Analysis Of F8 Gene Mutation Spectrum In 241 Non-inversion Hemophilia A Families Based On Multiplex PCR And Target Capture Sequencing Technology

BackgroundHemophilia A(Hemophilia A,HA)is an X-linked recessive inherited hemorrhagic disease,the most common type of severe hereditary Coagulation dysfunction,which is due to the F8 gene that encoding Coagulation factor Ⅷ(FⅧ)defect that lead to decrease or insufficiency of FⅧ synthesis,and the bleeding tendency is the main clinical manifestation of the disease.Commonly involved tissues include mucosa,muscle,subcutaneous,joints,and important organs.At present the most effective treatment for HA is still FⅧ replacement therapy.Infusion of exogenous FⅧ is needed for lifetime.However,the development of inhibitors is one of the most serious complications related to replacement therapy,which greatly lower the quality of therapeutic effect,and places a heavy burden on the family and society.Studies have shown that the mutation type of F8 gene is closely related to the severity of the disease,the bleeding phenotype,and the development of inhibitor.Therefore,genetic diagnosis in patients with hemophilia,their families,and carriers has important theoretical and practical significance for early diagnosis,early prevention,and prognosis management.In addition,gene therapy is expected to cure the disease,and understanding the mutation type of F8 gene will allow clinicians to analyze the therapeutic effect based on the genotype and potential inhibitor development risk.MethodsWe collected 241 non-inversion HA families diagnosed by Nanfang Hospital,and used multiplex PCR target capture sequencing technology to sequence all exons and exon-intron adjacent to 10bp of hemophilia-related genes in HA families.the The sequencing results was analyzed by self-developed integrated bioinformatics analysis system for annotation and interpretation,the pathogenicity of detected variants were classified adhere to the American Academy of Medical Genetics and Genomics(ACMG)guideline,then candidate causative pathogenic mutation were listed.The Ion Reporter software was used to analyze large deletion/duplication.Subsequently,the next-generation sequencing results were compared with Sanger sequencing results.ResultsOf 241 HA families enrolled in our court,a total of 186 F8 variants were detected in 222 HA families,and the detection rate was 92.12%(222/241).No potentially causative mutations were found in the F8 coding sequences of 19 HA families.The Sanger sequencing results were consistent with the NGS results.186 F8 risk variants were identified,with 139(139/186,74.73%)point mutations,44(44/186,23.66%)small InDels,and 3(3/186,1.61%)large deletions.Of these risk variants,117 mutations had been reported,69 mutations were novel,and 167 were pathogenic/likely pathogenic.In addition,there were three common mutations in this study:c.3780C>G(p.Asp1260Glu),c.6724G>A(p.Val2242Met),and c.3169G>A(p.Glu1057Lys)with the frequency of 6.22%(15/241),3.32%(8/241),2.90%(7/241),respectively in our court.In addition,grandfathers of the proband with no hemorrhagic phenotype in 3 special families were found,missense mutation may be one of the reasons causing phenotype heterogeneity.ConclusionIn this study,multiplex PCR target capture next-generation sequencing was used to detect F8 gene mutation in 241 non-inversion HA families.Our results showed the feasibility for non-inversion F8 gene mutation detected by multiplex PCR targeted capture sequencing.All F8 gene mutation types were tested.For the first time,69 novel mutations of the F8 gene were identified,and bioinformatics analysis was performed.Three special families with clinical phenotypic heterogeneity were found,mssense mutation may be one of the reasons causing phenotype heterogeneity.Our study enriched the mutation spectrum of F8 gene which would provide further understanding of genotype-phenotype relationship.