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目的:探讨2例遗传性凝血因子Ⅶ(Coagulation factor Ⅶ,FⅦ)缺陷症家系基因型与临床表型的关系。方法:用DNA直接测序法对先证者及其家庭成员FⅦ基因的全部外显子及其侧翼、启动子和3’非翻译区进行分析,寻找基因突变;将含插入突变序列克隆入pMD18-T TA克隆载体中,对所得两条染色体相应序列分别测序,以确定不同突变在染色体上的分布。用等位基因特异的聚合酶链反应(ASPCR)方法证实测序所发现的突变。结果:家系1先证者在8号外显子上发现一个杂合基因突变:11514C>T,导致Thr(ACG)359 Met(ATG)氨基酸替换,该突变来自其母亲;其父亲为11496G>A改变,导致Arg(CGG)353 Gln(CAG)杂合多态性。家系2先证者发现了三种杂合多态性:即—323插入10个核苷酸(—323P0/P10)、73G>A(G73A)及Arg 353 Gln均来自先证者的母亲和外祖父,三种多态性均位于先证者同一条染色体上。家系其他成员的基因型为野生型。AS-PCR证实了先证者及其家系成员的基因突变。结论:两个家系先证者的FⅦ缺陷症基因型与其临床表型无明显相关性。
Objective: To investigate the relationship between genotypes and clinical phenotypes of two cases of genetic variant factor Ⅶ (FⅦ) deficiency. Methods: All the exons of FⅦ gene, their flanks, promoter and 3 ’untranslated region of probands and their family members were analyzed by DNA direct sequencing to find the gene mutation. The inserted sequence was cloned into pMD18- T TA cloning vector, the corresponding sequence of the two chromosomes were sequenced to determine the distribution of different mutations in the chromosome. Allele-specific polymerase chain reaction (ASPCR) methods were used to confirm the mutations found in sequencing. RESULTS: A heterozygous gene mutation was found on exon 8 of the family 1 proband: 11514C> T, resulting in the amino acid replacement of Thr (ACG) 359 Met (ATG) from its mother; its father changed at 11496G> A , Resulting in a heterozygous polymorphism of Arg (CGG) 353 Gln (CAG). Family 2 probands found three heterozygous polymorphisms: the -323 insertions of 10 nucleotides (-323P0 / P10), 73G> A (G73A), and Arg353 Gln came from the proband’s mother and grandfather All three polymorphisms are located on the same chromosome of the proband. The other members of the family have a genotype of wild type. AS-PCR confirmed the gene mutation of probands and their family members. Conclusion: The genotypes of F Ⅶ deficiency in two probands have no significant correlation with their clinical phenotypes.