Gene Detection And Analysis For The Hemophilia B In Shan Dong Provice

Objective①Investigate and analysis the clinical date of 24 persons with hemophilia B to realize the current treatment status of the hemophilia B patients in shandong province.②To explore the mechanism of molecular pathology and find the pattern of FIX gene mutations,24 patients with HB were studied with gene sequencing and analysis.Methods①Collecting the clinical data of 24 cases of hemophilia B patients,including gender, age, age of onset, age of diagnosis, family heredity,the frequency of the replacement therapy within half a year, and to conduct a comprehensive physical examination and infectious markers examination;②Activated partial thromboplastin time（APTT）, prothrombin time（PT）, thrombin time（TT）, fibrinogen（Fib）, coagulation factors IX activity（FIX:C）, factor IX antigen （FIX:Ag） and coagulation factorsⅧactivity（FⅧ:C）were tested to make phenotypic diagnosis;③Genomic DNA was extracted from each HB disease members’peripheral blood leucocytes.Using primer Premier 5.0 software,eight pairs primers were designed to cover all eight exons areas and the flanking sequences of FIXGene respectively, according to the FIXGene reference sequences（the GenBank entry NC₀00023.10）.For the proposition and suspicious carriers,all regions of FIXgene,including all exons and the flanking sequences,were amplified by Polymerase Chain Reaction（PCR）.The products of PCR were sequenced by the dideoxy chain termination using ABI3730XL sequencer.Then the sequencing results were analyzed with software Chromas and BLAST,searching for mutations in these regions of the gene.Results①Total 24 HB patients were enrolled in this study:16 severe, and 8 moderate patients.②All the 24 patients’ FIX genes were detected mutations, and 18 different types of mutations were detected. Including missense mutation 13 species and 2 kinds of nonsense mutations,deletion of three.13 types of mutations for the reported mutations,five types of mutations are not reported in the world.③ Our study first discovered five new mutation types,including:c.200delA、 c.306delT、c.688-690delGGA、c.1025C>G、C.1157C>A.Conclusion①HB patients have apparent clinical phenotype heterogeneity. This heterogeneity reflects by variability in age at first bleed and first joint bleed, frequency of bleeding, joint deformities.②Gene analysis could directly explain molecular mechanism of HB,and also provided the foundation for further studies to the function of coagulation factor IX.There was obvious heterogeneity in FⅨ gene mutation and missense mutation was still the main way of mutation in our study.FⅨ gene mutation is a common explicit radical and their encoding amino acids on the role of protein function.This experiment found 5 kinds of new mutations,the results not only enrich the FⅨ gene mutations spectrum,but also provided the molecular basis for the pathogenesis.DNA sequencing is an efficient method for carriers and prenatal gene diagnosis.