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Review Pain genetics: past, present and future Jeffrey S. Mogil1,2 1 2 Department of Psychology, McGill University, Montreal, QC, H3A 1B1, Canada Alan Edwards Centre for Research on Pain, McGill University, Montreal, QC, H3A 1B1, Canada Chronic pain is a classic example of gene environment interaction: inflammatory and/or nerve injuries are known or suspected to be the etiology of most chronic pain syndromes, but only a small minority of those subjected to such injuries actually develop chronic pain. Once chronic pain has developed, pain severity and analgesic response are also highly variable among individuals. Although animal genetics studies have been ongoing for over two decades, only recently have comprehensive human twin studies and large-scale association studies been performed. Here, I review recent and accelerating progress in, and continuing challenges to, the identification of genes contributing to such variability. Success in this endeavor will hopefully lead to both better management of pain using currently available therapies and the development and/or prioritizing of new ones. Individual differences in pain and analgesia Chronic pain is probably the most prevalent human health problem, contributing to individual morbidity and mortality and imposing high societal costs [1]. Current management of chronic, non-cancer pain is far from optimal, with existing analgesics characterized by limited efficacy and a high adverse-effect burden. Decades of robust government and industry investment have yielded few basic research-toclinical translational successes, and many have looked to the molecular genetics revolution as a new way forward [2]. In fact, genetic approaches in pain research, as elsewhere, are now fully integrated into the research enterprise. For example, according to the PainGenes Database (http:// paingeneticslab.ca/4105/06_02_pain_genetics_database. asp) [3], at least 358 genes are thought to be relevant to pain or analgesia based on the phenotype of null mutant mice, and many hundreds of pain-regulated genes have been identified by gene expression profiling (microarray) studies [4]. In this review, I focus solely on mendelian genetics, as one of the greatest challenges to both pain treatment and analgesic development is robust interindividual variability. Individual differences are evinced on at least three different levels. First, there is great variability in the development of chronic pain syndromes per se, currently conceptualized by many as a transition from acute to chronic pain. Many chronic pain states are known or presumed to be secondary to traumatic or infectious injury to soft tissues and especially to nerves (i.e. neuropathic pain), but most individuals experiencing these insults do not develop chronic pain (e.g. [5]). In this sense, chronic pain is the prototypical example of a gene environment Corresponding author: Mogil, J.S. ([email protected]) 258 interaction: the insult is required, but so too are susceptibility factors that might be inherited. Second, assuming a chronic pain state exists, the intensity of the pain (and of other symptoms, signs and sequelae) is highly variable and often not at all predictable from the severity of the presumed injury or disease (e.g. radiographically determined joint degeneration in osteoarthritis [6]). Such variability can also be demonstrated quite easily for acute pain in the laboratory, with standardized noxious heat stimuli eliciting pain ratings near zero in some individuals and near maximal (‘worst pain imaginable’) in others (e.g. [7]). Variable pain ratings are associated with similar variability in pain-evoked cortical activations [8], suggestive of true differences in subjective perception of the stimulus. Finally, great variability exists in patients’ analgesic responses to both opioids (e.g. [9]) and non-steroidal anti-inflammatory drugs (NSAIDs) (e.g. [10]); however, analgesia genetics will not be considered in detail here. Of course, individual differences could be explained by environmental (including stochastic) as well as inherited factors. Familial aggregation and twin studies of chronic pain syndromes have recently been performed to