Analysis of a Chinese pedigree affected with Hereditary coagulation factor Ⅻ deficiency due to compound heterozygous variants of F12 gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical phenotypes and genetic variants of a Chinese pedigree affected with Hereditary coagulation factor Ⅻ (FⅫ) deficiency.@*METHODS@#A pedigree presented at the First Affiliated Hospital of Air Force Medical University on December 24,2021 was selected as the study subject. Activated partial thromboplastin time (APTT) and coagulation factor Ⅻ activity (FⅫ:C) were determine by a clotting method, and FⅫ antigen was detected with an ELISA assay. Following the extraction of genomic DNA, all exons and flanking regions of the F12 gene were subjected to Sanger sequencing. Clustalx-2.1-win, PROVEAN and Swiss-PDB Viewer software was used to analyze the conservation of amino acids at the variant sites, impact of of the variants on the amino acid substitutions and the protein structure.@*RESULTS@#The APTT of the proband has prolonged to 70.2 s. Her FⅫ:C and FⅫ:Ag have decreased to 12% and 13%, respectively. DNA sequencing revealed that the proband has harbored c.346G>A (p.Gly97Ser) and c.1583C>A (p.Ser509Tyr) heterozygous compound missense variants in exons 5 and 13 of the F12 gene, respectively. Her father and sister were heterozygous carriers for the c.346G>A (p.Gly97Ser) variant, whilst her mother and brother were heterozygous for the c.1583C>A (p.Ser509Tyr) variant.@*CONCLUSION@#The c.346G>A (p.Gly97Ser) and c.1583C>A (p.Ser509Tyr) compound heterozygous variants of the F12 gene probably underlay the pathogenesis of hereditary coagulation FⅫ deficiency in this pedigree.

Analysis of a Chinese pedigree affected with Hereditary FⅫ deficiency due to compound heterozygous variants of F12 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the laboratory phenotype and molecular pathogenesis in a Chinese pedigree affected with Hereditary coagulation factor Ⅻ (FⅫ) deficiency.@*METHODS@#A male proband admitted to Ningbo No.2 Hospital on July 17, 2021 due to chronic gastritis and members of his pedigree (7 individuals from three generations) were selected as the study subjects. Prothrombin time (PT), activated partial thromboplastin time (APTT), FⅧ activity (FⅧ: C), FⅨ activity (FⅨ: C), FⅪ activity (FⅪ: C), FⅫ activity (FⅫ: C), and FⅫ antigen (FⅫ: Ag) were determined. All of the exons, exon-intronic boundaries, as well as the 5'- and 3'-untranslated regions of the F12 gene were subjected to Sanger sequencing. Candidate variants were verified by cloning sequencing. The effect of candidate variants on the protein function was analyzed by bioinformatics software.@*RESULTS@#The proband, a 47-year-old male, had significantly prolonged APTT (180.0 s) and decreased FⅫ:C and FⅫ:Ag levels (< 1%). His father, mother, brother and two sons also showed certain degrees of reduction. Genetic testing revealed that the proband has harbored compound heterozygous variants of the F12 gene, namely c.1092_1093insC (p.Lys365Glnfs*69) in exon 10 and c.1792_1796delGTCTA (p.Val579Hisfs*32) in exon 14. His mother and elder son were heterozygous for the c.1092_1093ins variant, whilst his father, brother, and younger son were heterozygous for the c.1792_1796delGTCTA variant. Analysis of the promoter region of exon 1 also showed that the proband and both sons had harbored a 46T/T polymorphism, whilst other family members were 46C/T. Bioinformatic analysis suggested that the p.Val579 is a highly conserved site. Protein model analysis showed that, with the p.Val579Hisfs*32 variant, a benzene ring was added and the hydrogen bond of surrounding amino acids was changed. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.1792_1796delGTCTA was classified as a pathogenic variant (PVS1+PM2_Supporting+PM4).@*CONCLUSION@#The c.1092_1093insC (p.Lys365Glnfs*69) and c.1792_1796delGTCTA (p.Val579Hisfs*32) compound heterozygous variants of the F12 gene probably underlay the decreased FXII levels in this pedigree. Above finding has also enriched the mutational spectrum for FⅫ deficiency.

