The genetic mediation of individual differences in sensitivity to pain and its inhibition

Abstract

The underlying bases of the considerable interindividual variability in pain-related traits are starting to be revealed. Although the relative importance of genes versus experience in human pain perception remains unclear, rodent populations display large and heritable differences in both nociceptive and analgesic sensitivity. The identification and characterization of particularly divergent populations provides a powerful initial step in the genetic analysis of pain, because these models can be exploited to identify genes contributing to the behavior-level variability. Ultimately, DNA sequence differences representing the differential alleles at pain-relevant genes can be identified. Thus, by using a combination of “top-down” and “bottom-up” strategies, we are now able to genetically dissect even complex biological traits like pain. The present review summarizes the current progress toward these ends in both humans and rodents.

Pain is considered both a sensation and an emotion (1) and shows considerable complexity and subjectivity, especially when compared with other sensory systems. Physical injury is neither necessary nor sufficient for the experience of pain, and in both clinical and laboratory settings, the perception of pain bears a notoriously poor relationship to the intensity of the noxious stimulus. Mildly noxious stimuli can be perceived as very painful whereas very noxious stimuli can produce no pain whatsoever (2). Substantial advances in understanding why this might be have been made in the past few decades, including the discovery of pain-modulatory mechanisms (producing both analgesia and antianalgesia) and nervous system plasticity following exposure to noxious stimuli.

Even as our understanding of pain physiology has been facilitated by such paradigmatic advancements, pain perception in humans and animals nonetheless displays considerable, and unexplained, interindividual variability. The focus of this review is to examine the scope of individual differences in pain and analgesia in both humans and laboratory animals and to consider what is known and what remains to be determined regarding the genetic contributions to such variability. Null mutants (e.g., transgenic knockouts) will not be considered presently, because they have been recently reviewed (3).

The Scope of Individual Variability to Pain and Analgesia

Human Studies.

Variability in pain-related traits (or “phenotypes”)—experimental pain sensitivity, propensity to develop painful pathologies, and sensitivity to analgesic manipulations—has long been appreciated, although empirical validation of such variability is somewhat limited. In 1934, Libman (4) reported that pressure applied to the mastoid bone in the direction of the styloid process produces “marked” pain in 60–70% of his human patients, but no or little pain in 30–40%. Subsequent investigations over the next few decades with more quantitative methodology confirmed the presence of large individual differences in threshold sensitivity and tolerance to noxious pressure (5–8), heat (7, 9, 10), and electrical current (7, 11) applied to cutaneous tissues, and tolerance to visceral (6) and deep muscle (9, 10) pain. Impressive individual differences in sensitivity to opioid analgesics were also documented during this period, typified by Lasagna and Beecher (12) observing a “success rate” of only 65% of the standard clinical dose of morphine, 10 mg (see also ref. 13).

Modern studies have confirmed that interindividual variability in pain thresholds greatly exceeds intraindividual variability for pressure pain threshold (e.g., refs. 14 and 15), cold pressor tolerance (e.g., ref. 16), and pain associated with manual palpation of tender points in fibromyalgia sufferers (17). In the cold pressor test, Chen et al.(16, 18) consistently observe dichotomous responses, with a minority “pain-sensitive” group tolerating the test for a mean duration of 50 sec and a majority “pain-tolerant” group able to tolerate the test for the full 300-sec duration.

Similar findings have been obtained for opiate analgesics, which display large clinical (e.g., refs. 19 and 20) and experimental (e.g., ref. 21) variability in their efficacies, side effects, and tolerance liability. The analgesic actions of morphine on dental extraction pain also show evidence of bimodality, with morphine “responder” and “nonresponder” groups being easily identified at a range of doses (22). Individual differences in the actions of an opiate antagonist, naloxone, have also been demonstrated. Buchsbaum et al. (23), after dividing human subjects into pain-sensitive and pain-insensitive groups based on their sensitivity to electric shock to the forearm, noted that intravenous injection of 2.0 mg of naloxone produced hyperalgesia in the latter group and (paradoxically) analgesia in the former.

Animal Studies.

