OBJECTIVE: To identify F7 gene mutations in one pedigree with congenital coagulation factor VII (FVII) deficiency and explore the effect of F7 gene mutations on the structure and function of FVII. METHODS: Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured based on the one-stage assay, and PT-based one stage assay was used to detect the activity of FII, V, VII and X. Genomic DNA was extracted from the peripheral blood of family members. The sequences of all the exons and exon-intron boundaries of F7 were amplified by polymerase chain reaction (PCR) using specific primers followed by Sanger sequencing. PolyPhen-2, PROVEAN and Swiss-Pdb Viewer software were used to analyze the effect of mutations on the structure and function of FVII. RESULTS: The proband had a prolonged PT (33.8 s) due to low FVII activity (6.6%) and normal APTT, and had a history of epistaxis. The proband's mother and father displayed a slightly prolonged PT (13.2 and 13.9 s, respectively), and their FVII activity was 40.3% and 38.3%, respectively. The compound heterozygous c.722C>A (p.Thr241Asn) in exon 7 and c.1165T>G (p.Cys389Gly) in exon 8 of F7 gene were identified in the proband, and inherited from his father and mother, respectively. The p.Thr241Asn and p.Cys389Gly missense change were likely to have a damaging effect predicted by polyphen-2 and PROVEAN software. In silico modeling analysis showed that there was one hydrogen bond formed between wild-type Thr241 and Val249, two hydrogen bonds formed between mutant Asn241 and Val249 and between mutant Asn241 and Leu242, as well as one hydrogen bond and one disulfide bond formed between wild-type Cys389 and Leu370 and between wild-type Cys389 and Cys375, respectively. The hydrogen bond formed between mutant Gly389 and Leu370 and disulfide bond formed between mutant Gly389 and Cys375 both broke. CONCLUSIONS FVII deficiency in this family is caused by p.Thr241Asn and p.Cys389Gly mutation. In silico modeling may be a valuable tool for understanding amino acid residues from variants leading to congenital FVII deficiency.
Humans ; Factor VII/genetics* ; Mutation ; Pedigree ; Factor VII Deficiency/genetics* ; Prothrombin Time ; Partial Thromboplastin Time ; Male ; Female

OBJECTIVE: To detect potential mutations of the coagulation factor Ⅶ (F7) gene in a pedigree affected with hereditary FⅦ deficiency and explore its molecular pathogenesis. METHODS: The FⅦ antigen (FⅦ:Ag) was analyzed by an enzyme-linked immunosorbent assay (ELISA) method. Prothrombin time (PT), FⅦ activity (FⅦ:C) and other coagulant parameters were quantified with an one-stage clotting assay. The F7 gene was amplified by PCR and sequenced. Mutational sites were confirmed by reverse sequencing. Impact of amino acid substitution was assessed using SIFT and PolyPhen-2 software. Structure of the mutant protein was analyzed using Swiss-pdb Viewer software based on the three-dimensional structure in the Protein Data Bank. RESULTS: The propositus had prolonged PT (36.3 s), with FⅦ:C and FⅦ:Ag significantly reduced to 2% and 44%, respectively. Her father, mother, younger sister and daughter had slightly prolonged PT and reduced FⅦ:C (86%-120%). The FⅦ:Ag of her father and younger sister were also reduced. DNA sequencing revealed that the propositus has carried compound heterozygous mutations (Lys341Glu and IVS6-1G>A) of the F7 gene. Her father and younger sister were heterozygous for the IVS6-1G>A mutation, while her mother and daughter were heterozygous for the Lys341Glu mutation. Bioinformatics analysis indicated that Lys341Glu mutation may affect the stability and function of the FⅦ protein. CONCLUSION The Lys341Glu and IVS6-1G>A mutations probably underlie the reduced activity of FⅦ in this pedigree.
Factor VII ; genetics ; Factor VII Deficiency ; genetics ; Female ; Genetic Testing ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree

OBJECTIVETo provide mutation analysis and prenatal diagnosis for a family affected with congenital factor VII(FVII) deficiency.

