ALBERT

Description: Abstract 3365 Background: A recent study suggested an increased rate of inhibitor development among Hispanic compared to White hemophilia A (HA) patients; however, a possible mechanism was not proposed or explored. In light of findings implicating a role for race-specific distributions of factor VIII (F8) and class-II human-leukocyte antigen (HLA-II) haplotypes on the disparate risk of inhibitor development in the Black HA population, we sought to determine the distributions of F8 and HLA-II haplotypes among Hispanic Americans and compare them to that of Black and White Americans. Methods: Using archived genomic DNA samples from the 194 unrelated founders of the San Antonio Family Heart Study (SAFHS), a collection of 1431 people from 41 large Mexican American families, we re-sequenced all exons of F8 to genotype the known nonsynonymous-single nucleotide polymorphisms (ns-SNPs) and identify any novel ns-SNPs. We then performed high-resolution HLA-II genotyping to identify each founders' pair of HLA-DRB1 alleles to compare them against those found in other ethnic groups. Results: Among the 291 potentially distinct Mexican American X-chromosomes evaluated, we identified the H2 F8 haplotype, defined by D1241E, in 25.0% of the subjects, which is in between that observed in the White (7.4%) and Black (37.4%) populations. We also found H3 F8, defined by D1241E and M2238V, in two subjects, who represent the first non-Black individuals reported to carry this haplotype. Furthermore, we discovered a previously unreported ns-SNP (H1919N), whose minor allele was found in only one male and defines a ninth wild-type F8 haplotype (H9). Regarding HLA-II alleles, the distribution among Mexican Americans in the present study was quite different from those found in Black and White individuals in the National Marrow Donor Program registry (see Figure). Three of the 4 most common HLA-II alleles in the Mexican Americans (DRB1*0802, DRB1*0407 and DRB1*1406) were seen in 〈 1% of both Blacks and Whites. Discussion: This is the first study to report the haplotypic prevalence of F8 and HLA-II alleles in Mexican Americans, the largest Hispanic ethnic group in the United States. By informing specific wild-type factor VIII (FVIII) peptides for use in HLA-II binding and T-cell stimulation assays, these results may help to identify high risk combinations of FVIII therapeutics and individual HLA-II repertoires that contribute to the higher rate of inhibitor development observed in Mexican versus Caucasian American HA patients. Disclosures: Viel: Histonis, Incorporated: Employment. Howard:Haplomics, Inc.: Equity Ownership.

Description: Recombinant factor VIII (r-fVIII) is the most widely-used and effective therapy for hemophilia A (hA). Many patients unfortunately become refractory when the wildtype(wt) protein is seen as foreign and is targeted by functionally neutralizing anti-fVIII antibodies termed inhibitors. While inhibitors occur most often in severe hemophiliacs with complex fVIII mutations and a complete circulating absence of the fVIII protein, recent studies show patients with missense-mutations (mMt) have a higher incidence than previously thought and demonstrate r-fVIII can be immunologically targeted even when differing by only a single amino acid. Because mMt represent the most frequent overall etiology of hA (~39% of cases), common non-hemophilic protein variants of fVIII may represent an important novel modulator of inhibitor development in this setting. To determine the extent of such variation, we resequenced all coding regions of the fVIII loci (F8) in 137 healthy subjects from 7 ethnic groups, including 86 Caucasians and 16 African-Americans (AA). We identified 5 common nonsynonymous single nucleotide polymorphisms (nsSNPs). Surprisingly, whereas 4 were polymorphic in AA only 2 were variable in Caucasians, despite having examined 6x the number AA X-chromosomes. Since recent studies show AA patients have a 2-fold higher inhibitor incidence than Caucasians, thus establishing ethnicity as a risk factor in this complication, we were intrigued to find that minor alleles for 2 nsSNPs are restricted to AA and substitute amino acids in major B-cell inhibitor epitopes located in the A2 and C2 domains. To confirm these findings and accurately define the number and frequency of human haplotypes (H) (eg. distinct combinations in which the alleles of these 5 nsSNPs are linked in vivo) we resequenced F8 in a second study group that included 75 additional healthy AA. Here we show there are at least 7 distinct wt forms of the human fVIII protein by defining 7 haplotypes (H) from the 5 nsSNPs: H1, H2, …, H7. H1 exists in all ethnic groups, is the most common overall form of fVIII and represents 2 of the 3 r-fVIII concentrates used