Genetic analysis of a Chinese pedigree affected with Congenital coagulation factor XII deficiency due to a c.1A>G start codon variant of F12 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical characteristics and genetic etiology of a consanguineous Chinese pedigree affected with Congenital coagulation factor XII (XII) deficiency.@*METHODS@#Members of the pedigree who had visited Ruian People's Hospital on July 12, 2021 were selected as the study subjects. Clinical data of the pedigree were reviewed. Peripheral venous blood samples were taken from the subjects. Blood coagulation index and genetic testing were carried out. Candidate variant was verified by Sanger sequencing and bioinformatic analysis.@*RESULTS@#This pedigree has comprised 6 individuals from 3 generations, including the proband, his father, mother, wife, sister and son. The proband was a 51-year-old male with kidney stones. Blood coagulation test showed that his activated partial thromboplastin time (APTT) was significantly prolonged, whilst the FXII activity (FXII:C) and FXII antigen (FXII:Ag) were extremely reduced. The FXII:C and FXII:Ag of proband's father, mother, sister and son have all reduced to about half of the lower limit of reference range. Genetic testing revealed that the proband has harbored homozygous missense variant of c.1A>G (p.Arg2Tyr) of the start codon in exon 1 of the F12 gene. Sanger sequencing confirmed that his father, mother, sister and son were all heterozygous for the variant, whilst his wife was of the wild type. By bioinformatic analysis, the variant has not been included in the HGMD database. Prediction with SIFT online software suggested the variant is harmful. Simulation with Swiss-Pbd Viewer v4.0.1 software suggested that the variant has a great impact on the structure of FXII protein. Based on the Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics (ACMG), the variant was rated as likely pathogenic.@*CONCLUSION@#The c.1A>G (p.Arg2Tyr) variant of the F12 gene probably underlay the Congenital FXII deficiency in this pedigree. Above finding has further expanded the spectrum of F12 gene variants and provided a reference for clinical diagnosis and genetic counseling for this pedigree.

Analysis of F12 gene variants and molecular mechanisms in patients with coagulation factor Ⅻ deficiency / 中华医学遗传学杂志

OBJECTIVE@#To analyze the sequence of the F12 gene and molecular mechanism for 20 patients with coagulation factor Ⅻ (FⅫ) deficiency.@*METHODS@#The patients were selected from the outpatient department of the Second Hospital of Shanxi Medical University from July 2020 to January 2022. The activity of coagulation factor Ⅷ (FⅧ:C), factor Ⅸ (FⅨ:C), factor Ⅺ (FⅪ:C) and factor Ⅻ (FⅫ:C) were determined by using a one-stage clotting assay. All exons and 5' and 3' UTR of the F12 gene were analyzed by Sanger sequencing to detect the potential variants. Bioinformatic software was used to predict the pathogenicity of the variants, conservation of amino acids, and protein models.@*RESULTS@#The FⅫ:C of the 20 patients has ranged from 0.07% to 20.10%, which was far below the reference values, whilst the other coagulation indexes were all normal. Sanger sequencing has identified genetic variants in 10 patients, including 4 with missense variants [c.820C>T (p.Arg274Cys), c.1561G>A (p.Glu521Lys), c.181T>C (p.Cys61Arg) and c.566.G>C (p.Cys189Ser)], 4 deletional variants c.303_304delCA(p.His101GlnfsX36), 1 insertional variant c.1093_1094insC (p.Lys365GlnfsX69) and 1 nonsense variant c.1763C>A (p.Ser588*). The remaining 10 patients only harbored the 46C/T variant. The heterozygous c.820C>T(p.Arg274Cys) missense variant in patient 1 and the homozygous c.1763C>A (p.Ser588*) nonsense variant in patient 2 were not included in the ClinVar and the Human Gene Mutation Database. Bioinformatic analysis predicted that both variants were pathogenic, and the corresponding amino acids are highly conserved. The protein prediction models suggested that the c.820C>T (p.Arg274Cys) variant may affect the stability of the secondary structure of FⅫ protein by disrupting the original hydrogen bonding force and truncating the side chain, leading to changes in the vital domain. c.1763C>A (p.Ser588*) may produce a truncated C-terminus which may alter the spatial conformation of the protein domain and affect the serine protease cleavage site, resulting in extremely reduced FⅫ:C.@*CONCLUSION@#Among individuals with low low FⅫ:C detected by one-stage clotting assay, 50% have harbored variants of the F12 gene, among which the c.820C>T and c.1763C>A were novel variants underlying the reduced coagulating factor FⅫ.

