本文選題：青少年的成人起病型糖尿病(MODY) + GCK�。� 參考：《北京協和醫(yī)學院》2014年博士論文

【摘要】：第一部分中國青少年的成人起病型糖尿病(MODY)患者篩查 [目的] 探討中國青少年的成人起病型糖尿病患者(MODY)的臨床特點和分子遺傳學特征。 [方法] 設定血糖異�；颊哌M行MODY致病基因檢測的入組標準。按照該標準收集2010年至2014年在北京協和醫(yī)院疑診為MODY的糖尿病家系患者,系統性分析其臨床特點及實驗室檢查資料。根據患者的臨床特點判斷其可能的MODY類型,抽提相關家系成員的基因組DNA,聚合酶鏈式反應(PCR)擴增后進行MODY致病基因的直接測序。 [結果] 1.共入組33例家系,基因檢測發(fā)現11例MODY2家系和1例MODY3家系,共發(fā)現和診斷MODY2患者32例,MODY3患者2例,由GCK基因復合雜和突變引起的新生兒糖尿病患者1例。 2.發(fā)現不同的GCK基因突變位點11個(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)為新報道突變。發(fā)現HNF1A基因突變位點1個(P519L)。 3.對11例MODY2家系的研究顯示,起病時年齡跨度大、缺乏糖尿病典型癥狀以及較低的血甘油三酯水平是MODY2患者的臨床特征。大多數MODY2患者OGTT試驗有正常的胰島素分泌曲線。 4.中國MODY2患者和致病基因檢測陰性的早發(fā)糖尿病患者均具有較低的hsCRP水平,可能和其較高的rs1169288和/或rs2464196攜帶率有關。 [結論] MODY2和MODY3分別占中國MODY家系的33%和3%,大部分MODY患者其致病基因未明。 第二部分中國妊娠期糖代謝異常人群GCK基因突變初步篩查 [目的] 初步明確中國妊娠期血糖異常人群中葡萄糖激酶(GCK)基因突變情況。 [方法] 回顧性研究,選擇2005年7月至2008年5月在北京協和醫(yī)院進行妊娠期血糖篩查并進行了口服葡萄糖耐量(OGTT)試驗和糖化血紅蛋白(HbAlc)檢測的妊娠婦女。以空腹血糖在5.5～10.0mmol/L、OGTT試驗2h與Oh血糖差值小于4.6mmol/L且HbAlc值小于8.0%作為篩選條件,對滿足所有條件者進行GCK基因外顯子區(qū)和啟動子區(qū)-71GC的突變篩查。 [結果] 共納入577例受試者,符合GCK基因檢測條件者30例,可獲得標本數17例,發(fā)現1例GCK基因突變致青少年的成人起病型糖尿病2型(MODY2)患者和1處非編碼區(qū)新變異。該MODY2患者6號外顯子區(qū)c.626CT(NM_000162.3)突變導致第209位編碼氨基酸從蘇氨酸變?yōu)榧琢虬彼?p. T209M, NP_000153.1).推測中國妊娠期血糖異常人群GCK最小突變率為0.27%,估測中國總人群中MODY2的最小患病率為21/10萬。 [結論] 中國妊娠期血糖異常的人群中GCK基因突變并不常見。 第三部分GCK基因突變功能學研究 [目的] 探討不同葡萄糖激酶(GCK)基因突變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)的分子機制,以及K90R、M197V突變引起先天性高胰島素血癥(CHI)的分子機制。[方法] 構建攜帶有谷胱甘肽S轉移酶(GST)標簽的野生型和各突變型GCK質粒。體外表達和純化野生型和各突變型GST-GCK重組蛋白。酶偶聯分析法進行動力學和熱穩(wěn)定性分析。 [結果] 1.與野生型相比,R43C、R191W、E221K、R250H、A379E、K90R突變導致蛋白產量下降,M197V突變導致蛋白產量增加。 2.與野生型相比,K169N、R191W、A379E突變導致葡萄糖S0.5值顯著升高,催化常數(Kcat)顯著下降,R191W, A379E突變還導致ATP-Km值升高。總體相對活性指數(Ia)分別為0.001,0.037和0.233。 3.與野生型相比,E221K突變導致葡萄糖S0.5、ATP-Km值升高,Kcat下降,工。為0.477；R43C突變未導致葡萄糖S0.5、ATP-Km值的顯著改變,Kcat的顯著降低使其Ia為0.429。 4.R250H突變僅導致Kcat輕度下降,Ia與野生型無明顯差異；R275H突變導致葡萄糖S0.5和Kcat的輕度下降,Ia與野生型也無明顯差異。 5.K9OR和M197V突變導致葡萄糖S0.5和希爾系數(h)下降、ATP-Km升高,K90R突變同時導致Kcat輕度下降,總體Ia分別為1.620和4.690。 6.熱穩(wěn)定性分析顯示,K169N、R191W(?)A379E突變隨著孵育溫度升高或時間延長酶活性有明顯下降。 [結論] 1.酶動力學異常、催化活性下降和熱穩(wěn)定性下降是K169N、R191W和A379E突變導致高血糖的原因。 2.酶動力學異常、催化活性下降參與E221K突變導致的高血糖發(fā)�。淮呋钚韵陆祬⑴cR43C突變導致的高血糖發(fā)病。 3.酶動力學異常是M197V和K9OR突變導致先天性高胰島素血癥的原因。 4.酶動力學和熱穩(wěn)定性研究未能揭示R25OH和R275H突變導致高血糖的原因。
[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.
【學位授予單位】：北京協和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R587.1

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本文編號：1991181