disentangle these possibilities [11–17]. Heritability estimates range widely, but most studies demonstrated at least moderate heritability. Most existing twin studies have measured the concordance of the painful disease itself among twin pairs, but not the twin–twin correlation of pain ratings. This fact is relevant because genes underlying pain could be involved in either the predisposition to develop chronic pain syndromes or determining actual pain levels within those syndromes, or both; it is still unclear what the overlap between these categories is. Better estimates of the heritability of pain itself would come from acute pain tests under controlled laboratory conditions, and two recent studies have established the heritabilities of many such assays in the h2 = 22–60% range [18,19]. There is currently no extant twin study of analgesic heritability, although one is in progress [20]. Lessons from animal pain genetics Heritability of preclinical assays of pain is moderate-tohigh (0.30<h2<0.76; median: h2 = 0.46) [21], in excellent agreement with human twin studies using analogous stimuli [18,19]. In addition to quantitative variation in pain and analgesic sensitivity, strain differences in pain-related anatomy, electrophysiology and neurochemistry have been observed [22–24]. Genetic correlation studies using artificially selected (selectively bred) lines and inbred strain panels have revealed: (i) unique inheritance of different pain symptoms (e.g. thermal, mechanical, chemical) 0168-9525/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2012.02.004 Trends in Genetics, June 2012, Vol. 28, No. 6 Review Trends in Genetics June 2012, Vol. 28, No. 6 regardless of etiology; (ii) common inheritance of analgesic efficacy against the same pain symptom, regardless of the drug used; and (iii) an inverse relationship between pain sensitivity and analgesic response [22,23]. Recent studies have evaluated these claims in humans. Contradictory data have been gathered regarding the existence of genetic factors common to multiple pain states (i.e. pain ‘stoicism’ genes) [25,26] versus genetic dissociation of different pain modalities (i.e. thermal, mechanical or ischemic) [18,27–29]. This debate is far from academic, as it is common practice in human genetic association studies to pool across modality to preserve statistical power. Several groups [30–32] have observed that, as in mice, basal heat pain thresholds can predict response to clinical opioid analgesia in humans. It has been suggested that high basal pain sensitivity indicates reduced pain-inhibitory capacity, and that the latter may represent a risk factor for chronic pain development [33]. A test of this intriguing hypothesis awaits large prospective studies. Other major findings from animal pain genetics studies include the surprisingly common and robust observance of sex–strain interactions (see [34]), and the interaction of genotype differences in pain sensitivity with environmental factors, such as diet [35], conspecific aggression [36], social housing [37], experimenter (and other laboratory effects) [38] and circadian rhythmicity [39]. A growing number of quantitative trait loci (QTLs) associated with variable pain sensitivity among inbred strains have been discovered in mice and, in a handful of cases, the probable gene underlying the QTL has been identified (Table 1). It is notable that several of these QTLs have sex-specific effects [40–46]. Association studies have subsequently confirmed the relevance of some of the human analogs of these genes in clinical and/or human experimental pain states. Little attention has been paid to comparative genetics among animal species, but a few findings are noteworthy. First, the insensitivity to capsaicin pain and thermal hyperalgesia (i.e. increased sensitivity to noxious thermal stimuli after injury) of birds [47] and naked mole-rats [48] have been elucidated at the genetic level, identifying amino acid substitutions in the Trpv1 capsaicin receptor gene in the former [47] and a promoter deletion in the tachykinin, precursor 1 (Tac1; substance P) gene in the latter [49]. Naked mole-rats are also insensitive to acid, recently found to be the result of a variant Nav1.7 sodium channel