P.6.a.008 Neuropeptide S receptor gene variant and stressful life events: associations with alcohol use disorder and alcohol consumption

Abstract

Genetic variation in the Protein C gene (PROC) is associated with altered risk of adverse outcome for a number of diseases. Common single nucleotide polymorphisms (SNPs) in the promoter region and the adjacent 5’ region of PROC are associated with Protein C expression. We tested the hypothesis that common SNPs (minor allele frequency > 10%) between the frequently studied promoter SNPs -1654 (rs1799808) and -1641 (rs1799809), and the end of PROC intron 2 alter nuclear transcription factor binding.We used electrophoretic mobility shift assays with 25-mer oligonucleotides centered on each of the 10 SNPs assessed in this potential regulatory region of the Protein C gene to test for differential binding to nuclear factors isolated from Hep-G2 cells.We found that the G-allele oligo of the intron 2 SNP rs2069915[G/A] bound nuclear factors more avidly than the A-allele (p = 1.9 × 10-9, n = 24). Similarly, we found that the C-allele oligo of the intron 2 SNP rs2069916[C/T] bound nuclear factors more avidly than the T-allele, (p = 3.7 × 10-6, n = 19). Cold competition and supershift assays suggested that the protein differentially binding to the C-allele of rs2069916 was USF1. Notably, we observed minimal nuclear factor binding to oligos containing haplotypes of the previously reported -1654 and -1641 SNPs. Luciferase reporter assays that showed the A-T haplotype of rs2069915 and rs2069916 drives transcription significantly more than the C-G haplotype (t-test, P = 0.015, n = 12).Differential transcription factor binding occurs for common SNPs in the 5’ intronic regions of PROC which may contribute to PROC regulation and reported PROC SNP – phenotype associations.