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.

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...

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.