gene [50], also known to play an important role in some human monogenic pain disorders (see below). Robust species specificity of the genetic mediation of pain is also suggested by a recent meta-analysis of microarray (gene expression profiling) studies of chronic pain, in which almost no genes in the dorsal root ganglia that were consistently up- or downregulated by nerve or inflammatory injury in the rat could be confirmed as similarly regulated by those injuries in the mouse [4]. This is a disturbing finding, because extrapolation of genetic findings from one rodent species to humans is undermined by the inability to extrapolate from one rodent to another. Single-gene disorders of pain The ‘low-hanging fruit’ of human genetics are inherited mendelian disorders in which a single, mutated gene produces with high penetrance an obvious disease state. The mutations underlying such diseases can be elucidated via genetic linkage mapping. A handful of rare, monogenic disorders of pain are known, including the hereditary sensory and autonomic neuropathies (HSANs), in which Table 1. Pain-relevant QTLs in mice in which the gene affected by the underlying variant has been proposed Phenotype Heat pain f Inflammatory pain g Chronic pain h Location a 4:71 cM 4:80 Mb 7:33 cM 7:50 cM 9:60 cM 10:70 cM 12:110 cM 14:34 Mb 5:123 Mb 8:28 Mb Gene b Oprd1 Tyrp1 Trpv1 Calca Atp1b3 Avpr1a Yy1 Mapk8 P2rx7 Chrna6 Sex c < only Both Both <>, Both < only Both Both <>, <>, Evidence d Ph., Pos. cSNP, MUT Pos. Expr., KD, Mut., Ph. KD, Ph. Expr., Mut., Ph. Expr., Mut. Expr., Ph. cSNP, Ph. Expr., Mut. 15:80 Mb Cacng2 Both Expr., Mut. Refs [46,106] [4] [106] [32] [84] [45] [108] [107] [108] Wieskopf, J. et al., unpublished [109] Human? e Yes No No No No Yes No No Yes Yes Yes Refs [7] [45] [108] Wieskopf, J. et al., unpublished [109] a Mouse chromosome, followed by peak of linkage in either centiMorgans (cM) or Mb from the proximal end of the chromosome. b Genes encode the following proteins: Atp1b3, sodium-potassium ATPase, b3 subunit; Avpr1a, arginine vasopressin receptor 1A; Calca, calcitonin gene-related polypeptide; Chnra6, nicotinic cholinergic receptor, a6 subunit; Cacng2, voltage-dependent calcium channel, g2 subunit (stargazer); Mapk8, mitogen-activated protein kinase 8; Oprd1, delta-opioid receptor; P2rx7, P2X7 purinoceptor; Trpv1, transient receptor potential cation channel, V1; Tyrp1, tyrosinase-related protein 1; Yy1, yin-yang 1 transcription factor. c <: male; ,: female; both: linkage equally strong in male and female subjects; < only: linkage only observed in male subjects; <>,: linkage observed significantly more strongly in male versus female subjects. d cSNP: coding region SNP with functional consequences in some strains; Expr.: strain-dependent expression levels; KD: knockdown (via antisense or siRNA); Mut.: differential phenotype in mutant (transgenic knockout, transgenic knockin, congenic or spontaneous mutant) mouse; Ph.: effect on phenotype of protein-relevant pharmacological ligand; Pos.: inferred by correspondence of gene location with peak of linkage. e Yes: positive genetic association of a variant within the analogous human gene and a similar trait in humans subjects; No: no relevant human association study yet performed, or negative data obtained. f Assays include hot-plate test, hot water tail-withdrawal test and radiant heat paw-withdrawal (Hargreaves’) test. g Assay used was 5% formalin test. h Assays used were mechanical allodynia following spared nerve injury and autotomy following sciatic and/or saphenous nerve transection. 259 Review Trends in Genetics June 2012, Vol. 28, No. 6 Table 2. Single-gene pain disorders in which the responsible gene(s) have been identified Disorder a OMIM number Syndromes featuring absence of pain 243 000 CAIP 162 400 HSAN-1 613 640 613 708 614 116 201 300 HSAN-II 613 115 614 213 223 900 HSAN-III HSAN-IV (CIPA) HSAN-V Syndromes featuring pain Familial periodic fever FHM-I FHM-II FHM-III FMF Hereditary pancreatitis HNA PEPD Primary erythermalgia Gene Protein Refs b SCN9A SPTLC1 SPTLC2 ATL1 DNMT1 HSN2 FAM134B KIF1A IKBKAP [110] [111] [112] [113] [114] [115] [116] 256 800 608 654 NTRK1 NGFB Voltage-gated sodium channel, IXa (Nav1.7) Serine