目的：分析从2个血友病A家系中检出的2个未报道的内含子突变对剪接的影响,明确这2种突变的致病性,为遗传咨询提供依据。方法：通过检测相关的凝血指标,明确血友病A的诊断。进行F8相关检测,包括PCR扩增及测序分析F8的26个外显子及其侧翼序列、拷贝数检测、内含子1和内含子22倒位检测,明确致病突变。采用巢式PCR扩增外周血中的mRNA,从异位转录水平分析内含子突变对剪接的影响。针对6个短串联重复序列（short tandem repeats, STR）位点F8Up226、F8Up146、F8Int13、F8Int25、F8Down48和DXS1073进行家系遗传连锁分析,用SNaPshot SNP分型技术检测外周血、口腔黏膜细胞和毛囊细胞嵌合情况,分析突变来源。结果：家系1中血友病A先证者1的 FⅧ：C为0.9%,检测到F8的9号内含子c.1444-2dupA突变,mRNA分析显示该突变导致F8的10号和11号外显子缺失;家系2中血友病A先证者2的 FⅧ：C为5.1%,检测到F8的18号内含子c.5999-29T>G突变,mRNA分析显示该突变产生2种转录本,即缺失19号外显子的异常转录本和少量正常转录本。家系1无血友病A家族史,遗传分析显示突变来源于先证者的外公,但外公的血液、毛发和口腔样本嵌合检测均未检出突变。结论：c.1444-2dupA和c.5999-29T>G突变均为国际首次报道,突变导致了不同程度的剪接异常,分别引起重型和轻型血友病A。家系1的c.1444-2dupA为新发突变,可能是在先证者外公的精子形成过程中发生。
Objective: To study the molecular pathogenesis of two novel splice site mutations of F8 in hemophilia A and to provide the evidence for genetic counseling. Methods: Coagulation tests were performed to establish the diagnosis of hemophilia A. A serial of genetic tests were performed to analyze F8, including directly sequencing the PCR amplification products of 26 exons and flanking regions of F8, detection of copy number variations (CNVs) and detection of intron 1 and 22 inversion for defining the pathogenetic mutation. Nested PCR was applied to detect F8 ectopic transcripts in leukocyte to analyze the molecular pathogenesis of the splice site mutations. Multiplex fluorescent PCR was used to detect six STR locus (F8Up226, F8Up146, F8Int13, F8Int25, F8Down48 and DXS1073) for gene linkage analysis. Mosaic analysis was performed with peripheral blood cells, oral mucosal cells and hair follicle cells by SNaPshot SNP technique to analyze the origin of mutation. Results: Levels of FⅧ:C were 0.9% and 5.1% in probands 1 and 2, respectively. Proband 1 presented with a splice site mutation in intron 9 (c.1444-2dupA); mRNA analysis showed a smaller band, resulting from exons 10 and 11 skipping. Proband 2 had a T>G substitution in intron 18(c.5999-29T>G); mRNA analysis showed a smaller band and a weak normal size band. The smaller band presented exon 19 skipping. In pedigree 1, the mutation was inheri-ted from proband's grandfather who was with normal phenotype and genotype. Mosaic analysis showed no mosaic existing in his peripheral blood cells, oral mucosal cells and hair follicle cells. Conclusions: Mutations of c.1444-2dupA and c.5999-29T>G are first reported ,which cause severe and mild hemophilia A, respectively. c.1444-2dupA in pedigree 1 is a de novo mutation which might arise from sperm of proband's grandfather at meiosis.
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