| Background and ObjectiveHemophilia is a group of inherited bleeding diseases due to sever coagulation disorders result form the defects of some of blood clotting factors. Both man and women can be affected, but prevalent in males.The major forms include hemophilia A,hemophilia B, factor XI deficiency and other factors deficiency. Hemophilia is the most common in congenital hemorrhagic diseases as bleeding is the primary clinical manifestation.The most common types are hemophilia A and hemophilia B respectively caused by gene mutations of blood coagulation factor Ⅷ and blood coagulation factor IX, both of them are x-linked recessive heredity. Hemophilia A accounts for80%～85%in male hemophilia patients, as contrast, hemophilia B is only15%～20%with that male incidence is about1/30000far below the hemophila A and female patients are rare. Currently, the main treatment for hemophilia is plasma replacement therapy such as supplying the blood coagulation factors for patients to prevent or control their bleeding symptom. As an inherited disease, it will be possible to be cured off according to the treatment direct at its corresponding gene. Therefore, it is necessary to have further study in the types of genetic mutations to establish an effective genetic diagnosis system and provide evidences for therapy. Hemophilia B, also called Christmas Disease, its mechanism is that the reduction of FIX content or FIX dysfunction in plasma result from FIX gene deficiency make the procoagulant activity absent or reduced, which then lead to form the serious coagulation disorders. According to the activity of blood coagulation factor IX, it clinically divides into light, medium and heavy. In1985, Yoshitake etc finished the whole FIX gene sequence analysis. It is known that, FIX gene is3.5kb in length and consist of eight exons, seven introns, and its flanking sequence, and its eight exons explicitly encode its corresponding6structure domains of FIX protein. George Brownlee set up the HB mutations database firstly in1990. At present, the latest released FIX Mutation Database (12edition) enrolls2891cases and shows the962kinds of unique mutation including91patients with big loss or insertion in FIX gene. With the worldwide intensive HB study and the increase of new-found HB cases, the mutation database is updated everyday and more and more new mutations are found and recognized by human. On the basis of the distribution of mutants in intragenic protein domains and control regions in database shows that gene mutations in HB cases have obvious heterogeneity, which can occur in any position on exons, introns, promoter or its flanking sequences of FIX gene. Presently, the known FIX gene mutation types include missense mutation which take account for about60%, nonsense mutation, small insertion and deletion.Presently, gene diagnosis methods in HB patients and carriers are classified into the indirect method and direct method. Indirect methods such as restriction fragment length polymorphism (RFLP), short tandem repeat sequence (STRs) and single nucleotide polymorphisms (SNP) can be used to do gene diagnosis in HB patient whose genealogy has no clear family story and propositus, by combining multiple STR polymorphism sites. Direct methods, such as denaturing gradient gel-electrophoresis (DGGE) and single strand conformation polymorphism (SSCP), are using PCR to amplify the target gene and then doing the heteroduplex analysis to do the detection. For diffuse HB cases and carriers which have no family history or no propositus, gene diagnosis or prenatal diagnosis cannot be done out, even using linkage analysis through polymorphism sites. When abnormal things are found through direct detection methods like SSCP and DGGE etc, it still need to proceed further gene sequence analysis to determine the specific mutation, and that methods above have the disadvantages as complexity, time-consuming and low sensitivity by the way. In recent years, with PCR technology is becoming more and more mature and FIX gene is smaller and other relevant factors, many domestic and foreign research groups adopt the gene sequencing method instead of other choices to do HB gene diagnosis and all obtain good results. On the basis of literature materials and other study, this research design and synthesize8couples primers covering eight exons and its flanking sequence of FIX gene, use the polymerase chain reaction (PCR) and DNA sequencing technology on31HB cases to do FIX gene mutation detection, and compare sequencing results with normal series through the Chromas software in the Blast as to looking for specific genetic mutation position.Analysing the gene mutation results from FIX gene direct sequencing of all HB patients collected in this study, the specific position, type, amino acid