clinically. While H2 is the most common form in AA (44%) and possibly represents the other therapeutic r-fVIII molecule, we found that at least 20% will have AA-restricted fVIII proteins; H4 (4%), H5 (12%) and H7 (4%). Only 2 forms of fVIII were found in Caucasians, H1 (90%) and H2 (10%), in contrast, and neither were ethnically restricted. In summary, when combined with reports of at least 3 other nsSNPs, our findings establish as inaccurate the long held view that fVIII is basically a monomorphic protein in non-hemophiliacs. We hypothesize that greater immunologic barriers exist when r-fVIII is infused into patients with mMt in endogenous fVIII molecules containing one or more minor alleles of these nsSNPs. Moreover, due to the number and frequency of AA-restricted wt fVIII variants, we predict these nsSNPs contribute pharmacogenetically to the higher incidence of inhibitors in this ethnic group. As such, we have established a 6-site multi-center study to test this hypothesis using AA hA patients as the optimal group. So far we have enrolled 34 of 223 total AA hA patients at the participating centers and are obtaining blood samples from each for fVIII:C and fVIII:Ag measurements and F8 mutation detection. Because the allelic basis of hA has not been studied in AA, this mutation scan is essential to rule out the plausible alternative hypothesis that their higher inhibitor incidence is due to a distinct spectrum of molecular abnormalities. Ultimately, all patients with mMt will be tested to determine the presence and titer of anti-fVIII, using both Bethesda and ELISA assays, and the H of their mutant fVIII will be defined. The number of patients with inhibitors and AA-restricted forms (H4, H5 or H7) will then be compared to those with inhibitors but whose mMt are in haplotypes found in r-fVIII molecules (H1 or H2).

Description: Abstract 3366 Background/Aims: The hemophilia A (HA)-causing Factor (F)VIII gene (F8) mutation type is a well-established determinant of risk for the development of alloimmune inhibitors that neutralize replacement FVIII proteins in ∼20% of all HA patients. Studies have investigated variants of immune response genes to determine if they may account for the inter-individual variability in FVIII immunogenicity observed in patients with the same F8 abnormality, e.g., the intron-22 inversion. While some studies have found associations between inhibitor status and promoter polymorphisms in CTLA4, TNFA and/or IL10, others have not. If these promoter polymorphisms are indeed functional and truly influence inhibitor development, their alleles could modulate transcription initiation rates. The goal of this study was to investigate the possibility of cis-acting genotype-specific differences in mean steady-state mRNA levels encoded by CTLA4, TNFA and IL10. Methods: We examined the relationship of lymphocyte CTLA4, TNFA and IL10 mRNA levels with the genotypes of 265 SNPs located across their structural loci in 1189 Mexican American subjects in the San Antonio Family Heart Study. Expression profiles were generated using Illumina's HWG-6 BeadChips and genotypes came from the Illumina OmniExpress-12 BeadChip. Measured genotype association analyses that accounted for non-independence of family members and employed an additive model (in which testing to determine whether gene expression varies by genotype, with the model constrained so that each “dose” of the minor allele raises or lowers gene expression by an equal amount “beta”) were performed using the software package SOLAR. P-values were calculated using a 1 degree-of-freedom chi-square test comparing the likelihood of a model where the change in expression levels by genotype is estimated to the likelihood of a model where beta is constrained to zero. Results: None of the 265 genotyped SNPs within or near these three genes (i.e., 49 SNPs in IL10, 35 SNPs in CTLA4 & 181 SNPs in TNFA) function as cis-acting regulatory variants, as no significant genotype-specific associations with these genes' transcript levels were identified. Conclusions: We observed no evidence for cis-regulation of CTLA4, TNFA or IL10 in Mexican Americans, the largest and most rapidly growing minority population in the United States, despite having genotyped directly the previously implicated promoter polymorphisms in the current analysis (e.g., see Figure). Although Hispanic American HA patients were recently found to have a significantly higher risk for inhibitor development than White HA patients, it is possible that cis-acting functional variants in this minority population are rare and not well-represented by the common GWAS SNPs used for these analyses. Since linkage disequilibrium patterns between markers are population-specific, we are also currently genotyping these SNPs in a large cohort of African and Caucasian American HA patients. