OBJECTIVE: To analyze a Chinese pedigree with Hereditary coagulation factor Ⅻ (FⅫ) deficiency duo to variants of F12 gene and explore its molecular pathogenesis. METHODS: A patient who underwent laparoscopic cystectomy at the Department of Gynecology of the First Affiliated Hospital of Wenzhou Medical University in June 2012 was selected as the study subject. Coagulation factor indexes of the proband and her family members (5 individuals from three generations) were determined. All exons, flanking sequences, 5' and 3' untranslated regions of the F12 gene of the proband and her family members were analyzed by direct sequencing. Three bioinformatics software was used to analyze the conservation, pathogenicity and protein model of the variant. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No. 2012-17). RESULTS: The activated partial thromboplastin time (APTT), FⅫ activity (FⅫ:C) and FⅫ antigen (FⅫ:Ag) of the proband was 180.0 s, 1.0% and 2.1%, respectively. DNA sequencing revealed that she has harbored compound heterozygous variants of the F12 gene, namely c.712_713insT (p.Cys238Leufs *73) in exon 8 and c.1561G>A (p.Glu521Lys) in exon 13. Her mother and younger son were heterozygous for the p.Cys238Leufs*73 variant, while her older son was heterozygous for the p.Glu521Lys variant. Bioinformatic analysis suggested that Cys238 is highly conserved and p.Cys238Leufs*73 is a pathogenic variant, which eventually resulted in a truncated protein. CONCLUSION The c.712_713insT and c.1561G>A compound heterozygous variants of the F12 gene probably underlay the decreased FⅫ level in this pedigree, among which c.712_713insT (NM_000505) was unreported previously.
Adult ; Female ; Humans ; Male ; Middle Aged ; Base Sequence ; China ; Factor XII/genetics* ; Heterozygote ; Mutation ; Pedigree ; Factor XII Deficiency/genetics* ; East Asian People

OBJECTIVE: To investigate the molecular pathogenic mechanisms of a family with hereditary factor Ⅶ (FⅦ) deficiency. METHODS: A family (3 generations, 12 members) with hereditary FⅦ deficiency, in which the proband presented with menorrhagia and was admitted to the First Affiliated Hospital of Wenzhou Medical University in April 2023, was selected as the study subject. Clinical data of the family members were collected. Peripheral venous blood samples were collected from all 12 members for routine coagulation tests and genomic DNA extraction. All exons and flanking sequences of the F7 gene were amplified by PCR and analyzed by Sanger sequencing. Thrombin generation assay was performed to evaluate the coagulation potential of the proband and her parents. Multiple online bioinformatics software tools were used to analyze the conservation and pathogenicity of candidate variants identified in the proband. The pathogenicity of variant was classified according to the Standards and Guidelines for the Interpretation of Sequence Variants released by American College of Medical Genetics and Genomics (ACMG) (hereinafter referred to as ACMG guidelines). Homology modeling of the variant FⅦ protein was performed using homology modeling (SWISS-MODEL). Amino acid sequence alignment between wild-type and variant FⅦ proteins was conducted using MEGA v7, and spatial conformational differences were analyzed using PyMOL to assess the potential impact of the F7 gene variants on the structure and function of the FⅦ protein. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics No.: KY2022-R193). RESULTS: Coagulation tests showed that the proband's prothrombin time (PT) was significantly prolonged to 33.1 s, and both factor Ⅶ activity (FⅦ:C) and antigen (FⅦ:Ag) levels were reduced to 2%. Her parents, eldest sister, second sister, younger brother, and four children all showed mildly prolonged PT, with FⅦ:C and FⅦ:Ag levels approximately 50% of normal. Genetic sequencing identified compound heterozygous variants in the F7 gene of the proband: a heterozygous missense variant c.722C>A (p.Thr241Asn) in exon 7, and a heterozygous deletion variant c.1261_1261delA (p.Ile421Ser*fs75) in exon 8. Retrieval from domestic and international databases found no previous reports of the latter variant, suggesting it is novel. Familial co-segregation analysis confirmed that these variants were inherited from her father and mother, respectively. The thrombin generation assay demonstrated that the proband had a significantly decreased peak thrombin height (peak ratio: 29.5%), significantly increased thrombin lag time ratio and time-to-peak ratio (3.03 and 2.93, respectively), but only a mildly decreased endogenous thrombin potential (ETP) ratio of 90.7%. Online bioinformatics analysis indicated that threonine-241 (p.Thr241) in the FⅦ protein was not conserved, while isoleucine-421 (p.Ile421) was highly conserved. Both the p.Thr241Asn and p.Ile421Serfs*75 variant sites in the proband's F7 gene were predicted to be pathogenic. According to the ACMG guidelines, the p.Thr241Asn (PM3+PP1+PP3+PP4+PP5) and p.Ile421Ser*fs75 (PM2+PM4 +PP1+PP3+PP4) variants were both classified as "likely pathogenic". Structural analysis of the FⅦ protein indicated that the p.Ile421Ser*fs75 frameshift variant led to the substitution of Cysteine-428 by Alanine, preventing the formation of a critical disulfide bond between amino acid residues 400 and 428 present in the wild-type FVII protein. CONCLUSION The compound heterozygous variants p.Thr241Asn and p.Ile421Ser*fs75 in the F7 gene are likely the genetic etiology responsible for the reduced FⅦ levels in this hereditary FⅦ deficiency family.
Adult ; Female ; Humans ; Male ; Middle Aged ; China ; Factor VII/chemistry* ; Factor VII Deficiency/genetics* ; Heterozygote ; Mutation ; Pedigree ; East Asian People/genetics*