Analysis of A Pedigree with Hereditary Coagulation Factor Ⅻ Deficiency Caused by Compound Heterozygous Mutations / 中国实验血液学杂志

OBJECTIVE@#To analysis clinical phenotype and potential genetic cause of a family affected with hereditary coagulation factor Ⅻ deficiency.@*METHODS@#The prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), D-Dimer (D-D), coagulation factor Ⅻ activity (FⅫ:C) and coagulation factor Ⅻ antigen (FⅫ:Ag) were determined for phenotype diagnosis of the proband and his family members(3 generations and 5 people). Targeted capture and whole exome sequencing were performed in peripheral blood sample of the proband. Possible disease-causing mutations of F12 gene were obtained and further confirmed by Sanger sequencing. The corresponding mutation sites of the family members were analyzed afterwards. The online bioinformatics software AutoPVS1 and Mutation Taster was used to predict the effects of mutation sites on protein function.@*RESULTS@#The APTT of the proband was significantly prolonged, reaching 180.9s. FⅫ:C and FⅫ:Ag of the proband was significantly reduced to 0.8% and 4.17%, respectively. The results of whole exome sequencing displayed that there were compound heterozygous mutations in F12 gene of the proband, including the c.1261G＞T heterozygous nonsense mutation in exon 11 (causing p.Glu421*) and the c.251dupG heterozygous frameshift mutation in exon 4 (causing p.Trp85Metfs*53). Both mutations are loss of function mutations with very strong pathogenicity, leading to premature termination of the protein. AutoPVS1 and Mutation Taster software predicted both mutations as pathogenic mutations. The results of Sanger sequencing revealed that c.1261G＞T heterozygous mutation of the proband was inherited from his mother, for which his brother and his daughter were c.1261G＞T heterozygous carriers. Genotype-phenotype cosegregation was observed in this family.@*CONCLUSION@#The c.1261G＞T heterozygous nonsense mutation in exon 11 and the c.251dupG heterozygous frameshift mutation in exon 4 of the F12 gene probably account for coagulation factor Ⅻ deficiency in this family. This study reports two novel pathogenic F12 mutations for the first time worldwide.

Short-term prophylaxis for delivery in pregnant women with hereditary angioedema with normal C1-inhibitor / Profilaxia de curto prazo para o parto em grávidas com angioedema hereditário com inibidor de C1 normal

Abstract Objective To verify the efficacy of short-term prophylaxis for vaginal or cesarean section childbirth with plasma-derived C1-inhibitor concentrate in pregnant women. They should have hereditary angioedema (HAE) and normal plasma C1-inhibitor. Methods Case report of pregnant women diagnosed with HAE with normal C1- inhibitor who had been treated with intravenous C1-inhibitor concentrate for prophylaxis of angioedema attacks when hospitalized for delivery. The exon 9 of the Factor 12 (F12) genotyping gene was performed by automatic sequencing in all patients. Results Three cases of pregnant women with HAE with normal serum level of C1- inhibitor are reported. The genetic test detected the presence of a pathogenic mutation in the F12 gene. Deliveries occurred uneventfully and patients had no HAE symptoms in the following 72 hours. Conclusion C1-inhibitor concentrate could be useful to prevent angioedema attacks during and after delivery.

Resumo Objetivo Verificar a eficácia da profilaxia de curto prazo para o parto vaginal ou cesáreo com inibidor de C1 derivado de plasma concentrado em mulheres grávidas. Eles devem ter angioedema hereditário e inibidor normal de C1 no plasma. Métodos Relato de caso de gestantes diagnosticadas com angioedema hereditário com inibidor de C1 normal que foram tratadas com inibidor intravenoso de concentrado de C1 para profilaxia de ataques de angioedema quando hospitalizadas para o parto. O exon 9 do gene de genotipagem do fator 12 (F12) foi realizado por sequenciamento automático em todos os pacientes. Resultados Três casos de gestantes com angioedema hereditário com nível sérico normal de inibidor de C1 são relatados. O teste genético detectou a presença de uma mutação patogênica no gene F12. Os partos ocorreram sem intercorrências e as pacientes não apresentaram sintomas hereditários de angioedema nas 72 horas seguintes. Conclusão O concentrado de inibidor de C1 pode ser útil para prevenir ataques de angioedema durante e após o parto.

