ALBERT

Description: Abstract 2116 Plasma levels of von Willebrand Factor (VWF) are highly variable among healthy people and ~70% of this variability is heritable. Elevated levels of VWF are associated with thrombosis while low levels of VWF cause the common bleeding disorder, von Willebrand Disease (VWD). The genome-wide association study (GWAS) is a powerful tool for identifying common genetic variants contributing to complex traits, though less common variants are likely to be missed by this approach. We performed combined GWAS and linkage analysis in a cohort of healthy young college students. The study was approved by the University of Michigan IRB (IRBMED #2005-0080). 1189 healthy individuals (14-35 yrs of age) were recruited, comprising 507 sibships. VWF levels were determined by alphalisa assay (mean 109%, range 26 – 410%). VWF level heritability (h2) was 73% among siblings and 82% among monozygotic twins. Samples were de-identified and DNA was extracted from peripheral blood, quantified and genotyped using the Illumina Omni-1 quad SNP chip at the Broad Institute (MIT, Boston). 1182 individuals passed genotyping quality control with 〉97% call rates for 979,855 SNPs. Genotyping quality control was performed as part of the NIH's GENEVA program. GWAS on log transformed, age and sex adjusted VWF levels identified a major peak over the ABO locus (p

Description: Abstract 274 The ABO(H) carbohydrate blood group system is highly polymorphic in humans, with 〉200 ABO alleles described to date. The ABO gene encodes the glycosyltransferase, ABO, which has two main isoforms which generate blood group A or blood group B. Blood group O is the result of a non-functional ABO glycosyltransferase. ABO blood group is commonly imputed in genome-wide association studies where ABO is associated with phenotypes such as von Willebrand Factor level or myocardial infarction. We sought to determine the accuracy of a common ABO genotyping algorithm in a repository of 1092 individuals from a healthy sibling cohort study, the Genes and Blood Clotting Cohort (GABC). We utilized three published single nucleotide variants (SNVs) to assign ABO blood group (A2, B, and O) relative to the A1 reference allele: rs8176704 (G/A) to annotate A2, rs8176749 (C/T) to annotate B, and rs687289 (A/G) to annotate O. We detected ABO glycans in red blood cell-rich buffy coat samples using an adaptation of published ABO forensic dried blood spot techniques. Briefly, samples were diluted and applied to a nitrocellulose membrane, blotted with murine monoclonal anti-A (Immucor) or murine monoclonal anti-B (Immucor) antibodies followed by and streptavidin-conjugated donkey anti-mouse IgG HRP secondary (Jackson ImmunoResearch). Blots were developed, imaged using ImageQuant 350 (GE), and scored semi-quantitatively by two blinded independent observers relative to red blood cell-rich buffy coat reference panels from normal blood donors. Comparison of ABO imputed genotype to detected ABO glycans in red blood cell-rich buffy coat identified discrepancies in the predicted major ABO blood group assignment (i.e. blood group O, A, B, or AB) in 4.4% of individuals. Interestingly, A glycan density was generally low in individuals with a discrepant A phenotype, suggesting that these individuals harbor a less robust blood group A enzyme variant. There was no common pattern for genotype-phenotype discrepancies. Multiple types of errors (both presence and absence of ABO glycans) were observed, and divergent patterns of ABO glycans were observed between homozygotes for ABO SNVs, suggesting multiple ABO alleles or other genetic modifiers underlie the discordant calls. Interrogation of the Exome Variant Server (http://evs.gs.washington.edu/EVS/) found one-third of ABO nonsynonymous polymorphisms in the exome data set are novel, indicating a large proportion of the genetic variation at ABO has not been previously described. Furthermore, the majority of nonsynonymous variants in the EVS database for the genes encoding H antigen (the ABO acceptor structure), FUT1 and FUT2, were also novel. In summary, ABO(H) is more variable genetically than previously suspected. A commonly applied ABO SNV genotyping algorithm resulted in 4.4% frequency of major ABO blood group glycan discrepancy as detected in red blood cell-rich buffy coats, likely due in large part to the underlying genetic diversity at ABO. Further work is needed to deeply characterize the genetic diversity at ABO, FUT1, and FUT2 and correlate the spectrum of allelic variants