OBJECTIVE: To investigate the molecular mechanism for a family with Hereditary coagulation factor Ⅻ (FⅫ) deficiency. METHODS: The proband, a 63-year-old female, was admitted to the First Affiliated Hospital of Wenzhou Medical University in August 2024 for lumbar disc herniation. Coagulation tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), and FⅫ activity (FⅫ:C), were carried out for the proband and her family members (9 individuals from three generations) using a one-stage clotting assay. The level of FⅫ antigen (FⅫ:Ag) was determined with an Enzyme-linked immunosorbent assay (ELISA). Sanger sequencing was conducted to identify potential variants in the F12 gene. Multiple in silico tools were used to predict the conservation, hydrophobicity, and structural impact of the identified variants. Recombinant expression plasmids were constructed and transiently transfected into HEK293T cells. The recombinant FⅫ protein was analyzed using Western blotting (WB) and ELISA. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics No.: KY2022-R193). RESULTS: The proband showed a markedly prolonged APTT (160.3 s) and significantly decreased FⅫ:C (2%) and FⅫ:Ag (5%) levels. Analysis of the F12 gene sequence revealed a 46C/T genotype in the promoter region, a heterozygous c.1457G>A (p.Cys467Tyr) missense variant in exon 12, and a heterozygous c.1561G>A (p.Glu502Lys) missense variant in exon 13. Bioinformatic analysis showed that the p.Cys467 is highly conserved across various species, and the p.Cys467Tyr variant may affect local structural stability of the FⅫ protein. The p.Cys467Tyr variant had no effect on the transcription of the F12 gene. However, the variant has significantly decreased the FⅫ:Ag levels and FⅫ protein expression in the cell culture supernatant compared to the wild-type expression vector, while in the cell lysate, it is higher than the wild-type expression vector. In other words, the p.Cys467Tyr variant has probably caused a secretion defect of FⅫ protein. CONCLUSION The 46C/T genotype, the heterozygous p.Cys467Tyr missense variant, and the heterozygous p.Glu502Lys missense variant are associated with reduced plasma FⅫ levels in this pedigree. The p.Cys467Tyr variant, which was unreported previously, did not affect the synthesis of FⅫ but may have resulted in a secretion defect.
Humans ; Female ; Middle Aged ; Mutation, Missense ; Pedigree ; HEK293 Cells ; Male ; Factor XII/genetics* ; Adult ; Factor XII Deficiency/genetics*

