| The genetic analysis of a Chinese Pedigree with Combined F? and F? deficiencyObjectives:Combined factor ?(FV)and factor ?(F?)deficiency(F5F8D)is a rare bleeding disease in which most patients have spontaneous bleeding symptoms.We aimed to investigate the genetic changes in a Chinese F5F8D pedigree.Methods:The proband was a 30-year-old female patient with symptoms of menorrhagia and easy bruise.Her parents with no bleeding tendency were consanguineous marriage.The plasma coagulation activity and coagulation function of the proband and her parents were detected with coagulation assay.Exons and flanking sequences of LMAN1,MCFD2,F5 and F8 genes were amplified using polymerase chain reaction,the products sequenced directly.The results were checked through reverse sequencing,and the corresponding sites of the parents were analyzed.The effects of mutation on the structure and function of protein were analisied by softwares of Clustal-X 2.1,Polyphen2,SIFT,Mutation Teasers,and PyMOL.Result:The proband was diagnosed as F5F8D,based on the tests that her plasma FV:C,F?;C,PT,APTT and vWF:Ag were 9.9%,5.9%,17.7s,75s and 101.0%respectively,with corrected PT and APTT 13.5s and 31.3s respectively.The coagulation tests of her parents were within the normal range.A novel homozygous mutation c.398A>T(leading to p.D 133V)in exon4 of MCFD2 gene and a heterozygous mutation c.5012G>A(resulting to p.R1671H)in exon14 of F8 was detected in the proband,both of her parents had heterozygous mutation(c.398A>T),the mother also had a heterozygous mutation(c.5012G>A).The results showed the disorder was closedly related to c.398A>T mutation,the damage of p.R1671H mutation was undefined.However,the c.5012G>A mutation was reported to be the molecular pathogenesis of a severe hemophiliac,meaning that the mutation could propably decrease F ? level in plasma.Conclusion:Combined FV and F? deficiency is a rare bleeding disorder with coagulation factor V and VIII level decrease at the same time,we tried to detect four related gene and finally found that MCFD2 gene mutation c.398A>T accounted for the disease and leaded to bleeding complications,F8 mutation may account for the low level of coagulation factor VIII.To the best of our knowledge,the novel MCFD2 mutation c.398A>T was reported for the first time in the literature.More study need to be done to conform the mechanism of how the mutation disrupt the synthesis and secretion of FV and F?.The genetic analysis of coagulation factor XI(FXI)deficiency.Objective:To investigate the molecular pathogenesis of two coagulation factor XI(FXI)deficiency patients.Method:The diagnosis was validated by coagulant assays:APTT and correct test,PT,INR and coagulation factors activities.The patients' DNA were extracted and all exons and flanking sequences of FXI gene were amplified using PCR.After purified,the products were sent for sequencing directly,the mutations were detected by comparing with wild sequences and analysed using some bioinformatics software.Results:The two patients were diagnosed coagulation factor XI deficiency due to prolonged APTT and low activities of coagulation factor FXI,the results of APTT,FXI:C were 88.1s,1.1%and 107.1s,3.8%respectively.Genetic analysis discovered double heterozygous mutations g.1251-1G>A and g.1271de1T in the first patient and the sequencing results of TA plasmid clones showed that the two mutations are located on different single strands of chromosomes.Double heterozygous mutations g.1070A>G and g.1446C>G were discovered in the second patient resulting in Lys357Arg and Cys482Stop.Software analysis indicated the mutations probably brought amino acid sequence changed,protein features affected and splice site changed.Conclusion:Double heterozygous mutations g.1251-1G>A,g.1271 delT and g.1070A>G,g.1446C>G had been identified in two coagulation factor ? deficiency patients which might be the cause of their prolonged APTT and low F?:C.To the best of our knowledge,the novel four mutations are reported for the first time in the literature. |
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