Common variants in P2RY11 are associated with narcolepsy

Abstract

Growing evidence supports the hypothesis that narcolepsy with cataplexy is an autoimmune disease. We here report genome-wide association analyses for narcolepsy with replication and fine mapping across three ethnic groups (3,406 individuals of European ancestry, 2,414 Asians and 302 African Americans). We identify a SNP in the 3' untranslated region of P2RY11, the purinergic receptor subtype P2Y₁₁ gene, which is associated with narcolepsy (rs2305795, combined P = 6.1 × 10⁻¹⁰, odds ratio = 1.28, 95% CI 1.19-1.39, n = 5689). The disease-associated allele is correlated with reduced expression of P2RY11 in CD8(+) T lymphocytes (339% reduced, P = 0.003) and natural killer (NK) cells (P = 0.031), but not in other peripheral blood mononuclear cell types. The low expression variant is also associated with reduced P2RY11-mediated resistance to ATP-induced cell death in T lymphocytes (P = 0.0007) and natural killer cells (P = 0.001). These results identify P2RY11 as an important regulator of immune-cell survival, with possible implications in narcolepsy and other autoimmune diseases.

Figure 1

Risk locus on 19q13.2, showing gene organization and linkage disequilibrium in the region of interest (10,071,000-10,130,000). Top: D′/LOD-based LD plot using data from combined Chinese and Japanese populations (CHB-JPT). Bottom: D′/LOD-based LD plot for individuals of European ancestry only (CEU-TSI). D′ values are calculated from HapMap v3R2 CHB, JPT, CEU, and TSI populations. In addition, r² values between the original marker, rs4804122 (green) and the best transethnic marker rs2305795 (orange) derived from our own data are indicated. rs2305795 falls in the 3′UTR of P2RY11.

Figure 2

P2RY11 mRNA expression in peripheral blood mononuclear cells (PBMCs). A) Expression in PBMCs from 116 subjects with various rs2305795 genotypes (Mean + SEM, 60 patients and 56 controls; AA n=49, AG n=51, GG n=16). As no direct effect of disease status on P2RY11 expression was observed, subjects are grouped by genotype. The P2RY11 rs2305975AA genotype results in a 50% reduction in P2RY11 expression compared to the rs2305795GG genotype and is associated with increased risk of narcolepsy. B) P2RY11 expression by rs2305795 genotype in various immune cell subsets (Mean + SEM, n=7-8 normal controls per genotype category). NK cells= CD56⁺ natural killer cells; B Cells = CD19⁺ B cells; Monocy.=CD14⁺ monocytes; DCs=myeloid/plasmacytoid dendritic cells. Shown are Bonferroni corrected one-way ANOVA p-values.

Figure 3

PBMC cell death induced by ATP is inhibited by the stimulation of P2RY11 and varies by rs2305795 genotype. A) Effect of ATP on cell viability and dose-response of co-incubation with the P2RY11 specific agonist NF546 and antagonist NF340 (Mean + SEM, n= 7-8, rs2305795AG control subjects). Overall one-way ANOVA p-values are shown, with Tukey's post test: * significantly different from control with no ATP, p<0.01; ^# significantly different from treatment with 100 μM ATP but no NF546, p<0.01; ^$ significantly different from treatment with 100 μM ATP and 100 μM NF546, p<0.01. B) Effect of the rs2305795 genotype on the percent of cells rescued from ATP induced cell death by P2RY11 stimulation. 10 μM NF546 has a less potent effect on cell survival after ATP-induced cell death with the rs2305795AA genotype compared to the rs2305795GG genotype. Heterozygote subjects fall in between. Mean + SEM, n=9 subjects in each group.

Figure 4

Effect of ATP and P2RY11 co-stimulation on different immune cell subtypes. PBMCs were co-incubated with 100 uM ATP and the P2RY11 specific agonist NF546 in different doses and the effect on different cell fractions was determined by FACS. An effect by rs2305795 genotype was seen in T lymphocytes and NK cells but not in B lymphocytes and monocytes. Shown are mean + SEM, n=8/column, p-values are from one-way ANOVAs.