METHODSDNA was extracted from peripheral blood samples from the proband and his parents. All exons and flanking sequence of the FVII gene were amplified with PCR and subjected to direct sequencing. Prenatal diagnosis was performed by amniocentesis.

RESULTSA homozygous mutation (NM_000131.3) c.572-1G>A was identified in the proband. Both parents of the fetus were carriers of the mutation.

CONCLUSIONA method for molecular diagnosis of congenital factor VII deficiency was established and successfully applied for an affected family.

Factor VII Deficiency ; genetics ; Humans ; Infant, Newborn ; Male ; Mutation ; Prenatal Diagnosis

OBJECTIVETo identify potential mutations in a family affected with inherited factor Ⅶ (FⅦ) deficiency.

METHODSProthrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, FⅦ activity (FⅦ:C) and other coagulant parameters of the proband and 15 family members were measured. Potential mutations were screened in the pedigree by polymerase chain reaction and direct DNA sequencing.

RESULTSThe PT of the proband and his younger brother was significantly prolonged to 39.0 s and 30.1 s, respectively. FⅦ:C of the proband and his younger brother was obviously reduced to 2% and 3%, respectively. FⅦ:C of his grandmother, maternal grandmother, aunt, father, mother, maternal uncle and maternal aunt was all below the normal range (80%-108%), which measured 68%, 54%, 71%, 73%, 62%, 72% and 59%, respectively. The other coagulant parameters were in the normal range. Two heterozygous mutations, g.11349G>A and g.11482T>G, both reside in exon 8 of the F7 gene, have resulted in p.Arg304Gln and p.His348Gln substitutions, were identified in the proband. The same mutations were also found in the proband's younger brother. Four maternal members in this family (grandmother, mother, maternal uncle and maternal aunt of the proband) were heterozygous for the p.Arg304Gln mutation, while three paternal members (grandmother, aunt and father of the proband) were heterozygous for the p.His348Gln mutation.

CONCLUSIONThe proband had inherited two independent mutations of the F7 gene including g.11349G>A and g.11482T>G from his mother and father, respectively. The compound heterozygous mutation probably explains the low FⅦ concentrations in this pedigree.

Adult ; Base Sequence ; Blood Coagulation Tests ; Factor VII ; genetics ; metabolism ; Factor VII Deficiency ; blood ; genetics ; Female ; Genetic Testing ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Pedigree ; Young Adult

OBJECTIVETo identify potential mutations and explore the molecular mechanism underlying combined inherited coagulation factors VII(FVII) and X(FX) deficiency for a family featuring consanguineous marriage between maternal cousins.

METHODSProthrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, FVII activity (FVII:C), FX activity (FX:C), FVII antigen (FVII:Ag), FX antigen (FX:Ag) and other coagulant parameters of the proband and 5 family members were measured. Potential mutations in exons, exon-intron boundaries and 5', 3' untranslated sequences of F7 and F10 genes were screened by polymerase chain reaction and direct sequencing. Suspected mutations were confirmed by sequencing the opposite strand.

RESULTSPT and APTT of the proband were obviously prolonged to become 76.4 s and 60.2 s, respectively. FVII:C, FVII:Ag,FX:C and FX:Ag of the proband were obviously reduced to become 4%, 6%, 6% and 33%, respectively. Both PT and APTT of her grandmother, father, mother and daughter were slightly prolonged, which have measured 16.4 s, 15.8 s,16.9 s, 16.5 s, and 44.0 s, 42.1 s, 41.1 s, 43.5 s, respectively. And their FVII:C (34%, 39%, 31%, 40%, respectively), FX:C (50%, 58%, 47%, 42%, respectively) and FX:Ag (51%, 54%, 58%, 47%, respectively) were slightly reduced, while FVII:Ag was in the normal range. The coagulant parameters of her younger brother were within normal range. Two homozygous mutations, g.11267C to T in exon 8 of F7 gene, which resulted in an Arg277Cys substitution, and g.28139G to T in exon 8 of F10 gene which led to a Val384Phe substitution, were identified in the proband. The proband's grandmother, parents and daughter were heterozygous for both Arg277Cys and Val384Phe mutationss. Wild-type alleles of both F7 and F10 genes were also found in the younger brother.

