The Study Of Medical Relavent Findings Of 42 Healthy Individuals Using Whole Exome Sequencing And An Intronic Mutation C.6430-3C>G In The F8 Gene Causes Hemophilia A

Part I The study of medical relavent findings of 42 healthy individuals using whole exome sequencingBackground:Whole exome sequencing(WES)has been proved to be a very powerful approach to identify new disease genes and molecular diagnosis of unknown clinical cases.However,limited studies reported on if this approach could provide useful information and should be offered to the apparently healthy people.Methods:WES was performed on 42 healthy individuals at an Illumina HiSeq platform.1493 genes were analyzed for 303 monogenic disorders including inherited cancers,cardiac arrhythmias,hereditary diseases of metabolism,pregnancy screening of common genetic diseases,age-related diseases,neuromuscular diseases,skin disorders,and eye diseases.Variants of pathogenic,likely pathogenic as well as variants of unknown significance(VUS)were reported according to the current ACMG guidelines.Genetic counseling was provided.Results:Two primary findings of VUS matched to family histories were found.89 susceptible variants and 106 pathogenic variants were identified and reported,respectly.And 925 variants of unknown significance were found in total.132 Pathogenic or likely pathogenic(P/LP)variants were screened in 42 individuals who might or would be affected.And there were 63 P/LP variants which inherited in a recessive pattern.Conclusions:This study suggests that WES for apparently healthy people,will enable physicians to take appropriate measures for disease diagnosis,prevention and treatment in their patients.It will also help these individuals to prevent the genetic disease passing down to their offspring.Part II An intronic mutation c.6430-3C>G in the F8 gene causes hemophilia AObjective:Hemophilia A is a bleeding disorder caused by coagulation factor VIII protein deficiency or dysfunction,which is classified into severe,moderate,and mild according to factor clotting activity.An overwhelming majority of missense and nonsense mutations occur in exons of F8 gene,while mutations in introns can also be pathogenic.This study aimed to investigate the effect of an intronic mutation,c.6430-3C>G(IVS22-3C>G),on pre-mRNA splicing of the F8 gene.Methods:We applied DNA and cDNA sequencing in a Chinese boy with hemophilia A to search if any pathogenic mutation in the F8 gene.Functional analysis was performed to investigate the effect of an intronic mutation at the transcriptional level.Human Splicing Finder and Pymol were also used to predict its effect.Results:We found the mutation c.6430-3C>G(IVS22-3C>G)in the F8 gene in the affected boy,with his mother being a carrier.cDNA from the mother and pSPL3 splicing assay showed that the mutation IVS22-3C>G results in a two-nucleotide AG inclusion at the 3’ end of intron 22 and leads to a truncated coagulation factor VIII protein,with partial loss of the Cl domain and complete loss of the C2 domain.The in silico tool predicted that the mutation induces altered pre-mRNA splicing by using a cryptic acceptor site in intron 22.Discussion:The IVS22-3C>G mutation was confirmed to affect pre-mRNA splicing and produce a truncated protein,which reduces the stability of binding between the F8 protein and vWF carrier protein due to the loss of an interaction domain.