Analysisof Thiamin-Responsive Megaloblastic Anemia Syndrome Pathogenesis by Targeted Gene Disruption

硫胺素反应性巨幼细胞性贫血（TRMA ; OMIM 249270）是一种常染色体隐性遗传疾病，由巨幼细胞性贫血、糖尿病、感音神经性耳聋和硫胺素治疗反应定义。据推测，TRMA是由硫胺素焦磷酸合成所需的两种蛋白质（硫胺素焦磷酸激酶（TPK）和硫胺素转运蛋白）之一的畸变引起的。我们等鉴定了SLC 192 A为编码高亲和力硫胺素转运蛋白（thiamin transporter，THTR 1）的致病基因，本研究分离了小鼠硫胺素转运蛋白（thiamin transporter，mTHTR 1）的互补基因和基因组DNA。使用5 '-RACE，在小鼠肝细胞中鉴定了位于相对于翻译起始密码子的-175和-183的SLC 192 A的两个转录起始位点。我们计划建立SLC 192 A缺陷的小鼠模型，以了解TRMA的发病机制。另一方面，我们还分离了人TPK cDNA（hTPK 1），并分析了其酶学性质。此外，我们还鉴定了hTPK 1基因在HepG 2细胞中表达所需的最小5 '启动子区，该启动子区编码的序列在-540 ° C-490 ° C之间，并包含一个Sp1结合位点。该区域Sp1位点的突变导致启动子活性降低。用HepG 2细胞核提取物和人工合成的hTPK 1启动子区-514的491序列的24 bp寡核苷酸进行电泳迁移率变动分析，鉴定出特异性DNA-蛋白复合物。这些结果提示Sp1在调节hTPK 1表达中的重要性。

Thiamin-responsive megaloblastic anemia (TRMA ; OMIM 249270) is an autosomal recessive disorder defined by the occurrence of megaloblastic anemia, diabetes mellitus, sensorineural deafness, and responding to thiamin treatment. It has been speculated that TRMA is caused by the aberration of one of the two proteins both required for thiamin pyrophosphate synthesis, thiamin pyrophosphokinase (TPK) and thiamin transporter. We and others identified SLC192A as the disease gene that encodes the high affinity thiamin transporter (THTR1).In this study, we isolated the complementary and genome DNAs of mouse thiamin transporter (mTHTR1). Using 5'-RACE two transcriptional start sites for SLC192A located at -175 and -183 relative to the translation start codon were identified in mouse liver cells. We planed to generate a mouse model of SLC192A deficiency to understand the pathogenesis of TRMA. On the other hand, we also isolated human TPK cDNA (hTPK1) and analyzed the enzymatic properties. Furthermore, we identified the minimal 5'-promoter region required for the expression of hTPK1 gene in HepG2 cells which was found to be encoded in a sequence between -540〜-490,and included a Sp1 binding site. Mutation in Sp1 site in the region led to a decrease in the promoter activity. Using Electrophoretic mobility shift analysis with the nuclear extracts from HepG2 cells and the synthetic 24bp oligonucleotide corresponding to -514〜-491 sequence of hTPK1 promoter region, specific DNA-protein complexes were identified. These results suggested the importance of Sp1 in regulating the hTPK1 expression.