Cherie Cameron

A single nucleotide polymorphism at nucleotide -1793 in the von Willebrand factor (VWF) regulatory region is associated with plasma VWF:Ag levels.

von Willebrand Factor (VWF) is a large multimeric glycoprotein involved in the transport and protection of factor VIII and in mediating platelet–subendothelium and platelet–platelet interactions. We have documented the presence of a single nucleotide polymorphism (SNP) at nucleotide (nt) −1793 (G 0·36 or C 0·64) in the VWF 5′‐flanking sequence. This polymorphism is in strong linkage disequilibrium with the previously reported SNPs at nts −1234, −1185 and −1051 and, like this other group of polymorphisms, shows a significant association with plasma VWF levels. This association is more marked in subjects who are more than 40 years of age. Further, circumstantial evidence to support a role for the −1793 sequence in regulating VWF expression comes from our demonstration of differential binding of endothelial cell nuclear proteins, including the transcription factor NFκB, by this sequence. In summary, the association of the −1793 SNP with plasma VWF levels provides additional evidence for the role of the VWF regulatory region between nts −1793 and −1051 in controlling VWF expression.

Genetic linkage and association analysis in type 1 von Willebrand disease: results from the Canadian type 1 VWD study.

Summary.  Background: von Willebrand disease (VWD) is the most common bleeding disorder known in humans, with type 1 VWD representing the majority of cases. Unlike the other variant forms of VWD, type 1 disease represents a complex genetic trait, influenced by both genetic and environmental factors. Aim: To evaluate the contribution of the von Willebrand factor (VWF) and ABO blood group loci to the type 1 VWD phenotype, and to assess the potential for locus heterogeneity in this condition, we have performed genetic linkage and association studies on a large, unselected type 1 VWD population. Method: We initially collected samples from 194 Canadian type 1 VWD families for analysis. After the exclusion of families found to have either type 2 or type 3 VWD, and pedigrees with samples from single generations, linkage and association analysis was performed on 155 type 1 VWD families. Results and conclusion: The linkage study has shown a low heterogeneity LOD score of 2.13 with the proportion of families linked to the VWF gene estimated to be 0.41. Linkage was not detected to the ABO locus in this type 1 VWD population. In the family‐based association test, significant association was found between the type 1 VWD phenotype, the quantitative traits, VWF:Ag, VWF:RCo, and FVIII:C and the ABO ‘O’ and ‘A’ alleles and the VWF codon 1584 variant. There was also weak association with the −1185 promoter polymorphism and VWF:Ag, VWF:RCo, and FVIII:C plasma levels. These studies provide further evidence to support the role for genetic loci other than VWF and ABO in the pathogenesis of type 1 VWD.

In vivo evaluation of an adenoviral vector encoding canine factor VIII : High-level, sustained expression in hemophiliac mice

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

Influence of a GT repeat element on shear stress responsiveness of the VWF gene promoter

Summary.  Background: Plasma von Willebrand factor (VWF) is mainly derived from endothelial cells, cells that express a large repertoire of genes that are transcriptionally regulated by fluid shear stress. Endothelial VWF expression is not uniform throughout the vasculature, and levels are increased at regions associated with disturbed blood flow and steep gradients of shear stress. It is, however, unknown whether shear stress influences the regulation of VWF gene expression. Objectives: Our objective was to evaluate the effect of shear stress on endogenous endothelial VWF mRNA expression and VWF promoter (− 2722 to − 1224) activity and to determine whether genetic elements modulate this flow‐induced expression. Methods: A parallel plate flow chamber was used to expose endothelial cells to a shear level of 15 dynes cm−2 for 24 or 6 h. VWF mRNA expression was analyzed. Various VWF promoter constructs that each contain either SNP haplotypes 1 or 2 and either a 17‐GT or a 23‐GT repeat element were transfected into endothelial cells, and flow‐induced promoter activation was assessed. Results: When endothelial cells were exposed to shear stress, endogenous VWF mRNA expression increased 1.84‐fold and average VWF promoter activity was enhanced 3.4‐fold. Single nucleotide polymorphisms at − 2708 and − 2525, and the shear stress‐response element at − 1585, are not responsible for the shear stress‐induced increase. Rather a GT repeat element at − 2124 mediates the increase in activity, and the length of this polymorphic repeat element influences the magnitude of induction. Conclusions: Shear stress enhances VWF promoter activity and a polymorphic GT repeat element mediates the stress‐induced transactivation.

