Gene And Characterization Analysis Of A Family With Hereditary Blood Coagulation Factor ?

Objective: Hereditary blood coagulation factor ? deficiency is caused by the defect of factor ?(F?)function originated from congenital F7 gene mutations,which impacts the extrinsic coagulation pathway at the initial stage with clinical symptoms of hemorrhage.It is an autosomal recessive genetic disease and the propositus' parents are often consanguineous marriage.Hereditary blood coagulation factor ? deficiency is characterized by bleeding tendency,prolonged prothrombin time,normal activated thromboplastin time and decreased factor ? activity.In general,the patients with homozygous mutations display significantly decreased factor ? activity and heavy bleeding manifestation,and are attacked in their new born infancy.The incidence rate of this disease is very low,at about 1 per 500,000,without gender difference.Alexander et al.first reported in 1951 that about 18% of parents of children patients are consanguineous marriage.The gene coding F? protein is located on chromosome 13 q34,close to the upstream 2.8 kb of the coagulation factor X,with 1,2850 bp in length and 3141 bp mRNA.The cDNA is 2462 bp,which is made up of 9 exons and 8 introns.F? is one of blood coagulation factors dependent on vitamin K and belongs to a single sugar protease composed of 406 amino acid residues.F?is synthesized in the liver and the kidney,but stored in the kidney.Circulatory fibrinogen and its degradation products can stimulate F? synthesis.The molecular weight of F? protein is 4,5078 Dalton,with gamma carboxyglutamic acid residues as the N terminal,glutamic acid residue as C terminal.The molecule of F? is the serine protease domain structure including gamma carboxyglutamic acid residues,two epidermal growth factor(EGF)and a catalytic domain from the N terminal.According to F? activity and antigen level,hereditary F? defects can be divided into three categories:negative cross reaction substances(cross reacting material,CRM),positive cross reaction material(CRM)and cross reaction material(CRM).The clinical manifestations of the disease vary.Some may have severe hemorrhage,even intracranial hemorrhage,and some may not have hemorrhage.The varieties of F7 gene mutation has no relationship with F? activity,antigen level and clinical hemorrhage.Currently,there are 283 F? relevant mutations in the human gene mutation database(February 2014).Mutation types include missense,promoter,shear loci,nonsense,small insertions and deletions of mutations,in which there are 70% missense mutation,10% deletions,9% splice site mutation,6% promoter mutant.The update of F7 gene database and the findings of new pedigrees have important significance for the diagnosis,gene therapy and prognosis of this hereditary disease.This study examines one female patient with hereditary FVII deficiency and her family members,observes the gene mutation and clinical phenotype,and to investigate the molecular mechanism of the dysfunction.Methods:1 Genealogy and clinical data: The proband is a female at the age of 20.For tonsil operation,prolonged PT has been found in coagulation indicators examination.In routine physical examination there is no mucocutaneous bleeding with no abnormality of heart,lung and abdomen.She didn't display obvious bleeding tendency.In laboratory examination,blood routine,liver function,five items of hepatitis B are normal.As the family members,her patients has no bleeding tendency such as skin bleeding,bleeding gums and trauma.And her parents is not consanguineous marriage.2 Specimen: With the consent of the proband,her patients,their peripheral venous blood samples were collected,with 0.109 mol/L sodium citrate by 1:9 anticoagulant.After centrifugaltion of 3000 r/min,separation of blood cells and plasma were made and keep it in-80 ?.The plasma was used for blood coagulation index and the blood cells were used for genomic DNA extraction and PCR amplification.3 Plasma coagulation markers detection: prothrombin time(PT),activated partial thromoploastin time(APTT),fibrinogen(Fg)and blood coagulation factor activity including F?,F?,F?,F? activity were tested.4 Primers design: There are 12 pairs of PCR and sequencing primers.According to the F? gene sequences(Genbank J02933),12 pair of primers were designed with Primer 5 software to cover respectively the exons and flanks,5' and 3' non translation region sequencings of F7 gene.And exon 8was adopted segmented amplification method.Primer sequences is composed by Sangon Biological Engineering(Shanghai)Co.,LTD.Amplification products were identificated by 1.2% agarose gel electrophoresis.5 PCR amplification: 50 ul PCR reaction system includes 1 ul DNT templates,0.5 ul PCR primers of upstream and downstream,0.5 ul 25 mmol/L MgCl2,1 ul deoxyribose nucleotides(dNTPs),1.5 ul Taq DNA polymerase,0.5 ul tap enzyme and 35.5 ul water.PCR reaction is carried out in the Verity96 well produced by ABI in the United States.Reaction conditions:degeneration was for 3 minutes.According to the different primers they have the 55?