palmitoyltransferase, long-chain 1 Serine palmitoyltransferase, long-chain 2 Atlastin GTPase 1 DNA methyltransferase 1 Splice variant of WNK1 gene c Family with sequence similarity 134, B Kinesin family member 1A Inhibitor of k light polypeptide gene enhancer in B cells, kinase complex-associated protein Neurotrophic tyrosine kinase receptor 1 Nerve growth factor, b subunit 142 680 141 500 602 481 609 634 134 610/249 100 167 800 167 800 162 100 167 400 133 020 TNFRSF1A CACNA1A ATP1A2 SCN1A MEFV PRSS1 SPINK1 SEPT9 SCN9A SCN9A Tumor necrosis factor receptor superfamily, 1A Voltage-gated calcium channel, P/Q type, a1A Sodium-potassium ATPase, a2 subunit Voltage-gated sodium channel, Ia (Nav1.1) Familial Mediterranean fever gene (pyrin) Trypsin-1 Serine protease inhibitor, Kazal-type 1 Septin 9 Voltage-gated sodium channel, IXa (Nav1.7) Voltage-gated sodium channel, IXa (Nav1.7) [117,118] [119] [120,121] [122] [123] [124] [125] [126] [127] [128] [129] [130] a Based on a search of the Online Mendelian Inheritance in Man (OMIM) database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim) using the keyword ‘pain’. Syndromes are included if pain is reported as a defining symptom of the disorder in the ‘Description’ subsection. Excludes inherited dysplasias, myotonias, and Charcot–Marie–Tooth disease, all of which can be painful. Abbreviations: CAIP, channelopathy-associated indifference to pain; CIPA, congenital insensitivity to pain with anhidrosis; FHM, familial hemiplegic migraine; FMF, familial Mediterranean fever; HNA, hereditary neuralgic amyotrophy; HSAN, hereditary sensory and autonomic neuropathy; PEPD, paroxysmal extreme pain (familial rectal pain) disorder. b The reference provided is the first to identify unambiguously a causal gene variant. In many cases, prior linkage mapping studies existed in which the gene was hypothesized to be responsible. might even explain variability in more common pain states, the more direct approach to uncovering common disease-related gene variants is the genetic association study. This technique has now been adopted earnestly by clinical pain researchers, leading to an acceleration in published findings (Figure 1), and progress has been reviewed in depth elsewhere (e.g. [23,58,59]). A wide variety of genes has now been tentatively associated with both experimental and clinical pain states (Figure 2 and the 80 No. of findings pain sensibility is markedly absent (also known as congenital insensitivity to pain; [51,52]), the familial hemiplegic migraine (FHM) disorders [53], and neurological channelopathies presenting as paroxysmal pain disorders (primary erythromelalgia, paroxysmal extreme pain disorder and familial episodic pain syndrome) [54]. Genes and mutations responsible for many known disorders of this type have now been found (Table 2). These genes encode proteins of different functional classes (e.g. ion channels, enzymes, transcription factors and trophic factors), and perhaps the only common thread is the involvement in several disorders of the SCN9A gene encoding the Nav1.7 sodium channel, leading to its current status as a highpriority drug development target. Evidence that SCN9A might play a role in more common pain states was provided by the observation that there is significant association of the R1150W variant (rs6746030) with osteoarthritis, sciatica and post-amputation pain (but not post-discectomy pain or chronic pancreatitis pain) [55]. A recent study failed to replicate the osteoarthritis association in two separate cohorts, but found an association between the R1150W variant and multiple regional pain [56]. In the migraine genetics field, association studies have been largely unable to demonstrate the involvement of FHM genes in common migraines (e.g. [57]). 