The Study On The Clinical And Molecular Pathogenesis Of Hereditary Coagulation Factor Ⅺ Deficiency

Objective:Hereditary coagulation factor XI deficiency(FXID)is a rare hereditary hemorrhagic disease,also known as hemophilia C,which is an autosomal incomplete recessive genetic disease.It can occur in both men and women,and is mainly caused by the number or functional defect of coagulation factor XI caused by F11 gene mutation.The prevalence in the general population is estimated to be 1/10~6,which is more prevalent in the Ashkenazi and the Iraqi Jewish population.Most patients with FXI deficiency have no bleeding symptoms or have mild to moderate bleeding,and most of them show injury related bleeding.Especially for female patients,in addition to increased menstruation,they also show excessive bleeding after abortion or termination of pregnancy,postpartum hemorrhage and gynecological surgery related bleeding.The clinical bleeding phenotype has no significant correlation with FXI activity level and genotype,and it is difficult to predict the risk of bleeding after trauma or surgery.Therefore,some individuals with FXI deficiency may receive unnecessary replacement therapy in the perioperative period.Some replacement therapies,such as FXI concentrate and recombinant FVIIa,have been proven to increase the risk of thrombosis.In addition,transfusion of fresh frozen plasma FFP may also have the risk of transfusion related complications.On the contrary,untreated patients with FXI deficiency may increase the risk of bleeding and even endanger their lives during the perioperative period,bringing serious difficulties to clinicians and patients.Therefore,for individuals whose FXI activity is lower than normal range,it is important to make a clear clinical diagnosis and identify patients with bleeding tendency before trauma or surgery.Genetic diagnosis is of great significance to confirm the clinical diagnosis of hereditary FXI deficiency,and is helpful to the clinical management and genetic counseling of patients.At present,403 gene mutations have been reported in human gene mutation database and EAHAD factor XI(F11)gene mutation database,including point mutation,deletion,duplication,insertion and other mutation types,mainly missense mutation and nonsense mutation.This study analyzed the clinical manifestations,laboratory examinations and gene mutations of patients with hereditary factor XI deficiency,and explored its possible molecular pathogenesis through bioinformatics software,so as to summarize the clinical characteristics of patients with hereditary factor XI deficiency and provide a basis for clinical management and genetic counseling;At the same time,it is conducive to finding more new gene variations and supplementing the F11 gene mutation datas.In order to further study the specific molecular pathogenesis of hereditary coagulation factor XI deficiency,we also used CRISPR/Cas9 gene editing technology and homologous recombination repair(HDR)to construct a F11 site-directed mutagenesis cell line,which provides a research basis for further exploring the molecular pathogenesis of hereditary coagulation factor XI deficiency.Methods:1.The study on clinical manifestations,laboratory examinations and gene mutations of patients with hereditary factor XI deficiency(1)The clinical information of patients with hereditary factor XI deficiency were collected by telephone follow-up and consulting outpatient or inpatient medical records.(2)Coagulation test and coagulation factor activity was performed on Stago automatic blood coagulation instrument by clotting method.(3)Coagulation factor XI antigen was detected by enzyme-linked immunosorbent assay.(4)The presence of inhibitor in patients’plasma was detected by normal pooled plasma with abnormal plasma in a 1:1 ratio.(5)F11 gene mutation was detected by PCR amplification combined with Sanger direct sequencing.The sequencing results were analyzed by Chromas software and NCBI BLAST software.The suspected mutation was compared with the database,except SNP,to confirm F11 gene mutation.(6)The novel heterozygous variants were confirmed by T/A clone.2.Pedigree analysis of patients with novel gene mutations and preliminary discussion on pathogenesis of novel mutations(1)APTT,FXI activity and FXI antigen were detected by clotting method and enzyme-linked immunosorbent assay.(2)Clusterx-2.1-win software was used to analyze the amino acid conservatism of mutation sites for novel missense mutations.(3)Five online bioinformatic softwares(Poly Phen-2,Mutation taster,Fathmm,PROVIDEAN and SIFT)were used to analyze the pathogenicity of novel missense mutations.Mutation taster software was used to analyze the pathogenicity of deletion mutation.