AB0009 IDENTIFICATION OF A NOVEL POLYMORPHISM OF ERAP1 IN A GROUP OF ITALIAN PATIENTS WITH BEHçET SYNDROME

Abstract

Background The endoplasmic reticulum aminopeptidase protein 1 gene (ERAP1) was associated to several human diseases, including Behcet syndrome (BS), a multisystemic disorder with unknown etiology. ERAP1 protein is involved in immune response and its role can be influenced by gene single nucleotide variations (SNVs) with an unclear mechanism [1-5]. Objectives We aim to genotype ERAP1 whole structure searching for SNVs, in 50 consecutive BS patients and 50 sex and ethnically-matched healthy controls (HC) unrelated to each others and/or to BS patients. Methods We used both bioinformatics and molecular methodologies. Specific primers for the coverage of all ERAP1 regions were designed using the NCBI Primer-Blast tool. Genomic DNA was extracted from whole blood and amplified using in vitro PCR. Good-quality PCR amplicons were directly sequenced using the GATC Biotech Sanger sequencing service and bioinformatically analysed using Mutation Surveyor software and NCBI-Blast Nucleotide on line similarity search tool. SNV functional impact was predicted using on line PolyPhen-2 [6], while the 3D protein prediction was obtained using 3D Protean server [7]. Results Our study was performed recruiting an explorative cohort of BS patients from Southern Italy, a population characterized by low disease prevalence. We identified a novel heterozygous SNV at 18169 nucleotide position (NG_027839.1:g.18169A>T; HGVS nomenclature) within ERAP1 exon 3 (Fig. 1a). It was found in 7/50 (14% of cases) of BS patients and in none of HC. The novel polymorphism was submitted and released in GenBank Database (MK252970 accession number). The SNV was a missense variation responsible for the substitution glutamate (hydrophilic amino acid) to valine (hydrophobic amino acid) at 183 position (NP_057526.3:p.Glu183Val; HGVS nomenclature) (Fig. 1b). This is a conserved site involved in the substrate binding, due to its significant role in the anchorage of the N-terminal amine group of the peptides. Because of the different amino acid chemical features, the computational assessment of the protein structure was performed (Fig. 1c) recognizing a change in the protein energy and stability: the SNV was associated to a more stable protein chain (ΔE: -2.022), probably affecting the enzyme conformational state change and activity, as well as the substrate binding process. The amino acid change was predicted to be damaging with maximum score when PolyPhen-2 prediction software was queried (score: 1.00) (Fig. 1d). Conclusion We found a SNV not previously reported in literature in a relatively small group of Italian BS patients. Our data need to be tested in a larger case-control study. In particular, the link between our SNV and the protein stability is to be validated in future functional studies.