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Factor (F)VIII immunogenicity is the main obstacle to both successfully treating hemophilia and receiving FDA approval for new therapeutics. Since immune responses to allogeneically distinct proteins require T-cell activation and proliferation, at least one foreign peptide must be liberated from FVIII that can bind with high affinity to one or more of a patient's HLA-class-II (HLA-II) isomers. Due to the complex pathogenesis of inhibitor development, which involves numerous genetic variations and both treatment and product related variables, our ability to predict which patients will become alloimmunized to FVIII remains suboptimal. The role of FVIII glycosylation in inhibitor development appears to be an important, yet not well characterized, modifying influence; defining the mechanism has been complicated by the fact that some FVIII products are highly glycosylated with thousands of potential glycoforms. Here we describe studies of the spectrum of FVIII peptides eluted from HLA-II complexes and the impact of glycosylation on the proteolysis and/or binding of these peptides to a patient's individual HLA-II isomers, i.e. to further incorporate personalized medicine in hemophilia care. Methods: Immature monocyte-derived dendritic cells (DCs) from 12 unrelated donors were generated in vitro and matured in the presence of an equimolar pool of a full length (FL) and 4 B-domain deleted (BDD) rFVIII proteins free of VWF and other proteins. Following harvest and lysis, HLA-DP, -DQ, and -DR molecules were recovered using a specific immunoaffinity step. Peptides were then eluted from these complexes and sequenced by high resolution mass spectrometry (LC/MS/MS). The bound peptides were mapped to the FVIII reference sequence. The estimated binding affinity of FVIII peptides to the HLA-II alleles found in these donors were obtained from NetMHCIIpan and compared to the peptides observed to be bound. Results: The 12 DC donors express 29 distinct HLA-II isomers (6 DP, 11 DQ, 12 DR) from an overall HLA-II gene repertoire having the following alleles: DP: 01:03/02:01, 01:03/03:01, 01:03/04:01, 01:03/04:02, 02:01/01:01, 02:02/05:01 DQ: 01:01/05:03, 01:02/06:02, 01:02/06:09, 01:03/05:01, 01:03/06:03, 01:03/06:04, 02:01/02:01, 02:01/02:02, 03:01/03:02, 03:02/03:03, 05:01/03:01 DR: 01:01, 03:01, 04:01, 04:04, 07:01, 09:01, 11:01, 11:04, 13:01, 13:02, 14:01, 15:01 These 29 HLA-II isomers were found to have 77 bound peptides that covered 1,202 of the 2,332 total amino acid residues in the NCBI reference FL-FVIII protein, whose domain structure and consensus N-linked glycosylation (NLG) sites are shown in panel A of the Figure. The HLA-II bound peptides were mapped to the FVIII regions from which they derive (panel B). One of the 20 NLG sites known to be glycosylated and 3 of the 4 known non-glycosylated NLG sites were found to be in a bound peptide. A NetMHCIIpan analysis predicted that no FVIII nonamer containing a consensus NLG site binds strongly to any of the 12 DR isomers; but, it also clearly showed that more peptides bind with weak or strong affinity as peptide length increases (data not shown). Conclusion: Despite finding 3 of FVIII's 4 non-glycosylated consensus NLG sites in the tightly bound HLA-II/peptide complexes, only 1 of the 20 glycosylated NLG sites was found among these bound peptides. The near complete absence of N-linked glycans in the presented HLA-II bound peptides is compelling, but incomplete, evidence that glycosylation influences the immunogenicity of FVIII. Potential mechanisms may include effects on internalization, proteolysis and/or HLA-II binding. Further direct studies are required to determine if these post-translational modifications affect proteolysis and/or HLA-II binding; core peptides that can be proteolyzed and/or bound in the absence of glycosylation or with alternative glycan conformations may provide more evidence that glycosylation can modulate immunogenicity. We hypothesize that HLA-II bound and unbound FVIII peptides constitute a novel immune-response-related biomarker that will improve the accuracy of inhibitor risk prediction by allowing pertinent patient-specific pharmacogenomic inputs to be analyzed in a more biologically relevant manner. References: Karosiene et al. NetMHCIIpan-3.0, a pan-specific MHC-II prediction method including all 3 human MHC-II isotypes: HLA-DR, -DP and -DQ. Immunogenetics, 2013. Disclosures Hofmann: CSL Behring: Employment. Zollner:CSL Behring: Employment. Powell:CSL Behring: Employment. Maraskovsky:CSL Behring: Employment. Howard:Baxter: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Haplomics, Inc.: Patents & Royalties; CSL Behring: Consultancy, Honoraria, Research Funding.

