血友病A家系的单体型基因连锁分析

目的：该研究开展温州地区血友病A（HA）家系的可变数目串联重复序列（VNTR）多态性、短串联重复序列（STR）多态性和限制性片段长度多态性（RFLP）单体型基因连锁分析，为HA遗传咨询和生育指导提供症状前、携带者基因诊断方面的依据。方法：针对HA先证者及其有关家系成员，进行单体型基因连锁分析。采用PCR检测凝血因子VIII（FVIII）基因外的DXS52（St14）位点的VNTR多态性，检测FVIII基因外的DXS15（CA）n、DXS9901（GT）n、DXS1073（GT）n位点和内含子1（GT）n、13（CA）n、22（GT）n（AG）n、24（GT）n位点的STR多态性并经毛细管电泳确证。另外采用PCR产物限制酶切检测FⅧ基因的内含子18、19、22位点的RFLP。结果：以2个HA家系为例报道研究结果。家系一先证者年幼弟弟肯定为正常人，先证者母亲、外祖母为携带者，先证者小姨肯定为携带者而其年幼儿子肯定为正常人。家系二先证者外祖母肯定不是携带者，先证者的X染色体来自外祖父，但已知外祖父不是患者，那么按照最大风险估计，母亲的那条来自外祖父的X染色体在外祖父生殖细胞中FVIII基因发生了突变，因此母亲是携带者。家系二先证者年幼妹妹是携带者，将来有生育患儿的风险，但先证者大姨及其年幼女儿不是携带者。结论：该研究的HA家系的VNTR-PCR、STR-PCR和PCR/RFLP单体型基因连锁分析，特别是对症状前男孩的诊断、对未曾有患病后代的女性携带者的检出，具有非常重要的实际意义，可以为遗传咨询和生育指导提供可靠依据。

Heplotype gene linkage analysis for hemophilia A families

Objective: To develop variable number tandem repeat (VNTR) polymorphism, short tandem repeat (STR) polymorphism and restriction fragment length polymorphism (RFLP) heplotype gene linkage analysis for hemophilia A (HA) pedigrees in Wenzhou, further to provide evidences of gene prognosis of males and female carriers for HA genetic counseling and family planning guidance. Methods: For the HA probands and related family members, haplotype gene linkage analysis was processed. PCR was used to analyze the VNTR polymorphism in extragenic site DXS52 (St14) of clotting factor VIII (FVIII) gene, to analyze the STR polymorphism in extragenic sites DXS15 (CA)n, DXS9901 (GT)n, DXS1073 (GT)n and in intragenic sites intron 1 (GT)n, 13 (CA)n, 22 (GT)n (AG)n, 24 (GT)n of FVIII gene, and the STR polymorphism was identified by capillary electrophoresis. Also PCR/RFLP was used to analyze the RFLP in intragenic sites intron 18, 19 and 22 of FVIII gene. Results: The results of two HA pedigrees were taken as examples. In pedigree 1, the very young brother of proband was normal, the mother and grandmother of proband were carriers, the young aunt of proband was carrier and her very young son was normal. In pedigree 2, the grandmother of proband was not carrier, the X chromosome of proband was interited from his grandfather. Since the grandfather was not HA patient, supposing the most serious situation, it was deduced that a mutation of FVIII gene had happened to the X chromosome of the mother which interited from the grandfather when it was in germ cell of grandfather, thus the mother of proband was carrier. In pedigree 2, the very young sister of proband is carrier, and there will be risk for her sons, but the eldest aunt of proband and her very young daughter are not carriers. Conclusion: The VNTR-PCR, STR-PCR and PCR/RFLP haplotype gene linkage analysis of HA pedigrees of this study, especially the presymptomatic gene diagnosis of males, the gene diagnosis of female carriers without HA offspring, will have their very important clinical implications, can provide reliable evidences for genetic counseling and family planning guidance.