CONCLUSIONA homozygous Arg277Cys mutation and a Val384Phe mutation have been respectively identified in the F7 and F10 genes, which can explain the low levels of FVII and FX in this family. The former has been inherited from the consanguineous parents.

Adult ; Aged ; Consanguinity ; Factor VII Deficiency ; genetics ; Factor X Deficiency ; genetics ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; Phenotype

OBJECTIVETo identify and characterize a missence mutation Ser250 Phe underlying coagulation factor Ⅶ (FⅦ) deficiency in a Chinese patient and his family.

METHODSThe FⅦ gene (F7) was analyzed by DNA sequencing, and the FⅦ levels (including antigen and activity) in patient's plasma were determined with enzyme-linked immunoabsorbent assay (ELISA) and one stage prothrombin time based method. In addition, a FⅦ-250 Phe mutant corresponding to the identified mutation was expressed in HEK293 cells, and a subcellular localization experiment in CHO cells was performed to clarify the molecular mechanism of FⅦ deficiency caused by the FⅦ-250 Phe mutation.

RESULTSThe patient had a prolonged prothrombin time (PT: 36.5 s) and low levels of both FⅦ antigen and activity (130.2 ng/mL and 4.0%, respectively). Two heterozygous mutations were identified in the F7 gene (NG-009262.1), which included a g.15975 G>A mutation at the splice receptor site of intron 6 (IVS6-1G>A) and a novel g.16750 C>T mutation in exon 8, which resulted in replacement of Ser (TCC) 250 with Phe (TTC)250 in the vicinity of a charge-stablizing system. By gene expression experiments, the antigen and activity levels of FⅦ-250 Ser and FⅦ-250 Phe in the culture medium were (37.77 ± 2.30) ng/mL and (4.02 ± 0.52) ng/mL, respectively. ELISA and Western blotting analyses indicated that expression of the mutant FⅦ-250 Phe and wild type FⅦ-250 Ser was (130.51 ± 2.32) ng/mL and (172.45 ± 2.25) ng/mL, respectively. FⅦ-250 Phe was found in endoplasmic reticulum and Golgi apparatus, suggesting that the mutant FⅦ-250 Phe could be normally synthesized in the cells but was inefficiently secreted.

CONCLUSIONCompound heterozygous mutations in F7 gene (g.15975G>A and g.16750C>T) may be responsible for the FⅦ deficiency in this patient. The novel FⅦ 250 Phe can be transported from endoplasmic reticulum to Golgi apparatus, but may be degraded or inefficient.

Adult ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Enzyme-Linked Immunosorbent Assay ; Factor VII ; genetics ; physiology ; Factor VII Deficiency ; genetics ; HEK293 Cells ; Humans ; Male ; Mutation, Missense

Factor VII Deficiency ; diagnosis ; genetics ; Female ; Humans ; Middle Aged ; Pedigree ; Phenotype

OBJECTIVETo investigate potential mutations and clinical features of 9 unrelated patients with inherited coagulation factor VII (FVII) deficiency.

METHODSClinical diagnosis was validated by assaying of coagulation parameters including prothrombin time, activated partial thromboplastin time, FVII activity and specific antigens. All exons, exon-intron boundaries, and 5' and 3' untranslated regions of F7 genes were amplified with PCR. Potential mutations were detected by direct sequencing of purified PCR products. Suspected mutations were confirmed by sequencing of the opposite strand.

