Mutation Screening And Functional Analysis Of F8 Gene In Patients With Severe Hemophilia A

Objective: Hemophilia A is an X-linked recessive bleeding disorder caused by the deficiency or fuctional defects in coagulation factor ?.Affected individuals are almost exclusively males with an incidence of 1 in 5000 live-born males.Patients often suffer spontaneous or traumatical bleeding.Severe haemorrhage,low F? coagulant activity(<1%)and high cost of replacement therapy are often shown in patients with severe hemophilia A.Therefore,it is important to provide genetic diagnosis for severe patients.F8 gene is considered as the main pathogenic gene for hemophilia A and more than 3000 mutations with high genetic heterogeneity have been reported in human gene mutation database(HGMD),including nonsense mutation,missense mutation,splicing mutation,insertion,deletion and inversion.Intron 22 inversion and intron 1 inversion which are reproducible mutations with high incidence are main molecular pathogenesis for severe hemophilia A.In the present study,inversion detection,mutation screening and functional analysis in F8 gene were performed in 35 patients with severe hemophilia A in order to enrich the existing F8 mutation spectrum and provide new clues for molecular mechanism on severe hemophilia A.Methods: Long distance polymerase chain reaction(LD-PCR)and multiplex PCR were performed in 35 patients with severe hemophilia A to detect intron 22 inversion and intron 1 inversion,respectively.Direct DNA sequencing in F8 gene was carried out for patients without intron inversion,following functional analysis for novel c.670+1G>A splicing mutation.Wild-type and mutant-type F8 minigene expression vectors were constructed and transfected into human embryonic kidney cell HEK293 T respectively,following reverse transcription-polymerase chain reaction(RT-PCR)and DNA sequencing to verify the splicing in vitro.Results: 1.Intron 22 inversion was positive in 27 out of 35 severe hemophilia A patients,which accounted for 77.1%(27/35).2.Intron 1 inversion was negative in all 35 severe hemophilia A patients(0/35).3.Four mutations in F8 gene,including c.185C>G nonsense mutation(p.Ser62Ter),c.2951C>A nonsense mutation(p.Ser98Ter),c.7016G>T missense mutation(p.Arg2339Met)and c.670+1G>A splicing mutation,were identified in 6 out of 8 non-inversion patients.Novel mutations included c.2951C>A nonsense mutation(p.Ser98Ter)identified in one patient and c.670+1G>A splicing mutation identified in two patients.However,c.185C>G nonsense mutation(p.Ser62Ter)identified in two patients and c.7016G>T missense mutation(p.Arg2339Met)identified in one patient had been previously reported.No mutations in F8 gene were identified in two patients with severe hemophilia A.4.Bioinformatic prediction showed that c.670+1G>A mutation could lead to the loss of the normal splicing donor site(c.670+1G)and the activation of the cryptic splicing donor site(c.670+177G).Cell experiments in vitro confirmed that the RT-PCR products with normal splicing showed an expected band of 399 bp after transfected with wild-type F8 minigene expression vector.However,the RT-PCR products showed an extra expansion after transfected with mutant-type F8 minigene expression vector.Subsequently,DNA sequencing revealed that abnormal splicing occurred in the mutant-type F8 minigene expression vector,which resulted in the retainment of 176 additional nucleotides at the 5? end of intron 5.Conclusions: 1.Intron 22 inversion in F8 gene is the main molecular mechanism for severe hemophilia A.2.c.2951C>A nonsense mutation and c.670+1G>A splicing mutation are novel pathogenic mutations for severe hemophilia A.3.c.670+1G>A splicing mutation can lead to abnormal splicing and result in the retainment of 176 additional nucleotides at the 5? end of intron 5.