Yoshida Kei, Mushimoto Yuichi, Tanase-Nakao Kanako, Akiba Kazuhisa, Ishii Kanako, Urakami Tatsuhiko, Sugihara Shigetaka, Kikuchi Toru, Fukami Maki, Narumi Satoshi
Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan.
Department of Pediatrics and Child Health, Nihon University School of Medicine, Tokyo, Japan.
Clin Pediatr Endocrinol. 2021;30(4):179-185. doi: 10.1297/cpe.30.179. Epub 2021 Oct 1.
We previously performed next-generation sequencing-based genetic screening in patients with autoantibody-negative type 1 diabetes, and identified the p.Leu168Pro mutation in Here,we report the clinical course of the patient and the results of functional characterization of this mutation. The proband had bilateral renal hypodysplasia and developed insulin-dependent diabetes during childhood. The pathogenicity of Leu168Pro-HNF1B was evaluated with three-dimensional structure modeling, Western blotting, immunofluorescence analysis and luciferase reporter assays using human embryonic kidney 293 cells. Three-dimensional structure modeling predicted that the Leu168 residue is buried in the DNA-binding Pit-Oct-Unc-specific (POU) domain and forms a hydrophobic core. Western blotting showed that the protein expression level of Leu168Pro-HNF1B was lower than that of wild-type (WT) HNF1B. Immunofluorescence staining showed that both WT- and Leu168Pro-HNF1B were normally localized in the nucleus. The cells transfected with WT-HNF1B exhibited 5-fold higher luciferase reporter activity than cells transfected with an empty vector. The luciferase activities were comparable between WT-HNF1B/Leu168Pro-HNF1B and WT-HNF1B/empty vector co-transfection. In conclusion, Leu168Pro is a protein-destabilizing mutation, and the destabilization is likely due to the structural changes involving the hydrophobic core of POU. The disease-causing Leu168Pro mutation is a loss-of-function mutation without a dominant-negative effect.
我们之前对自身抗体阴性的1型糖尿病患者进行了基于新一代测序的基因筛查，并在[具体基因名称未给出]中鉴定出p.Leu168Pro突变。在此，我们报告该患者的临床病程以及此突变的功能特征研究结果。先证者患有双侧肾发育不全，并在儿童期发展为胰岛素依赖型糖尿病。利用人胚肾293细胞，通过三维结构建模、蛋白质免疫印迹法、免疫荧光分析和荧光素酶报告基因检测对Leu168Pro-HNF1B的致病性进行了评估。三维结构建模预测Leu168残基埋藏于DNA结合的Pit-Oct-Unc特异性（POU）结构域中，并形成一个疏水核心。蛋白质免疫印迹法显示Leu168Pro-HNF1B的蛋白质表达水平低于野生型（WT）HNF1B。免疫荧光染色显示WT-HNF1B和Leu168Pro-HNF1B均正常定位于细胞核。转染WT-HNF1B的细胞比转染空载体的细胞表现出高5倍的荧光素酶报告基因活性。WT-HNF1B/Leu168Pro-HNF1B与WT-HNF1B/空载体共转染后的荧光素酶活性相当。总之，Leu168Pro是一个使蛋白质不稳定的突变，这种不稳定可能是由于涉及POU疏水核心的结构变化所致。致病的Leu168Pro突变是一个功能丧失性突变，无显性负效应。
