Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
PLoS One. 2012;7(6):e38906. doi: 10.1371/journal.pone.0038906. Epub 2012 Jun 20.
Type 2 Maturity Onset Diabetes of the Young (MODY2) is a monogenic autosomal disease characterized by a primary defect in insulin secretion and hyperglycemia. It results from GCK gene mutations that impair enzyme activity. Between 2006 and 2010, we investigated GCK mutations in 66 diabetic children from southern Italy with suspected MODY2. Denaturing High Performance Liquid Chromatography (DHPLC) and sequence analysis revealed 19 GCK mutations in 28 children, six of which were novel: p.Glu40Asp, p.Val154Leu, p.Arg447Glyfs, p.Lys458_Cys461del, p.Glu395_Arg397del and c.580-2A>T. We evaluated the effect of these 19 mutations using bioinformatic tools such as Polymorphism Phenotyping (Polyphen), Sorting Intolerant From Tolerant (SIFT) and in silico modelling. We also conducted a functional study to evaluate the pathogenic significance of seven mutations that are among the most severe mutations found in our population, and have never been characterized: p.Glu70Asp, p.His137Asp, p.Phe150Tyr, p.Val154Leu, p.Gly162Asp, p.Arg303Trp and p.Arg392Ser. These seven mutations, by altering one or more kinetic parameters, reduced enzyme catalytic activity by >40%. All mutations except p.Glu70Asp displayed thermal-instability, indeed >50% of enzyme activity was lost at 50°C/30 min. Thus, these seven mutations play a pathogenic role in MODY2 insurgence. In conclusion, this report revealed six novel GCK mutations and sheds some light on the structure-function relationship of human GCK mutations and MODY2.
2 型青年发病型糖尿病（MODY2）是一种单基因常染色体疾病，其特征为胰岛素分泌和高血糖的原发性缺陷。它是由 GCK 基因突变引起的，这些突变会损害酶的活性。在 2006 年至 2010 年期间，我们研究了来自意大利南部的 66 名疑似 MODY2 的糖尿病儿童的 GCK 突变。变性高效液相色谱法（DHPLC）和序列分析在 28 名儿童中发现了 19 种 GCK 突变，其中 6 种是新的：p.Glu40Asp、p.Val154Leu、p.Arg447Glyfs、p.Lys458_Cys461del、p.Glu395_Arg397del 和 c.580-2A>T。我们使用 Polymorphism Phenotyping（Polyphen）、Sorting Intolerant From Tolerant（SIFT）和计算机建模等生物信息学工具评估了这 19 种突变的影响。我们还进行了一项功能研究，以评估在我们人群中发现的七种最严重突变中的七种突变的致病性意义，这些突变从未被描述过：p.Glu70Asp、p.His137Asp、p.Phe150Tyr、p.Val154Leu、p.Gly162Asp、p.Arg303Trp 和 p.Arg392Ser。这七种突变通过改变一个或多个动力学参数，使酶的催化活性降低超过 40%。除了 p.Glu70Asp 之外的所有突变都显示出热不稳定性，实际上在 50°C/30 分钟时超过 50%的酶活性丧失。因此，这七种突变在 MODY2 的发生中起着致病作用。总之，本报告揭示了六种新的 GCK 突变，并阐明了人类 GCK 突变与 MODY2 之间的结构-功能关系。
