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

范可尼-比克尔综合征（FBS）是一种常染色体隐性遗传病，表现为肝肾糖原积累、范可尼肾病、葡萄糖和半乳糖利用受损。最近有报道称，在FBS患者中，编码葡萄糖转运体GLUT2的基因发生了一些突变。通过PCR、直接测序、限制性内切片段长度分析和亚克隆，我们研究了3名日本患者，发现了4个新的突变：剪接位点突变（IVS2-2A>G）、无义突变（Q287X）和两个错义突变（L389P和V423E）。以前没有报道发现突变型GLUT2的杂合子表现为肾性糖尿。患者1的母亲和兄弟为V423E杂合突变，表现为肾性糖尿。我们推测GLUT2可能是家族性肾性糖尿症的不完全外显的候选基因。如果某些突变型GLUT2蛋白具有显性负作用，由突变型和野生型蛋白组成的寡聚物可能导致运输活性的取消。为了证明这一假设，我们试图在表达显性阴性GLUT2亚基的小鼠中产生GLUT2的功能性敲除和Fanconi-Bickel综合征。为了产生GLUT2的显性阴性结构，在cDNA序列中引入突变，将423位的valine (V)残基改变为glutamate (E)。转基因载体将GLUT2-V423E cDNA插入CAG启动子下游。切除表达单元（cre - glut2: CAG-loxP-DsRed-loxP-IRES-EGFP），纯化后按标准程序微注射到受精卵中。通过分析尿糖选择表达GLUT2-V423E的转基因小鼠。

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.