change are obtained. The gene mutation detection analysis of these31cases is meaningful to clear and definite the mechanism of FⅨgene mutant, and provides the molecular basis for the development of gene therapy, and also provides necessary supplements for the world HB mutations database at the same time, and lastly is an integral part of the worldwide study on HB.Materials and methods1.research object 31cases of patients in HB with unrelated families, are collected from the hematology department and the gynecology and obstetrics and gynecology department of Southern Hospital. All patients present different degrees of bleeding phenomenon in clinic, and their FIX activity (factor IX activity, the F IX:C) are all inferior to5%.2.specimen collection and DNA extraction2ml of each HB patients’peripheral blood is collected by vein, meantime adding edetic acid to effect on anticoagulation. In laboratory, genomic DNA is extracted with peripheral blood DNA extracted kit (American Life company) and then DNA is saved in-20℃backup.3.primer design and PCR amplificationAccording to the F IX Gene sequences (Gene Bank K02402), and referring to the related literatures, we design and synthesize eight couples primers respectively to cover all eight exons areas and their flanking sequences of F IX Gene. The synthesization work of primers is done by the Yingjun primers biological technology Co., LTD from Shanghai.All PCR are carried out under the following conditions:5pmol (final concentration for0.1mM) of each primer,5nmol (final concentration for0.1mM) of each dNTPs,5ul of10×Taq PCR Buffer (including100mM Tris-HCl,15mM MgCl2and500mM KC1, pH8.3),2U of Taq DNA polymerase enzyme (from TAKARA company), and50-200ng of human genomic DNA. The final volume is adjusted to50ul with water. All PCR amplification reactions are performed on a Gene Amp PCR system9700(PE Applied Biosystems). The polymerase chain reaction (PCR) conditions for8couples primers designed by our research are follows:an initial denaturation step at95℃for5min followed by30PCR cycles of denaturizing at94℃for30sec; annealing at59℃for30sec;extension at72℃for1min; a final extension at72℃for10.4.PCR products electrophoresis and sequencingAn electrophoresis of the amplification products are conducted on1%agarose gels buffer stained with ethidium bromide (EB), in which5ul reaction products were mixed with1ul loading, with the gels run at80V for20min. PCR products fit the sequencing requirements are sent to do the DNA sequencing which is completed by Yingjun biological technology Co., LTD from Shanghai. We use Chromas and Blast and other biological softwares to analysis the sequencing results at last.The results and discussion1ResultsThrough the Chromas software comparing sequencing results with normal series on Blast,31cases of HB patients have been detected the mutations using DNA sequencing. By consulting the related articles and mutation database, test results of these31samples in this study show that, FIX gene mutation sites scattered without hot zone, and the types of mutations is various including point mutation, insertion and deletion. Most of mutations are point mutations, and except a whole gene deletion, the remains are short deletion mutations in the deletion mutation types.34kinds of mutations are found in these31cases, including1case of triple mutations,6cases of double mutations, and also13mutations are new recognized.2.DiscussionSo far, the FIX gene and its protein structure and function have been studied clearly. FIX gene locates in Xq27.1, approximately33.5kb in length, and is consist of eight exons, seven introns and control areas of its flanking sequences. Gene mutation types of HB are various and common in point mutation, short deletion (less than30bp) and insertion,80%of which are single-base changes. Different from the HA, the sporadic rate of HB could reach30%-50%. The HB mutation database was established firstly in1990. New defects are frequently being discovered following with the accumulation of number of cases. According to the reported distribution of mutations, mutation can occur in all regions in the FIX gene including poly A signal area and is obviously heterogeneous.As a whole, except6cases with deletion (1patients with whole gene loss and5cases with short loss), the rest are taken place with point mutations and account for80.6%which reveal that point mutation is the most widespread. Secondly, the HB mutation database shows that the distribution of mutations in8exons is rather different.Because of encoding the EGF domain combined with Ca2+and catalytic domain, the mutation incidence in4th exon and8th exon is higher than others. Though there are only31patients in our study, mutations in a total of11cases occur at4th exon and8th exon with a proportion of35.5%. And