Molecular characterization of congenital hyperinsulinism/hyperammonemia caused by glutamate dehydrogenase gene defects

先天性高胰岛素血症(CHH)是由谷氨酸脱氢酶(GDH)调节失调引起的。我们研究了两名患有CHH的日本患者的GDH基因。与患者2相比，患者1出现起病晚、轻度低血糖和轻度高氨血症。在淋巴细胞GDH活性方面，患者1的基础GDH活性比对照组高2倍，对GTP抑制轻度不敏感。患者2在对照组中对GTP抑制和ADP的变构刺激表现出严重的不敏感。遗传学研究发现，在患者1和2中，L413V和G446D分别存在杂合和从头突变。COS细胞表达研究证实，这两个突变均为致病基因。由于突变型GdH基因的剂量效应，在COS细胞表达系统中强调了L413V和G446D对GTP抑制的不敏感性。生化和遗传学结果显示，L413V的GDH值低于G446D，这与临床表型一致。根据牛GdH的结构，G446D位于枢轴螺旋及其周围的GTP结合部位，而L413V位于天线状结构的α-螺旋。这些不同的突变部位对谷氨酸脱氢酶有不同的影响。类天线结构对GDH的活性起着重要的作用。

Congenital hyperinsulinism and hyperammonemia (CHH) is caused by dysregulation of glutamate dehydrogenase (GDH). We characterized the GDH gene in two Japanese patients with CHH. Patient 1 showed late-onset and mild hypoglycemic episodes and mild hyperammonemia, compared with patient 2. In GDH activity of lymphoblasts, patient 1 showed 2-fold higher basal GDH activity than control subjects and mild insensitivity for GTP inhibition. Patient 2 showed severe insensitivity for GTP inhibition, and similar allosteric stimulation by ADP in the controls. Genetic studies identified heterozygous and de novo L413V and G446D mutations in patients 1 and 2, respectively. COS cell expression study confirmed that both mutations were disease-causing gene. The insensitivity for GTP inhibition in L413V and G446D was emphasized in COS cell expression system as a result of the dosage effect of mutant GDH gene. L413V showed less impairment of GDH than G446D based on biochemical and genetic results, which was consistent with the clinical phenotype. Based on the structure of bovine GDH, G446D was located in GTP binding site of pivot helix and its surroundings, while L413V was located in α-helix of antenna-like structure. These different locations of mutations gave different effects on GDH enzyme. The antenna-like structure plays an important role in GDH activity.