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Intron-22-inversion patients express the entire Factor VIII (FVIII)-amino-acid sequence intracellularly as 2 non-secreted polypeptides and have a positive “intracellular (I)-FVIII-CRM” status. Mutations conferring a positive I-FVIII-CRM status are associated with low inhibitor risk and are pharmacogenetically relevant because inhibitor risk may be affected by the nature of the therapeutic FVIII-protein (tFVIII), the affinity of any tFVIII-derived foreign peptide (tFVIII-fp) for any HLA class-II isomer (HLA-II) comprising individual major histocompatibility complex (MHC) repertoires, and the stability of any tFVIII-fp/HLA-II complex. We hypothesize that mutations conferring a completely or substantially negative I-FVIII-CRM status are pharmacogenetically irrelevant because inhibitor risk is high with any tFVIII and individual MHC repertoire.

Background Although autosomal recessive hematologic disorders are individually rare and difficult to ascertain, studies involving one or more homozygous affected children and their unaffected heterozygous parents have led to expanded understanding of known and discovery of previously unknown processes. The son and daughter of two Salvadoran parents were diagnosed with congenital thrombotic thrombocytopenic purpura (cTTP) at 6 and 2 years of age, respectively, after presenting with fever, respiratory symptoms, hemolytic anemia, and thrombocytopenia and being found to have ADAMTS13 activities Methods Prior to a plasma infusion, blood samples were collected from the children and parents. Genomic DNA was isolated for polymerase chain reaction (PCR), and direct Sanger sequencing of all ADAMTS13 exons and flanking intronic segments was performed; all variants identified were confirmed by bidirectional sequencing of a second, independently generated amplicon. Total RNA was isolated and the steady-state level of ADAMTS13 mRNA was measured using a quantitative real-time PCR (q-RT-PCR)-based assay. ADAMTS13 was characterized enzymatically using the fluorogenic FRETS-VWF73 substrate and antigenically by ELISA. Results Both children were found to be homozygous and parents to be heterozygous for the previously described, cTTP-causing ADAMTS13 single-base-substitution mutation 20506C>T, a missense mutation that encodes cDNA-nucleotide 2518 (c.2518C>T) and ADAMTS13 residue 692 (692Arg>Cys [692R>C]) (Fig. A). As expected, the children's ADAMTS13 antigen and activity levels were undetectable, although notably, steady-state levels of the ADAMTS13 mRNA were >2.5-fold higher in the daughter than in the son. The re-sequenced regions of the ADAMTS13 loci segregating within this family contained 26 additional SNVs, seven of which were nonsynonymous (ns) including two previously unreported ns-SNVs: 27852C>T (c.3362C>T; 972Arg>Trp) and 33325G>A (c.3733G>A; 1096Arg>His) (Fig. A, left panel). The parents' genotypes differed at nine positions, including three ns-SNVs, creating two distinct, non-mutant haplotypes (designated I and III) at the gene, mRNA and protein levels. The q-RT-PCR assay revealed >4-fold higher steady-state mRNA levels in the father compared to the mother (p Discussion We capitalized on the fortuitous finding of children with complete homozygosity across ancestrally-related ADAMTS13 alleles harboring a null-type, loss-of-function mutation, as this enabled the substantially different levels of gene expression and function observed in the parents to be attributed to their two previously unreported, SNV-based, ADAMTS13 haplotypes. Additional investigation at the molecular, biochemical, cellular, and organismal levels will be necessary to determine which of the myriad potential individual SNV- and/or haplotype-based mechanisms are responsible for the observed parental differences in circulating ADAMTS13 antigen and activity. Disclosures: Kim: Haplomics, Inc.: Membership on an entity’s Board of Directors or advisory committees; Baxter: Honoraria. Marder:Baxter: Research Funding. Howard:Haplomics, Inc.: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Baxter: Research Funding.

Abstract 3349 Introduction: The protease ADAMTS13 (A Disintegrin and Metalloprotease with Thrombo Spondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder Thrombotic Thrombocytopenic Purpura (TTP). TTP has been in particular observed in transplant patients following treatment with Cyclosporin A (CsA), a drug which is frequently administered in patients to suppress allogeneic immune response. However, a mechanistic explanation for such a link has remained unclear. Here, we report that cyclophilin-B mediated peptidyl-prolyl cis–trans isomerase (PPIase) activity, which is also targeted by CsA, plays an important role in the production of active ADAMTS13. Methods and Results: An in vitro transient expression system was used to study the effects of CsA on the synthesis and activity of ADAMTS13. We found that exposing HEK293 cells to CsA results in a dose-dependent reduction in the level of secreted ADAMTS13, but not the ubiquitously-expressed housekeeping protein Hsp70. Moreover, treatment with other immunosuppressive drugs, such as rapamycin or tacrolimus/FK506 did not impact the secretion of ADAMTS13. CsA is a known inhibitor of several cellular substrates, including cyclophilin B (CypB), which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. We show a direct, functional interaction between CypB and ADAMTS13 using co-immunoprecipitation analysis. Specifically, ADAMTS13 and CypB could be detected in association together in untreated cells, but not in those treated with CsA, where only ADAMTS13 could be detected. ADAMTS13 levels are also reduced in the setting of siRNA-mediated CypB knockdown, suggesting a direct link between CypB expression/activity and ADAMTS13 maturation. We have found that CsA leads to intracellular conformational changes in the ADAMTS13 protein, evidenced by a unique trypsin digestion pattern of ADAMTS13 from CsA-treated cells relative to untreated cells. Additionally we found that treatment of cells with N-acetyl-Leu-Leu-norleucinal (ALLN), a proteasome inhibitor, resulted in about a 2.5 fold rescue in ADAMTS13 expression in the setting of CsA treatment. This observation suggests that CsA treatment leads to an enhanced proteasomal degradation of ADAMTS13, possibly due altered conformation. Although CsA did not influence the overall level of intracellular ADAMTS13, it resulted in diminished specific proteolytic activity, further suggesting improper ADAMTS13 folding. Finally, we show that plasma samples from CypB knockout mice have reduced levels of circulating ADAMTS13. Conclusions: Our findings indicate that cyclophilin-mediated activity is an important factor in ADAMTS13 secretion and activity. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization process mediated by CypB in its proper folding and maturation. We suggest that CsA treatment disrupts the interaction between ADAMTS13 and CypB. Although this may not be the sole mechanism by which CsA ultimately lowers levels of secreted ADAMTS13, this work altogether provides a novel mechanistic explanation for CsA-induced TTP in transplant patients. Disclosures: No relevant conflicts of interest to declare.

