Objective of this study was to identify gene mutation involved in a patient with type 2A von Willebrand disease (vWD). The bleeding time, vWF:Ag, FVIII:C, RIPA and multimeric assay were used for phenotypic diagnosis. All of the 52 exons and the exon-intron boundaries of vWF gene were amplified by polymerase chain reaction (PCR) and direct sequencing was carried out. The results indicated that the levels of vWF:Ag, FVIII:C and RIPA decreased in this patient, the vWF multimer with high and intermediate molecular weight was absent in plasma. The sequencing of genomic DNA revealed a C4738G (L1580V) missense mutation in the vWF gene from the patient. In conclusion, the C4738G (L1580V) missense mutation effecting the form of vWF multimer was responsible to molecular mechanism in this patient with vWD.
Adult ; Humans ; Male ; Mutation, Missense ; von Willebrand Disease, Type 2 ; genetics ; von Willebrand Factor ; analysis ; genetics

Type 2N von Willebrand disease (vWD) can be confused with hemophilia A due to decreased factor VIII levels and a bleeding tendency, and differential diagnosis is of importance for providing the optimal treatment and genetic counseling. For the accurate diagnosis of type 2N vWD, von Willebrand Factor (vWF) function tests, multimer assay and gene mutation analysis are needed. The patient was a 38-yr-old Nepalese woman with a history of bleeding manifestations from childhood, such as hemarthrosis, intramuscular hematoma, and menorrhagia. Family history revealed that her mother and elder brothers also had bleeding manifestations from childhood. When she had a laparotomy in 1991, she was diagnosed as hemophilia A with factor VIII level of 3.6% and was transfused with whole blood, factor VIII and cryoprecipitates. In June 2007, she was admitted to our hospital for further evaluation of bleeding tendency. Blood tests revealed normal CBC; bleeding time, 2 min; PT, 14.9 sec (11-14 sec); aPTT, 51.2 sec (24-38 sec); and factor VIII, 4.9% (50-150%). The prolonged aPTT was corrected by 1:1 mixing test to the levels of 106% and 84%, respectively, before and after 2 hr-incubation at 37degrees C. No abnormalities were found in the vWF antigen level (71.3%), ristocetin cofactor assay (130.4%), and multimer assay. Direct DNA sequencing of the VWF gene revealed homozygous missense mutation located in exon 19, c.2446C>T (p.Arg816Trp), confirming the diagnosis of type 2N vWD.
Adult ; Amino Acid Substitution ; Asian Continental Ancestry Group/genetics ; Base Sequence ; Female ; Genotype ; Homozygote ; Humans ; Nepal ; von Willebrand Disease/blood/*diagnosis/genetics ; von Willebrand Factor/analysis/*genetics

OBJECTIVETo analyze phenotype and genotype of three Chinese pedigrees with von Willebrand disease (vWD), and explore the molecular mechanism.

METHODSBleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor (vWF): ristocetin cofactor (RCof) (vWF:RCof), vWF antigen (vWF:Ag), vWF activity (vWF:A) test, vWF collagen binding assay (vWF:CB), vWF and Factor VIII (FVIII) binding assay (vWF:FVIII:B) and multimer analysis were used for phenotype diagnosis. Genomic DNA was extracted from the peripheral blood (PB). All the 52 exons and flanking sequences of the probands' vWF gene were amplified by PCR and analyzed by direct sequencing.

RESULTSAPTT were prolonged in all three probands, while BT were normal excepting for proband 3. Plasma RIPA, vWF:RCo, vWF:Ag, vWF:A and vWF:CB were decreased in different extents. In multimer analysis, proband 3 lost the large and intermediate molecular weight multimers, while proband 1 and 2 were normal. Gene analysis in the three probands revealed three heterozygous missense mutations of 144067 G→A (R2287Q) in exon 39, 110374G→A (R1374H) and 110770C→T (S1506L) in exon 28 and heterozygous polymorphism 110667G→A (D1472H) in exon 28, respectively.