In the concluding comment of Libman’s (4) seminal study of human pain variability, he posits that studies of animals may prove of value, because “in all the work hitherto performed it has been taken for granted that sensitiveness is the same in all animals of a given species” (p. 341). Indeed, this is the working assumption of much biological research: that findings obtained from some presumed randomly bred sample of Rattus rattus will be representative of the “universal rat,” and subsequently generalizable to all rats, and mice, and maybe to all humans. It remains an empirical question as to just how untenable this assumption is. Unfortunately for pain researchers (but fortunately for pain geneticists!), the existing data regarding the nociceptive and analgesic sensitivity of laboratory rodent populations reveal great, and in some cases qualitative, variability.

Rodent populations of use for genetic analysis can either be produced (e.g., inbred strains, recombinant inbred strains, artificially selected lines, transgenics) or identified (e.g., spontaneous mutants). The characteristics of these genetic models have been reviewed elsewhere (e.g., ref. 24). The most studied genetic rodent models of relevance to pain include the recombinant inbred (RI) CXBK mouse strain (25, 26), the High Analgesia/Low Analgesia (HA/LA) mouse lines selectively bred for swim stress-induced analgesia (SIA) (27, 28), the High Analgesic Response/Low Analgesic Response (HAR/LAR) mouse lines selectively bred for levorphanol analgesia (29), the High Autotomy/Low Autotomy (HA/LA) rat lines selectively bred for autotomy (30), and the normotensive Wistar Kyoto versus Spontaneously Hypertensive Rat (WKY/SHR). The former models have been thoroughly reviewed (24). The literature regarding the relationship between nociception and genetic or experimentally induced hypertension has also been recently reviewed (31).

Strain differences.

Although less well studied at present, the comparison of inbred strain responses is more relevant to the issue of the scope of individual differences than the aforementioned models. Inbred strains are derived by repeated (>20 generation) full-sibling (i.e., brother × sister) mating (32). Mating of individuals with common ancestors increases the probability of offspring inheriting two copies of the same allele identical-by-descent. During inbreeding, therefore, genetic heterozygosity is progressively lost as alleles of initially segregating genes are fixed into a homozygous state.

Tables 1 and 2 present some existing data regarding inbred (and outbred) strain differences of relevance to pain in rats and mice, respectively. The only obvious generalizations that can be made from Table 1 are the nociceptive sensitivity of the Lewis (LEW) inbred rat strain and the sensitivity to a wide variety of analgesic manipulations of the outbred Sprague–Dawley (SD) strain. Multistrain comparisons (“strain surveys”) are far more common in the mouse because of the ready availability of over 30 major inbred strains. Obvious generalizations from mouse strain surveys are thus harder to make. One exception is the voluminous research demonstrating the relative sensitivity of the DBA/2 (D2) strain to opioid analgesia compared with the C57BL/6 (B6) strain (not shown in Table 2, but reviewed in refs. 33 and 34). Although D2 mice display high, and B6 mice display low magnitudes of analgesia, the B6 strain is markedly more sensitive than the D2 strain to other opioid-mediated phenomena, including locomotor activation, learning/memory, and muscular rigidity (Straub tail).

Table 1.

