Molecular Mechanism Of Protein C Deficiency Caused By Mutations PROC Gene N355S,G392E,T314A

Clinical characteristics and genetic mutation analysis of three patients with hereditary protein C deficiencyObjective:To carry out various coagulation tests and genetic diagnosis of three patients characterized by deep vein thrombosis.Methods:The peripheral blood of patients were collected.Bleeding and coagulation indexes,plasma anticoagulant protein activity,and the level of protein C（PC）were analyzed.Next-generation sequencing（NGS）technology（exome sequencing）was used to detect genetic mutations in patients.Bioinformatics software was used to analyze the function of mutation sites,and ClusterX-2.1-win multiple sequence alignment was used to analyze the conservation of amino acid mutation sites.Results:Protein C activity（PC:A）measured by the chromogenic substrate method in the three patients was significantly reduced,while the content of PC antigen（PC:Ag）was within the normal range,Therefore,these patients were diagnosed as type Ⅱ PC deficiency.Except for the obvious increase of D-D and FDP in patient Ⅰ,the other coagulation,anticoagulation and fibrinolysis function test indexes,such as PS:A,APTT,PT,TT,FDP,PLG:A,D-D,AT:A were normal.Mutations in PROC was detected by NGS sequencing,which indicated patients Ⅰ had c.1064 A>G led to ASN355SER（N355S）,Patient Ⅱ had c.1175 G>A causes GLY392GLU（G392E）,and Patient Ⅲ had c.940 A>G leads to THR314ALA（T314A）.Multiple sequence alignment suggests that the three amino acid sites are highly conserved in different species.Conclusion:Three patients with deep vein thrombosis were diagnosed as hereditary protein C deficiency based on coagulation tests and NGS exon sequencing.The three mutation sites are closely related to the occurrence of venous thrombosis in patients,and they are new mutations that have not been reported at home and abroad.These three amino acid sites are highly conserved between species,and amino acid substitution may have a profound impact on the structure and function of proteins.Molecular Mechanism of Protein C Deficiency Caused by Mutations of PROC Gene N355S,G392E,T314AObjective:To determine the molecular mechanism of functional defect of protein C（PC）caused by point mutations of human protein C gene（PROC）N355S,G392E and T314A.Methods:The wild-type（WT）and mutant plasmids(PC_WT,PC_N355S,PC_G392E,PC_T314A)of PROC gene were constructed and transiently transfected into HEK293 cells.The expression of mutant proteins in vitro were tested.The mRNA level changes of WT and mutant PC after 24 h of transfection were detected by real-time PCR.Western blot and ELISA were used to detect the changes of intracellular and extracellular protein levels of WT and mutant PC.The supernatant of cells transfected for 24-48 h was concentrated by ultrafiltration.The protein in the concentrated solution was quantified,and PC activation and enzyme kinetics tests were performed.The effect of mutation on PC protein structure was analyzed by Pymol software.Results:The plasmids were identified by enzyme digestion and sequenced to confirm the correctness of the sequence.They were named PC_WT,PC_N355S,PC_G392E and PC_T314A respectively.The relative expression abundance of PROC mRNA in PC_N355S,PC_G392E and PC_T314A plasmid transfected groups was 1.14 ±0.46,0.96 ± 0.08,and 1.08 ± 0.17,respectively.There was no significant difference compared with that in PC_WT plasmid transfected group（1.02 ± 0.24）（P>0.05）.Western blot analysis of the lysates of transfected cells showed that the content of PC_T314A recombinant protein was slightly lower and the bands were relatively lighter.The ELISA results of the concentrated cell culture supernatants showed that the PC:Ag levels of PC_N355S and PC_G392E were 98.8%± 2.4%and 101.4%±3.1%,respectively,which had no difference compared with PC_WT,while PC_T314A decreased compared with PC_WT（PC:Ag:88.6%±3.2%）（P<0.05）.The results of enzyme kinetic test showed that APCN355s(K_m=338.3 ± 43.2,V_max=2.015 ±0.12),APC_G392E(K_m = 292.2 ± 28.4,V_max=1.893 ± 0.07)and APC_T314A(K_m=299.5 ±24.6,V_max=1.775 ± 0.06)showed an increase in K_m and a decrease in V_max compared with APC_WT(K_m=238.2 ± 4.58,V_max=3.205 ± 0.06),suggesting that these mutation affects the ability of activated PC to cleave substrates.The structural model suggests that the amino acid substitutions of N355S,G392E and T314A mutations collide with the surrounding amino acid groups and distort the surrounding structure,which may have adverse effects on the folding and biological function of PC.Conclusion:The N355S,G392E and T314A mutations in the PROC gene cause functional defects in PC by weakening the binding between PC and substrate.These three mutations have caused serious spatial collisions in the protein structure,affecting the folding of PC and the reactivity of active sites.