Investigation of mechanism for the genesis of polymorphisms of the coagulation factor XIII and PGM1 genes

我们证明了凝血因子XIII“a”亚基遗传多态性的分子基础和五个外显子的新序列多态性。发现4个F13A等位基因在外显子12和外显子14的三个位点被核苷酸取代。我们开发了PCR-RFLPs，通过它可以在基因组水平上检测到这些替换。除了这三个位点外，我们还在高加索人群中发现了外显子2、5、8、12和14的新多态性位点，而在日本人中，这些位点除了外显子2位点外均为非多态性位点。这些多态位点被发现以单倍型的形式传播。在理论上72种可能的组合中，共有18种单倍型被发现出现在高加索人群中，这意味着频繁的重组事件而不是反复发生的点突变导致了单倍型的产生。此外，我们还发现了14个不同的变异等位基因和1个缺陷等位基因的序列差异。14个变异等位基因的核苷酸变化导致的氨基酸取代与其产物在IEF凝胶中的迁移率变化一致。发现缺陷等位基因是由F13A基因5内含子剪接受体位点的单核苷酸突变引起的剪接异常引起的。本研究获得的数据表明，F13A基因区域存在重组热点。我们现在正在研究内含子和5‘和3’侧翼区域的序列。

We demonstrated molecular basis of the genetic polymorphism of the coagulation factor XIII "a" subunit and novel sequence polymorphisms in five exons. Four F13A alleles were found to be defined by nucleotide substitutions at the three sites of exon 12 and exon 14. We developed PCR-RFLPs by which these substitutions can be detected at the genomic level. In addition to the three sites, we characterized novel polymorphic sites of exons 2,5,8,12 and 14 in Caucasian populations whereas these sites were non-polymorphic in Japanese except for the site of exon 2. These polymorphic sites were found to be transmitted as haplotypes. A total of 18 haplotypes out of theoretically 72 possible combinations were found to occur in the Caucasian populations, implying that frequent recombination events rather than reccurent point mutations lead to production of the haplotypes. Furthermore, we revealed sequence differences for 14 distinct variant alleles and one deficient allele by characterizing their complete sequences. Amino acid substitutions resulting from the nucleotide changes of the 14 variant alleles were consistent with mobility shift of their products in IEF gels. The deficient allele was found to result from abnormal splicing caused by a single nucleotide mutation at the splice-acceptor site in intron 5 of the F13A gene.The data obtained in this study suggest that there are recombination hot-spots in the F13A gene region. We are now investigating the sequences of introns and 5'-and 3'-flanking regions.