Description: Regardless of advances in prenatal diagnosis, carrier detection and gene therapy for hemophilia-A, new patients with bleeding diatheses due to inadequate plasma FVIII activity (FVIII:C) levels will still require specialized management at treatment centers. In the ‘post-genome era’ the possibility exists for personalized medicine, in which an individual’s genetic information will be used to tailor prophylactic and/or treatment regimens that will optimize patient outcomes. As listed in the HAMSTeRS database, ~1,000 distinct loss-of-function F8 variants, representing all mutation types including inversions, insertion/deletions and single nucleotide substitutions (SNSs), have been associated with deficiencies of FVIII. To estimate how soon a complete catalogue of every possible mutation affecting FVIII:C levels may become available, we first determined the theoretical number of potential missense and nonsense F8 alleles, whether loss-of-function or not, based on each possible SNS in the coding region as compared to the reference sequence. While the impact of a missense change on FVIII:C, if any, is not always obvious, in contrast to premature-termination codons (PTCs), which are almost always deleterious, findings from a recent resequencing study raises the possibility that non-hemophilic structural differences between a patient’s endogenous FVIII protein and the infused “wildtype” molecule may increase risk of alloimmunization during replacement therapy. Wildtype FVIII contains 2,351 amino acid (aa) residues: 2,332 in the mature protein and 19 in the signal peptide (SP). Appropriate SNSs within the codons for 793 of these residues would create a PTC (UAA, UAG, UGA). Since three distinct base substitutions are possible at each of the three codon positions, 996 nonsense alleles could theoretically arise naturally. As only 123 distinct nonsense mutations are listed in HAMSTeRS,

Description: INTRODUCTION Mass spectrometry (MS) is a potentially useful tool for the study of the hemostasic system and its imbalances that lead to bleeding and thrombotic disorders. By harnessing the high throughput and broad scope of MS, vast data may be available to investigators and clinicians to help predict or manage hemostatic events. Although utilizing MS to evaluate coagulation proteins appears promising, amino acid (AA) substitutions resulting from genetic variation may yield a spectrum of mass-to-charge ratios (m/z) that can impede accurate protein identification. The goals of this study were to describe 1) the proteins present in a blood sample that might be involved in or otherwise affect coagulation, 2) the realm of variations that might occur with a single nucleotide substitution (SNS) in the reference coding sequences of these proteins, and 3) the variation of peptide fragments of these proteins when only one of the nucleotides is a variant. METHODS We obtained protein lists from the NCBI BioSystems database for the terms: Blood Clotting Cascade, Complement Cascade, Formation of Fibrin Clot, Hemostasis, Platelet Activation, Platelet Aggregation Plug Formation, Platelet Degranulation, Platelet Homeostasis, and Thrombin Signaling (e.g., http://www.ncbi.nlm.nih.gov/biosystems/198840). We linked the Symbol (gene) to the CCDS ID (consensus coding sequence, http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi ). For each nucleotide, we enumerated the effect of a SNS relative to the other three nucleotides. We then generated every peptide of length 5-20, determined the change in mass based on the average, as opposed to the monoisotopic, mass of the substituted AA, and assessed whether the peptide was unique among those in the system. Finally, we determined whether possible N-linked glycosylation sites were preserved, destroyed, or created by SNS. We considered a putative site N[^P][S|T], that is N in position 1, not P in position 2, and either a S or T in position 3. RESULTS The proteins in the biosystems that were also in the CCDS database comprised 517 distinct Symbols and 951 distinct CCDS ID's, comprising 2,180,352 codons. The duplicate Symbols include transcript variants, for instance, the Symbol F8 linked to CCDS ID's 35457.1 and 44026.1, thereby diminishing