A study on gene mutation of coagulation factor Ⅺ protein secretion disorder and its mechanism / 中华检验医学杂志

Objective:To investigate the molecular pathogenesis of a newly discovered gene mutation in a family with hereditary coagulation factor Ⅺ(FⅪ) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in September 2021 due to "calculus of intrahepatic duct". The patient had no symptoms of spontaneous bleeding.The clinical data and blood samples of the proband and her family members (10 persons in 3 generations) were collected.The activated partial thromboplastin time (APTT) and FⅪ activity (FⅪ:C) were performed by the one-stage clotting assay. FⅪ antigen (FⅪ:Ag) were detected by enzyme linked immunosorbent assay (ELISA). Genomic DNA extracted from peripheral blood cells of subjects was used as template to analyze F11 gene mutation by DNA direct sequencing. Bioinformatics software was used to analyze the effects of mutations on protein structure and function. Wild-type and mutant FⅪ protein expression vectors were constructed and transient transfected into HEK293T cells. The total RNA was extracted from positive transfected cells and then reversely transcribed into cDNA. The mRNA expression level of F11 gene in transfected cells was detected by real-time fluorescence quantitative PCR (qRT-PCR). The content of FⅪ:Ag and the expression of FⅪ protein in transfected cell lysates and culture supernatant were detected by ELISA and western blot.Results:The APTT of the proband was significantly prolonged to 107.9s (reference range 29.0-43.0s), while FⅪ:C and FⅪ:Ag were significantly decreased to 2% (reference range 84%-122%) and 5% (reference range 76%-127%), respectively. Gene sequencing analysis indicated that the proband had c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation in exon 6 and 13 of the F11 gene, respectively. Bioinformatics analysis showed that the amino acids at site 161 of FⅪ protein were threonine (Thr) in the matrix composed of five different species, indicating that Thr161 site was highly conserved among homologous genes in different species. p.Thr161Met heterozygous mutation affected the stability of local intermolecular structure of FⅪ protein. In vitro expression experiments of p.Thr161Met mutation showed that FⅪ protein had a normal synthesis in the cells but secretion dysfunction.Conclusions:c.536C>T (p.Thr161Met) heterozygous missense mutation and c.1556G>A (p.Trp501Ter) heterozygous nonsense mutation were mainly responsible for the decrease of FⅪ in this family. p.Thr161Met mutation was first reported in the world and did not affect the normal synthesis of FⅪ protein, but caused secretion dysfunction.

Investigation on gene defects in a pedigree with inherited coagulation factor Ⅺ deficiency / 中华检验医学杂志

Objective To investigate the gene defects of a pedigree with inherited coagulation factor Ⅺ (FⅪ) deficiency by analyzing its phenotype and molecular genetic characteristics. Methods A pedigree with inherited FⅪ deficiency was enrolled in this study. The activated partial thromboplastin time (APTF), prothrombin time (PT), FⅪ activity (FⅪ: C) and FⅪ antigen (FⅪ: Ag) were determined for phenotype diagnosis. Fifteen exons and their flanks of F11 gene from the proband's genomic DNA were amplified by polymerase chain reaction (PCR), and the PCR products were directly sequenced to analyze the F11 gene mutation. The PCR products amplified from genomic DNA from the proband, her parents and 100 healthy donors were digested with restriction enzyme BssSI to exclude gene polymorphism and confirm the mutation site. The cleavage site in the signal peptide was predicted by the SignalP software. Results The values of APTT, PT, FⅪ: C and FⅪ: Ag of the proband were 69.5 s, 12.3 s, 2.6% and 2.5%, respectively, indicating that this case was cross-reacting material (CRM) negative. The same values of healthy controls were 35 s, 13 s, 100% and 100%, respectively. As compared with Genbank AY191837 sequence, four variants in F11 exons were found. G3733C heterozygous mutation in exon 2 causod Gly to Arg substitution at-1 amino acid position in signal peptide (G-1R). The G3733C mutation in exon 2 introduced a new BssSI enzyme digestion site. Further analysis of the 100 randomly collected DNA samples from the normal population excluded the possibility of G3733C as a polymorphism. CI6642T heterozygous mutation in exon 8 introduced a premature stop codon at 263 amino acid position (Q263Term). Conclusions G-1R mutation and Q263Term compound heterozygous mutation in F11 gene are the mechanism of FⅪ deficiency for the proband. G-1R mutation is a novel F11 gene mutation causing inherited FⅪ deficiency.

Mutation analysis of coagulation factor Ⅺ gene of inherited factor Ⅺ deficiency / 中华检验医学杂志

Ⅺ deficiency in Chinese Han population. Conclusion The 13 mutations of the F Ⅺ gene which were found in this study may unravel the molecular pathogenesis of the F Ⅺ deficiency in Chinese Han population.

Laboratory diagnosis of lupus anticoagulant associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ) deficiency:report of 13 cases / 中华检验医学杂志

Objective To explore laboratory diagnosis tests for lupus anticoagulant associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency.Methods We investigated the coagulation factors'activity,coagulation factors'antibodies,anticardiolipin antibodies,lupus anticoagulant and thrombin generation in thirteen patients of lupus anticoagulant associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency,ten hemophilia A patients, forty-eight healthy people. In these thirteen patients,there were five patients with schizophrenia and had been taking chlorderazin for a long time,two patients with antiphospholipid syndrome, one patient with pemphigus, five patients in which causes of disease were unknown.Among these patients,two patients has haemorrhage,eleven patients don't have reduced.The antibodies of these coagulation factors and LA were positive.Six patients in this group were ACA poslhve,and seven patients were ACA negative.The lag time of eleven non-haemorrage patients with LA positive associated by F Ⅷ,F Ⅸ and F Ⅺ deficiency was(5.60±1.18)min,higher than that of control group which was(1.88±0.25)min.This differenee had statistically significant(t=10.05,P＜0.01).The time to peak was(8.11±1.91)min,significantly higher than that of control group which was (4.10±0.36)min(t=8.83,P＜0.01).The endogenous thrombin potential(ETP)was(1 640.87±higher than that of hemophilia A patients which was(38.50±6.20)%(t=9.77,P＜0.01).The time to peak of hemophilia A patients was(8.01±1.81)min,significantly higher than that of control group which Was(4.10±0.36)min(t=8.20,P＜0.01).The lag time of control group and hemophilia A patients was (1.88±0.25)min and(2.01±0.84)min,respectively.There was no significant difierence of the lag time between these two groups(t=1.26,P＞0.05).Conclusions Diseases like lupus anticoagulant associated bv coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency and inherited hemophilia should use APTT,PT,should be used as confirmation tests. LA and ACA may serve as indication test for identifying the causes of these diseases. ETP and ETP percentage are quite good parametem for predicting the bleeding risk of patients with LA positive associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency.