A Strategy for the Molecular Diagnosis in Hemophilia A in Chinese Population

Abstract

Hemophilia A is an x-linked recessive inherited bleeding disorder. So far, more than 1,885 disease-causing mutations of factor VIII gene have been identified. Clinic confers a great challenge for the molecular diagnosis. We aim to make a better strategy for the molecular diagnosis in Hemophilia A. First, factor VIII intron 22 inversion and intron 1 inversion mutations were detected using Inversion-PCR and double-tube multiple PCRs. And then, non-inversion mutations were analyzed by denaturing high performance liquid chromatography and/or direct sequencing. Novel mutations were further analyzed the conservation and 3D structures by a B domain deleted crystallographic model and bioinformatics. Finally, we can indirectly confirm the diagnosis by linkage analysis for the patients with the confusing diagnosis by the techniques mentioned above. Eleven patients with the factor VIII Inv 22 were found, and the remaining 16 patients were found with 11 different mutations, of which 3 was novel mutations affecting A1, B domains and splicing site. Moreover, the prenatal diagnosis was performed on 14 fetuses. Ten fetuses were successfully confirmed to be normal, 1 fetus to be a heterozygote with factor VIII c.3275-3276 ins A and 3 fetuses to be hemizygotes with factor VIII Inv 22 mutation.

Genetic diagnosis in Hemophilia A from southern China: five novel mutations and one preimplantation genetic analysis

“…We used a modified I‐PCR technique to detect factor VIII Inv22 , and intron 1 inversion was detected according to the literature . After excluding subjects with intron 22 and intron 1 inversions, all 26 exons, including the flanking intronic sequences, promoter, and 3′ UTR were amplified using primers and PCR conditions described previously . The amplified fragments were purified and sequenced commercially (BGI, Guangzhou, China).…”

Application of indirect linkage analysis and direct genotyping to hemophilia A carrier detection in Sichuan, China

“…We also randomly selected five families to confirm the diagnoses by the indirect method. All the exons of the FVIII gene were amplified with the primers described by He et al (2013).…”