In the follow-up analysis of rs7958311 and CPI, similar effects were observed in both the BrePainGen and Tromsø6 data sets. There was a significant effect of genotype and time but no interaction, suggesting that the effect of genotype was consistent across time points (2-way analysis of variance, genotype effect P < 0.0001 and time effect P < 0.0001 for both data sets; Fig. 2). In the post hoc comparisons (the Dunnett multiple comparison test), homozygous minor allele carriers (AA) differed from the main allele homozygotes (GG) (the Dunnett multiple comparison test, GG vs AA, adjusted P = 0.0001 for both data sets). There were no differences between heterozygotes and main allele homozygotes (GG vs AG, P = ns), suggesting a possible nonadditive effect.
In the Tromsø data set, the effect of sex and gene-by-sex interactions were tested using PLINK software. There was a significant effect of sex when AUC% was analyzed in the whole sample (P = 6.66E-33, β = −4.521; Fig. 1) and the subsequent analyses were performed for each sex separately. Gene-by-sex interactions were not significant (P > 0.05).
In the BrePainGen cohort, associations between rs7958311 variants and postoperative pain during the first postoperative week were assessed. There was a significant effect of genotype (P = 0.0046) and time (P < 0.0001) but no interaction (P > 0.05). The post hoc analysis showed that the effect was driven by minor allele homozygotes (AA) who reported lower pain intensity compared with the main allele homozygotes (GG) (the Dunnett test for multiple comparisons, AA vs GG: P = 0.027). Heterozygous carriers did not differ from main allele homozygotes (the Dunnett test for multiple comparisons, AG vs GG: P = 0.98) (Fig. 4A).
In the Tromsø 6 data set, associations between rs7958311 variants and chronic multisite pain were assessed. Minor allele carriers were less likely to have multisite pain and MAF was lower in multisite pain patients (21%) compared with the controls (27%) (P = 0.047, β = −0.27 ± 0.14; odds ratio = 0.75, confidence interval 95 = 0.57-0.99) (Fig. 4B).
The main effects of the rs7958311 genotype were seen in pain intensity. Cold pain intensity AUC% is affected by both the primary afferent drive, the role of which may be the most prominent at the beginning of the task, and pain-modulatory mechanisms which are more likely to activate at a later stage. The lack of time-interaction in the post hoc test suggests that the genotype effect was already present at the beginning of the test. P2X7 expression in the nervous and immune systems, involved in modulation of pain (gene atlas, http://biogps.org), and upregulation in nerves and immune cells in patients with neuropathic pain suggest at least 3 potential sites of action: peripheral immune cells, central immune cells, and neurons.13,44
As P2RX7 is expressed in several brain areas and is associated with neuropsychiatric phenotypes, supraspinal mechanisms should also be considered.7,43,45 Modulation of gene expression can mediate SNP effects and reveal potential site of action. In previously published in vitro assays, both rs208294 and rs7958311 have shown effects consistent with and potentially contributing to their functional profiles (GOF and LOF, respectively).57,60 These effects were dependent on other co-occurring variants. The GTEx database showed eQTL associations between rs7958311 and P2RX7 expression in human tissues, most significant for the brain (hippocampus, P = 0.015, β = −0.17), although the effect was not sufficient to survive multiple comparison corrections. Interestingly, rs7958311 decreased the expression of P2RX4 in whole blood (P = 1.4e-7, β = −0.23) and fibroblasts (P = 0.000059, β = −0.17) and these effects remained significant after accounting for multiple comparisons (https://gtexportal.org). Thus, rs7958311 is involved in the regulation of P2RX4 expression as a cis-eQTL. An association was detected because of the close proximity of P2RX4 and P2RX7 genes which are structurally related, have overlapping functions, form heteromers, and interact functionally.16 Although the mechanisms and physiological relevance of regulation of P2RX4 expression by rs7958311 remain to be revealed, they are likely to emphasize direct P2RX7-mediated effects: as P2RX4 compensates for the lack of P2X7 function, its downregulation by rs7958311 could provide cumulative or synergistic effect.
Despite several advantages (clearly defined phenotypes, large and homogenous cohorts, and 2 independent samples), our study also has limitations that can impact the effect of rs7958311. Cold withdrawal time data were right-censored, particularly in the Tromsø 6 data set, resulting in loss of statistical power. The additive genetic model of regression was applied, which might not be the most sensitive or powerful approach.41,42,47 Homozygous rs7958311 minor allele carriers differed from the main allele homozygotes in the follow-up CPI analysis and based on the shape of Kaplan–Meier CWT curves. Heterozygous carriers, although representing a larger group and having more weight in the statistical analysis, did not differ, suggesting that both minor allele copies are required for the effect and thus a recessive model could be a better fit with the data. Although our sample size is quite large for the experimental pain field, it is still modest compared with larger genome-wide association studies. Increasing the sample size could increase statistical power.
The authors have no conflict of interest to declare.
The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7-PEOPLE-2013-COFUND) under grant agreement no 609020—Scientia Fellows (O.K.) and FP7-HEALTH-2013 under grant agreement no 602919 (E.K.), and Helsinki University Hospital Research Funds (TYH2008225, TYH2010210, and TYH2012212 to E.K.). This work was also supported by The Norwegian Research Council (grants no 177725 to AS and no 231187/F20 to B.S.W.) and South-Eastern Norway Regional Health Authority (grant no 2015064 to C.S.N.).
The authors are grateful to the study participants, research nurses Eija R. Ruoppa, RN and Minna Kaiponen, RN, and the technical staff of the sixth Tromsø Study for excellent assistance throughout the project and the valuable work. The authors thank R. Sirokov (MSc) and R. Airo (MSc) for their excellent assistance and support in bioinformatical aspects.
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