Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these “silent” variations can have a significant impact on protein expression and function and should no longer be considered “silent”. Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.

Haemophilia B is an X-linked recessive disorder caused by deficiency of functional coagulation factor IX, which results almost exclusively from mutations in the F9 gene. We sought to determine features, which could distinguish between mutations that cause severe disease symptoms from those that cause non-severe disease symptoms. Towards this objective, we have performed a statistical analysis of reported point mutations in F9. These include: potential local changes in mRNA free energy, codon usage, charge and type of mutated amino acid, location of the mutation with regard to protein secondary structure and functional domain and amino acids' evolutionary conservation scores. Wilcoxon signed-rank tests showed highly significant differences between severe and non-severe disease causing mutations in their effect on free energy of small mRNA fragments and evolutionarily conserved amino acids. Our results suggest that information at the mRNA level as well as conservation of the amino acid correlate well with disease severity. This study demonstrates that computational tools may be used to characterize the severity of haemophilia B associated with point mutations and suggests their utility in predicting the outcome of sequence changes in recombinant proteins.

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.

Single-nucleotide variations defining previously unreported ADAMTS13 haplotypes are associated with differential expression and activity of the VWF-cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura familyAlthough autosomal recessive haematological disorders, such as congenital thrombotic thrombocytopenic purpura (cTTP), 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 molecular-genetic characteristics. We present an in-depth examination of ADAMTS13 haplotypes, mRNA levels and protein expression, activity, and enzyme kinetics in this case study of two Salvadoran children with cTTP -the first reported cases of this disease in individuals originally from Central America -and their parents.The propositi -a son and a daughter born to asymptomatic, non-consanguineous (but possibly distantly related) parents from the same town in El Salvador -were diagnosed with cTTP at ages 6 and 2 years, respectively. Although they developed haemolytic anaemia and thrombocytopenia 2 d (son) and 17 months (daughter) after birth, their cTTP diagnosis was not rendered until both were hospitalized simultaneously with fever, respiratory symptoms, haemolytic anaemia and thrombocytopenia. Peripheral blood smears for both demonstrated schistocytes, and their von Willebrand factor-cleaving protease (VWF-CPase) activities were <1% without ADAMTS13 IgG antibodies. Fresh frozen plasma (FFP) infusion induced rapid (within 48 h) normalization of their blood counts and resolution of the microangiopathic changes. Both children have been treated with prophylactic infusions of FFP (10 ml/kg every 2Á5 weeks) without long-term neurological or renal sequelae.Following approval by institutional review boards and receipt of informed consent, blood samples were collected from the children, prior to FFP administration, and from both parents. Genomic DNA and total RNA were extracted from peripheral blood leukocytes using QIAamp DNA Blood Maxi and PAXgene Blood RNA kits, respectively (Qiagen; Germantown, MD, USA). All ADAMTS13 exons, at least 50 base pairs (bp) of each flanking intron junction,~500 bp 5' of the promoter, and~200 bp of 3'-flanking genomic DNA were amplified using polymerase chain reaction (PCR), cleaned using Exo-SAP-IT (Affymetrix; Santa Clara, CA, USA), and subjected to direct bi-directional Sanger sequencing followed by capillary electrophoresis and analysis on ABI-3730 instruments. Reverse transcription was performed (Shomron et al, 2010), and plasma VWF-CPase antigen was measured using the Technozym ADAMTS13 enzyme-linked immunosorbent assay (Technoclone; Vienna, Austria) and the Victor X3 multilabel plate reader (PerkinElmer; Waltham, MA, USA). Fluorogenic FRETS-VWF73 (Peptides International; Louisville, KY, USA) was prepared and assayed (Sauna et al, 2009). The kinetic characteristics of ADAMTS13 were obtained using GraphPad Pri...

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 <1% without neutralizing IgG antibodies. They remain without long-term neurologic or renal sequelae following prophylactic infusions of fresh plasma (10 mL/kg every 2.5-weeks). The purpose of this study was to characterize and correlate single-nucleotide variations (SNVs) in each parent's, non-mutant ADAMTS13 allele with its mRNA and protein expression, activity, and enzyme kinetics. 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<0.001; Fig. B). Plasma ADAMTS13 activity and antigen levels were ∼2-fold greater in the father than in the mother (p=0.00164 and p=0.0633, respectively), but the specific activities of these structurally distinct ADAMTS13 proteins were notably almost identical (253.5 vs. 256.2 U/μg). Moreover, initial velocity kinetic analysis using the Michelis-Menten equation demonstrated that the Vmax of the father's ADAMTS13 was twice that of the mother's (1.4 vs. 0.7; p < 0.0001) while its affinity for substrate was one-third that of her ADAMTS13 (Km = 0.3 vs. 0.1; p = 0.0585). 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.

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.