Rat strain differences of relevance to pain

Trait	Parameters	Administration	Stimulus	Strain Difference*	Ref.
Nociceptive sensitivity
Thermal			TW	WAG > F344	117
			TF	LE = LEW = WIS > F344 = SD	118
			HP	LEW > F344	119
Electrical			FJ	F344 > SD	120
Mechanical			CD	LEW > F344	121
			VF	F344 = LEW = WIS > SD	42
Chemical			CFA	LEW > SD	122
			CFA	LEW > AVN	123
Neuropathic			NT	SD > WKY	124
			NT	LE = SAB = SD = WKY > LEW	125
			NT	BUF = SD > BN > WIS > LEW	126
Analgesia
Morphine	0–15 mg/kg	i.p.	FJ	SD > F344†	120
	0–10 mg/kg	i.p.	TF	SD > WIS	127
	0–10 mg/kg	i.p.	TW	F344 > WAG	117
	0–20 mg/kg	i.p.	FT	F344 > LEW	41
	0–10 mg/kg	i.p.	TF	LE = SD ≥ LEW = WIS ≥ F344	118
	0–10 mg/kg	s.c.	HP	SD > WKY	128
Codeine	50–400 μmol/kg	s.c.	TF	SD > DA	116
Clonidine	0–60 μg/kg	i.p.	FT	SD > WKY‡	129
	10 μg	i.c.v.	TW	WAG > F344	130
TRH	1 mg/kg	i.p.	TW	WAG > F344	131
Serotonin	0–300 μg/kg	i.v.	TF	SD > WKY	132
	0.5 μg	i.c.v.	TW	F344 > WAG	130
Footshock	1.5 mA		TW	F344 > SD§	133
Restraint	30 min in tubes		TF	LE = SD > F344 = LEW = WIS	118
Acupuncture	100 Hz, 1–3 mA		TF	P77PMC > WIS	134
Analgesic tolerance
Morphine	14 × 5–10 mg/kg	i.p.	TF	SD > WIS	127
	8 × 10 mg/kg	s.c.	HP	WKY > SD	128

Strain Abbreviations: BN, Brown Norway; BUF, Buffalo; DA, Dark Agouti; F344, Fischer 344; LE, Long–Evans (outbred); LEW, Lewis; SAB, Sabra (outbred); SD, Sprague–Dawley (outbred); WAG, Wistar Albino Glaxo (WAG/GSto); WIS, Wistar (outbred); WKY, Wistar Kyoto. Genealogical origins of all inbred rat strains can be found at http://www.informatics.jax.org/bin/strains/search. 

Other Abbreviations: CD, colorectal distention; CFA, complete Freund’s adjuvant; FJ, flinch–jump test; FT, formalin test; HP, hot-plate test; i.c.v., intracerebroventricular, i.p., intraperitoneal; NT, sciatic and saphenous nerve transection; TF, radiant heat tail-flick test; TRH, thyrotropin-releasing hormone; TW, hot water tail-immersion/withdrawal test. 

^*Only studies with significant strain differences are reported. Excluded are studies involving selected lines [including the spontaneously hypertensive rat (SHR)] and mutants. 

^†Morphine analgesia was significantly attenuated by pretreatment with p-chlorophenylalanine in SD, but not F344 rats. 

^‡Clonidine analgesia was naloxone-reversible in SD rats but naloxone-insensitive in WKY rats. 

^§F344 rats also developed increased conditioned analgesia to footshock relative to SD rats. 

Table 2.

Mouse strain differences of relevance to pain

Trait	Parameters	Administration	Stimulus	Strain Difference*	Ref.
Nociceptive sensitivity
Thermal			HP	B6 = ICR = SW > BALB	135
			HP	B6 > D2 >C3H	136
			HP	B6 > 7 others	62
			HP	RIIIS > 9 others > AKR	59
			HT	B6 > 9 others > AKR	59
			TW	B6 > 9 others > RIIIS	59
Chemical			AC	CBA > C3H = D2 > B6	137
			AC	C3H = CBA > 6 others	62
			AC	C3H > 9 others > 129	59
			FT	13 others > A	138
Neuropathic			NT	OF1 = BALB > NMRI > B6CBAF1	139
			NT	C3HeB > ICR	140
			NT	B6 > 8 others > AKR = C58	59
Hypersensitivity
Thermal			CAR	BALB > 9 others > SM	59
			PNI	AKR > 9 others > C58	59
Mechanical			PNI	129 > 9 others > CBA	59
Analgesia
Morphine	0–8 mg/kg	s.c.	HP	CF-1 > CFW	141
	0–320 mg/kg	i.p.	HP	SW > B6 = BALB = ICR	135
	0–2 mg/kg	s.c.	AC	A = B6 = D2 > C3H = ICR = SW	142
	75 mg pellet		TF	SW > ICR	143
	75 mg pellet		HP	C3H = D2 > CD-1	144
	5 mg/kg	s.c.	TF	BALB > CD-1 > SW	145
	5 mg/kg	s.c.	HP	BALB > CD-1 > SW	145
	0–100 mg/kg	s.c.	HP	BALB = CBA = D2 > AKR = B6 = C3H	62
	0–10 mg/kg	s.c.	AC	B6 = D2 > C3H = CBA > AKR	62
	0–12 μg	i.c.v.	TW	C3H > 9 others > SWR†	58
U-50,488	5 mg/kg	s.c.	TF	CD-1 > SW > BALB	145
NalBzoH	50 mg/kg	s.c.	TF	CD-1 > SW > BALB	145
D-phenylalanine	0–400 mg/kg	i.p.	HP	B6 = C3H > BALB = D2	146
Nitrous oxide	25–75%		AC	A > 8 others > D2	147
Alprazolam	0–100 mg/kg	i.p.	TF	BALB > SW > B6 = CD-1	148
Nicotine	0–10 mg/kg	i.p.	TF	CD-1 > CF-1	149
Epibatidine	0–0.1 mg/kg	s.c.	TW	A > 7 others > C3H	150
Acetaminophen	0–250 mg/kg	s.c.	AC	AKR > 6 others > BALB	(unpublished data)
Analgesic tolerance
Morphine	9 × 50–100 mg/kg		HP	B6 = BALB = ICR > SW	135
	75 mg pellet		TF	SW > ICR	143
	75 mg pellet		TF	CD-1 > 129‡	151

