Study On The Molecular Pathogenesis Of Protein C Deficiency Caused By PROC Gly74Ser Mutation And Haemophilia A Caused By Aberrant Intron 22 Inversion

In this study,we performed investigation on the clinical phenotype,genotype and pedigree study in a pedigree with inherited protein C deficiency and a pedigree with haemophilia A caused by aberrant intron 22 inversion.Further protein expression and kinetics analysis were performed to clarify the molecular pathogenesis of the PROC Gly74 Ser mutation.Protein C is a vitamin K-dependent serine protease zymogen in plasma which upon activation by thrombin in complex with thrombomodulin(TM)down-regulates the clotting cascade.Activated protein C(APC)exerts its anticoagulant function through protein S-dependent degradation of factors Va and VIIIa.We recently identified a venous thrombosis patient whose plasma level of protein C antigen is normal,but its anticoagulant activity is only 34% of the normal level.Genetic analysis revealed that the proband carry a novel heterozygous mutation c.346G>A,p.Gly74Ser(G74S)in PROC.Thrombin generation assay indicated that the anticoagulant activity of the proband’s plasma has been significantly impaired.We expressed protein C-G74 S in mammalian cells and characterized its properties in established coagulation assays.We demonstrate that the protein C variant can be normally activated by the thrombin-TM complex and the resulting APC mutant also exhibits normal amidolytic and proteolytic activities toward both FVa and FVIIIa in the absence of protein S.However,it was discovered the protein S-dependent catalytic activity of APC variant toward both procoagulant cofactors has been significantly impaired.Protein S concentration-dependence of FVa degradation revealed that the capacity of APC variant to interact with the cofactor has been markedly impaired.The same results were obtained for inactivation of FVa-Leiden suggesting that the protein S-dependent activity of APC variant toward cleavage of Arg-306 site has been adversely affected.These results provide insight into the mechanism through which G74 S substitution in APC causes thrombosis in the proband carrying this mutation.Haemophilia A(HA)is a common X-linked recessive bleeding disorder and almost one half of patients with severe HA are caused by intron 22 inversion(Inv22)in the F8.Inv22 is constituted by 4 types of non-allelic homologous recombination(NAHR)between one intragenic homologous region(int22h-1)and two extragenic homologous regions(int22h-2 orint22h-3),including type I(involving int22h-1 and int22h-3),type II(involving int22h-1 and int22h-2),int22h-mediated deletions(Del22)and int22h-mediated duplications(Dup22).The aberrant band patterns of Inv22 refer to the non-canonical rearrangements mediated by int22 h other than genetic variants described above.Inv22 is considered to be almost exclusively of meiotic originin germ cells during spermatogenesis and only one mosaic Inv22 female carrier with the mutation possibly occurring during mitosis of the embryo has been reported so far.We identified a novel complex recombination mediated by int22 h copies in a sporadic severe HA pedigree by AQ-PLP and located the sequences flanking the breakpoint region using genome walking technique,AccuCopy technique,gene chip and real-time PCR.The disease causing genetic variant registered an 18.1 kb deletion including part of int22h-1 through the intron 23 of F8 and a 113.3 kb duplication of part of int22h-2 through the intron 1 of TMLHE inserted in the religated region of the F8.Two intrinsically linked mechanisms of recombination-dependent DNA replication: microhomology-mediated break-induced replication(MMBIR)followed by break-induced replication(BIR)might be responsible for the incident of the complex recombination during early embryogenesis of the proband’s mother.