CONCLUSIONThe three heterozygous mutations (R2287Q, R1374H and S1506L) and an heterozygous polymorphism (D1472H) are genetic defects of the hereditary vWD of the three pedigrees respectively. R2287Q is a novel mutation reported for the first time in the literature.

Adult ; Child ; DNA Mutational Analysis ; Female ; Genotype ; Heterozygote ; Humans ; Male ; Pedigree ; Phenotype ; von Willebrand Diseases ; diagnosis ; genetics ; von Willebrand Factor ; genetics ; metabolism

BACKGROUND: We intended to find the mutations of von Willebrand factor (VWF) gene as the most important contributing factor of von Willebrand disease (VWD) in Korean patients. METHODS: In 40 known vWD patients mutations of vWF gene were sought by direct sequencing of PCR products targeting exons 18, 19, 20, 26, 28 and 52 frequently implicated as the locations of mutation. For factors other than VWF gene contributing to VWD phenotype, we tested ABO blood group and measured ADAMTS13 activity in VWD patients. RESULTS: Twenty-seven cases (67.5%) were type 1 vWD, 3 cases (7.5%) type 3, and 5 cases (12.5%) type 2A. Three cases were type 2A or 2B (7.5%) and 2 cases were suspected to be type 2N (5.0%). Among them six candidate missense mutations were found: V1279I, R1306W, R1308C, and V1316M were previously reported in type 2B and type 1 vWD, and C858W and T1477I were novel findings. All patients were heterozygotes. Blood group O was overly represented in VWD patients, while ADAMTS13 activity of the patients was not significantly different from that of normal control. CONCLUSIONS: Mutation of VWF gene detected by genetic studies can significantly improve the diagnostic accuracy, especially in subtype assignment of VWD. Two novel mutations, C858W and T1477I associated with VWD were found and expected to contribute to the elucidation of its pathophysiology.
ABO Blood-Group System ; ADAM Proteins/analysis ; Heterozygote ; Humans ; Korea ; *Mutation, Missense ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Sequence Analysis, DNA ; von Willebrand Disease/classification/diagnosis/*genetics ; von Willebrand Factor/analysis/*genetics

The aim of current study was to detect intron 22 inversion of factor VIII gene in severe hemophilia A (HA) patients and screen the carriers of the gene inversion. Fifty-five cases of severe HA were involved and factor VIII gene inversion was detected and identified by long distance-PCR (LD-PCR) and 0.6% agarose gel electrophoresis. The 11 kb and 12 kb bands indicate the factor VIII gene inversion and non-inversion, respectively. Occurring of both 11 kb and 12 kb bands indicates a carrier of the inversion. The results showed that factor VIII gene inversion existed in 22 out of 55 cases, which accounted for about 40% of total detected patients. Five carriers of factor VIII gene inversion were diagnosed from the members in 15 families. In conclusion, LD-PCR assay is a simple, rapid and accurate method for detection of factor VIII gene inversion, and this approach is helpful in screening, carrier testing, and prenatal diagnosis of severe hemophilia A.
Adolescent ; Adult ; Antigens ; analysis ; Child ; Child, Preschool ; Chromosome Inversion ; Factor VIII ; genetics ; Hemophilia A ; blood ; genetics ; Humans ; Infant ; Male ; Polymerase Chain Reaction ; methods ; von Willebrand Factor ; immunology

To study the mechanism of thrombogenesis and search new anti-thrombotic agent, the cDNA for human vWF A1 domain was high-level expressed in E. coli and recombinant protein of vWF A1 with biologic activity was obtained. The gene encoding A1 domain was amplified by PCR from plasmid containing full length cDNA of human vWF. After confirming by DNA sequencing analysis, the recombinant expression plasmid pQE31-vWF/A1 was constructed and introduced into E. coli M15 strain, then induced by IPTG; the expressed protein was purified with Ni-NTA agarose, identified by Western blotting. The results showed that the 854 bp DNA fragment was obtained by PCR from the plasmid containing full length cDNA for human vWF and its sequence was identical to the published sequence. High level expression of A1 protein was yielded after 5 hour-induction, which amounted to 30% of total bacteria protein in inclusion body. Western blot demonstrated it possessed good antigenicity and high specificity. It is concluded that cDNA for vWF/A1 had been cloned successfully, high level expression of A1 protein was achieved in E. oli. This study will provide a basis for the further clinical and basic research on the role of vWF in thrombosis and hemostasis.
Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Escherichia coli ; genetics ; Humans ; Polymerase Chain Reaction ; Recombinant Proteins ; analysis ; von Willebrand Factor ; analysis ; biosynthesis ; genetics