Identification of compound heterozygous variants of F12 gene in a pedigree affected with inherited coagulation factor XII deficiency / 中华医学遗传学杂志

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.

Factor XIII-guided treatment algorithm reduces blood transfusion in burn surgery / Algoritmo de tratamento guiado pelo fator XIII reduz a transfusão sanguínea na cirurgia de queimados

Abstract Background and objectives: Major burn surgery causes large hemorrhage and coagulation dysfunction. Treatment algorithms guided by ROTEM® and factor VIIa reduce the need for blood products, but there is no evidence regarding factor XIII. Factor XIII deficiency changes clot stability and decreases wound healing. This study evaluates the efficacy and safety of factor XIII correction and its repercussion on transfusion requirements in burn surgery. Methods: Randomized retrospective study with 40 patients undergoing surgery at the Burn Unit, allocated into Group A those with factor XIII assessment (n = 20), and Group B, those without assessment (n = 20). Erythrocyte transfusion was guided by a hemoglobin trigger of 10 g.dL-1 and the other blood products by routine coagulation and ROTEM® tests. Analysis of blood product consumption included units of erythrocytes, fresh frozen plasma, platelets, and fibrinogen. The coagulation biomarker analysis compared the pre- and post-operative values. Results and conclusions: Group A (with factor XIII study) and Group B had identical total body surface area burned. All patients in Group A had a preoperative factor XIII deficiency, whose correction significantly reduced units of erythrocyte concentrate transfusion (1.95 vs. 4.05, p = 0.001). Pre- and post-operative coagulation biomarkers were similar between groups, revealing that routine coagulation tests did not identify factor XIII deficiency. There were no recorded thromboembolic events. Correction of factor XIII deficiency in burn surgery proved to be safe and effective for reducing perioperative transfusion of erythrocyte units.

Resumo Justificativa e objetivos: A cirurgia no grande queimado causa hemorragia de grande porte e disfunção da coagulação. Os algoritmos de tratamento guiados por ROTEM® e fator VIIa reduzem as necessidades de hemoderivados, mas falta evidência em relação ao fator XIII. A deficiência do fator XIII altera a estabilidade do coágulo e diminui a cicatrização. Este estudo avalia a eficácia e a segurança da correção do fator XIII e sua repercussão nas necessidades transfusionais na cirurgia do queimado. Métodos: Estudo retrospectivo randomizado de 40 doentes submetidos à cirurgia na Unidade de Queimados alocados em grupo A com estudo do fator XIII (n = 20) e grupo B sem estudo (n = 20). A transfusão eritrocitária foi guiada por gatilho de hemoglobina de 10 g.dL-1 e os outros hemoderivados por testes de coagulação de rotina e ROTEM®. A análise do consumo de hemoderivados incluiu unidades de eritrócitos, plasma fresco congelado, plaquetas e fibrinogênio. A análise dos biomarcadores da coagulação comparou os valores pré e pós-operatórios. Resultados e conclusões: O grupo A (com estudo de fator XIII) e o grupo B apresentaram área de superfície corporal total queimada idêntica. Todos os doentes do grupo A revelaram déficit pré-operatório de fator XIII, cuja correção reduziu significativamente a transfusão de unidades de concentrado eritrocitário (1,95 vs. 4,05, p = 0,001). Os biomarcadores de coagulação pré e pós-operatórios foram semelhantes entre os grupos, revelaram que os testes de coagulação de rotina não identificam o déficit de fator XIII. Sem eventos tromboembólicos registrados. A correção do fator XIII na cirurgia do queimado revelou-se segura e eficaz na redução da transfusão perioperatória de unidades de eritrócitos.

Phenotypic and genetic analysis of two pedigrees affected with hereditary coagulation FXII deficiency / 中华医学遗传学杂志

