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

发布时间：2018-06-07 12:30

本文选题：青少年的成人起病型糖尿病(MODY) + GCK　；参考：《北京协和医学院》2014年博士论文

【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.507GC)、 Y215X(c.645CA)、R250H(c.749GA)、W257X(c.771GA)、G261R(c.781GC)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-71GC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.626CT(NM_000162.3)突变导致第209位编码氨基酸从苏氨酸变为甲硫氨酸(p. T209M, NP_000153.1).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。
[Abstract]:Part one screening of adult onset diabetes mellitus (MODY) in Chinese adolescents
[Objective]
Objective to investigate the clinical characteristics and molecular genetic characteristics of adult onset diabetes mellitus (MODY) in Chinese adolescents.
[method]
The criteria for setting the MODY pathogenicity test for patients with abnormal glycemia were set up. According to this standard, the patients who were suspected to be MODY in the Peking Union Medical College Hospital from 2010 to 2014 were collected. The clinical characteristics and laboratory examination data were systematically analyzed. According to the clinical characteristics of the patients, the possible MODY types were judged and the related family members were extracted. The genome of DNA was amplified by polymerase chain reaction (PCR) and then directly sequenced by MODY gene.
[results]
1. in the group of 33 families, 11 MODY2 families and 1 MODY3 families were detected by gene detection. 32 cases of MODY2 patients, 2 cases of MODY3 patients, 1 cases of neonatal diabetes caused by GCK gene complex and mutation were found and diagnosed.
2. R43C, T168A, K169N, R191W, Y215X, E221K, R25OH, G261R, M235T, W257X, A379E) were found in different GCK gene loci.
3. studies of 11 MODY2 families showed that the onset of a large age, a lack of typical symptoms of diabetes, and a lower level of triglyceride were the clinical features of MODY2 patients. Most of the MODY2 patients had normal insulin secretion curves in the OGTT test.
4. both Chinese MODY2 patients and early onset diabetic patients with negative pathogenetic genes have lower hsCRP levels and may be associated with higher rs1169288 and / or rs2464196 carrying rates.
[Conclusion]
MODY2 and MODY3 accounted for 33% and 3% of the MODY families in China respectively, and most of the MODY patients had unknown genes.
The second part is a preliminary screening of GCK gene mutation in pregnant women with abnormal glucose metabolism in China.
[Objective]
The mutation of glucokinase (GCK) gene in Chinese pregnant women with abnormal blood glucose was preliminarily defined.
[method]
A retrospective study was conducted in the Peking Union Medical College Hospital from July 2005 to May 2008. The pregnancy women were screened for gestation and were tested by oral glucose tolerance (OGTT) test and glycosylated hemoglobin (HbAlc). The fasting blood glucose was 5.5 to 10.0mmol/L, the OGTT test of 2H and Oh was less than 4.6mmol/L and HbAlc was less than 8%. The mutation screening of GCK gene exon region and promoter region -71GC was performed on all eligible patients.
[results]
A total of 577 subjects were included in a total of 30 cases with GCK gene test conditions, and 17 specimens were obtained. 1 cases of GCK gene mutations in adult onset diabetes 2 (MODY2) and 1 non coding regions were found. The c.626CT (NM_000162.3) mutation of the exon 6 of the MODY2 patient resulted in the change of the 209th encoded amino acids from threonine to the threonine. Methionine (P. T209M, NP_000153.1). The minimum mutation rate of GCK in Chinese gestational glycemic population was 0.27%. The minimum prevalence rate of MODY2 in Chinese population was estimated to be 21/10 million.
[Conclusion]
GCK gene mutations are not common in Chinese people with abnormal blood glucose during pregnancy.
Functional study of the third part of GCK gene mutation
[Objective]
To investigate the molecular mechanisms of different glucokinase (GCK) mutations R43C, K169N, R191W, E221K, R250H, R275H and A379E in juvenile onset diabetes type 2 (MODY2), as well as the molecular mechanism of K90R, M197V mutation causing congenital hyperinsulinemia (CHI).
The wild type and various mutant GCK plasmids carrying the glutathione S transferase (GST) label were constructed. The recombinant protein of the wild type and each mutant GST-GCK was expressed and purified in vitro. The kinetic and thermal stability analysis was carried out by enzyme coupling analysis.
[results]
1. compared with wild type, R43C, R191W, E221K, R250H, A379E and K90R mutations cause protein production to decline, and M197V mutation causes protein production to increase.
2. compared with the wild type, K169N, R191W and A379E mutations lead to a significant increase in the S0.5 value of glucose, and a significant decrease in the catalytic constant (Kcat). R191W, A379E mutation also leads to a higher ATP-Km value. The overall relative activity index (Ia) is 0.001,0.037 and 0.233., respectively.
3. compared with the wild type, the E221K mutation leads to the glucose S0.5, the ATP-Km value, the Kcat decrease, the work. It is 0.477. The R43C mutation does not lead to the significant change of the glucose S0.5, the ATP-Km value, and the significant decrease of Kcat to 0.429..
4.R250H mutation only resulted in a slight decrease in Kcat and no significant difference between Ia and wild type; R275H mutation resulted in a slight decrease in glucose S0.5 and Kcat, and there was no significant difference between Ia and wild type.
The mutation of 5.K9OR and M197V resulted in a decrease in glucose S0.5 and Hill's coefficient (H), an increase in ATP-Km, and a slight decrease in Kcat at the same time, and the total Ia was 1.620 and 4.690. respectively.
6. thermal stability analysis showed that K169N, R191W (A379E) mutation decreased significantly with incubation temperature increasing or time prolonging.
[Conclusion]
1. the abnormality of enzyme kinetics, the decrease of catalytic activity and the decrease of thermal stability are the causes of hyperglycemia caused by mutations in K169N, R191W and A379E.
2. enzyme kinetics abnormality, catalytic activity decline in E221K hyperglycemia induced by mutation, catalytic activity decline in R43C mutation induced hyperglycemia.
3. abnormal enzyme kinetics is the cause of congenital hyperinsulinemia caused by M197V and K9OR mutation.
4. enzyme kinetics and thermostability studies failed to reveal the causes of hyperglycemia induced by R25OH and R275H mutations.
【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

【参考文献】