Volume 16 Supplement 1
Fine mapping and functional study of the systemic lupus erythematosus-associated NMNAT2/SMG7 locus
© Zhao et al.; licensee BioMed Central Ltd. 2014
Published: 18 September 2014
NMNAT2 (rs2022013 located at intron 1) was identified as a SLE risk locus in a European-derived population in a genome-wide association study (GWAS). NMNAT2 (nicotinamide mononucleotide adenylyltransferase 2) is expressed mainly in the brain, regulating energy metabolism. Proximal to the SLE-associated NMNAT2 variant is SMG7, encoding a component of the mRNA quality control pathway that regulates spliceosomal machinery such as Sm and snRNP via alternative splicing. We fine mapped the NMNAT2/SMG7 region in multiple ancestries and explored functional consequences of the identified variants.
Materials and Methods
We genotyped/imputed 313 SNPs covering an ~550 kb NMNAT2/SMG7 region in 15,424 case-control subjects from European-Americans (EA), African Americans, Asians and Amerindian/Hispanics, assessed SNPs for association with SLE using a logistic regression model adjusted for sex and ancestry, and used haplotype-based conditional testing to distinguish independent associations. Quantitative real-time PCR and luciferase reporter assays were used to examine allelic differences in SMG7 expression and transcription activity. PBMCs from SLE patients (n = 13) were cultured with or without siRNA targeting SMG7, GAPDH (positive control) or siRNA with a nontargeting sequence (NC, negative control), and culture supernatants were measured by ELISA for levels of antinuclear antibody (ANA) and cytokines/chemokines.
We confirmed association at rs2022013 and identified two independent signals in EA only: intron 1 of NMNAT2 tagged by rs12146097 (P = 1.5 × 10-10, OR = 1.38); and multiple SMG7 SNPs tagged by rs2275675 (P = 5.7 × 10-8, OR = 1.22). Expression quantitative trait locus data showed SLE-risk alleles of NMNAT2/SMG7 variants consistently associated with decreased mRNAs of SMG7, but not NMNAT2, in cell lines, suggesting SMG7 is a more likely risk gene for SLE. The rs2275675 risk allele was associated with decreased SMG7 mRNAs dose dependently in PBMCs of 86 SLE patients and 119 controls (P = 0.001 and 6.84 × 10-8, respectively), and reduced transcription activity in two transfected cell lines (P ≤ 0.004). SMG7 mRNA levels in PBMCs correlated inversely with ANA titers in 68 SLE patients (P = 0.0089, r = -0.31). Compared with culture supernatants of SLE PBMCs treated with NC-siRNA, those treated with SMG7- siRNA showed increased ANA (P < 0.0001) and CCL19 (P = 0.0002; a ligand for CCR7 promoting movement/ interaction of B-Th cells and antibody production).
We confirmed the previous GWAS NMNAT2 association and identified independent SMG7 association with SLE in an EA population. The SLE-risk alleles are dose-dependently associated with decreased SMG7 mRNAs, and SMG7 reduction increases ANA and CCL19 production in PBMC cultures of SLE patients, suggesting that dysfunction in mRNA surveillance conferred by SLE-associated SMG7 variants contributes to SLE manifestations.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.