OBJECTIVE@#To carry out phenotypic and genotypic analysis for two Chinese pedigrees affected with coagulation factor XII (F XII) deficiency.@*METHODS@#Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT), and blood coagulation factor VIII, IX, XI, XII activity (FVIII:C, FIX:C, FXI:C, FXII:C) were determined with one stage clotting assay on a STAGO coagulation analyzer. FXII antigen was determined with an enzyme linked immunosorbent assay (ELISA). The 14 exons and their flanking sequences of the F12 gene were subjected to PCR amplification and Sanger sequencing. The conservation and structure of mutant protein were analyzed with MegAlign software and PYMOL software.@*RESULTS@#The APTT of the probands was significantly prolonged, while their FXII:C and FXII:Ag were significantly reduced. Genetic analysis of the proband has revealed three novel mutations in the F12 gene, including g.5972G>A splice site mutation in intron 5, g.8810_8814delGTCTA in exon 14, and g.6259G>A (p.Pro182Leu) in exon 7. In addition, a previously known mutation IVS13-1G>A has been found.@*CONCLUSION@#Four mutations have been identified in the two Chinese pedigrees, among which three were novel. Above mutations probably played a role in the defect of FXII in the two pedigrees.

Oral Rivaroxaban Treatment for L-asparaginase-induced Deep Thrombophlebitis in Lower Extremity / 임상소아혈액종양

We are reporting our experience of oral rivaroxaban (Xarelto(R)) treatment for L-asparaginase (L-ASP)-induced deep vein thrombophlebitis in the lower extremity developed during childhood acute lymphoblastic leukemia (ALL) chemotherapy, with a brief review of the literature. A 16-year-old boy was admitted to our institution with right lower leg pain and gait difficulties. He was diagnosed with ALL and started chemotherapy protocol. He had been under a chemotherapy course of delayed intensification (DI)-1. We began antibiotics treatment for possible inflammation including cellulitis of the leg and planned an MRI scan. The MRI scan indicated thrombophlebitis of the right posterior calf deep veins. Subsequent DVT CT and coagulation profiles showed other abnormal findings. Coagulation factor assay were noted with decreased levels of multi factors; Factor II 45%, Factor IX 35.3 %, Factor X 30%, Factor XI 19%, Factor XII 22%, and anti-coagulants levels were decreased also with variant degrees; Protein C Activity 51%, Protein C Ag 54.5%, Protein S Activity 35%, Protein S Antigen, total 27.1%, Protein S Antigen, free 41.7%. Low molecular heparin (LMWH) treatment was initiated and the patient was switched to oral rivaroxaban (Xarelto(R)). After 6 weeks treatment, abnormal coagulation profiles and MRI scan showed improvement. Furthermore, the patient had no other symptoms or recurrence of thrombotic events. There was no significant adverse reaction to rivaroxaban in this patient.

Identification of a novel heterozygous mutation in a pedigree with hereditary coagulation factor XII deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify potential mutation underlying hereditary coagulation factor XII (FXII) deficiency in a pedigree and explore its molecular pathogenesis.</p><p><b>METHODS</b>Activated partial thromboplastin time (APTT), FXII activity (FXII:C) and FXII antigen(FXII:Ag) and other coagulant parameters of the proband and 5 family members were measured. Potential mutations in the 14 exons and intron-exon boundaries of the FXII gene were screened with polymerase chain reaction (PCR) and direct DNA sequencing. Suspected mutations were confirmed with reverse sequencing. Corresponding PCR fragments from other family members were also sequenced.</p><p><b>RESULTS</b>APPT of the proband and his son were significantly prolonged to 121.5 s and 98.5 s, respectively. FXII:C and FXII:Ag of the proband and his son have reduced to 5%, 6.8% and 9%, 12.2%, respectively. Plasma plasminogen activity (PLG:A) in both individuals was slightly higher than the normal reference range. FXII:C of his second daughter and grandson were slightly reduced to 64% and 60%. FXII:C of the other family members were all in the normal range (72%-113%). A heterozygous missense mutation, g.8597G>A, was identified in exon 13 of the FXII gene in the proband, which resulted in an p.Asp538Asn substitution. For the promoter regions of the FXII gene, the genotype of the proband was 46TT. The same mutations and 46T/T were also found in the proband's son but not in other members of the family. The genotypes of the proband's spouse, eldest daughter and grandson were 46CT, and his second daughter was 46TT.</p><p><b>CONCLUSION</b>The heterozygous mutation of g.8597G>A identified in exon 13 of FXII gene is a novel mutation. Heterozygous p.Asp538Asn mutation and 46TT in the FXII gene can cause hereditary FXII deficiency, which was probably responsible for the low FXII concentrations in this pedigree.</p>

Oral Rivaroxaban Treatment for L-asparaginase-induced Deep Thrombophlebitis in Lower Extremity / 임상소아혈액종양

