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.
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