RESULTSAll probands have featured prolonged prothrombin time, with FVII activity ranging between 2.0% to 6.0%. The titers of FVII antigen were significantly reduced in 7 probands. Eight mutations, including 6 missense mutations, 1 deletion and 1 insertion, were identified, among which 3 (Gln100Leu, Ser269Pro and g.11520_11521insT) were not described previously. Six mutations have located in the protease domain. All mutations were inherited, and consanguineous marriages were reported in 5 families. Mutations g.27_28delCT, Cys329Gly, Arg304Trp and His348Gln have been identified in unrelated families. There was a lack of correlation between the mutations and their clinical features. Two individuals with homozygous His348Gln mutations and 1 individual with homozygous Arg304Trp mutation were only mildly affected or asymptomatic. Two patients, who have respectively carried homozygous and heterozygous deletions of g.27_28delCT, were moderately affected and asymptomatic. In 4 patients carrying double heterozygous mutations, 1 (Ser269Pro and Cys329Gly) was asymptomatic, 2 (Arg304Trp and Cys329Gly, Arg277Cys and g.11520_11521insT, respectively) had a mild bleeding tendency, whilst 1 (Gln100Leu and His348Gln) has a moderate bleeding diathesis.

CONCLUSIONThere seem to be hotspots of F7 gene mutations in ethnic Han Chinese populations. And there is a lack of correlation between particular types of mutations and clinical phenotypes.

Adolescent ; Adult ; Aged ; Base Sequence ; Blood Coagulation Disorders, Inherited ; genetics ; Child ; Factor VII ; genetics ; Factor VII Deficiency ; genetics ; Female ; Heterozygote ; Homozygote ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Young Adult

OBJECTIVETo 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).

METHODSOne 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.

RESULTSNo 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).

CONCLUSIONPolymorphisms 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.

Adult ; Aged ; Aged, 80 and over ; China ; Factor VII ; genetics ; Factor XII ; genetics ; Female ; Fibrinogen ; genetics ; Genetic Predisposition to Disease ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; genetics ; Polymorphism, Genetic

OBJECTIVETo analyze the gene mutation in two pedigrees of inherited coagulation factor VII (FVII) deficiency, and investigate the relationship between the genotype and phenotype.

METHODThe coagulation function and coagulation factors activity of probands were detected for phenotype diagnosis, all exons and junctions of FVII gene from the family members' genomic DNA were amplified using polymerase chain reaction (PCR), and detected the gene mutation by direct sequencing. Mutations were confirmed by reverse sequencing.

RESULTThe prothrombin time (PT) of proband 1 was 265.2 s, FVII:C was 22% and the PT of proband 2 was > 120 s, FVII:C was 1%. Homozygous 17844G→A mutation in No. 8 exon of FVII gene was identified in the proband 1 resulting in Gly343Ser, and heterozygosity for the same mutations were confirmed in his parents and a sister. The proband 2 was compound heterozygous, one mutation was the same as the proband 1 but was a heterozygosity that can also found in his mother and brother; the other heterozygosity mutation was located on No. 8 exon 18055G→A that resulted in Gln413Arg which was inherited from his father.

CONCLUSIONNo. 8 exon of FVII gene encodes catalytic domain. Mutation found in those domain could change the FVII catalytic domain spatial structure, affected FVII function and stability, and the sufferer of homozygote and compound heterozygous may have clinical bleeding tendency. Almost no clinical findings in simple heterozygotes, however, a few of heterozygotes could have a tendency of bleeding because of genetic polymorphism which would reduce the FVII:C.

Blood Coagulation Disorders ; blood ; genetics ; Child, Preschool ; DNA Mutational Analysis ; Factor VII ; genetics ; Factor VII Deficiency ; blood ; genetics ; Heterozygote ; Homozygote ; Humans ; Infant ; Male ; Molecular Sequence Data ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Prothrombin Time