60 40 20 0 1975 1980 1985 1990 1995 2000 2005 2010 2015 Year of publication TRENDS in Genetics Candidate gene association studies of pain Although genes responsible for rare inherited disorders may provide excellent targets for drug development and 260 Figure 1. Number of pain-relevant candidate gene association study findings in humans by year of publication. See online supplementary material for details of each finding. Review Trends in Genetics June 2012, Vol. 28, No. 6 (a) (f) Acute pain ADRB2 COMT HTR2A IL1RN KCNJ6 MAOA MAOB OPRM1 Inflammatory pain CFTR HLA (all) IL1 IL6 MEFV SPINK1 0 0 5 10 15 5 20 No. of findings (b) (g) Back pain CACN2D3 CASP9 CILP COL1A1 COL9A2 COL9A3 GCH1 IL1A IL1B IL1RN IL6 KCNS1 SCN9A 0 5 10 15 20 No. of findings (c) 10 15 20 15 20 No. of findings Musculoskeletal pain ACAN ANP32A ASPN COL6A4P1 COMT DIO2 EDG2 ESR1 GDF5 HFE IL1RN P2RX7 PCSK6 SCN9A SMAD3 TRPV1 0 Cancer pain 5 10 No. of findings ARRB2 COMT GCH1 IL6 IL8 NFKBIA OPRM1 PTGS2 STAT6 TNF (h) Neuropathic pain ADRA1A CACNG2 HLA (all) 0 5 10 15 20 KCNS1 P2RX7 SCN9A No. of findings TRPA1 TRPV1 (d) Experimental pain 0 5 10 15 20 No. of findings ADRA2C AVPR1A COMT DRD3 DRD4 FAAH GCH1 KCNS1 MAOA MC1R OPRD1 OPRM1 SLC6A3 SLC6A4 TRPA1 TRPV1 (i) Visceral pain CYP2D6 ESR1 GC GNB3 GSTM1 IL16 0 5 10 15 20 No. of findings (e) MIF PGR PTGS1 SERPINE1 TACR1 TPRV1 Idiopathic (functional) pain 5 0 10 15 20 No. of findings ADRA1D ADRA2A ADRB2 CNR1 COMT GSTM1 HTR2A HTR3E IL1 IL10 IL1RN MBL2 MC1R MTHFD1 MTRR SHMT1 SLC6A4 SOD2 TNF (j) Widespread pain ADRB2 APOE CHRBP COMT DRD4 HTR2A MC2R POMC SCN9A SERPINA6 SLC6A4 0 5 10 15 20 No. of findings 0 5 10 15 20 No. of findings TRENDS in Genetics Figure 2. Number of pain-relevant candidate gene association study findings in humans by phenotype (see online supplementary material for details). Genes with at least one positive association are shown in green; negative associations are shown in red. supplementary material online, a searchable and sortable compendium of individual association findings, positive and negative, from 1976 to 2011), as well as pain endophenotypes, such as cortical activation in the so-called ‘pain matrix’, wind-up (i.e. increased pain owing to temporal summation), and conditioned pain modulation (i.e. counter-irritation). In most cases, genes were chosen for study based on their known or presumed role in pain 261 Review Trends in Genetics June 2012, Vol. 28, No. 6 physiology, and/or because of their well-studied and highfrequency variants. In other cases, the genes were originally identified in animals (mice, rats or Drosophila) using whole-genome QTL, mutant screening or microarray profiling; these have been especially heuristic. Nonetheless, pain genetics has so far been dominated by investigations of an exceedingly restricted number of genes, with 10 genes (or gene complexes) accounting for over half of the extant findings (Figure 3). As in other fields, replication of these associations has been inconsistent at best, with follow-up studies finding no association, association in the opposite direction phenotypically, association in some but not other cohorts, or associations but with different single nucleotide polymorphisms (SNPs) or haplotypes from those originally reported; potential reasons for this have been the subject of considerable debate [60–62]. Only one true meta-analysis has been performed so far, on the N40D (A118G) polymorphism of the OPRM1 gene encoding the m-opioid receptor. Although the preclinical data are solid, no overall statistical relationship was found between OPRM1 genotype and opioid requirements or pain levels in patients [63]. In addition to the general problems of sample size, design, subject ascertainment, selective publication bias (‘winner’s curse’), population substructure confound, and so on, the pain field struggles with the ‘lumping/splitting’ problem hinted at previously. It is not at all clear whether one should expect to find genes broadly affecting multiple pain syndromes or symptoms, or whether completely different sets of genes ultimately underlie, say, back pain versus post-herpetic neuralgia, or even thermal versus mechanical hypersensitivity experienced by the same patient. A more subtle point is this: if one does find a gene variant with broad effects on chronic pain, is it more likely that the gene participates in pain physiology per se, or in the physiology of psychological modulators of pain (e.g. Key: COMT OPRM1 GCH1 HLA (all) SLC6A4 HTR2A IL1A,B IL1RN TRPV1 TNF All others TRENDS in Genetics Figure 3. Popular candidate genes in pain genetic research. Studies of variants within these 10 genes outnumber studies of all other genes combined. Abbreviations: COMT, catechol-O-methyltransferase; GCH1, GTP cyclohydrolase 1; HLA, human lymphocyte antigen (major histocompatibility complex); HTR2A, serotonin receptor, 2A; IL1A,B, interleukin-1 receptor a or b; IL1RN, interleukin-1 receptor antagonist; OPRM1, m-opioid receptor; SLC6A4, solute carrier family 6, member 4 (serotonin transporter); TRPV1, transient receptor potential cation channel, V1; TNF, tumor necrosis factor. 