with ABO(H) carbohydrate phenotypes. Such high resolution ABO and H classification would likely advance our understanding of the complex physiologic processes influenced by ABH blood groups, including disorders of hemostasis and thrombosis, vascular disease, infectious disease susceptibility, complications of pregnancy, and allogeneic exposures. Disclosures: Ginsburg: Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children's Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Description: Abstract 3313 Plasma VWF levels vary by approximately five-fold in healthy populations and are influenced by both environmental and inherited factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis. Increased levels of VWF occur with age, may rise acutely due to inflammation or infection, and may serve as a biomarker for endothelial dysfunction and atherosclerosis. Genetic factors are thought to account for 60 – 70% of the variance in VWF antigen levels with ∼30% of this effect due to ABO blood type. VWF multimers are assembled in the Golgi apparatus where the VWF propeptide is cleaved to form mature VWF. Because the half life of plasma VWF propeptide (ppVWF) is ∼5X faster than the mature VWF antigen, the ratio of ppVWF/VWF:Ag in plasma primarily reflects clearance rates of mature VWF while alterations in biosynthesis and/or secretion of VWF affect the level of both ppVWF and VWF:Ag. In order to identify additional quantitative trait loci (QTL) for VWF antigen variation and altered clearance of VWF:Ag, we performed genetic analyses in a healthy young sibling cohort, the Genes and Blood Clotting Study (GABC, n=1,152). The cohort was SNP genotyped with an Illumina Omni-1 array resulting in 763,195 SNPs available for analysis after extensive QC. Plasma VWF antigen (VWF:Ag) and VWF propeptide levels were determined by AlphaLISA (Perkin Elmer, Waltham, MA), using polyclonal anti-human VWF antibodies (DAKO, Glostrup, Denmark) or monoclonal anti-ppVWF antibodies (Blood Center of Wisconsin, Milwaukee, WI). The median levels were 108.1 IU/dL, 99.97 IU/dL and 1.0 for VWF:Ag, ppVWF and ppVWF/VWF:Ag respectively. The estimated heritability (h2) was 64.5%, 55.72%, and 64.26% for VWF:Ag, ppVWF, and ppVWF/VWF:Ag respectively. A genome-wide association study (GWAS) for VWF:Ag in this cohort revealed significant signals at the ABO and VWF loci, consistent with previously published reports. However, GWAS for ppVWF levels demonstrated a signal only at 12p13, overlying the VWF gene itself. The top SNP, rs1800378 (beta 4.297, p-value 1.58E–11), encodes a nonsynonymous substitution (His484Arg) in the VWF propeptide. In contrast, GWAS for ppVWF/VWF:Ag showed a strong signal at 9q34, the ABO locus, (top SNP rs687289, beta −0.2131, p-value 1.88E–62) and no significant signal at 12p13. These results suggest that ABO is the major common determinant of variability in VWF clearance rates and a common SNP, rs1800378, is associated with VWF propeptide levels. We next utilized the sibling structure in this cohort to perform linkage analyses. Linkage analysis for VWF:Ag identified a novel locus at 2p12-2q13 in addition to the expected signal at ABO. The effect size of this chromosome 2 locus on VWF variation (19.2% variance explained) was comparable to the effect of the ABO locus (24.5%). Linkage analysis for ppVWF demonstrated novel loci at 7q21 (LOD score 5.03) and at 11q21 (LOD score 4.12), with no significant signal on chromosome 2. In contrast, linkage analysis for ppVWF/VWF:Ag identified signals at 2p12 (LOD score 3.17), 8p22 (LOD score 3.48) and 11q22 (LOD score 3.41). Taken together, these results identify a number of potential candidate loci for genetic regulation of plasma VWF through alteration of synthesis, storage, secretion or clearance mechanisms. We hypothesize that signals present in linkage but not association are due to VWF regulatory genes with high allelic heterogeneity, harboring many genetic variants that are individually rare, but in aggregate present in a large portion of sibships. Segregation of these variants among sibs could then explain the strong signal by linkage, but lack of signal by association in unrelated individuals. Replication of the strong 2p12-2q13 signal in both VWF:Ag and ppVWF/VWF:Ag linkage studies suggests that the responsible variants at this locus affect VWF levels through altered clearance. The novel QTLs for ppVWF/VWF:Ag levels on chromosome 8p22 and 11q22 are also likely to work through altered clearance mechanisms while the novel signals for