The −1185 A/G and −1051 G/A dimorphisms in the von Willebrand factor gene promoter and risk of myocardial infarction

Elevated plasma von Willebrand factor (VWF) levels are associated with coronary artery disease, although the precise mechanism for this is unclear. Recently, four linked dimorphisms in the VWF gene promoter were demonstrated to influence plasma VWF level. We conducted a case–control study of 525 acute myocardial infarction (MI) cases and 451 control subjects, all aged  75 years, to assess the potential contribution of two of these dimorphisms (−1185 G/A and −1051 A/G) to the risk of MI. The frequency of the −1185A/−1051G haplotype, associated with elevated VWF levels, was similar in the case and control groups, yielding a haplotypic odds ratio for MI of 0·93 (95% CI 0·77, 1·12, P = 0·43), and there was no significant association between the −1185A/−1051G haplotype and the risk of MI in any subgroup analysed. We therefore conclude that possession of the −1185A/−1051G haplotype does not confer an increased risk for MI.

Differential termination of primer extension: a novel, quantifiable method for detection of point mutations

SummaryWe have developed a novel technique that is useful for the rapid detection of previously characterized point mutations in the human genome. The method relies on the differential termination of primer extension being performed simultaneously on normal and mutant template molecules in the presence of a selectively limited complement of deoxynucleotide triphosphates. We have used this technique to determine the carrier status of two potential carriers of a hemophilia B mutation at codon 145 in the factor IX gene. In addition, the technique has been used to quantify the levels of mutant sequence in several tissues of a hemophilia B patient who is a somatic mosaic for a point mutation at codon 350.

Androgen effects on factor IX expression: in-vitro and in-vivo studies in mice.

A role for steroid hormones has been proposed for the post‐pubertal factor IX increment of ∼25% seen in both normal males and females, as well as in the post‐pubertal phenotypic recovery seen in haemophilia B Leyden. We have evaluated androgen receptor binding to the factor IX promoter and have assessed transcriptional activation of the factor IX gene in hepatocytes through transient transfection studies and through expression of factor IX in a murine model of androgen insensitivity. Whereas transfection of the androgen receptor alone did not activate expression from the factor IX promoter, co‐transfection with the CCAAT enhancer binding protein resulted in a synergistic 17‐fold enhancement of transcriptional activity. Using liver nuclear extracts and recombinant androgen receptor protein we have confirmed binding of this protein to the factor IX proximal promoter and disruption of binding with a mutation at nucleotide −26. Finally, studies in normal and testicular feminized male mice showed different developmental patterns of factor IX expression. In normal mice, expression recapitulates that seen in humans, with early post‐natal levels being ∼50% of the adult values and with a post‐pubertal increment of ∼25%. In contrast, testicular feminized animals did not show a significant post‐pubertal increment of factor IX. These studies provide further support for the role of androgen receptor binding to the factor IX promoter in regulating the developmental expression of factor IX.

Absence of allelic loss on chromosome 5q by RFLP analysis in preleukemia

Thirty-eight patients with various forms of myelodysplastic syndrome (MDS) were studied for the loss of restriction fragment length polymorphism (RFLP) heterozygosity on chromosome 5q as inferential support for the presence of a growth regulatory locus in this area of the genome. Conventional chromosomal analysis was performed in addition to RFLP studies of constitutive and granulocyte DNA using five polymorphisms from chromosome 5. Allelic loss in granulocyte DNA was identified in only one patient in whom monosomy 5 had already been defined cytogenetically. These results suggest that DNA sequence loss from chromosome 5q other than that observed cytogenetically is a rare event in MDS. Thus the potential involvement of a growth regulatory gene(s), from this area of the genome, in the leukemogenic process most likely involves a more subtle genetic change.