Objective:Molecular mechanisms underlying compound heterozygous mutations in a patient with inherited antithrombin (AT) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in November 2018 with a one-day history of sudden syncope and limb twitching. Peripheral venous blood was collected from the proband and members of his lineages, totaling nine persons across three generations, and a family lineage survey was conducted. AT activity (AT:A) was measured using a chromogenic substrate assay, while AT antigen (AT:Ag) was detected through an immunoturbidimetric assay. Mutation sites were identified by means of Sanger sequencing of the SERPINC1 gene, and silico tools were applied to predict the mutational conservation and hydrophobicity changes. Recombinant plasmid expression vectors were constructed and transfected into HEK293T cells for in vitro overexpression studies. The recombinant AT protein was characterized using Western Blotting, ELISA, and cellular immunofluorescence assays.Results:The proband was a 21-year-old man with type Ⅰ AT deficiency. His AT:A was 33%, along with a corresponding reduction in AT:Ag. The genetic analysis revealed there was a heterozygous insertion mutation at c.318_319insT (p.Asn107*) and a heterozygous missense mutation at c.922G>T (p.Gly308Cys) in exons 2 and 5, respectively. These mutation sites were entirely conserved among the homologous species. Additionally, hydrophobicity studies showed that the p.Gly308Cys mutation will decrease the hydrophilicity of amino acid residues 307-313. The in vitro expression studies indicated a reduction of approximately 46.98%±2.94% and 41.35%±1.48% in the amount of recombinant protein AT-G308C in transfected cell lysates and culture supernatants, respectively. Treatment with the proteasome inhibitor (MG132) restored the cytoplasmic levels of AT-G308C protein to a level similar to that of wild-type protein. However, neither cell lysate nor culture supernatant demonstrated the presence of the recombinant protein AT-N107*. Conclusions:The heterozygous insertion mutation of p.Asn107* and the heterozygous missense mutation of p.Gly308Cys have been associated with reduced AT levels in proband. The p.Asn107* heterozygous insertion mutation may initiate the degradation of mRNA via nonsense mutation-mediated mechanisms, which would remove the defective transcripts, as well as the p.The Gly308Cys heterozygous missense mutation may cause the AT protein to undergo proteasome-dependent degradation by modifying the hydrophobicity of nearby residues in the cytoplasm.

Objective:To investigate the relationship between inherited protein S deficiency (PSD) and cerebral venous sinus thrombosis (CVST) by phenotype and gene mutation analysis of 2 inherited PSD pedigrees with nonsense mutations.Methods:Retrospective analysis of clinical and imaging data of 2 patients diagnosed with CVST who were treated in the Department of Neurology, the First Affiliated Hospital of Wenzhou Medical University in July and October 2023 was carried out. The peripheral blood samples were collected from proband 1 and her family members (3 subjects, 2 generations in total), and proband 2 and his family members (8 subjects of 3 generations in total). Their protein S (PS) activity (PS:A), the content of total PS antigen (TPS:Ag) and free PS antigen (FPS:Ag) were measured for definite diagnosis. Polymerase chain reaction was done in all 15 exons of the active PROS1 gene and its 5′ and 3′ untranslated regions and the amplification products were analyzed by direct sequencing. The results were compared with human PROS1 reference sequences, using Chromas software to find the mutation sites. Results:The proband 1 is a female, and the proband 2 is a male. Both of them had young onset and the clinical presentation of CVST. The PS:A level was reduced to 29% in the proband 1 and reduced to about 35% in her mother; PS:A was reduced to 21%-27% in the proband 2 and his 6 family members; a decline in the same proportion of TPS:Ag and FPS:Ag was found in the 2 probands and their family members, therefore they were primarily diagnosed as typeⅠPSD. Gene analysis showed that the proband 1 and her mother had a nonsense mutation of c.1680T>A in exon 14 (p.Tyr560 *) of the PROS1 gene; the proband 2 and his 6 family members had a nonsense mutation of c.1687C>T in exon 14 (p.Gln563 *) of the PROS1 gene. Conclusion:The reduced protein S levels in PSD patients and their family members may be associated with the p.Tyr560 * and p.Gln563 * nonsense mutations of the PROS1 gene, and the clinical manifestations of CVST in PSD patients may be related to these 2 nonsense mutations.