ppVWF levels on 7q21 and 11q21, not significant in the VWF:Ag linkage study, may affect efficiency of cleavage or clearance of ppVWF. Identification of the underlying genes at these novel loci and their mechanism of action could lead to improved understanding of the severity, penetrance and treatment for von Willebrand Disease as well as the risk for venous thromboembolic disease associated with elevated VWF. Disclosures: Ginsburg: Shire Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children's Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Description: von Willebrand disease (VWD) type 1 is characterized by incomplete penetrance and variable expressivity. Individuals with very low levels of VWF (〈 20 IU/dl) typically have mutations in the VWF gene. However, no such mutations can be identified in a significant subset of type 1 VWD patients, particularly those with VWF levels 〉 30 IU/dL. ABO is the best-characterized genetic modifier of VWF levels, accounting for up to 30% of the genetic variability and blood type O is overrepresented in patients with VWD type 1. VWF is highly polymorphic with over 1,100 reported single nucleotide variants (SNVs). The impact of many of these changes remains unknown. We hypothesize that common variants in VWF affect VWF levels and may play a role in the diagnosis of VWD type 1. We genotyped 94 tagging SNPs through VWF in a large Amish family (n=445) and study their association with VWF levels. We performed single SNP association tests using age, gender and ABO status as covariates and multiple testing corrections were implemented. We observed significant associations (p value ≤ 0.0005) between VWF levels and twelve tagging SNPs: rs2362479 (telomeric to VWF), rs10849363 and rs7964554 (intron 4); rs216308 (intron 24); rs216310 and rs216311 (exon 28); rs11612370 and rs11612384 (intron 33); rs216330, rs11064003, and rs2239162 (intron 37); and rs216293 (intron 38). To replicate the association of these SNPs with VWF levels in a larger Caucasian population, we conducted association analyses in three population-based cohorts of healthy individuals: 1) the Zimmerman Program for Molecular and Clinical Biology of VWD (ZPMCB-VWD) control cohort (n=150), 2) the Genes and Blood-Clotting Study (GABC) cohort (n=934) and 3) the Trinity Student Study (TSS) cohort (n=2,145). All association analyses were modeled including age, gender, rs687289 (a proxy marker of blood group O) and adjusted for multiple testing. Three SNP associations were replicated in the ZPMCB-VWD control cohort. An additional four SNP associations were replicated in the ZPMCB-VWD control and TSS cohorts. Associations of the intron 37 variants rs11064003 and rs2239162 with VWF levels were replicated in all cohorts and reached genome wide significance with p values of 1.34 x 10 -11 and 7.35 x 10 -15 respectively in the TSS cohort. We then genotyped the 12 candidate SNPs that showed significance in the original family in a cohort of 310 Caucasian VWD type 1 index cases (ZPMCB-VWD). We compared the minor allele frequency (MAF) of the tagging SNPs between the ZPMCB-VWD control cohort (n=150) and the ZPMCB-VWD type 1 index case cohort (n=310). The minor allele of rs7964554 (associated with lower VWF levels) was more common (46.2%) in patients with VWD type 1 than in controls (34.0%), and that difference was statistically significant (p=0.0005). The minor alleles of rs11612370, rs11612384, rs11064003, and rs2239162 (all associated with higher VWF levels) were less common (p=0.018, 0.009, 0.012, and 0.0004, respectively) in VWD type 1 index cases compared to controls. In this population if the subject has VWD type 1, then the odds of being heterozygous for the minor allele of rs7964554 were 1.47 (95% CI: 1.08, 2.00; p=0.014). Similarly, the odds of being heterozygous for the minor allele of rs2239162 were 0.64 (95% CI: 0.45, 0.91; p=0.014). In summary we have identified several common variants in VWF that have a strong effect on VWF levels and more importantly are either under or over represented in patients with VWD type 1 indicating that may influence its diagnosis. Disclosures: Shapiro: Baxter: Consultancy, Global steering committees Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Global steering committees, Global steering committees Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Inspiration: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL Behring: Research Funding; Biogen Idec: Research Funding. Di Paola:CSL Behring: Consultancy; Pfizer: DSMB, DSMB Other.