262 anxiety, depression, anger or catastrophizing)? There is some evidence to suggest the latter (e.g. [64–66]) and, although this makes no difference whatsoever to the issue of explaining variability, it might diminish interest in that gene as a target for drug development. Multi-gene association studies Despite the current explosion of interest, human pain genetics has lagged behind genetics efforts in other fields of biomedicine, for reasons including its subjective quantification, relatively low funding levels, a dearth of familial aggregation and twin studies (until recently), and the relative lack of interested epidemiologists, geneticists and biostatisticians [67]. Migraine genetics is the exception, with at least three genome-wide association studies (GWAS) having been published so far [68–70], but migraine genetics has been reviewed extensively by others and will not be considered here. GWAS of disorders with pain as a major symptom, including endometriosis [71], Crohn’s disease [72] and osteoarthritis [73,74], have been published, but pain intensity in these studies was neither measured nor analyzed. Only one GWAS has been published so far with pain itself as a dependent measure, relating to pain levels after third molar extraction and inhibition of that pain by the NSAID ketorolac [75]. Although a true GWAS, with all subjects genotyped using the Affymetrix 500K SNP chip, the total sample size was only n = 112, and the only association surviving correction for multiple comparisons was with a SNP in an unannotated gene (LOC400680) in linkage disequilibrium with the ZNF429 gene encoding a zinc finger protein. A modified GWAS (using DNA-pooled ‘good’ versus ‘poor’ responders) of opioid (morphine, oxycodone and fentanyl) analgesia in patients with cancer has recently been reported, with the strongest association by far with rs12948783, upstream of the rhomboid 5 homolog 2 (RHBDF2) gene of unknown function [76]. No polymorphisms in any known opioid receptor genes were found to be associated. Of interest is the fact that the strongest effect by far in this study was European country of origin (P<2.210–16), suggesting the importance of ethnic differences. Although not a GWAS, preliminary results of a particularly ambitious multi-center, multi-gene study of temporomandibular disorder (TMD; an orofacial chronic pain disorder affecting 5–10% of the population [77,78]) have just been published. Called OPPERA (Orofacial Pain: Prospective Evaluation and Risk Assessment), it comprised a case–control study and large prospective cohort study of 3200 initially TMD-free individuals recruited at four study sites, of which 260 have already developed diagnosed TMD during the initial 5-year study period. A panel of 3295 SNPs representing 23 high-priority and 358 trait-relevant gene targets (the ‘Pain Research Panel’) was genotyped in 166 chronic TMD cases and 1442 controls, supplemented by an additional 182 cases and 170 controls from a non-OPPERA cohort [79]. No associations achieved strict experiment-wide significance, and only two existing associations [with catechol-O-methyltransferase (COMT) and 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A)] were supported; however, these two involved different SNPs than reported previously. By contrast, several new suggestive associations Review were evinced, to genes known to play important roles in pain physiology [e.g. calcium channel, voltage-dependent, alpha 2/delta subunit 1 (CACNA2D1), nerve growth factor (NGF) and tachykinin, precursor 1 (TAC1)] and to genes with no currently known function in pain [e.g. calcium/calmodulindependent protein kinase IV (CAMK4), cholinergic receptor, muscarinic 2 (CHRM2), interferon-related developmental regulator 1 (IFRD1), G protein-coupled receptor kinase 5 (GRK5) and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor; NR3C1)]. Attempts are ongoing to increase the statistical power and confirm some of these associations in independent TMD cohorts and cohorts of other patients with chronic pain. Ongoing work investigating the murine analogs of some of these genes has found strong evidence for their involvement in pain in the mouse (L.M., A.K., S.B.S., R.E.S., J.