Development and application of molecular diagnostic method of hemophilia A

Abstract

Objective To develop and validate a method for detecting factor 8 gene (F8) mutations in hemophilia A patients by Ion Torrent semiconductor sequencing. Methods Intron 22 and intron 1 inversions of F8 gene were identified by long distance PCR (LD-PCR), other mutations in the F8 gene were identified by Ion Torrent sequencing.Candidate variants were validated by Sanger sequencing. Sanger sequencing was applied to screen HA carriers from 11 female family members in the 8 pedigrees. One pregnant woman was offered prenatal diagnosis via analyzing the fetal DNA obtained through amniocentesis. Results Four missense mutations (c.1331A>C, 1648C>T, c. 6506G>A, c. 6544C>T), two frameshift mutations (c.2393_2394insT, c. 6320delG), one splicing mutation (IVS5+ 5G>A), one nonsense mutation (c.43C>T) and one Inv22 mutation were identified in all nine probands respectively. Among 11 female family members, 10 females were identified to be HA carriers, and one didn't carry the maternal pathogenic mutation. Prenatal diagnosis result showed that the fetus inherited the wild-type maternal allele and was predicted to be unaffected by HA. Conclusion The targeted Ion Torrent sequencing is a reliable and efficient method to detect F8 mutations in patients with Hemophilia A disease.(Chin J Lab Med, 2016, 39: 496-500) Key words: Hemophilia A; Factor Ⅷ; Mutation