0.5and Kcat, there were no siginificant differences in Ia when they compared with wild type.5. Both mutants K90R and M197V resulted in decreased S0.5, Hill coefficient and increased ATP-Km. Mutant K90R also resulted in mild decrease in Kcat. The Ia for both were1.620and4.690, respectively.6. Thermostability analysis revealed that mutants K169, R191W and A379E were thermal instability.Conclusion 1. Abnormal enzymatic kinetics, decreased catalytic activity and thermal stability are the causes of K169N, R191W and A379E mutation to develop hyperglycemia in patients of MODY2.2. Abnormal enzymatic kinetics and decreased catalytic activity involve in the pathogenesis of mutant E221K. R43C mutation promotes the development of MODY2by reduced catalytic activity.3. Abnormal enzymatic kinetics are the causes of M197V and K90R mutation to develop hypoglycaemia in patients of CHI.4. Results of enzymatic kinetics and thermostability analysis do not reveale the pathogenesis of R250H and R275H mutation to develop hyperglycemia.更多还原