We are reporting our experience of oral rivaroxaban (Xarelto(R)) treatment for L-asparaginase (L-ASP)-induced deep vein thrombophlebitis in the lower extremity developed during childhood acute lymphoblastic leukemia (ALL) chemotherapy, with a brief review of the literature. A 16-year-old boy was admitted to our institution with right lower leg pain and gait difficulties. He was diagnosed with ALL and started chemotherapy protocol. He had been under a chemotherapy course of delayed intensification (DI)-1. We began antibiotics treatment for possible inflammation including cellulitis of the leg and planned an MRI scan. The MRI scan indicated thrombophlebitis of the right posterior calf deep veins. Subsequent DVT CT and coagulation profiles showed other abnormal findings. Coagulation factor assay were noted with decreased levels of multi factors; Factor II 45%, Factor IX 35.3 %, Factor X 30%, Factor XI 19%, Factor XII 22%, and anti-coagulants levels were decreased also with variant degrees; Protein C Activity 51%, Protein C Ag 54.5%, Protein S Activity 35%, Protein S Antigen, total 27.1%, Protein S Antigen, free 41.7%. Low molecular heparin (LMWH) treatment was initiated and the patient was switched to oral rivaroxaban (Xarelto(R)). After 6 weeks treatment, abnormal coagulation profiles and MRI scan showed improvement. Furthermore, the patient had no other symptoms or recurrence of thrombotic events. There was no significant adverse reaction to rivaroxaban in this patient.

Gene analysis in four inherited coagulation FⅫ deficiency pedigree / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the genotype and pathogenesis in four Chinese pedigrees with Factor Ⅻ deficiency.</p><p><b>METHODS</b>Activated partial thromboplastin time (APTT), FⅫ procoagulant activity (FⅫ∶C), FⅫ antigen（FⅫ∶Ag）and other coagulant parameters were detected. The FⅫ deficiency Pedigree members,all exons,boundary introns including the splice junctions of the FⅫ gene were amplified with Polymerase chain reaction (PCR). Expression plasmids were constructed by mutagenesis based on the wild-type and transfected into COS7 cells. FⅫ∶C and FⅫ∶Ag of the expression levels were tested in the supernatant and cell lysate.</p><p><b>RESULTS</b>The four probands presented prolonged APTT with all the values of FⅫ∶C and FⅫ∶Ag were low to 2% and 1%, respectively. There were common 46C/T polymorphism in the promoter regions of FⅫ gene in four pedigrees. Proband A was heterozygous for two mutations, g.5741-5742delCA (His101Gln) and g.7142insertC (Lys346Gln). Proband B was a heterozygous deletion mutation g.6800-6808del9bp. The results of the transfection revealed that FⅫ∶Ag in cell lysates and conditioned media protein FⅫ6800-6808del9bp were 85.6% and 51.9%. The FⅫ∶C in the conditioned media was 56.4%. Proband C was a heterozygous mutation g.8699G>A(Gly542Ser). Proband D was a homozygous mutation 8699G>A, whose parents with consanguineous marriage.</p><p><b>CONCLUSION</b>Four mutations, g.5741-5742delCA, g.7142insertC, g.6800-6808del9bp and g.8699G>A with 46C/T polymorphism in the promoter regions of FⅫ gene, were identified in the four Factor Ⅻ deficiency pedigrees. The two mutations g.5741-5742delCA and g.6800-6808del9bp were first found in China. FⅫ 6800-6808del9bp expressed in vitro suggested that almost normal proteinum synthesis but defect proteinum secretion.</p>

