Humans ; Factor XIII/genetics* ; Factor XIII Deficiency/genetics* ; Mutation ; Pedigree

OBJECTIVETo explore F (13) A gene mutation in a pedigree with hereditary coagulation factor XIII (FXIII) deficiency.

METHODSThe FXIII deficiency was diagnosed by clot solubility test and other standard laboratory clotting tests. All exons, exon-intron boundary sequences of F(13) A gene were amplified by PCR and the products were sequenced directly. Any mutation identified by direct sequencing was confirmed by reverse sequencing. The mutation identified in the proband was screened in the family members.

RESULTSThe assays of PT, Qiulan, fibrinogen leveling, platelet counts, bleeding time were normal and the clot solubility test was positive in the proband. The homozygous deletion of 33 nucleotides (127067de133) in exon 10 of F(13) A gene which resulted in deletion of 11 amino acids in FXIIII A protein with 720aa residues was identified in the proband. Family studies showed that the mutation was inherited from the parents both of whom carried the heterozygous deletion mutation.

CONCLUSIONThe homozygous 127067de133 mutation of F(13) A gene is responsible for the disorder of the pedigree.

Adolescent ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Heterozygote ; Homozygote ; Humans ; Male ; Pedigree ; Sequence Deletion

OBJECTIVETo identify the genetic defect of inherited coagulation factor (F) deficiency in a Chinese family and to explore its molecular mechanism.

METHODSThe activity and antigen of plasma F were measured by photometric test and enzyme-linked immunosorbent assay, and rocket-electrophoresis, respectively. All the exons and exon-intron boundaries of the FA subunit gene were amplified by PCR and then DNA sequencing was performed. Restriction endonuclease analysis was used for the PCR products of the family members and 80 healthy donors to exclude gene polymorphism.

RESULTSRapid dissolution of the proband's fibrin clot occurred within 30 minutes, and antigen of his plasma F was significantly decreased, two compound heterozygous missense mutations (a C to T transition at nucleotide 177,246 which caused Arg703Trp, and a A to G transition at nucleotide 177,286 which caused His716Arg) in exon 15 of FA subunit gene were found. The possibility of gene polymorphism was excluded by restriction endonuclease analysing. Each of these two missense mutations was respectively found in his mother and father. Molecular modeling based on 3D crystallographic data predicted that the mutant protein decreased stability and was likely to be rapidly degraded.

CONCLUSIONSThe inherited F deficiency in the Chinese family is caused by two compound heterozygous missense mutations-Arg703Trp and His716Arg in the FA subunit, which to our knowledge, are reported for the first time.

Base Sequence ; Child ; Exons ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Heterozygote ; Humans ; Male ; Molecular Sequence Data ; Mutation, Missense ; Pedigree

OBJECTIVETo explore gene defect of hereditary coagulation factor XIII deficiency.

METHODSPCR and gene sequencing or ARMS-PCR were used to detect the FXIIIA gene of peripheral white blood cell (PBC) from two Chinese hereditary coagulation factor XIII deficiency family members and 60 normal subjects respectively. The level of FXIIIA gene mRNA was tested by RT-PCR.

RESULTS(1) Nucleotide sequence analysis of the two probands' and their family members' DNA revealed that all of the three patients had homozygous missense mutation in FXIII A subunit gene. Proband 1 had a C to G transition at nucleotide (nt) 1 241 in exon 10 and proband 2 and his sister a C to T transition at nt 232 in exon 3 of FXIII A gene, which resulted in the substitution of Ser413 with Trp and Arg 77 with Cys, respectively. Family study showed that the two mutations were inherited from the parents who were correspondingly heterozygotes at nt 1 241 or nt 232. (2) The two mutations were not found in the normal subjects. (3) The FXIIIA gene mRNA level in the two probands was a little decreasing.

CONCLUSIONIt is the two novel mutations that results in FXIIIA deficiency. The two mutations of FXIIIA gene may affect its function or alter protein folding. The defective FXIII which is unstable and degraded rapidly in cytoplasm may be the main cause of FXIII deficiency.

Blood Coagulation Disorders, Inherited ; genetics ; Child ; Exons ; genetics ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Female ; Heterozygote ; Humans ; Pedigree ; Point Mutation ; Polymerase Chain Reaction ; methods

OBJECTIVETo analyze the expressed mRNA of the factor subunit A (FA) in monocyte in a hereditary factor (F) deficiency family.

METHODSThe F A mRNA of the proband and the other family members was analyzed by RT-PCR, semi-quantitative RT-PCR, cloning and sequencing. The three dimensional structure of the protein was predicted by SWISS-MODEL and viewed by RASMIOL.

