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疾病，由巨幼细胞性贫血、糖尿病和感音神经性耳聋的发生定义，对硫胺素治疗有反应。硫胺素焦磷酸激酶（thiamin pyrophosphokinase，TPK）或硫胺素转运系统极有可能是该异常蛋白的候选者。在患者（包括一个日本家庭）中发现了硫胺素转运蛋白基因（THTR-1）的突变。另一方面，我们分离了一个小鼠硫胺素焦磷酸激酶的cDNA克隆（mTPK 1），使用小鼠表达序列标签数据库分析，两步聚合酶链反应程序和功能互补筛选与酿酒酵母硫胺素焦磷酸激酶缺陷突变体（thi 80）相结合。随后使用mTPK 1克隆通过菌落杂交分离人cDNA（hTPK 1）。hTPK 1 ORF编码的蛋白质含有243个氨基酸残基，与mTPK 1蛋白的同源性为89%。hTPK 1 mRNA在人各种组织中广泛表达，但表达水平很低，培养的TRMA成纤维细胞中hTPK 1 mRNA含量不受硫胺素浓度的影响。此外，我们通过FISH技术将hTPK 1基因定位于7 q34。预期使用hTPK 1 cDNA的基础科研将增强对人硫胺素代谢的理解。

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.