Identification of a novel mutation of factor XII gene in a family with coagulation FXII deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze genetic mutation and molecular pathogenesis in a family affected with inherited coagulation factor XII(FXII) deficiency.</p><p><b>METHODS</b>Activated partial thromboplastin time (APTT), FXII procoagulant activity (FXII:C), FXII antigen (FXII:Ag) and other coagulants were measured. For affected members of the family, exons 1-14 and flanking intronic regions of the FXII gene were amplified with polymerase chain reaction (PCR) and sequenced thereafter. Expression plasmids containing mutant FXII cDNA was constructed and transfected into COS7 cells transiently. Expressions of FXII:Ag and FXII:C were analyzed.</p><p><b>RESULTS</b>The proband has manifested a prolonged APTT of 108.1 s (reference range: 27.0-41.0 s). Her husband has a normal APTT. Other members of the family had slightly increased APTT. The FXII:C and FXII:Ag of the proband have both dropped to about 0.01 (reference range: 0.72-1.13). The FXII:C levels of her husband, son, daughter and grandchild were 0.57, 0.24, 0.14, 0.16, respectively. And the FXII:Ag levels in her husband, son, daughter and grandchild were 0.55, 0.27, 0.15, 0.21, respectively. The proband and her daughter have both carried a heterozygous deletional mutation 6800-6808delAGCTGGGAG (6800-6808del9bp) in exon 9. For the promoter region of the FXII gene, the genotypes of the proband, her son, daughter and grandchild was TT, whilst that of her husband was CT. Expression study has shown that, whilst the mutant FXII protein has accumulated in the cells similar to wild-type protein, its secretion has reduced approximately by half.</p><p><b>CONCLUSION</b>A novel deletional mutation 6800-6808del9bp has been identified in the FXII gene. Although mutant FXII protein can still accumulate in cells, its secretion has become insufficient. The 6800-6808del9bp mutation and 46T/T have both contributed to the pathogenesis of FXII deficiency in the family, but may have not been the sole cause.</p>

Association between gene polymorphisms and myocardial infarction in Han Chinese of Yunnan province / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine frequencies of genetic polymorphisms of coagulation factor VII (FVII), coagulation factor FXII (FXII), fibrinogen (FBG) and 9p21 in ethnic Han Chinese from Yunnan province, and to assess the association between such polymorphisms and onset of myocardial infarction (MI).</p><p><b>METHODS</b>One hundred and forty-two patients with MI and 192 healthy controls were analyzed. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and pyrosequencing were used to determine the genotypes of FVII, FXII, FBG and 9p21.</p><p><b>RESULTS</b>No significant difference was found in the frequencies of R353Q, 5'F7, C46T, -148C/T, rs1333049 and rs4977574 loci between the two groups (P> 0.05). However, the frequencies of AA of -455G/A, T and TT of rs1333040, T and TT of rs10116277 and G and GG of rs2383207 were significantly higher in MI group compared with the controls (P< 0.05), whilst the frequencies of CT of rs1333040 and GT of rs10116277 were significantly lower in MI group compared with the controls (P<0.05).</p><p><b>CONCLUSION</b>Polymorphisms of FVII, FXII, -148C/T of FBG and rs1333049 of 9p21 were not associated with myocardial infarction. Polymorphisms of -455G/A of FBG and rs1333040, rs10116277 and rs2383207 of 9p21 may be associated with MI in ethnic Han Chinese from Yunnan province.</p>

Analysis of an hereditary coagulation factor XII deficiency in a consanguineous pedigree / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze genetic mutation and explore its molecular pathogenesis for an hereditary coagulation factor XII(F XII) deficiency in a pedigree featuring consanguineous marriage.</p><p><b>METHODS</b>Activated partial thromboplastin time (APTT), F XII procoagulant activity (F XII:C), F XII antigen (F XII:Ag) and other coagulant parameters were assayed. For the proband and his family members, exons 1-4, introns including the splice junctions of the F XII gene were amplified with polymerase chain reaction (PCR). The PCR product was purified and sequenced. The mutations were confirmed by sequencing the complimentary strand.</p><p><b>RESULTS</b>The proband has featured prolonged APTT at 157.5 s (reference range, 27.0-41.0 s). The APTT of his son has increased slightly at 48.3 s. The remaining members of the family were in normal range. F XII activity and F XII antigen of the proband were significantly decreased (<1%). The F XII activity of his wife, daughter, son and mother was also dropped to about 51%, 21%, 21% and 50%, respectively, and so was the F XII antigen (42%, 32%, 37% and 48%, respectively). Homozygous missense mutation of G→A transition at position 8699 in exon 14 resulting in Gly542Ser was identified in the proband. His mother, son and daughter were heterozygous for Gly542Ser. In the promoter regions of F XII gene, the genotype of the proband and the other members was 46T/T.</p><p><b>CONCLUSION</b>Homozygous missense mutation Gly542Ser was found in a pedigree of hereditary F XII deficiency. The homozygous missense mutation might have resulted from his parents by consanguineous marriage. Gly542Ser and 46T/T have contributed to the pathogenesis of the hereditary factor XII deficiency pedigree.</p>