Strain Abbreviations (substrain identifiers are omitted; in most cases, inbred strains were obtained from The Jackson Laboratory: B6, C57BL/6; BALB, BALB/c; C3H, C3H/He; CD-1, Hsd:ICR (outbred); CF-1, Hsd:NSA (outbred); CFW, HsdWin:CFW1 (outbred); D2, DBA/2; ICR, Institute for Cancer Research stock (many suppliers; outbred); SW, Swiss Webster (outbred). Genealogical origins of all inbred mouse strains can be found in Festing (37) or at http://www.informatics.jax.org/bin/strains/search. 

Other Abbreviations: AC, abdominal constriction (writhing) test; CAR, carrageenan; FT, formalin test; i.c.v., intracerebroventricular; i.p., intraperitoneal; i.v., intravenous; HP, hot-plate test; NalBzoH, naloxone benzoylhydrazone (a κ₃-opioid agonist); NT, sciatic and saphenous nerve transection; PNI, peripheral nerve injury (Chung model); TF, radiant heat tail-flick test; TW, hot water tail-immersion/withdrawal test; VF, von Frey fiber test. 

^*Only studies with significant strain differences are reported. Excluded are studies involving selected lines, mutants, recombinant inbred (RI) strains, and non-extant populations. Also excluded are studies specifically comparing the B6 and D2 strains. 

^†Strain differences observed varied with sex. 

^‡No analgesic tolerance whatsoever developed in the 129/SvEv substrain used. 

Qualitative strain differences.

In addition to the quantitative strain differences compiled in Tables 1 and 2, some very intriguing qualitative strain differences of relevance to pain have been noted. For instance, a number of investigations have suggested that certain strains activate opioid analgesic systems after exposure to stress, whereas other strains produce approximately equivalent amounts of SIA, but of a non-opioid (i.e., naloxone-insensitive) character (35–37). Vaccarino et al. (38) reported that naloxone injection produced paradoxical analgesia on the formalin test in BALB/c mice, but not B6 or outbred CD-1 mice. Fujimoto and colleagues (39) have demonstrated convincingly that heroin analgesia is mediated by μ-opioid receptor activation in outbred Institute for Cancer Research stock (ICR) mice, but by δ-receptor activation in outbred Swiss Webster (SW) mice. The same workers have recently (40) identified μ-, δ-, and κ-type heroin responders among inbred mouse strains. Vaccarino and Couret, Jr. (41) observed that the presence of formalin-induced pain during tolerance induction wholly prevented tolerance development in the Fischer 344 (F344) strain but not in the LEW strain. Lee et al. (42) observed a complete blockade of neuropathic mechanical allodynia after treatment with the α-adrenergic receptor antagonist, phentolamine, in LEW rats; this treatment was wholly ineffective in F344 rats. The authors concluded that this neuropathy was sympathetically maintained in the former strain only. Finally, Proudfit’s laboratory has demonstrated (43) that electrical stimulation-produced analgesia is reversed by α₂-adrenergic antagonists in SD rats from the now defunct Sasco (Omaha, NE), but not in SD rats from Harlan–Sprague–Dawley. This difference may be explained by the differential projection routes and dorsal horn termination fields (laminae VII–X versus laminae I–IV, respectively) of pontospinal noradrenergic neurons in these substrains (44).

