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Mutation analysis of the GLUT2 gene in patients with Fanconi-Bickel syndrome

Fanconi-Bickel综合征患者GLUT2基因突变分析

基本信息

批准号：
12670722
负责人：
OHURA Toshihiro
金额：
$ 2.05万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670722/
关键词：
Fanconi-Bickel syndrome Glycogen storage disease type XI dominant negative effects Renal glucosuria GLUT2 Transgenic mice Cre loxP system ファンコニービッケル症候群

项目摘要

Fanconi-Bickel syndrome (FBS) is an autosomal recessive disorder manifesting hepatorenal glycogen accumulation, Fanconi nephropathy, impaired utilization of glucose and galactose. Recently several mutations in a gene encoding a glucose transporter, GLUT2, have been reported in patients with FBS. Using PCR, direct sequencing, restriction fragment length analysis, and subcloning, we studied three Japanese patients and found four novel mutations : a splice-site mutation (IVS2-2A>G), a nonsense mutation (Q287X), and two missense mutations (L389P and V423E).No previous reports have found that the heterozygotes with mutant GLUT2 to show renal glucosuria. The mother and brother of patient 1, who were heterozygous for the V423E mutation, manifested renal glucosuria. We speculate the possibility of GLUT2 as a candidate gene for familial renal glucosuria with incomplete penetrance. If some mutant GLUT2 proteins have a dominant-negative effect, an oligomer composed of mutant and wild-type proteins could result in abolition of transport activity.To prove this hypothesis, we were trying to generate functional knockout of the GLUT2, and Fanconi-Bickel syndrome in mice expressing a dominant-negative GLUT2 subunit. To generate the GLUT2 dominant-negative construct, mutation were introduced to the cDNA sequence to change the valine (V) residue at position 423 to glutamate (E). For the transgene vector, GLUT2-V423E cDNA was inserted downstream of the CAG promotor. The expression unit (CDRE-GLUT2 : CAG-loxP-DsRed-loxP-IRES-EGFP) was excised, purified, and microinjected into fertilized eggs by standard procedure. Transgenic mice expressing GLUT2-V423E will be selected by analyzing urine sugar.

Fanconi-Bickel综合征（FBS）是一种常染色体隐性遗传疾病，主要表现为肝肾糖原累积、Fanconi肾病、葡萄糖和半乳糖利用受损。最近，在FBS患者中报告了编码葡萄糖转运蛋白GLUT 2的基因中的几个突变。本研究采用PCR、直接测序、限制性片段长度分析和亚克隆等方法，对3例日本患者进行了研究，发现了4种新的突变：1种剪接位点突变（IVS 2 -2A>G）、1种无义突变（Q287 X）和2种错义突变（L389 P和V423 E）。患者1的母亲和兄弟为V423 E突变杂合子，表现为肾性糖尿。我们推测GLUT 2可能是家族性肾性糖尿伴不完全性糖尿的候选基因。如果某些突变的GLUT 2蛋白具有显性负效应，则由突变蛋白和野生型蛋白组成的寡聚体可能导致转运活性的取消。为了证明这一假设，我们试图在表达显性负GLUT 2亚基的小鼠中产生GLUT 2的功能敲除和Fanconi-Bickel综合征。为了产生GLUT 2显性阴性构建体，将突变引入cDNA序列以将位置423处的缬氨酸（V）残基改变为谷氨酸（E）。对于转基因载体，将GLUT 2-V423 E cDNA插入CAG启动子的下游。切下表达单位（CDRE-GLUT 2：CAG-loxP-DsRed-loxP-IRES-EGFP），纯化，并通过标准程序显微注射到受精卵中。通过分析尿糖选择表达GLUT 2-V423 E的转基因小鼠。