~60? annealing 35 s,72 ? extension 40-50 s,72 ? repair extension5-8 min,with a total of 35 cycles.The PCR products are observed by 1%agarose electrophoresis under 150 v,100 mA for 20 min.6 Purification and sequencing analysis of PCR products: PCR products after tapping are sent to Sangon Biological Engineering(Shanghai)Co.LTD.to measure sequencing after purification with double DNA chain termination method on the 3730 xl(ABI)sequencing machine.By comparing the sequence results with the J02933 sequence published by the national biological information center in the USA,the gene mutations were seeked.To confirm the found gene mutation,the reverse sequence were sequencing with Chromas software.7.Construction and analysis of molecular models: To predict the function of the signal peptide sequence through on line database http://www.cbs.dtu.dk/services/SignalP/ to infer its influence on the function of the FVII protein.Results:1 The coagulation index detection: Compared with the normal people,the proband's PT value is significantly prolonged and F?: C is significantly decreased by 1.1%,with normal APTT value and normal activities of factor?,factor? and factor?.Other family members have normal PT and APTT,with normal F?: C,and other blood coagulation factor activities.the proband's prolonged PT could be totally corrected by normal plasma,which showed that there was inhibitors to FVIIa in her plasama.2 Sequencing results: Comparing F? gene mutations of the patients and their family members with the published J02933 sequence of the NCBI gene pool as a standard,(1)heterozygous 556 th nucleotide mutations T/G occurred in the proband's and his father's exon lA of F7 gene,with codon CTG turning into CGG,corresponding leucine(L)into arginine(A),Leu556 Arg,while the her mother didn't have this mutation.(2)8401th nucleotide mutation C/T occurred in the proband's and his father's exon 5 of F7 gene,with CAC turning into a CAT,but the corresponding amino acids are the histidine(H),synonymous mutations,while her mother still didn't have this mutation.So this is polymorphism.(3)11814th nucleotide insertion mutation occurred in 3'the translation section of the proband' F7 gene,inserting in two As,while her parents didn't have this mutation.(4)12432th nucleotide G/A mutation occurred in the 3' the translation section,which has the effect on the termination of translation and the final exon splicing.The proband and his father has this mutation,while the mother didn't have.3 Prediction of mutation on protein function: As a result of 556 th nucleotide heterozygous mutation of the proband's F7 gene,amino acid here takes place of the change of L12 R,which is just located in the hydrophobic region of the F? protein signal peptide.Through general and specific changes of signal peptide region before and after the F? protein mutations,we find that the change of L12 R may leads to segmentation of the different sections of the signal peptide region,and has a certain influence on the normal function of the signal peptide region,then maybe result in reduction of the ability for F?protein initially translated products to enter into the endoplasmic reticulum(ER),and reduction of the mature proteins and F? activity.Conclusions:1 Homozygous mutations or double heterozygotes have varying degrees of hemorrhage,and simple heterozygous almost has no clinical symptoms.2 First report of a case with L12 R mutation of F7 gene: Heterozygous556 th nucleotide mutations T/G occurred in exon lA of the proband's F7 gene,which leads to L12 R amino acid changes in the signal peptide region.Function prediction finds such mutation may have some impact on segmentation of the different areas of signal peptide region,which affects the normal function of the signal peptide region.And it eventually results in the ability of the initially translated products of FVII protein to enter into the endoplasmic reticulum decreased.And then it brings about the decreases of the mature FVII protein and the reduction of the activity..