Single nucleotide polymorphisms associated with P2X7R function regulate the onset of gouty arthritis

<div><p>Background</p><p>Gout is an inflammatory disease that is caused by the increased production of Interleukin-1β (IL-1β) stimulated by monosodium urate (MSU) crystals. However, some hyperuricemia patients, even gouty patients with tophi in the joints, never experience gout attack, which indicates that pathogenic pathways other than MSU participate in the secretion of IL-1β in the pathogenesis of acute gouty arthritis. The ATP-P2X7R-IL-1β axis may be one of these pathways.</p><p>Objective</p><p>This study examines the role of Adenosine triphosphate (ATP) in the pathogenesis of gout and the association of ATP receptor (P2X7R) function with single nucleotide polymorphisms and gout arthritis.</p><p>Methods</p><p>Non-synonymous single nucleotide polymorphisms (SNP) loci of P2X7R in Chinese people were screened to compare the frequencies of different alleles and genotype distribution of selective SNPs in 117 gouty patients and 95 hyperuricemia patients. Peripheral white blood cells were purified from the peripheral blood of 43 randomly selected gout patients and 36 hyperuricemia patients from the total group. Cells were cultured with MSU or MSU + ATP, and supernatants were collected for the detection of IL-1β concentrations using enzyme-linked immunosorbent assay (ELISA).</p><p>Results</p><p>1. Eight SNP loci, including rs1653624, rs10160951, rs1718119, rs7958316, rs16950860, rs208294, rs17525809 and rs2230912, were screened and detected, and rs1653624, rs7958316 and rs17525809 were associated with gout arthritis. 2. IL-1β concentrations in supernatants after MSU + ATP stimulation were significantly higher in gouty patients than in the hyperuricemia group [(131.08 ± 176.11) pg/ml vs. (50.84 ± 86.10) pg/ml]; Patients (including gout and hyperuricemia) carrying the susceptibility genotype AA or AT of rs1653624 exhibited significantly higher concentrations of IL-1β than patients carrying the non-susceptibility genotype TT [(104.20 ± 164.25) pg/ml vs. (21.90 ± 12.14) pg/ml]; However, no differences were found with MSU stimulation alone.</p><p>Conclusions</p><p>ATP promotes the pathogenesis of gouty arthritis via increasing the secretion of IL-1 β, and its receptor (P2X7R) function associated single nucleotide polymorphisms may be related to gouty arthritis, which indicates that ATP-P2X7R signaling pathway plays a significant regulatory role in the pathogenesis of gout.</p></div

Association between the number of susceptible genotypesand the risk of gout.

<p>Association between the number of susceptible genotypesand the risk of gout.</p

Allele and genotype frequencies and genetic model of SNPs in the P2X7R gene in gout and hyperuricemia patients.

<p>Allele and genotype frequencies and genetic model of SNPs in the P2X7R gene in gout and hyperuricemia patients.</p

The eight selected non-synonymous coding SNPs in the P2X7R gene.

<p>The eight selected non-synonymous coding SNPs in the P2X7R gene.</p

IL-1β concentrations in the supernatant of cultured peripheral leukocytes.

<p>* <i>P</i><0.05. (<b>A)</b> There were no differences in IL-1β concentrations between the gout and hyperuricemia groups following MSU stimulation alone, but IL-1β concentrations increased significantly in gout patients following MSU + ATP stimulation. (<b>B)</b> MSU + ATP stimulation significantly elevated IL-1β in patients with rs1653624 who carried the gout susceptibility genotype AA or AT; there was no difference IL-1β concentrations after MSU stimulation alone. (<b>C)</b> There were no significant differences between patients who carried the gout susceptibility genotype of rs7958316 or rs17525809 after MSU + ATP stimulation. (<b>D)</b> IL-1β concentrations in patients who carried the rs7958316 susceptibility genotype (TT) increased significantly after MSU + ATP stimulation compared to patients who carried the rs17525809 susceptibility genotype (AA or AG), but there were no differences in patients who carried the rs17525809-non-susceptible genotypes (GG).</p

Linkage disequilibrium and sequence conservation of SNPs in <i>ETS1</i>.

<p>Shown are LD for significant SNPs in and around <i>ETS1</i> detected by GWAS (A), and replicated SNPs in the 3′-UTR and downstream of the gene (B), and sequence conservation for sequences around SNP rs1128334 among different species (C).</p

Quantile-Quantile plot of expected (x-axis) and observed (y-axis) −log₁₀(<i>P</i> value) distribution in our GWAS analysis.

<p>(A) Considering all the available SNPs. (B) SNPs in and around <i>HLA locus</i>, and <i>TNFSF4</i>, <i>STAT4</i>, <i>TNFAIP3</i>, <i>IRF5</i>, <i>BLK</i>, as well as <i>BANK1</i> were excluded from analysis.</p

Involvement of <i>ETS1</i> in Th17 cell and B lymphocyte development and autoimmunity.

<p>Involvement of <i>ETS1</i> in Th17 cell and B lymphocyte development and autoimmunity.</p

Allelic expression of <i>ETS1</i> on SNP rs1128334 in PBMC of healthy individuals.

<p>PBMC cDNA processed from 33 healthy individuals heterozygous on rs1128334 were used for allelic expression detection of <i>ETS1</i> by pyrosequencing. (A) A case example of detection on the “A” allele and the “G” allele from both DNA and cDNA samples from the same individual. (B) The ratio of G/A allelic detection for both DNA and cDNA samples. The median G/A ratio for DNA is 1.09 (95% CI: 1.08–1.11) and the median G/A ratio for cDNA expression is 1.32 (95% CI: 1.21–1.43), <i>P</i><0.0001 by paired student's <i>t</i> test.</p

<i>ETS1</i> Haplotype analysis on SLE association.

<p><i>ETS1</i> Haplotype analysis on SLE association.</p