RESULTS(1) A large in frame deletion from codons 11 to 279, spanning from exon 2 to 7 of F A (DelCD11-279), was identified in the proband at mRNA level and a truncated protein is predicted composed of 464 amino acids. Compared with the normal and the other families, the proband showed lower level of F A mRNA in RT-PCR. (2) SWISS-MODEL analysis showed that the truncated protein lacked the β-sandwich and a part of catalytic core, resulting in loss of the normal catalytic domains.

CONCLUSIONDelCD11-279 of F A mRNA is associated with hereditary F deficiency. The reduced expressing level of F A gene is one of the causes resulting in F deficiency in the patients.

Adolescent ; DNA Mutational Analysis ; Exons ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Female ; Humans ; Male ; Pedigree ; RNA, Messenger ; genetics ; Sequence Deletion

OBJECTIVETo identify the gene mutation type of an inherited coagulation factor XIII (FXIII) deficiency pedigree.

METHODSPCR and DNA sequencing were used to identify the mutations in the 15 exons and the flank sequence of FXIII gene in the proband. The identified mutations were validated by allele specific PCR, PCR restriction fragment length polymorphism technique or DNA sequencing in the family members and 100 healthy volunteers.

RESULTSArg77Cys and Argl74stop double heterozygous mutations were discovered in the proband. The pedigree analysis showed that Arg77Cys missense mutation inherited from her father, and Arg174stop from her mother. The Arg77Cys missense mutation in exon 3 was not found in her husband and the other 100 healthy volunteers.

CONCLUSIONA novel Arg174stop nonsense mutation was discovered in human FXIII gene. A simple DNA assay based on PCR for detection of this mutation was developed. The congenital FXIII deficiency in the proband might be caused by the coinheritance of the Arg77Cys missense mutation in exon 3 and the Arg174stop nonsense mutation in exon 4.

Adult ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; DNA Mutational Analysis ; Exons ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree

OBJECTIVETo perform phenotypic diagnosis, genetic diagnosis and prenatal diagnosis of inherited coagulation factor XIII (FXIII)deficiency in a Chinese family also provide a review of inherited coagulation F XIII deficiency.

METHODSThe activity levels of F XIII (F XIII:C) of proband and family members were measured by clot solubility test and REA-chrom F XIII kit. The antigen levels of F XIII(FXIII:Ag) were measured by enzyme-linked immunosorbent assay. Thrombelastography (TEG) test was used to make a comprehensive evaluation of coagulation status in the proband. All 15 exons and exon-intron boundaries of the F13A1 gene were amplified by PCR, and DNA sequencing was performed then. The mutation identified in the proband was screened in the family members. Furthermore, the related literatures were reviewed to provide a profile of clinical manifestation, gene mutations, the relationship between the mutations and phenotype, and treatments of inherited coagulation F XIII deficient cases.

RESULTSThe clot solubility test was positive in the proband. The FXIII:Ag level of the proband was less than 1% and the FXIII:C level was below the lower limit of detection (<3%). Two compound heterozygous missense mutations (p.Arg662* and p.Trp665*) were identified in the proband. Family study showed that the two mutations were both inherited from the parents. The fetus also carried two compound heterozygous mutations, the same as the proband, and was diagnosed with severe F XIII deficiency. As reported in the literatures, most mutations were missense mutations and nonsense mutations, and no hot spot was found. The clinical pattern of F XIII deficiency varied among patients, with potentially fatal consequences from severe bleeding complications.

CONCLUSIONBetter understanding of F XIII biochemical properties and function and developing of FXIII laboratory assays and genetic detection could prevent missed diagnosis, and patients moght benefit from better care.

Asian Continental Ancestry Group ; Base Sequence ; DNA Mutational Analysis ; Enzyme-Linked Immunosorbent Assay ; Exons ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Female ; Heterozygote ; Humans ; Introns ; Mutation, Missense ; Pedigree ; Phenotype ; Polymerase Chain Reaction ; Pregnancy ; Prenatal Diagnosis

