Characterization of Coding Synonymous and Non-Synonymous Variants in ADAMTS13 Using Ex Vivo and In Silico Approaches

Abstract

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.

Genetic variants in ADAMTS13 as well as smoking are major determinants of plasma ADAMTS13 levels

“…These 2 common ADAMTS13 variants have been examined in 2 previous studies. One study examined expression and secretion of Q448E in cell culture and found results similar to those of our study but an increased specific activity of this variant compared with wild-type, 39 and another article examined both the Q448E and A732V variants expressed in cell culture and found similar decreases in secretion into culture media and ;25% reduction in enzyme activity as measured by a collagen binding assay. 40 However, both previous studies used transient transfection methods which may have altered patterns of synthesis and secretion compared with our experiments in cell lines with stable expression from the same locus.…”

Rare Variants in the ADAMTS13 Von Willebrand Factor–Binding Domain Contribute to Pediatric Stroke

“…1 However, an independent causal relationship between ADAMTS13 levels could not be established because of the unavailability of prospective studies. Recent studies aiming at the characterization of single nucleotide variants in ADAMTS13 through mRNA studies and in silico approaches 9,10 suggest that single nucleotide variants in ADAMTS13 influence its gene expression in a complex fashion, thus explaining interindividual variability in ADAMTS13 activity beyond nonsynonymous coding mutations affecting vWF-cleaving properties.…”

Single-nucleotide variations defining previously unreportedADAMTS13haplotypes are associated with differential expression and activity of the VWF-cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura family

“…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 Prism software.ADAMTS13 sequencing revealed both children to be homozygous and their parents to be heterozygous for the previously described, cTTP-causing, single-base-substitution mutation 20506C > T (Table IA) (Levy et al, 2001;Snider et al, 2004;Hing et al, 2013). The children's VWF-CPase antigen and activity levels were undetectable, although steady-state levels of the ADAMTS13 mRNA were >2Á5-fold higher in the daughter than in the son.…”

“…While prior studies have shown that the synonymous (s)-and ns-SNVs 3944C > T (c.864C > T ⇒ 140Ala > Ala) and 23789C > T (c.3143C > T ⇒ 900Ala > Val), respectively, are associated in vitro with no or only minor effects on VWF-CPase secretion compared to their more frequent wildtype alleles (Plaimauer et al, 2006;Edwards et al, 2012), the seven other allelically distinct, parental ADAMTS13 potential quantitative-trait nucleotides (QTNs) have never been experimentally investigated, individually or in combination with each other. The candidate functional SNVs, 11610G > C (IVS10[-32G > C]) and 18320G > A (IVS15[-26G > A]), are located in intronic regions known to contain cis-elements comprising 3'-splice junctions, and their alleles may, for example, influence precursor mRNA processing differentially.…”