Description: Homeostatic processing of von Willebrand factor (VWF) in the circulation relies on proteolysis of ultra-large VWF (ULVWF) multimers by the metalloprotease ADAMTS13. Deficiency in ADAMTS13 results in Thrombotic Thrombocytopenic Purpura (TTP) due to the accumulation of thrombogenic ULVWF. ADAMTS13 is secreted into the circulation in its active form and has no known inhibitor. Regulation of ADAMTS13 activity occurs in part through plasma ADAMTS13 levels and by the hemodynamic exposure of the scissile bond in VWF. To identify the genetic determinants of ADAMTS13 levels we performed genome-wide association studies (GWAS) and linkage analysis in two independent cohorts of young healthy individuals. This study recruited 3206 young healthy individuals of European descent who participated in the Genes and Blood Clotting Study (GABC, n=940) and the Trinity Student Study (TSS, n=2266). All subjects provided written informed consent and all genotyping and phenotyping was performed on de-identified samples. Details of the genotyping and data cleaning process for these cohorts were previously described. (Desch KC et al. PNAS 2013, 110:588-593) ADAMTS13 levels were measured by a custom AlphaLISA (Perkin-Elmer, Waltham, MA) assay using polyclonal anti-ADAMTS13 antibodies (a gift from X. Long Zheng, University of Pennsylvania). In GABC, the median ADAMTS13 levels were 115.4 IU/dL and demonstrated a 2.0-fold variation between the 5th and 95th percentiles. Heritability of ADAMTS13 levels was estimated at 77% in GABC and 43% in the TSS. ADAMTS13 levels were associated smoking in both cohorts and was associated with an 8.9% increase in levels of ADAMTS13 (P 〈 1.5E-11, GABC). GWAS identified 10 SNPs in GABC and 33 SNPs in TSS that were significantly associated with ADAMTS13 levels (P

Description: Background: ABO(H) is a carbohydrate blood group system expressed in multiple tissues including red blood cells, blood vessels, and mucosal surfaces. ABO is the largest known genetic modifier of plasma VWF level (VWF:Ag). It has been hypothesized that the effect of ABO on VWF is mediated by H glycan density. FUT2, the gene underlying Secretor phenotype, encodes a glycosyltransferase synthesizing H antigen in mucosal tissues, and variation in the gene has also previously been associated with VWF:Ag, but past studies have been conflicting. To clarify these relationships, we studied the relationship between VWF:Ag, ABH glycans, and FUT2 genotype. Methods: The primary study group was a representative cohort of US blood donors from the Retrovirus Epidemiology Donor Study (REDS, N=499). A validation cohort of unrelated individuals was created from the Genes and Blood Clotting Study (GABC), healthy siblings between ages 14 and 35 years from University of Michigan, Ann Arbor, by choosing a random individual from each family (N=488). VWF:Ag was determined by ELISA in platelet poor plasma. Forensic techniques were adapted to detect ABH glycans in whole blood (REDS) or RBC-rich, frozen buffy coat (GABC). A and B glycans were detected using anti-A or anti-B (Immucor). A biotinylated Ulex europaeus agglutinin (UEA lectin, Vector Labs) was used to detect H. Relative A, B, and H antigen density was quantified on dot blots with ImageQuant (GE). In REDS, functional FUT2 alleles (Secretor) were determined by Sanger sequencing of FUT2 exon 2. FUT2 copy-number was assayed with real-time quantitative PCR. In GABC, FUT2 genotypes were determined through SNP genotyping (Illumina). In REDS, genotype data was phased (BEAGLE) to identify functional haplotypes. In GABC, functional alleles were inferred from the genotypes of two SNPs (rs601338, rs1047781) that determined secretor status in nearly all samples in REDS (see below). Multivariate regression was applied to VWF:Ag as a function H antigen density and O vs. non-O blood group, both separately and within the same model. All models were adjusted for age, gender, and self-reported ethnicity. Results: ABO blood group frequencies in both cohorts were similar to that of the US population. VWF:Ag differed significantly between ABO blood groups with lower values in blood group O versus non-O (REDS: ratio = 0.75, 95% CI [0.71, 0.80], p

Description: Deep vein thrombosis and pulmonary embolism, collectively referred to as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Genetic factors account for 50-60% of VTE risk and a recent meta-analysis of genome-wide association studies confirmed that common variants in F5, ABO, and seven other loci are associated with VTE. Rare mutations in the anticoagulant genes PROC, PROS1 and SERPINC1 have been linked to VTE in family studies. In order to identify new genetic variants altering the risk for VTE, we performed whole exome sequencing (WES) in 373 unrelated individuals of European ancestry with unprovoked VTE and compared results to a previously sequenced control cohort of 5784 unrelated Europeans. To avoid variant calling bias, only SNVs from exons with 〉10X coverage and less than 5% difference in coverage between cases and controls were included, removing 11,813 of 188,689 intervals. We used an emerging framework for a "collapsing" analysis on genes, defining qualifying variants on the basis of annotation and minor allele frequency