Objective:To analyze 12 antithrombins (AT) gene mutations that cause AT deficiency and discuss the relationship between the SERPINC1 gene. mutations and venous thrombotic events.Methods:This study belongs to case series of observational studies. Collected the clinical data of 12 AT deficiency cases in the First Affiliated Hospital of Wenzhou Medical University from April 2014 to April 2021 and collected the blood samples before treatment. The AT activity (AT: A) and AT antigen (AT: Ag) was detected by chromogenic substrate and immunoturbidimetry, respectively. The 7 exons and flanking sequences of the SERPINC1 gene were sequenced directly by PCR, the suspected mutations were validated by reverse sequencing. Analyzed the correlation between the SERPINC1 gene. mutations and venous thrombotic events and figured out the proportion.Results:The AT: A of the 12 patients all decreased significantly, ranging from 30% to 66%, and the AT: Ag of the 7 patients decreased accordingly, showing type Ⅰ AT deficiency, and the AT: Ag of the other 5 patients were normal, presented type Ⅱ AT deficiency. 12 mutations were found including 6 heterozygous mutations which were discovered for the first time: c.456_458delCTT(p.phe121del), c.318_319insT(p.Asn75stop), c.922G>T(p.Gly276Cys), c.938T>C (p.Met281Thr), c.1346T>A(p.Leu417Gln)and c.851T>C(p.Met252Thr). All 12 patients had venous thrombosis, and 3 cases including 2 compound heterozygotes and 1 single heterozygote all suffered from deep venous thrombosis (DVT) when they were younger without obvious triggers. The other 9 patients all combined with the other thrombotic factors including old age, hypertensive, smoking, pregnancy, and prolonged immobilization.Conclusion:Patients with AT deficiency caused by SERPINC1 gene defects are prone to venous thrombosis, especially combined with other thrombotic factors.

Objective:To explore the molecular pathogenesis of hereditary protein C (PC) deficiency due to a p. Gly86Asp variant of the PROC gene through in vitro expression experiment.Methods:Wild type and Gly86Asp mutant expression plasmids of PC were constructed and respectively transfected into HEK 293FT cells. Total RNA was extracted from the transfected cells, and the expression of PROC gene was determined by quantitative real-time PCR (qRT-PCR). PC antigen (PC: Ag) in the supernatant of cell culture and cell lysate was determined by enzyme-linked immunosorbent assay (ELISA), and the level of PC protein was detected by Western blotting. Results:qRT-PCR has detected no significant difference in the transcription level of wild-type and mutant-type PC. Compared with the wild type, the level of mutant PC: Ag in the supernatant and cell lysate were 81.3%±2.6% and 110.0%±2.8%, respectively. No difference was detected in the molecular weight between the wild-type and mutant-type PC by Western blotting. The PC content of mutant type was higher than wild-type in cell lysate, while the opposite was found with the cell culture supernatant.Conclusion:The impaired secretion by mutant PC may be the molecular mechanism of PC deficiency caused by the p.