The interaction among collagen, von Willebrand factor (vWF) and glycoprotein Ib axis is the first step in hemostasis and thrombosis, especially under high shear condition. To develop a new remedy of anti-thrombosis, mRNA from endothelial cells was extracted, and reverse transcription PCR was adopted to amplify DNA of interest. After sequencing, recombinant expression vector was constructed. The amplified DNA fragment of vWF domain A3 was inserted into expression vector with 6 x his taq, pET20b(+), the recombinant was transformed into E coli (strain DE3) and induced by IPTG. Recombinant vWF-A3 was designated as a recombinant fragment comprising residues 918 - 1114 of mature vWF subunit. It was purified through Ni-NTA resin column and refolded in Tris buffer containing GSH and GSSG. The results showed that rvWF-A3 was expressed successfully in E coli (strain DE3), accounting for 46% of total bacterial protein with its purity of over 95%. It was identified that rvWF-A3 is capable to bind collagen and inhibit the wild vWF binding to collagen by competition. It is concluded that rvWF-A3 fragment might be an effective antithrombotic agent for preventing arterial thrombosis.
Cloning, Molecular ; Collagen ; metabolism ; Escherichia coli ; genetics ; Humans ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; von Willebrand Factor ; chemistry ; genetics ; metabolism

This study was aimed to investigate the impact of vWF A1381T polymorphism (rs216311) and ABO blood group on von Willebrand factor level in plasma. 120 healthy volunteers, aging from 19 to 33 years (average 24) were recruited. The vWF:Ag level in plasma was determined by ELISA. vWF gene A1381T polymorphisms were analyzed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequenced when necessary. The data were grouped by gender, blood group and/or genotype. The difference of plasma vWF level between male and female were analyzed by independent sample t test. One way ANOVA were used to analyze the difference of vWF level in each blood group of genotype while factorial design ANOVA were used to test the difference of vWF level in plasma between A1381T genotype and/or ABO blood groups. The results showed that analysis of plasma vWF level in 120 volunteers of both male (60) and female (60) demonstrated no statistical difference (t = 1.039, p = 0.301). The vWF level was lower in blood type O group than that in non-O group (p < 0.001); the plasma vWF level in AA mutant of vWF A1381T gene polymorphism was lower than that in AG and GG mutant (p = 0.003 and 0.019, respectively). In blood type O group, the vWF plasma level in AG mutant of vWF A1381T gene polymorphism resulted in non-difference (p = 0.070) compared with AA or AG mutant, while there was significant difference in vWF of plasma level when contrast tests were applied (t = 2.321 and p = 0.028, respectively). In non-O group, the plasma vWF level in AG mutant of vWF A1381T gene polymorphism were significantly different from AA mutant (p = 0.032). It is concluded that plasma vWF level unrelated with gender but interrelates with ABO blood groups. Plasma vWF level in vWF gene A1381T polymorphism with AA mutant is significantly lower than that with AG and GG mutant. In blood type O group, plasma vWF level in vWF gene A1381T polymorphism with AG mutant is higher than that with AA and GG mutant. In non-O group, the vWF plasma level in A1381T gene polymorphism with AG mutant is significantly higher than that with AA mutant. This change may be beneficial to understand some diseases, especially cardio-cerebral vascular diseases.
ABO Blood-Group System ; genetics ; Adult ; Blood Grouping and Crossmatching ; Female ; Genotype ; Humans ; Male ; Plasma ; chemistry ; Polymorphism, Genetic ; Young Adult ; von Willebrand Factor ; analysis ; genetics