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.

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.

Abstract 1185 Introduction: Synonymous mutations, previously called ‘silent’ mutations, are now widely acknowledged to be associated with various disease states by causing changes in protein expression, conformation, and function. A number of synonymous mutations in factor IX (Val107Val, Arg116Arg, and Gln191Gln) were discovered in patients presenting with mild hemophilia B. Further, the use of viral vectors harboring codon-optimized F9 for the treatment of hemophilia B is currently being evaluated. The synonymous mutations which are used in codon-optimized vectors are largely considered harmless and are therefore generously employed to boost expression levels of factor IX (FIX). Previously, we have shown that introducing synonymous mutations may cause changes in other characteristics apart from protein expression, such as in protein function and conformation. Methods: To evaluate the properties of FIX protein which contain synonymous mutations, we produced and characterized a panel of F9 variants harboring a single and or a combination of synonymous mutations, and compared them wild-type F9 (NCBI RefSeq NM_000133.3). One codon-optimized construct differed from wild-type F9 in over 50% of nucleotides. For each point mutation, we calculated relative synonymous codon usage, determined local mRNA structure and stability, and analyzed protein structure computationally. Concurrently, we transiently and or stably transfected HEK293 and liver HUH7 cells with each vector. We examined mRNA (using RT-PCR and sequencing) and protein expression levels (using ELISA and western blotting techniques), as well as activity using aPTT and chromogenic assays. Further, we examined the conformation of the expressed protein by examining differential binding patterns of conformation-specific monoclonal antibodies and analysis by trypsin digestion and native PAGE. Results: The disease-associated synonymous mutations (Val107Val, Arg116Arg, and Gln191Gln) resulted in altered FIX expression and activity with evidence of stability and conformational differences compared to wild-type FIX. Preliminary experiments reveal that the propeptide of the Val107Val mutant is less efficiently cleaved than the wild-type. Further, we were able to demonstrate, with a cell-free translation system, that Val107Val FIX is translated at a significantly decreased rate in vitro compared to wild-type FIX (30–40% less efficiently), offering a mechanistic explanation for the altered protein properties observed in disease-associated constructs. The reduced translation rate associated with the Val107Val synonymous mutation is accompanied by a reduced codon usage. In contrast, other constructs, including the codon-optimized F9, had markedly increased expression level with negligible difference in specific activity. Conclusions: Single synonymous mutations (e.g. Val107Val, Arg116Arg, and Gln191Gln) in F9 may precipitate hemophilia B because they result in markedly altered protein properties (expression, activity, and conformation). Codon-optimized vectors, which consist of a number of synonymous mutations, result in the quantitative gain in expression of FIX. However, other properties, particularly those of pharmacokinetic significance, need to be evaluated to ensure that introduced synonymous mutations have positive rather than negative effect on the resultant protein. Results from computational analysis of characteristics such as mRNA stability, codon usage, and secondary structures of FIX aligned with in vitro analyses. This work leads to a better understanding of ways in which synonymous mutations precipitate disease. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination policy. Disclosures: No relevant conflicts of interest to declare.

Abstract 2197 Hemophilia B is characterized by structural and functional defects in coagulation factor IX (FIX) caused by mutations in the F9 gene. Various mutations (nonsense, missense, etc.) are known to be associated with the disease, including a synonymous V107V mutation reported recently by Knobe and colleagues (Knobe et al., Hemophilia, 2008). However the mechanism by which this synonymous mutation contributes to the disease has not yet been elucidated. Earlier we have shown that synonymous codon substitutions in the mRNA of the multidrug resistance protein (MDR1) may change the conformation of the protein and result in altered functionality (Kimchi-Sarfaty et al., Science, 2008). Here we have performed in silico analyses of the synonymous codon substitution (GTGàGTA) leading to the V107V polymorphism and found that it may change the mRNA structure, stability, codon usage, and 3D structure of the encoded protein. We hypothesize that changes in codon usage might affect the rhythm of protein translation and thus result in slightly altered FIX conformation. In vitro analyses of FIX mRNA and protein expression supported our in silico analyses. The GTGàGTA (V107V) synonymous mutation results in reduced expression levels as well as an encoded protein with a slightly different conformation compared to wild-type FIX. These results show that the V107V polymorphism is not silent and might cause mild hemophilia B. This work sheds further light on ways in which synonymous mutations impact disease. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination policy Disclosures: No relevant conflicts of interest to declare.