Iwahana H, Yoshimoto K, Shigekiyo T, Shirakami A, Saito S, Itakura M
Otsuka Department of Clinical and Molecular Nutrition, University of Tokushima, Japan.
Am J Hum Genet. 1992 Dec;51(6):1386-95.
The molecular and genetic basis of a compound heterozygote for dys- and hypoprothrombinemia was analyzed. Abnormal nucleotide sequences of the human prothrombin gene were screened by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion and mutated primer-mediated PCR-RFLP. A single nucleotide substitution responsible for dysprothrombinemia of prothrombin Tokushima was detected, as were three polymorphisms. The mutation for hypoprothrombinemia was detected by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion in exon 6, near MboII-RFLP and NcoI-RFLP. Sequencing of PCR-amplified genomic DNA revealed a single base insertion of thymine (T) at position 4177. The resulting frameshift mutation caused both an altered amino acid sequence from codon 114 and a premature termination codon (i.e., TGA) at codon 174 in exon 7. Because exon 7 encodes the kringle 2 domain preceding the thrombin sequence, this frameshift leads to the null prothrombin phenotype. The inheritance of the hypoprothrombinemia gene from the father to the proband was proved by PCR-SSCP with endonuclease digestion and mutated primer-mediated PCR-RFLP.
对凝血酶原血症和低凝血酶原血症复合杂合子的分子和遗传基础进行了分析。通过聚合酶链反应-单链构象多态性（PCR-SSCP）结合内切酶消化和突变引物介导的PCR-RFLP技术，筛选人凝血酶原基因的异常核苷酸序列。检测到导致凝血酶原德岛型凝血酶原血症的一个单核苷酸替代以及三个多态性位点。通过在靠近MboII-RFLP和NcoI-RFLP的外显子6中进行PCR-单链构象多态性（PCR-SSCP）结合内切酶消化，检测到低凝血酶原血症的突变。对PCR扩增的基因组DNA进行测序，发现在4177位有一个胸腺嘧啶（T）的单碱基插入。由此产生的移码突变导致从第114密码子开始的氨基酸序列改变，并在第7外显子的第174密码子处产生一个提前终止密码子（即TGA）。由于第7外显子编码凝血酶序列之前的kringle 2结构域，这种移码导致无凝血酶原表型。通过PCR-SSCP结合内切酶消化和突变引物介导的PCR-RFLP技术，证实了低凝血酶原血症基因从父亲遗传给先证者。