Heritability of Pain-Related Traits

That individual differences in pain-related traits exist, of course, does not imply that these differences are necessarily attributable to genetic factors. Familial aggregation of pain pathologies and extremes of pain sensitivity has been repeatedly noted in humans, but such findings have almost uniformly been attributed to shared environmental variance and/or familial modeling (e.g., refs. 45–49). To separate genetic and environmental factors, twin studies have been conducted (some even featuring adoption), comparing the concordance rates of pain-related traits in monozygotic (identical) versus dizygotic (fraternal) twins (see ref. 50). Heritability (i.e., the proportion of overall phenotypic variance accounted for by genetic factors) has been estimated as 39–58% for migraine (51–53), 55% for menstrual pain (54), 50% for back pain (55), and 21% for sciatica (56).

Only one report exists of a twin study of nonpathological, basal pain sensitivity. MacGregor et al. (57) assessed forehead pressure pain thresholds in 269 monozygotic pairs and 340 dizygotic pairs, and observed only a slight excess correlation in monozygotic versus dizygotic twins (r = 0.57 vs. 0.51, respectively). This excess corresponds to a heritability of this trait of only 10% and suggests that the twin correlations in pain thresholds are largely due to shared environmental factors.

In contrast to this last study in humans, heritability estimates for nociceptive and analgesic sensitivity in mice are fairly high, ranging from 28% to 76% (29, 58–60)—certainly well within the range considered for further genetic analysis. Because none of the murine nociceptive assays used are similar to the forehead pressure pain test used by MacGregor et al. (57), it is difficult at the present time to evaluate whether humans and mice truly differ in the contribution of genes to pain sensitivity. Even if genetic factors are ultimately demonstrated to play only a minor role in the determination of individual pain sensitivity in humans, genetic studies of pain may still prove highly valuable. Such studies, for example, may illuminate those components of pain processing circuitry in mice and humans that are especially amenable to alteration, knowledge likely to be useful for the development of novel analgesic strategies.

Genetic Correlations Among Pain Phenotypes

The tools of classical genetics can be used, even in advance of the identification of the relevant genes, to determine whether traits share genetic mediation (see ref. 61). The fact that a given gene can influence more than one trait is known in genetic parlance as pleiotropy. Pleiotropic actions of genes result in the genetic correlation of traits, because allelic variation in a gene will influence all traits in which that gene participates. The determination of genetic correlation has proven to be very heuristic, leading to novel theories regarding the underlying physiological mediation of traits. A number of genetic correlations of pain-related phenotypes have been noted using selected lines and inbred strains (see ref. 24). I would like to focus on two intriguing findings, as follows.

Genetic Correlation of Nociception and Opiate Analgesia.

It has been demonstrated by several groups that a negative genetic correlation exists between initial nociceptive sensitivity and subsequent morphine analgesia (27, 62, 63). That is, mice that are initially sensitive to noxious stimuli tend to exhibit modest analgesic responses to morphine, whereas mice that are relatively resistant to basal nociception exhibit robust morphine analgesia. In two separate studies using multiple inbred strains, this correlation was estimated as r = −0.63 to −0.85 (62) and r = −0.61 (58). Thus, mice are “doubly advantaged” or “doubly disadvantaged” with respect to nociception and its opiate inhibition. Interestingly, this negative correlation (albeit with n = 2 only) can also be observed when comparing the sexes.

Genetic Correlations Among Nociceptive Assays.

