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

Fanconi-Bickel综合征(FBS)是一种常染色体隐性遗传病，表现为肝肾糖原蓄积、Fanconi肾病、葡萄糖和半乳糖利用受损。最近，有报道称，在FBS患者中，编码葡萄糖转运蛋白GLUT2的基因发生了几个突变。应用聚合酶链式反应、直接测序、限制性内切酶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个新的突变：1个剪接点突变(IVS2-2A和GT；G)、1个无义突变(Q287X)和2个错义突变(L389P和V423E)。患者1的母亲和兄弟是V423E突变杂合子，表现为肾性葡萄糖尿。我们推测GLUT2基因可能是不完全外显性家族性肾性糖尿的候选基因。如果一些突变的GLUT2蛋白具有显性负效应，由突变型和野生型蛋白组成的寡聚体可能导致运输活性的丧失。为了证明这一假说，我们试图在表达显性负性GLUT2亚单位的小鼠中产生GLUT2的功能性敲除和Fanconi-Bickel综合征。为了产生GLUT2显性负性结构，对其进行了突变，将第423位的Valine(V)残基改变为Glamate(E)。转基因载体将GLUT2-V423E基因插入到CAG启动子下游。将表达单位(CDRE-GLUT2：CAG-loxP-DsRed-loxP-IRES-EGFP)切割、纯化后按标准程序显微注射到受精卵中。通过尿糖分析筛选出表达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.