The present study was aimed to investigate the molecular mechanisms responsible for the pathogenesis of severe factor XIII (FXIII) deficiency. Site-directed mutagenesis was conducted to obtain human FXIIIA expression plasmids bearing the mutations. Wild type FXIIIA recombinant plasmid (pcDNA3.1-FXIIIA-wt) and 2 mutant FXIIIA recombinant plasmids (pcDNA3.1/FXIIIA/77mut, pcDNA3.1/FXIIIA/174mut) were transfected into the cultured COS-7 cells using lipofectamine 2000 transfection reagent, respectively. FXIII activities were measured by the Berichrom(®) FXIII chromogenic assay. The expression levels of FXIIIA mRNA were detected by real-time RT-PCR. The recombinant FXIIIA mutants were determined by using Western blot and ELISA. The results showed that the normalized mRNA levels of 2 mutants in transfected COS-7 cells were 0.82 ± 0.21 and 0.76 ± 0.17, respectively. The relative levels of both mRNA transcripts were not significantly decreased as compared with the wild type (1.06 ± 0.51). FXIII activity and FXIIIA antigen levels in concentrated media of cell expressing the wild type protein were (24.0 ± 2.9)% and (13.2 ± 2.3)%, respectively. FXIII activity and FXIIIA antigen levels in cell lysates containing the wild type recombinant protein were (61.6 ± 30.4)% and (32.8 ± 14.5)%, respectively. However, the antigen levels and activity of 2 mutants were severely decreased as compared to the wild type. It is concluded that both mutations severely disturb the normal expression of FXIIIA protein. The reduction of expression levels and decreased activities of the 2 mutants provides a convincible explanation for the deficiency phenotype in the index case.
Animals ; COS Cells ; Cercopithecus aethiops ; Codon, Nonsense ; Factor XIII Deficiency ; genetics ; Factor XIIIa ; genetics ; Genotype ; Humans ; Mutagenesis, Site-Directed ; Mutation, Missense ; Reverse Transcriptase Polymerase Chain Reaction

Change in fibrin stabilizing activity of factor XIII A subunit (FXIII-A) caused by a specific mutation, Val34Leu, is recently implicated to incidences of pathophysiology of thrombosis. In an effort to understand the effect of Val34Leu on enhanced catalytic role of FXIII-A, wild type human factor XIII A (HFXIII-A) and mutant HFXIII-A: HFXIII-A (V34L), HFXIII-A (V35L) and HFXIII-A (V34L/V35L) cDNA were expressed in E.coli system where the purified recombinant FXIII-A (rFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet. Using these rFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated rFXIII-A were characterized. rFXIII-A (V34L) and rFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type rFXIII-A and V35L variant. However, the activated rFXIII-A and mutants showed the identical catalytic efficiency as measured by in vitro assay. These results suggest that ready activation caused by a specific mutation of neighboring thrombin cleavage site(s) in the activation peptide of FXIII-A like V34L resulted in the real-time amount of the activated factor XIII-A that could influence the outcome of fibrin stabilization in vivo such as alpha2- plasmin inhibitor crosslinking to fibrin, a reaction known to be dependent on the initial concentration of active factor-XIII-A.
Blood Coagulation Tests ; Catalysis ; Enzyme Activation/genetics ; Escherichia coli/genetics ; Factor XIII/*genetics/*metabolism ; Fibrin/metabolism ; Human ; Immunoblotting ; Leucine/genetics ; Mutagenesis, Site-Directed ; *Polymorphism (Genetics) ; Recombinant Proteins/genetics/metabolism ; Thrombin/metabolism ; Valine/genetics

The polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recognized as a risk factor for primary intracerebral hemorrhage (PICH). In addition, FXIII Val34Leu has a significant ethnic heterogeneity. FXIII Val34Leu was detected in 41.7-54.8% of the Westerners, but in 2.5% of the Asians. We aimed to evaluate the prevalence of FXIII Val34Leu in patients with PICH and in healthy controls among Koreans. We recruited 58 in-patients with PICH, defined by brain computed tomography or magnetic resonance imaging, and 48 controls matched for age, sex, and risk factors for cerebrovascular diseases. Genomic DNA was extracted from blood. A 183-bp fragment of exon 2/intron B of the factor XIII Asubunit gene was amplified by polymerase chain reaction (PCR). The factor XIII genotype was determined through a single-stranded conformational polymorphism. Fifty-eight patients and 48 controls showed the same band patterns on SSCP. In addition, we directly sequenced six random-selected DNA segments using DNA auto-sequencer. In conclusion, the results of this study suggest that FXIII Val34Leu be absent or rare both in patients with PICH and in healthy controls among Koreans.
Adult ; Aged ; Aged, 80 and over ; Cerebral Hemorrhage/epidemiology/*genetics/prevention & control ; Electrophoresis, Polyacrylamide Gel/methods ; Factor XIII/*genetics ; Female ; Humans ; Korea/epidemiology ; Leucine/genetics ; Male ; Middle Aged ; Polymerase Chain Reaction/methods ; *Polymorphism, Genetic ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA ; Valine/genetics