the uniqueness of the peptides (transcript variants share some, if not most, of the reading frames). All of the codons were susceptible to an AA substitution; at least one variant nucleotide substation in position 1 or 2 of the codon always resulted in an AA substitution. SNS caused premature termination signals (stop codons) in 240,100 of these codons. Table 1 details the variations. A map of the N-glycosylation sites is available for each protein, although this may not affect MS directly. Of the 83,510 potential N-Linked Glycosylation sites, a SNS disrupted the putative AA sequence in 53,067 (64%). A SNS created a novel potential N-Linked Glycosylation site at 52,787 loci. Table 1. Wild-type Only Wild-type and Variants Peptide Length Peptides Distinct Peptides Peptides Distinct Peptides Relative Change in Mass 5 722,174 292,485 24,470,567 2,464,873 0.052250 10 717,564 350,570 47,911,535 20,982,641 0.025968 15 712,954 355,446 71,052,562 31,777,153 0.017273 20 708,344 357,177 93,893,423 42,465,377 0.012939 CONCLUSIONS Variant peptides due to a single SNS per peptide greatly outnumber wild-type peptides. The ability to identify a protein based on uniqueness of one of its peptides increases as the peptide size increases, but AA variations in those peptides that arise from one SNS will require 1) increased mass resolution and 2) both a search algorithm and database that accounts for the possible variations. Patients with hemostatic or thrombotic disorders may be more likely to have a variant, and these results highlight the need to know the genetic sequence associated with proteins being analyzes by MS if this technology is to be adopted for research and clinical purposes. The inclusion of currently identified SNPs and the effect of INDELs that preserve the reading frame is ongoing. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4422 Background/Aims: FVIII gene (F8) mutation type is a risk factor for FVIII inhibitors, which develop in ~20-30% of hemophilia A patients following FVIII replacement therapy. Studies have investigated immune response genes to determine why FVIII is immunogenic in some, but not all, patients with the same F8 abnormality. While some studies have found associations between inhibitor status and promoter polymorphisms in CTLA4, TNFA, and/or IL10, others have not. If these promoter polymorphisms influence inhibitor development, their alleles could modulate transcription initiation rates. The goal of this study was to investigate cis-acting genotype-specific differences in mean steady state mRNA levels. Method: We examined the relationship of lymphocyte CTLA4, TNFA, and IL10 mRNA levels with the genotypes of 49 SNPs located across their structural loci in 1189 Mexican American participants of the San Antonio Family Heart Study (SAFHS). Expression profiles were generated using Illumina's HWG-6 BeadChips and genotypes came from the Illumina Human HapMap 550 SNP panel. Measured genotype association analyses that accounted for non-independence of family members and employed an additive model were performed using the software package SOLAR. Result: Except for C(-13780)A, located upstream of CTLA4, which was modestly associated with CTLA4 mRNA levels (p

Description: The correlation between vitamin K-dependent coagulation factors (CF) and lipids has been previously established, with Factor VII notably receiving attention in the investigation of atherosclerosis. Current evidence suggests that plasma phospholipids (PL) may enhance the procoagulant reactions and may increase the availability of fat soluble vitamin K. PL may also affect the clearance of CF. There appears to be a concomitant decrease in CF as the result of lipid lowering therapies. While investigating single nucleotide polymorphisms (SNP) of the Factor IX (fIX) gene as potential quantitative trait loci (QTL) for fIX activity level (fIX:C) in the GAIT study, we observed an apparent relationship between two SNPs, G-793A and C-698T, with plasma cholesterol concentration. As expected, a similar relationship existed with low density lipoprotein (LDL) concentrations. In the current study, we examine these fIX promoter SNPs, which exhibit near perfect linkage disequilibrium (ρ=0.998, p