[Survey of the relationship between of thrombophilic factors and the rate and severity of bleeding in patients with severe hemophilia A]

Hemophilia is the most common disorder of deficiencies in thrombotic factors. In most patients, severities of symptoms are in proportion of the seriousness of deficiency in the thrombotic factors. But in some patients with severe hemophilia, having less than 1% in factor level; the clinical symptoms are lower and slighter than the other hemophilic patients. Even in some cases, thrombotic events in the severe hemophilic patients have been accrued. The underlying causes of these findings are unknown. The aims of this study were to determine the role of some factors and also the type of genetic mutation of these factors in the rate and severity of bleeding and also the symptoms of the severe hemophilic patients. Sixty hemophilia A patients [FVIII<1%] with having records in Hemophilic Patients Center were divided on the basis of the received factor rate items per year, bleeding score, orthopedic score and radiologic score, in three groups with mild, moderate and sever clinical presentations [each group with 20 patients]. And then the mutation tests in the gene of Leiden V factor, PG20210A, MTHFR, level of thrombotic factors [II, V, VII, VIII, IX, X, XI, XII, XIII, and Fibrinogen], Protein C, S, Antithrombine III, Phospholipid Ab and Hemosisteine and number of platelets were performed. Data using the x2, Fischer, ANOVA and Tukey test and SPSS-14 were analyzed. Of the studied factors, the differences among 3 groups from view point of daily activity [P<0.002], antithrombine III [P<0.013], number of platelets [P<0.007], protein C [P<0.013] and level of factor XII [P<0.01] was significant. No significant differences were found in three groups in other tested factors. In this study, there were significant differences only in the daily activity, antithrombine III, number of platelets, protein C and level of factor XII. Thus, further studies are required to determine the role of other factors that may contribute to differences in the clinical presentations of the severe hemophilic patients

In silico Prediction of Angiogenesis-related Genes in Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and a typical hypervascular tumor. Therefore, it is important to find factors related to angiogenesis in the process of HCC malignancy. In order to find angiogenesis-related factors in HCC, we used combined methods of in silico prediction and an experimental assay. We analyzed 1457 genes extracted from cDNA microarray of HCC patients by text-mining, sequence similarity search and domain analysis. As a result, we predicted that 16 genes were likely to be involved in angiogenesis and then the effects of these genes were confirmed by hypoxia response element(HRE)-luciferase assay. For instant,we classified osteopontin into a potent angiogenic factor and coagulation factor XII into a significant anti-angiogenic factor. Collectively, we suggest that using a combination of in silico prediction and experimental approaches, we can identify HCC-specific angiogenesis-related factors effectively and rapidly.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 æg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 æM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 æM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 æM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM)

The 46C/T Polymorphism of Coagulation Factor XII in Healthy Korean Population and in Patients with Ischemic Cerebrovascular Disease / 대한진단검사의학회지

BACKGROUND: In Orientals, the levels of the plasma coagulation factor XII (FXII) were only half of those in Caucasians. Recently, nucleotide polymorphism of the 46C to T substitution in the 5'-untranslated region was reported and this led to lower levels in plasma FXII. Some inherited FXII deficiencies were reported to show thromboembolic predisposition. We investigated the interaction of this polymorphism with FXII activity and the association of this polymorphism among ischemic cerebrovascular disease (CVD) patients among Koreans. METHODS: Blood was obtained from 334 healthy Korean adults and 163 patients. We measured FXII activities by the aPTT method. Molecular analysis using the allele-specific restriction with endonuclease Hga I was performed. RESULTS: Genotype frequencies of our controls were 7.8%, 47.3%, and 44.9% for C/C, C/T, and T/T. There were no significant differences in genotype frequencies between the controls and the patients (P=0.800). FXII activities of our controls were strongly dependent on the genotype: C/C, 129.4%; C/T, 92.4%; T/T, 50.2% (P60 years old), the prevalence of the T/T genotype increased significantly in the old-age category of patients (P=0.046). CONCLUSIONS: Koreans had a 69% frequency of T alleles with low levels of FXII activity. We could not prove any association between this polymorphism and ischemic CVD, but the prevalence of the T/T genotype in old-age patients was significantly higher than in the age-matched controls. This finding suggests that the 46C/T polymorphism of FXII might have an influence on the morbidity of ischemic CVD in the old-age group.