摘要目的：探讨1例永久性新生儿糖尿病患者的致病基因及其致病机制。方法对2013年8月北京协和医院诊治的1例永久性新生儿糖尿病患者，分析其临床特点并抽提相关家系成员的基因组DNA，聚合酶链反应(PCR)扩增后进行葡萄糖激酶(GCK)基因直接测序。构建野生型和突变型质粒，体外表达并纯化GCK重组蛋白，进行酶动力学和热稳定性分析。两组间数据比较采用t检验。结果在该新生儿糖尿病患者发现GCK基因c.571 C>T(R191W)和c.1136 C>A(A379E)复合杂和突变。功能学分析显示，与野生型相比，R191W和A379E突变导致蛋白产量明显下降[分别为(86±9)比(48±8)mg/L、(86±9)比(54±5)mg/L，t=5.56、5.36，均P<0.01]；当ATP为饱和浓度时酶促反应速度达到1/2最大反应速度时的葡萄糖浓度(S0.5)显著升高[分别为(7.63±0.21)比(35.27±2.20) mmol/L、(7.63±0.21)比(13.30±0.44) mmol/L，t=-21.70、-20.32，均P<0.001]；当葡萄糖为饱和浓度时酶促反应速度达到1/2最大反应速度时的ATP浓度(ATP-Km)显著升高[分别为(0.30±0.01)比(0.42±0.01) mmol/L、(0.30±0.01)比(0.54±0.04)mmol/L，t=-17.02、-10.68，均P<0.001]；突变蛋白对葡萄糖的催化常数显著下降[分别为(20.9±2.1)/s比(6.5±1.0)/s、(20.9±2.1)/s比(10.5±1.1)/s，t=10.61、7.58，均P<0.01]。突变蛋白的热稳定性下降，表现为同一孵育温度下或同一温度更短时间内酶活性的快速丧失。结论 GCK基因c.571 C>T (R191W)和c.1136 C>A(A379E)复合杂和突变是该新生儿糖尿病的致病原因。突变导致的蛋白表达下降、酶对葡萄糖和ATP的亲和力下降以及热稳定性的下降可能是其致病机制。

abstractsObjective To identify the cause and its pathogenic mechanism in one case with permanent neonatal diabetes mellitus(PNDM). Methods One case of PNDM diagnosed in Peking Union Medical College Hospital was analyzed for clinical features. Genomic DNA was extracted, followed by amplification with polymerase chain reaction and direct sequencing of glucokinase (GCK) gene. Wild and mutant plasmids were constructed and then expressed in E.coli. The recombinant proteins were purified, then tested to clarify their enzyme kinetics and thermal stability. Data between two groups were analyzed by t test. Results Compound heterozygous mutation c.571 C>T(R191W) and c.1136 C>A(A379E) in GCK gene were detected in this child. Compared with the wild type, mutants R191W and A379E had lower protein yield ((86±9) vs (48±8) mg/L;(86±9) vs (54±5) mg/L;t=5.56, 5.36, both P<0.01), lower appetency for glucose(the half-saturating substrate concentration (S0.5) for glucose:(7.63±0.21) vs (35.27±2.20)mmol/L;(7.63±0.21) vs (13.30 ± 0.44)mmol/L, t=-21.70,-20.32, both P<0.01), lower appetency for ATP(Km:(0.30 ± 0.01) vs (0.42 ± 0.01) mmol/L;(0.30±0.01) vs (0.54±0.04) mmol/L,t=-17.02,-10.68, both P<0.01) and lower catalytic ability&nbsp;((20.9 ± 2.1)/s vs (6.5 ± 1.0)/s;(20.9 ± 2.1)/s vs (10.5 ± 1.1)/s; t=10.61, 7.58, both P<0.01). The both mutants also showed increased thermal instability. Conclusion Compound heterozygous mutations R191W and A379E in GCK gene promote the development of PNDM by affecting enzyme kinetics of glucokinase as well as lowering protein stability.