Epistasis with HLA DR3 implicates the P2X7 receptor in the pathogenesis of primary Sjögren's syndrome

Abstract

Introduction: The aim of this study was to examine the association between functional polymorphisms in the pro-inflammatory P2X7 receptor and the Ro/La autoantibody response in primary Sjögren's syndrome (pSS).

Methods: Twelve functional P2RX7 polymorphisms were genotyped in 114 pSS patients fulfilling the Revised American-European Consensus Criteria for pSS, and 136 controls. Genotyping of the A1405G (rs2230912) polymorphism was performed on a replication cohort consisting of 281 pSS patients and 534 controls. P2X7 receptor function in lymphocytes and monocytes was assessed by measurement of ATP-induced ethidium+ uptake. Serum IL-18 levels were determined by ELISA.

Results: The minor allele of P2RX7 A1405G is a tag for a common haplotype associated with gain in receptor function, as assessed by ATP-induced ethidium+ uptake. A positive association between 1405G and anti-Ro±La seropositive pSS patients was observed in Cohort 1. Although not replicated in Cohort 2, there was a consistent, significant, negative epistatic interaction effect with HLA-DR3 in seropositive pSS patients from both cohorts, thereby implicating this gain of function variant in the pathogenesis of pSS. Serum IL-18 was elevated in seropositive pSS patients, but was not influenced by P2RX7 A1405G.

Conclusions: The P2RX7 1405G gain-of-function haplotype may be a risk factor for seropositive pSS in a subset of subjects who do not carry HLA risk alleles, but has no effect in subjects who do (epistasis). Potential mechanisms relate to autoantigen exposure and inflammatory cytokine expression. The observed elevation of IL-18 levels is consistent with P2X7 receptor activation in seropositive pSS patients. Collectively these findings implicate P2X7 receptor function in the pathogenesis of pSS.

Figure 1

Common (>5% frequency) P2RX7 haplotypes identified in the study population. Grey squares represent the major alleles and black squares the minor alleles, for each single nucleotide polymorphism. Numbering of polymorphisms are based on the original mRNA sequence [Y09561.1; GenBank]. Six major haplotypes were identified with a combined frequency of 70%.

Figure 2

Human peripheral blood mononuclear cells were analysed for ATP-induced ethidium uptake using time-resolved flow cytometry. Monocytes were identified by an anti-CD14 APC antibody and T lymphocytes were identified by an anti-CD3-PE antibody. Ethidium uptake (25 μM) was measured in response to P2X7 receptor stimulation by 1mM ATP. (A) and (C) are representative ethidium uptake curves from genotyped individuals. (B) and (D) are scattergrams of collated data from 5 to 18 subjects from each genotype group (number of subjects shown in brackets) with ATP-induced ethidium uptake calculated from the slope of the linear portion of the uptake plot over 1 minute. *P < 0.05 from one-way analysis of variance with Dunnett's post hoc test.

Figure 3

Boxplots of serum IL-18 levels (pg/mL) in controls (n = 36), autoantibody negative pSS (n = 18), and autoantibody positive primary Sjögren's syndrome (pSS) (n = 54). Serum IL-18 levels were significantly different between the three groups (P = 0.0003, Kruskal-Wallis test), and highest in autoantibody-positive pSS.