(4)Swiss-Model was used for homologous modeling,and Pymol software was used to analyze the protein molecular structure in mutant and wildtype protein of the novel mutations,so as to explore the possible molecular pathogenesis of the novel mutations.3.Construction of F11c.A1159G(p.Arg387Gly)site-directed mutagenesis cell line by CRISPR/Cas9 gene editing technology(1)Construction of px330-Sg RNA plasmid:Sg RNA was designed and synthesized on ZHANG LAB website.The restriction endonuclease Bpi I digests the px330 plasmid,the digested product is recovered on gel and ligated with annealed Sg RNA using T4 DNA ligase.The linked product was transformed,and the plasmid DNA was extracted.(2)Construction of LA-p MD-18T-p GK-puro-mcherry-RA donor plasmid:According to the position of sg RNA and point mutation,the primers of left and right homologous arms were designed.The DNA of patients with point mutation was used as a template to PCR amplify the left and right homologous sequences.The PCR product was recovered and purified by gel.The PCR products and p MD-18T-p GK-puro-mcherry plasmids were digested by restriction enzymes respectively.The digested products were recovered by gel and purified,and then linked with T4 DNA ligase.The linked products were transformed,and the plasmid DNA was extracted.(3)Establishment of F11c.A1159G(p.Arg387Gly)site-directed mutagenesis cell line:p X330-Sg RNA plasmid and LA-p MD-18T-p GK-puro-mcherry-RA donor plasmid were co-transfected into Hep G2 cells using lipofectamine 2000 transfection reagent.After drug resistance screening,clones were selected for culture.Collect cells,extract genomic DNA,identify positive clones by PCR amplification and Sanger sequencing,expand the culture after single cloning,and obtain mutant stable cell line after identification.Results:1.The study on clinical manifestations,laboratory examinations and gene mutations of patients with hereditary factor XI deficiency(1)Clinical manifestations and laboratory examinations of patients with FXI deficiencyAmong 17 unrelated patients,8 were male and 9 were female,with a median age of57(11-81)years.In 17 patients,APTT was significantly prolonged and FXI:C was reduced,with median APTT was 73.8(43.9-85.2)S and median FXI:C was 1.8(0.2-31.3)%.The FXI antigen was also decreased in all patients.Prolonged APTT could be corrected by normal pooled plasma,and the activity of other coagulation factors(FⅡ,FⅤ,FⅦ,FⅧ,FⅨ,FX,FXII)were normal.Among them,there were 15 cases of severe FXI deficiency(FXI:C<20%)and 2 cases of partial FXI deficiency(FXI:C20%～60%).The main reason for presentation in 17 patients was that the prolonged APTT found in preoperative examination.10 patients had no bleeding symptoms,and 7 patients had mild to moderate bleeding,with the main clinical manifestations being gingival bleeding,oral bleeding,skin ecchymosis,nosebleed,hematuria,bleeding after tooth extraction,increased menstruation,bleeding after abortion,bleeding after ligation and bleeding after trauma.17 patients were evaluated with ISTH-BAT bleeding score,and the ISTH-BAT median bleeding score was 0(0-4).11 patients underwent surgery,4 patients had no bleeding,4 patients received fresh frozen plasma(FFP)for prevention before surgery,1 patient received FFP after surgery,1patient needed surgical treatment for bleeding after ligation,and 1 patient needed hemostatic drugs for bleeding after tooth extraction.9 female patients had 22 pregnancies and 16 vaginal deliveries without bleeding symptoms.There were 6 miscarriages,among which 1 case had a large amount of bleeding after abortion and needed to be treated with oral hemostatic drugs.1 patient had increased menstruation.(2)Analysis of gene mutation in patients with FXI deficiencyIn the 17 patients,15 patients were complex heterozygous mutation,1 patient was homozygous mutation,and 1 patient was heterozygous mutation.A total of 21 different mutations were detected,including 12 missense mutations(12/21,57.1%),7 nonsense mutations(7/21,33.3%),1 splice mutation(1/21,4.8%),and 1 deletion mutation(1/21,4.8%),mainly missense and nonsense mutations.The frequency of nonsense mutation p.Arg54*was 15.6%(5/32),followed by missense mutation p.Gly400Val(4/32,12.5%),nonsense mutation p.Gln263*(3/32,9.4%),missense mutation p.Cys482Trp(2/32,6.3%)and nonsense mutation p.Trp501*(2/32,6.3%).Out of 21mutations,9 were located in the catalytic domain of FXI protein,5 in the Apple4 domain,2 in the Apple1 domain,2 in the Apple3 domain,2 in the signal peptide domain and 1 in the Apple2 domain,and the mutations were mainly distributed in the catalytic domain(9/21,42.3%)and the Apple4 domain(5/21,23.8%).Of the 21 mutations,15 were reported and 6 were first reported at home and abroad.Among the 6 novel mutations,4 missense mutations are p.Pro338Leu、p.Cys38Tyr、p.Cys542Tyr、p.Arg369Gly,1 deletion mutation c.1048del C and 1 nonsense mutation p.Gly336*。