Study On Congenital Coagulation Factor ? Deficiency Caused By Compound Heterozygous Mutations Of P.Thr241Asn And C.681+1G>T

Background and ObjectiveCongenital factor ??F??deficiency,caused by mutation of F? gene?F7?,is a rare bleeding disorder.It shows autosomal recessive inheritance.Patients with congenital F? deficiency are classified,depending on the manifestations,into major bleeders,minor bleeders and asymptomatic subjects.The results of routine coagulation tests of patients with congenital F? deficiency manifest as a prolonged prothrombin time?PT?,elevated international normalized ratio?INR?,reduced F? coagulant activity?F?:C?and/or F? antigen?F?:Ag?and nomal activated partial thromboplastin time?APTT?.Previous studies had showed that there was a weak or no correlation between F?:C,genotype and clinical bleeding symptom.These conditions may indicate that the pathogenic mechanism of this disease is relatively complex,leading to misdiagnosis and miss-diagnosis clinically,and probably resulting in increased bleeding risk or bleeding complication in this part of patients in surgical process.A pedigree with congenital F? deficiency was collected,with the purpose of seeking the type of F7 genotype that caused the disease,analyzing correlation among F?:C level,genotype and bleeding symptom,and preliminarily discussing the molecular pathogenic mechanism resulted from gene mutation.Thromboelastography?TEG?was used to assess the bleeding risk of the congenital F? deficiency.MethodsClinical data of a proband with congenital F? deficiency and his family members were collected.The coagulation tests and activity of coagulation factors were performed by blood samples.The TEG was used to evaluate the process of clot formation and fibrinolysis in whole blood sapmle.Then high-throughput sequencing was performed in samples of proband and his family members.After discovered mutation sites of target gene,primer pairs for the target gene was designed by Primer Premier 3.0 software.Mutation sites were confirmed by a generation sequencing,and the results were aligned by utilizing Chromas Lite 2.01 software and genomic reference sequence?NG₀09262.1?published by genbank of NCBI.The conservation of amino acid of human and homologous species was aligned with application of ClustalX2.1 software.The conformational analysis of the corresponding mutational amino acid in F7 protein was carried out by Swiss-PdbViewer 4.10 and PyMOL2.2.0 software.The prediction of pathogenicity in mutational amino acid was made by means of five bioinformatics tools online.ResultsThe results of coagulation tests and activity of coagulation factors in proband showed prolonged PT,increased INR,reduced prothrombin activity?PTA?,markedly decreased F?:C.The mixing study demonstrated that the prolonged PT in proband is completely corrected by the addition of the normal pool plasma.Moreover,these results indicated that F?:C and F?I:C were mildly low in proband's father,F?:C was gently reduced in proband's mother,F?I:C was slightly decreased in proband's sister.TEG displayed the results that kinetics time?K?was slightly increased,Angle,maximum amplitude?MA?and comprehensive index?CI?were declined in proband;K was mildly elevated,Angle,MA and CI were a little reduced in proband's father;K was slightly rised in proband's sister.Sequencing showed that there were two heterozygous mutations in proband,missense mutation c.722C>A?p.Thr241Asn?in exon 8 of F7 and splice site mutation c.681+1G>T in intron 7 of F7,only one heterozygous missense mutation Thr241 Asn in proband's father,and only one heterozygous splice site mutation c.681+1G>T in proband's mother,no mutation in proband's sister.Compared the conservation of amino acid of human with homologous species,Thr241 was less conservative.Modeling of mutational amino acid in F7 protein showed that there was no increased or reduced hydrogen bond between Asn241 and Val249 or other amino acids,after wild-type Thr241 mutated to Asn241.However,Thr241 Asn resulted in obvious fold and spatial conformational change of the peptide chain that consisted of amino acids at position 177¹89.Prediction of five bioinformatics tools online indicated that the missense mutation of Thr241 Asn was possibly deleterious or influenced the function of F7 protein.Conclusion1.The compound heterozygous mutations of c.722C>A?p.Thr241Asn?and c.681+1G>T in F7 gene is the molecular pathogenic mechanism leading to congenital factor ? deficiency.It is speculated that the compound heterozygous mutations change the molecular spatial configuration of the protein,thus affecting the function of F? protein.2.The level of F?:C is probably not related to bleeding symptom in patients with congenital factor ? deficiency.3.Thromboelastography can assess the bleeding risk of the congenital F? deficiency.