mutations in lth exon,6th exon and7th exon are less and only take account for16.1%.CpG dinucleotide area is considered to be mutation hot spot, Liu Jingzhong etc also confirmed it by using PCR method and GAWTS technology while mainly transition is the C-T/A. Seven cases in the study are detected in the CpG hot spots with types of transitions are C-T, G-A and G-T, accounting for22.6%of all patients, and it demonstrates the CpG double nucleotide as mutation hot spot again.Toyozumi and other people have made sure that the192th (FIX192) in lth intron was a two nucleotide polymorphisms (SNPS) site, which exists in normal Japanese. Wang Ningsui etc discovered this site and calculated that gene frequencies of A and G of FIX-192is respectively0.81and0.19in Chinese people. In the study, mutations of four samples occur in FIX192, which further verify this polymorphism site in the same place of FIX gene.At the same time, the only point mutation in this polymorphism sit is discovered both in two cases of HB patients including all sequences of8exons and splice sites, while patients were diagnosed as HB, there is a reason to suspect that the pathogenic mutations could be in the intron areas which haven’t been sequenced. Therefor, it need to amplify the sequences covering the large fraction of7introns and3’un-translating area to confirm the gene mutated sites by DNA sequencing.Referring to the latest HB mutations database, this research has revealed that13kinds of mutations are new-found and internationally reported for the first time. Four kinds of these new mutations are missense mutations occurred in exons, with amino acid changes for L to Q, Y to D, S to P and V to G respectively. The protein structure changes due to amino acid changes can lead to the reduction or missing of protein function, and then cause coagulant function obstacle which lead to the occurrence of hemophilia. Among the deletion mutations, besides del T which has been reported as a deficiency resulting from the framework shift, other three types are new, among which both del GCTGAAACCA and GCTAAACA lead to reading frame shift happened in exons and del ATTT at splice site. Splice site is an important part between the exons. Mutations occurred in splice site can cause abnormal splicing and finally result the coagulation disorders while protein structure and function are abnormal during splicing. In addition, six species of base mutations as T6320A, C6828T, G6640A, A29753G, G30321T and T17517G take place at the sequence positions outside the exons. Matching with intron/exon splice sequences, it can be sure that6320th base belongs to splice sequences and T6320A must be pathogenic mutation. The remaining5mutations are not in splice sites and whether each of them is a new polymorphism site or a pathogenic mutation, we should do further research for them.As compared to the HA, HB is a rare disease, the crowd incidence is only about1/30000. And our country is a developing country, the understanding of the disease in people compared with the developed countries are a large gap, HB patients can’t get diagnosed and treated, patients get medical treatment is a very little number. Domestic research organization and person study in HB also belong to the minority group, and the related literature about the Chinese HB report is quite limited. Although international has established HB mutations database, the contained number of cases has a few thousand cases, and the gene mutations present diversity, and send out rate is high in HB. In view of the limited HB research resources, the FIX gene mutation analysis on31HB patients is meaningful and guiding to the mutation screening for carriers and prenatal genetic diagnosis. Enriching the mutations spectrum, the discovery of new FIX gene mutations is not only conducive to realize the importance of some amino acid residues of FIX gene to the FIX activity and clear and definite the pathogenic mechanism, but also provide scientific evidences for the later gene therapy.Conclusion1. CpG dinucleotide area is a mutation hot spot.2. In our country crowd, the192th (FIX192) in1th intron is a two nucleotide polymorphisms (SNPS) site.3. The mutations occurring in exons like point mutation, deletion and insertion may lead to amino acid change or termination, can be determined as the pathogenic mutations. Mutations in splice site are sure to be disease-causing mutations.4.The cases which have no other gene mutation except a point mutation at polymorphism site need to conduct DNA sequencing for detecting disease-causing mutation. Gene mutation in introns should be identified whether it is a pathogenic mutation or a polymorphism site, and also need to undertake DNA sequencing in sequences outside the exons. |
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