2.Pedigree analysis of patients with novel gene mutations and preliminary discussion on pathogenesis of novel mutations(1)Laboratory examinations and gene mutations in family members of novel mutationsLaboratory examinations and gene mutation analysis were performed in family members of 4 novel mutations(p.Pro338Leu,p.Cys542Tyr,c.1048del C,p.Arg369Gly).The APTT of the four probands was significantly prolonged,their FXI activity and antigen level were significantly reduced,and the FXI activity and antigen level in their heterozygous mutation members were also lower than the normal range.In addition to a novel mutation,the four probands were combined with a different mutation that had been reported,suggesting that they were complex heterozygous mutation.Because the first three probands with novel mutations were older at the time of diagnosis and their parents were all dead,we could not analyze their parents.We speculated that the two mutations of the probands might be inherited from their parents respectively.The fourth proband was a complex heterozygote with a novel missense mutation p.Arg369Gly and a known missense mutation p.Gly400Val,whose father was a heterozygote with mutation p.Gly400Val,and whose mother was also a heterozygote with p.Arg369Gly.It indicated that the mutation p.Gly400Val of proband was inherited from the father and the novel mutation p.Arg369Gly was inherited from the mother.(2)Comparison of activated partial thromboplastin time(APTT)and FXI activity in patients with different mutation typesPatients with FXI deficiency and their family members were divided into complex heterozygous and homozygous mutation group and heterozygous mutation group.APTT value and FXI activity level between the two groups were compared.The APTT of complex heterozygous and homozygous mutation group was higher than that of heterozygous mutation group,and the FXI:C in the complex heterozygous and homozygous mutation group was lower than that in the heterozygous mutation group,with statistical significance(P<0.0001).(3)Bioinformatics analysis of novel gene mutations(1)Clusterx-2.1-win software was used to compare the amino acid sequences of 4 novel missense mutations(p.Pro338Leu,p.Cys38Tyr,p.Cys542Tyr,p.Arg369Gly)between humans and other homologous species(Mus musculus,Rattus norvegicus,chimpanzees,macaques,dogs,cattle,pheasants,tropical Xenopus).The results showed that Pro338,Cys38,Cys542,Arg369 were highly conservative between humans and homologous species.(2)The online bioinformatic softwares were used to predict the pathogenicity of 4 novel missense mutations(p.Pro338Leu,p.Cys38Tyr,p.Cys542Tyr,p.Arg369Gly)and 1deletion mutation c.1048del C.The results suggested that these mutations might be pathogenic mutations.(3)Swiss-Model software was used for homologous modeling,and Pymol software was used to analyze the protein molecular structure in mutant and wildtype protein of the novel mutation.The results showed that the novel missense mutations(p.Pro338Leu,p.Cys38Tyr,p.Cys542Tyr,p.Arg369Gly)not only changed its local structure,but also changed its interaction with other molecules.The novel deletion mutation c.1048del C,located in exon 10 of the F11 gene,suffered a frame shift mutation p.Leu332Phe*22 due to the deletion of base C at position 1048,forming a truncated protein of 352 amino acids in length,with 21 amino acids at the end also replaced,resulting in the absence of a small portion of Apple4 and the entire catalytic structural domain at the C-terminus,thus affecting the the structure of the FXI protein.p.Gly336*was a novel nonsense mutation with a premature stop codon at amino acid position 336,which maked the amino acid after 335 completely lost and produced a truncated protein,leading to the structural change of FXI protein.3.Construction of F11c.A1159G(p.Arg387Gly)site-directed mutagenesis cell line by CRISPR/Cas9 gene editing technology(1)The p X330-Sg RNA plasmid and LA-p MD-18T-p GK-puro-mcherry-RA donor plasmid were successfully constructed,and the sequencing results showed that they were correct.(2)The stable cell line model of homozygous mutation F11C.A1159G(p.Arg387Gly)was successfully screened.The sequencing map showed that base 1159 in exon 11 of F11gene was mutated from A to G.Conclusion:1.The clinical and laboratory characteristics of 17 unrelated patients with FXI deficiency were summarized and analyzed.Most patients had no bleeding symptoms,some patients had mild to moderate bleeding.The haemorrhagic phenotype of patients with FXI deficiency is heterogeneous,and has no significant correlation with the level of FXI activity and genotype.2.17 patients with FXI deficiency were analyzed for gene mutation,15 of them were complex heterozygous mutations,1 was homozygous mutation and 1 was heterozygous mutation.3.A total of 21 different mutations were detected in 17 patients,mainly missense mutations and nonsense mutations.15 were reported mutations and 6 were novel mutations,which enriched the F11 gene mutation database.4.Through gene mutation analysis in family members of novel mutation patients,pedigree maps of novel mutation patients were formulated.5.Bioinformatics analysis showed that the novel mutation sites had important biological characteristics and played an important role in FXI protein.6.The F11 site-directed mutagenesis cell model was successfully constructed by CRISPR/CAS9 gene editing technology combined with homologous recombination repair(HDR),which laid a foundation for further research on the molecular pathogenesis of hereditary factor XI deficiency.