We recently tested 11 inbred strains on 12 separate measures of nociception in common use in the mouse (59, 60). The assays used can be placed on a number of dimensions, including etiology (nociceptive, inflammatory, neuropathic), modality (thermal, chemical, mechanical), duration (acute, tonic, chronic), and location (cutaneous, subcutaneous, visceral). We reasoned that inbred strain variation could be exploited to identify clusters of genetically correlated nociceptive assays. Similar genetic mediation implies similar physiological mediation of assays, suggesting that they measure the same “type” of pain as defined mechanistically. Essentially, we were attempting to produce a natural rather than artificial taxonomy of nociception in the mouse, similar to that called for by Woolf et al. (64).

The results of this effort were variously expected and surprising. By using multivariate analyses, we identified three obvious clusters of pain tests, in which within-cluster genetic correlations greatly exceeded between-cluster correlations: “thermal” (Hargreaves’ test, hotplate test, tail-immersion/withdrawal test, and, surprisingly, autotomy), “chemical” (acetic acid abdominal constriction, magnesium sulfate abdominal constriction, acute- and tonic-phase formalin test), and “mechanical + hypersensitivity” (von Frey test, carrageenan thermal hypersensitivity, peripheral nerve injury thermal, and mechanical hypersensitivity) (59, 60). Thus, the stimulus modality dimension accounted for the obtained genetic correlations to a far greater degree than any other factor. The presence or absence of neuropathy or inflammation was found to be essentially irrelevant as was the site or duration of the stimulus.

Identification of Pain-Related Genes

The holy grail of pain genetics, of course, is the actual identification of pain-related genes and the polymorphisms within or near such genes that account for trait variability. Note that “pain-related gene” could be broadly defined as any gene encoding a protein of known pain relevance or of a gene whose null mutant exhibits a pain-related phenotype. Defined in this way, a large number of pain-related genes are known. However, if more properly defined as one in which allelic variation directly produces individual differences or pathology, only a handful of pain-related genes have been identified.

Techniques.

Essentially, there are two ways to identify genes associated with trait variability: (i) linkage analysis, including classical model-based linkage techniques and allele-sharing methods in humans and test-crosses in animals, and (ii) association studies (reviewed in ref. 65). Linkage analyses follow familial inheritance patterns, whereas association studies compare allele frequencies in defined populations. Given the increasingly large number of genes already cloned and mapped in Homo sapiens and Mus musculus, linkage studies may lead immediately to the identification of candidate genes, which can then be studied by using the latter approach once allelic variants are found. Candidate genes, of course, can also be evaluated by using nongenetic means, by investigating the physiology of the proteins they encode. Failing the identification of an already cloned candidate gene, positional cloning techniques (e.g., ref. 66) can be used to narrow the ≈20-centimorgan (cM)-wide chromosomal region identified by linkage down to the <1-cM-wide region required to realistically attempt DNA sequencing.

Human Studies.

A handful of single gene pain pathologies have recently been, or are on the verge of being, explained on the DNA sequence level.

Congenital insensitivity to pain.

Over 40 cases of congenital insensitivity to pain (CIP) with preservation of all other sensory modalities have been reported since the original description of a carnival performer known as The Human Pincushion (see ref. 67). Recently, the genetic basis of this neuropathy (CIP with anhidrosis; hereditary sensory and autonomic neuropathy type IV) was elucidated. Based in part on the striking similarities between CIP and the phenotype of null-mutant mice lacking the Ntrk1 gene encoding the high affinity, nerve growth factor-specific tyrosine kinase receptor, Indo et al. (68) considered the homologous human gene, NTRK1 (previously known as TRKA), as a candidate for CIP. Direct sequencing of the coding region of TRKA in four unrelated CIP patients revealed three separate, exonic mutations: a single base (C) deletion, an A → C transversion, and a G → C transversion (68).

Other sensory neuropathies.

Nicholson et al. (69) performed a microsatellite marker genome screen on 102 members of four Australian kindreds with multiple individuals with hereditary sensory neuropathy type I, a disease featuring loss of all sensory modalities but especially pain and temperature. Linkage was established to a series of markers encompassing a 5-cM region of chromosome 9. An obvious candidate gene, NTRK2, encoding the tyrosine kinase receptor type 2 that is bound by brain-derived neurotrophic factor and neurotrophin-4, was excluded by informative recombination events. Positional cloning efforts facilitated by the development of a yeast artificial chromosome-based transcript map are ongoing (70).

