Isolation and Characterization of Thiamin Pyrophosphokinase cDNA and the Expression in Thiamin-Responsive Megaloblastic Anemia

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病，表现为对硫胺素治疗有反应的巨幼细胞性贫血、糖尿病和感觉神经性耳聋。硫胺素焦磷酸激酶(TPK)或硫胺素转运系统极有可能是这种异常蛋白的候选蛋白。在这些患者中，包括一个日本家庭，发现了硫胺素转运蛋白基因(Thtr-1)的突变。另一方面，我们通过小鼠表达序列标签数据库分析、两步聚合酶链式反应和与酿酒酵母硫胺素焦磷酸激酶缺陷型突变体(Thi80)的功能互补筛选相结合的方法，分离到一个小鼠硫胺素焦磷酸激酶基因克隆(MTPK1)。利用mTPK1克隆进行克隆杂交，获得人hTPK1基因。HTPK1 ORF编码的蛋白质包含243个氨基酸残基，其序列与mTPK1产物的同源性为89%。HTPK1在人体各组织中的表达水平很低，且培养的TRMA成纤维细胞中的mRNA含量不受培养液中硫胺素浓度的影响。此外，我们还利用FISH技术将hTPK1基因的染色体定位定位于7q34。预计利用hTPK1基因进行的基础科学研究将加深对人类硫胺素代谢的了解。

Thiamin-responsive megaloblastic anemia (TRMA ; OMIM 249270) is an autosomal recessive disorder defined by the occurrence of megaloblastic anemia, diabetes mellitus, and sensorineural deafness, responding to thiamin treatment. It is highly possible that thiamin pyrophosphokinase (TPK) or thiamin transport system is a candidate for the aberrant protein.Recently, the TRMA gene was identified by positional cloning. Mutations in the patients, including a Japanese family, were found in a thiamin transporter gene (THTR-1). On the other hand, we isolated a mouse thiamin pyrophosphokinase cDNA clone (mTPK1) using a combination of mouse expressed sequence tag database analysis, two-step polymerase chain reaction procedure and functional complementation screening with Saccharomyces cerevisiae thiamin pyrophosphokinase-deficient mutant (thi80). The human cDNA (hTPK1) was subsequently isolated by colony hybridization using the mTPK1 clone. The predicted protein encoded by the hTPK1 ORF contained 243 amino acid residues whose sequence showed 89% identity with that of mTPK1 product. The hTPK1 mRNA was widely expressed in various human tissues at very low level, and the mRNA content in cultured TRMA fibroblasts was unaffected by the thiamin concentration of the medium. In addition, we assigned the chromosomal localization of the hTPK1 gene to 7q34 by FISH procedure. It is expected that basic scientific research using the hTPK1 cDNA will enhance the understanding of human thiamin metabolism.