-S.A., D.L., L.G., N.S., W.M., J.S.M., and L.D., unpublished). Although not part of OPPERA, this same group has just published another large multi-gene association study of fibromyalgia using the 350gene Pain Research Panel [80]. One gene, TAAR1 (encoding the G-protein coupled trace amine-associated receptor 1), showed evidence of association in both cohorts examined. There was no overlap with OPPERA-associated genes. OPPERA itself represents a perfect microcosm of current enthusiasms and frustrations with human genetic association studies. Candidate gene guesses are overwhelmingly proven wrong [81], supporting the value of blinded genome-wide approaches in both animals and humans. Replication is difficult, and a disappointingly small percentage of genetic and trait variance has so far been explained. On a more positive note, truly heuristic biological information has been gained in some cases, and it is hard to envisage how this information would have been obtained using any other approach. How many pain genes are there? As research into the genetics of pain and analgesia accelerates, a reasonable question to start asking is how many pain genes are there to find? Part of the difficulty in answering is a matter of definition: are pain genes only those genes contributing to pain variability? What about those contributing to susceptibility to painful diseases (e.g. arthritis), but not pain within those diseases? What about genes involved in, say, the development of nociceptors, their housekeeping functions, or genes related to very common neurochemical signaling systems (e.g. glutamate or GABA) with functions well beyond pain? Defined via significant transgenic knockout mouse phenotypes [3] or significant regulation in chronic pain states [4], many hundreds of pain genes in rodents have already been tentatively discovered. Presumably many more await discovery. Just how many genes will affect pain variability enough to be easily identified as such depends on: (i) their average effect size (which could be very weak indeed [82]); (ii) the breadth versus specificity of their effects; (iii) how applicable the common disease-common variant hypothesis is to pain [58]; and (iv) practical issues, such as the number and size of phenotyped patient cohorts. Effect sizes of pain genes are often not reported and are probably systematically overestimated [83]. Some murine pain QTLs appear to have exquisitely specific effects; for example, the Nociq1 QTL that has been shown to be mediated by Trends in Genetics June 2012, Vol. 28, No. 6 expression levels of Atp1b3 (encoding the b3 subunit of the sodium-potassium pump) affects responses in the early (0– 10 min) phase of the formalin test, but not afterwards and in no other preclinical assay [84]. It seems somewhat clear by now that pain does not have its own APOE or BRCA1; there is no genetic association in the field so far that has been consistently replicated or explained a large proportion of trait variance. Attention in the pain field, as elsewhere, is starting to turn to more complex approaches. Beyond genetics: epistasis, gene environment interactions, small RNAs, epigenetics and other -omics Both linkage and association study methodologies attempt to isolate the additive effect of DNA variants. It is well appreciated that trait variance is also impacted by gene gene interactions (epistasis), gene environment interactions and environmental factors per se, the effects of which might be mediated by epigenetic regulation. The first few examples of these phenomena in the pain field have recently been demonstrated. Two studies [85,86] observed interactions between the V158 M (G1947A) variant of COMT and the N40D (A118G) variant of OPRM1 in the determination of pain-related morphine consumption. In both cases, although the genotypic effect of the individual variants were non- or only marginally