FⅧ基因内含子22倒位及x染色体非随机灭活导致的女性血友病A

Haemophilia A in a female with a novel INV22 mutation and skewed X-chromosome inactivation

目的对1例女性血友病A（HA）患者进行产前诊断，探讨其分子发病机制。方法利用测定FⅧ活性（FⅧ：C）、出血时间（BT）、血管性血友病因子（VwF）等指标进行HA表型诊断；用长链PCR及序列特异PCR进行FⅧ基因内含子22及1倒位检测，对FⅧ基因的所有外显子及其侧翼序列进行测序。联合FⅧ基因内外的8个多态性位点进行遗传连锁分析，DXS52（ST14）位点多态性以PCR产物凝胶电泳法检测，7个微卫星位点（F8Civs13、CA22、DXS15、DXS9901、G6PD、DXS1073、DXS1108）的多态性以多重荧光PCR法检测，产前诊断加用性别位点（Amelo）。利用甲基化敏感的限制性内切酶HpaⅡ对基因组DNA进行酶切，荧光PCR法对HpaⅡ酶切前后的基因组DNA进行人类雄激素受体基因（HUMARA）中CAG重复序列的扩增，荧光扫描法对扩增产物进行检测，根据酶切前后PCR产物的峰值变化来分析判断x染色体的随机灭活模式。结果先证者的FⅧ：C为2．1％，其余表型检测结果均正常，其家系成员表型检测结果均正常。FⅧ基因内含子22倒位检测示先证者为22倒位阳性携带者，家系其他成员及胎儿均为22倒位阴性；先证者FⅧ基因内含子1倒位检测结果为阴性，其FⅧ基因测序未发现突变。遗传连锁分析发现先证者的x染色体分别遗传自其父亲、母亲；其胎儿为女性且遗传了先证者来源于其母亲的1条x染色体。先证者为x染色体非随机灭活，其来自母亲的x染色体被大部分灭活，而来自父亲的x染色体大部分被保留活性。结论FⅧ基因内含子22倒位及x染色体非随机灭活导致了该例女性血友病A的发生，其胎儿为正常女性。

Objective To make genetic and prenatal diagnosis of a female with Haemophilia A. Methods The FⅧ： C, BT and VWF were detected to make phenotypic diagnosis. LD-PCR was adopted for screening the intron 22 inversion and PCR was adopted for the screening the intron 1 inversion. The coding and boundary sequences of FⅧ gene were analyzed by PCR and DNA sequencing. Eight combined polymorphic markers（ Amelo, FSIVS13, CA22, DXS15, DXS9901, G6PD, DXS1073 and DXSll08 ） were applied for linkage analysis of the family by multiplex fluorescent PCR. The polymorphism of DXS52 （ST14） was analyzed by PCR and electrophoresis. Assessment of X inactivation was performed using an Hpa Ⅱ- polymerase chain reaction （PCR） assay for the X-linked human androgen receptor gene （HUMARA）. Results The female HA patient showed severe FⅧ deficiency （FⅧ： C 2. 1% ） and other phenotypic tests were normal. Her family members showed normal in all tests. The female proposita was found to be a carrier of FⅧ gene intron 22 inversion, but her family members as well as her fetus showed negative results. Except this inversion,no other mutation was found then. The female inherited two X chromosomes from both her parents＇ and her fetus inherited the maternally derived X chromosome from the female proposita according to the linkage analysis. Furthermore, X-inactivation pattern of the female was unbalanced and her maternally derived X chromosome was inactived mostly while the majority of her paternal derived one kept active. Conclusions The severe haemophilia A in the proposita resulted from the de novo FⅧ intron 22 inversion which most probably arose in the paternal germ line, associated with a skewing pattern of inactivation of the maternally derived X chromosome. Her fetus is normal female.