Objective:To explore the genetic basis for a patient with factor XIII (FXIII) deficiency.Methods:All exons of the F13A1 and F13B genes were amplified by PCR and sequenced directly. The sequencing was performed with a reverse primer if a variant was found. Conservation of variant site was analyzed by the ClustalX software. Four online bioinformatic software including MutationTaster, PolyPhen-2, PROVEAN and SIFT were used to predict the function of the mutation site. The Swiss-PdbViewer software was applied to analyze the changes in the protein model and intermolecular force. Results:The proband was found to harbor a novel c. 515G>C (p.Arg171Pro) variant of the F13A1 gene. The corresponding amino acid Arg171 is conserved among homologous species. Bioinformatic analysis indicated that Arg171Pro variant may affect the protein function. Protein model analysis showed that in the wild-type, there is one hydrogen bond between Arg171 and Pro27; one hydrogen bond between Arg171 and Thr28; two hydrogen bonds between Arg171 and Glu102. When Arg171 was mutated to Pro171, the three hydrogen bonds between Arg171 and Pro27, Glu102 are all disappeared and formed a new benzene ring which might affect the stability of the protein structure. No variant was found in the F13B gene. Conclusion:The Arg171Pro variant may account for the decreased FXIII level. Above finding has enriched the spectrum of F13A1 gene variants.

OBJECTIVE: To explore the molecular basis for a Chinese pedigree affected with hereditary coagulation factor VII (FVII) deficiency. METHODS: The coding regions of F7 gene were amplified by PCR and sequenced. Suspected variants were confirmed by reverse sequencing and validated in other members from the pedigree. Pathogenicity of the variants was analyzed with multiple bioinformatic tools. RESULTS: Genetic analysis revealed that the proband has carried compound heterozygous c.985T>C (p.Ser329Pro) and c.1091G>A (p.Arg364Gln) variants in exon 8 of the F7 gene. Her mother, brother and son were heterozygous for c.985T>C (p.Ser329Pro), while her father was heterozygous for c.1091G>A (p.Arg364Gln). Phylogenetic analysis suggested that both p.Ser329 and p.Arg364 are highly conserved among homologous species. Online bioinformatic software predicted both variants to be deleterious. Protein model analysis suggested that the Pro329 side chain may form a new hydrogen bond with Leu333. The Pro benzene ring may clash with Glu325 in the p.Ser329Pro variant model. The p.Arg364Gln variant have two additional hydrogen bonds compared with wild type Arg364. Both variants may lead to alteration of the protein structure. CONCLUSION The p.Ser329Pro and p.Arg364Gln variants in exon 8 of the F7 gene probably account for the reduced FVII in this pedigree.

OBJECTIVE: To explore the molecular pathogenesis for a pedigree affected with hereditary coagulation factor XII (FXII) deficiency. METHODS: Potential variant of the F12 gene was analyzed by PCR and Sanger sequencing. Expression plasmids were constructed by site-directed mutagenesis based on the wild-type and transiently transfected into 293T cells. FXII:C and FXII:Ag of the expression products were determined in the supernatant and cell lysate. Western blotting was used to verify the identify of the protein. RESULTS: Gene sequencing revealed that the proband has carried 46TT genetype and heterozygous p.Glu502Lys variants in exon 13, and a heterozygous p.Gly542Ser variant in exon 14 of the F12 gene. Transfection experiment suggested that the FXII:C and FXII:Ag of p.Glu502Lys variant in the supernatant were 28% and 24%, compared with the wild-type (100%) and FXII:Ag of cell lysates was 39% compared to the wild-type (100%). The FXII:C and FXII:Ag of p. Gly542Ser variant in the supernatant were 32% and 17% and the FXII:Ag of cell lysates was 59%. CONCLUSION The 46TT genetype, p.Glu502Lys and p.Gly542Ser variants of the F12 gene probably underlie the low FXII level in the proband. As shown by in vitro experiment, the p.Glu502Lys and p.Gly542Ser variants can both inhibit the synthesis and secrection of the FXII protein.
Exons ; Factor XII ; genetics ; Factor XII Deficiency ; genetics ; Heterozygote ; Humans ; Pedigree