significant, the joint genotype at both genes considered simultaneously achieved significance. Although the results of the two studies are not directly comparable and it remains unclear whether the gene effects were truly interactive and not just additive, epistasis between pain genes is probably a common phenomenon. Recent work has uncovered and elucidated a gene sex environment interaction of relevance to pain. A promoter SNP (rs10877969) upstream of the arginine vasopressin receptor 1A (AVPR1A) gene significantly affects capsaicin pain sensitivity, but only in men and, moreover, only when those men report subjective stress at the time of capsaicin testing [45]. This gene was identified via QTL mapping in mice, and a reanalysis of the murine data sets uncovered the fact that Avpr1a also had an analogous sex- and stressspecific effect on pain in this species. It is conceivable that any number of failures to replicate genetic association study findings may be ultimately explainable in terms of interactions between gene variants and organismal factors and environmental testing conditions. One of the more intriguing recent developments in cellular genomics is that naturally occurring noncoding miRNAs can profoundly inhibit gene expression by degrading mRNAs and regulating epigenetic machinery [87]. A handful of recent studies have documented changes in miRNA levels in pain states [88]. Causational evidence of miRNA involvement in pain comes from the demonstration that conditional deletion in nociceptors of the miRNAproducing enzyme, Dicer, blocks inflammatory pain hypersensitivity [89], and that small interfering-RNA-mediated knockdown of one miRNA, miR-103, produces hypersensitivity to pain in naı̈ve rats [90]. There is currently great interest in the possibility that the study of epigenetic mechanisms might provide novel insight into pain chronicity. Indeed, the concept that changes in chromatin structure might underlie 263 Review acute-to-chronic pain transitioning, pain ‘priming’ effects [91–93] and effects of neonatal pain experience on adult pain sensitivity (e.g. [94,95]), is compelling. Although no one has yet shown this to be true, the first evidence of epigenetic regulation by chronic pain in rodents is appearing in the literature (reviewed in [96]). Changes in the expression of pain-related genes [e.g. glutamate decarboxylase 2 (Gad2), potassium voltage-gated channel, Shal-related subfamily, member 3 (Kcnd3), methyl CpG binding protein 2 (Mecp2), Oprm1, Scn9a and secreted protein, acidic, cysteine-rich (Sparc)] are correlated with histone modifications [97–101], chronic pain states feature abnormal DNA methylation and histone acetylation [98,100,101], and treatment with histone deacetylase (HDAC) inhibitors is analgesic [99,102,103]. The value of these initial demonstrations is limited by their correlational nature and the non-specificity of HDAC inhibitors, but studies of the epigenetics of pain are likely to yield many surprises in the near future. Although the focus so far has been on genomics and transcriptomics, other ‘-omics’ techniques continue to develop, and have the theoretical advantage of being closer to real biological actions than are DNA and mRNA. Two painrelevant papers have just published. In one, tissue metabolites were profiled by mass spectrometry (metabolomics), identifying dysregulation in sphingomyelin–ceramide metabolism in neuropathic rats and suggesting inhibition of N,N-dimethylsphingosine production as a novel analgesic strategy [104]. In the other, cadaverous nerves from patients with complex regional pain syndrome (type 2) were subjected to proteomic analysis, identifying an absence of zinc-binding metallothioneins in these sufferers of chronic pain [105]. Concluding remarks Despite its delayed entry, pain genetics is now proceeding at a particularly rapid pace. As is not unusual in science, increasing knowledge has revealed the size of the problem to be far larger than anticipated. At the present time, I am not optimistic about pain geneticists explaining enough trait variance in clinical pain states or analgesic response to serve as a guide to individualized pain therapy any time soon. 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