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.
XIE Xiaoling, MA Siyu, WU Xi, LU Yeling, WANG Xuefeng, DING Qiulan
. Molecular pathogenesis of two novel splice site mutations of F8 in hemophilia A[J]. Journal of Diagnostics Concepts & Practice, 2018
, 17(01)
: 32
-37
.
DOI: 10.16150/j.1671-2870.2018.01.006
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