Description: The conversion of plasminogen (PLG) to plasmin is a critical step in the activation of the fibrinolytic system. Due to its broad substrate specificity, plasmin plays a pleotropic role in the circulation and extravascular space, controlling the degradation of fibrin clots as well as potential functions in regulating cell migration and angiogenesis. PLG activation is regulated through the interaction of tPA or uPA with plasminogen activator inhibitor type one (PAI-1). Genome-wide association studies (GWAS) have identified polymorphisms associated with the plasma levels of tPA and PAI-1, but to date, no studies have directly examined the control of PLG levels. In order to identify genetic determinants of PLG levels we performed GWAS in a cohort of 3244 young healthy individuals (557 sibships) who participated in the Genes and Blood Clotting Study (GABC) or the Trinity Student Study (TSS).(Desch KC et al. PNAS 2013, 110:588-593) All subjects provided written informed consent. Details of the genotyping and data cleaning process for these cohorts are as previously described. (Desch KC et al. PNAS 2013, 110:588-593) PLG levels were measured in both cohorts by AlphaLISA (Perkin-Elmer, Waltham, MA). Levels were natural log transformed and adjusted for participant age, gender and BMI (TSS only) prior to use in heritability estimates and GWAS, which were performed using the PLINK, EMMAX, MERLIN, GCTA and METAL software packages. The median PLG levels were 101.7 IU/dL and 104.7 IU/dL in the GABC and TSS cohorts respectively and had a 1.7 fold variation between the 5th and 95th percentiles. The narrow-sense heritability using intra-class correlation was 59.7% based on 1277 siblings in GABC and TSS, which was similar to the estimate of 60.2% based on the genotype data and the MERLIN software package. GWAS for PLG in the European only subset of GABC (n=940) did not reveal any genome-wide significant signals. However, GWAS for PLG levels in TSS (n=2304) revealed 9 significant SNPs on chromosome 6q26 at the PLG (plasminogen) and LPA (lipoprotein A) locus that together explained 7.8% of the variance in PLG levels according to the GCTA method, Figure 1. The top SNP was rs4252129 (p = 3.47E-25) which results in a single amino acid substitution (R523W) in PLG. The next strongest associations were with two SNPs near LPA, rs1084651 and rs783149 (p = 1.60E-13 and 1.07E-12 respectively). These SNPs are located between LPA and PLG (Figure 1). To determine if the LPA SNPs were independent from the top PLG SNP, a conditional analysis was performed using rs4252129 as a covariate. Rs1084651 and rs783149 remained significant (p= 7.73E-16 and 6.48E-15 respectively), verifying that they represent a second, independent association signal. Meta-analysis of both cohorts (using 723,715 common SNPs) confirmed the 6q26 signal and revealed another significantly associated region on 19q13.41, Figure 2. Two SNPs, rs10412972 and rs11084102 (p = 2.16E-09 and 1.14E-08, respectively) are intergenic variants 4.8 and 12.6 Kb downstream from SIGLEC14. The significant SNPs in the meta-analysis, 6 on 6q26 and 2 on 19q13.41, explained 5.8% of the variance for PLG. The top SNP in the TSS GWAS, rs4252129, had a minor allele frequency (MAF) of 1.4% in GABC and was not included in the GABC GWAS or meta-analysis. In summary, our data identify two loci (at PLG/LPA and SIGLEC14) that harbor genetic variants contributing to the variation of plasma PLG levels in healthy young adults. The strongest association was observed for a non-synonymous SNP in the PLG gene itself, with a MAF of 1.3% in the HapMap CEU population and 2.2% in TSS. The minor allele (W523) is associated with a 14.1% decrease in mean plasma PLG level. This low frequency, non-synonymous SNP, or another SNP in LD with it, may be the functional mutation leading to decreased PLG levels through unknown mechanisms. Whether the significant SNPs near LPA affect lipoprotein A or PLG is not clear. However, the association with causal variants at LPA is possible and consistent with previous biochemical studies demonstrating competitive inhibition of PLG binding to endothelial surfaces by lipoprotein A, or an effect of lipoprotein A on endothelial PAI-1 expression or the PAI-1 interaction with tPA/uPA. Finally, these studies identified two significant SNPs near SIGLEC14. This gene encodes a sialic acid lectin type receptor protein, closely related to SIGLEC 5 recently implicated in the clearance of von Willebrand factor. Disclosures: Ginsburg: Shire plc: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaeuticals: Membership on an entity’s Board of Directors or advisory committees; Catalyst Biosciences: Membership on an entity’s Board of Directors or advisory committees; Merck pharmaceuticals: Consultancy; Boston Children's Hospital: From Baxter to Children's Hosp (VWF), From Baxter to Children's Hosp (VWF) Patents & Royalties; University of Michigan: From Baxter to University of Michigan (ADAMTS13) Patents & Royalties.