We analyzed the association of bleeding severity with eight candidate gene haplotypes within pedigrees of twelve index cases of VWD type 2 (six type 2M, three type 2A and three type 2B), using a covariance components model for multivariate traits (Mendel 5.5: QTL Association). In addition to the 12 index cases, these pedigrees included 58 affected and 55 unaffected relatives, as characterized by plasma Ristocetin cofactor (VWF:RCo), levels, VWF antigen (VWF:Ag) levels, ristocetin induced platelet agglutination (RIPA), VWF multimeric analysis and specific mutations of VWF. A bleeding severity score was derived from a detailed questionnaire. The severity of bleeding episodes was ranked from 0 to 5 in each of eleven bleeding categories: epistaxis, cutaneous symptomatic bleeding, bleeding from minor wounds, oral cavity bleeding, gastrointestinal bleeding, bleeding associated with tooth extraction, surgery, postpartum hematoma, muscle hematoma, hemarthrosis, and menorrhagia. Donors were genotyped using specific fluorescent-labeled oligonucleotides in a melting curve analysis (LightTyper, Roche, Inc.). VWF:RCo levels, followed closely by VWF:Ag levels, had the strongest influence on bleeding severity score. ITGA2 promoter haplotype -52T and ITGA2B haplotype 1 (Ile843) were each associated with increased bleeding severity scores (p=0.039 and p = 0.024, respectively), and the association of both remained significant when bleeding severity score was adjusted for age (p=0.012 and p = 0.010, respectively). These associations persisted even if menorrhagia, the only gender-associated bleeding category, was excluded from the analysis. This is the first report of a statistically significant association of ITGA2 haplotype -52T with risk for bleeding in vivo, a finding that is consistent with the significant attenuation of ITGA2 transcription by this promoter haplotype in in vitro studies. No statistically significant association was observed with the major haplotypes of six other candidate genes, GP1BA, ITGB3, GP6, VWF, FGB, and IL6. Increased plasma VWF:Ag levels were associated with VWF haplotype 1 (−1793G) (p=0.04), although the impact of this VWF haplotype 1 association was not of itself sufficiently strong to directly influence bleeding severity score.

These results establish that genetic differences in the expression or activity of integrin receptor subunits alpha-2 and alpha-IIb can influence the bleeding phenotype of VWD type 2. Together with the results of our previous studies, these findings demonstrate that platelet receptor gene haplotypes can influence bleeding tendency not only in VWD type 1 but also in VWD type 2. Interestingly, the influence of GP6 haplotype b (Pro219) on bleeding seen in VWD type 1 was not evident in this study of VWD type 2 pedigrees. Further studies on additional VWD type 1 and type 2 pedigrees, particularly pedigrees with diverse racial and/or ethnic backgrounds, will enable us to distinguish the variety and extent of genetic differences that can influence bleeding tendency in this disease.

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