Another hereditary sensory neuropathy (type II), featuring loss of pain sensation and autoamputation, was recently subjected to genetic analysis in a consanguineous family with two affected sisters (71). These investigators used exclusion mapping, a mixed linkage/association strategy, to exclude a variety of known neurotrophin-related genes as candidates for this disorder.

Migraine.

An important recent finding of relevance to a more prevalent painful condition, migraine, has been the attribution of familial hemiplegic migraine and migraine-like episodic ataxia type 2 to mutations in the P/Q-type, calcium channel α1 subunit gene, CACNL1A4 (72). Previous linkage studies mapped the gene for these disorders to chromosome 19p13 (e.g., ref. 73). By using a technique called exon trapping, Ophoff et al. (72) cloned the 47-exon-long CACNL1A4 gene in this region, and identified four different missense point mutations in affected individuals that segregated with the disease in five families. Although familial hemiplegic migraine and episodic ataxia type 2 are rare, genetic factors are known to play a role in “normal” migraine as well, and common allelic variants of this or other ion channel genes may (or may not, ref. 74) contribute to its etiology (75). Of interest as well is a report of a familial migraine susceptibility locus on the X chromosome, which may explain the preponderance of this condition in females (76).

Pathologies linked to human lymphocyte antigens.

Other preliminary genetic investigations of painful pathologies with familial aggregation—including reflex sympathetic dystrophy/complex regional pain syndrome (77), rheumatoid arthritis (e.g., ref. 78), and fibromyalgia (e.g., ref. 79)—have demonstrated at least provisional linkage to or association with various human lymphocyte antigen regions or antigens. This system does not seem to be linked, however, to familial predisposition to discogenic low-back pain (80). It is expected that full-scale genetic investigations of these and other complex pain traits are ongoing or imminent. What are less likely to occur are investigations of nonpathological pain traits, owing to the added complexity introduced by a continuous phenotype. For genetic investigation of “normal” pain sensitivity and sensitivity to analgesia, animal studies provide much-needed statistical power.

Animal Studies.

At the present time, three published studies exist (although many more are ongoing in my laboratory and others) demonstrating linkage of a pain-related trait to chromosomal locations in the mouse. A two-step quantitative trait locus (QTL) mapping approach has been chosen in each case (see refs. 81 and 82 for a detailed description). First, RI strains of the 26-strain BxD set, developed by Taylor (83) from an F₂ intercross between B6 and D2 mice, were phenotyped to identify provisional linkages. These linkages were then independently confirmed or disconfirmed by using new (B6 × D2) F₂ hybrids.

Morphine analgesia.

Belknap and Crabbe (84) tested BxD RI strains for a number of systemic morphine responses, including analgesia on the hot-plate test. Of eight broad chromosomal regions provisionally found to be linked with morphine analgesic magnitude, two have subsequently been confirmed beyond the level of “suggestive linkage” (P < .0016) as proposed by Lander and Kruglyak (85): the Mpmv5 region (0–20 cM) of mouse chromosome 10 (86) and the Myo5a region (30–50 cM) of mouse chromosome 9 (87).

The results to date of this ongoing QTL mapping study nicely illustrate the utility of the approach. In the Mpmv5 region lies the Oprm gene (8 cM) encoding the mouse μ-opioid receptor type. Oprm is an obvious candidate gene for morphine analgesic magnitude, implicated via pharmacological (see ref. 88) and transgenic (89, 90) studies. Allelic variation at this QTL accounts for 28–33% of the observed genetic variability, and F₂ mice inheriting two copies of the D2 allele at this QTL exhibit 4-fold more analgesia from a 16 mg/kg dose of morphine than do F₂ mice inheriting two copies of the B6 allele (86). This QTL has been statistically associated with other opioid traits as well, including morphine consumption (91) and whole-brain [³H]naloxone binding (86).

One may have expected a priori that the gene encoding the μ-opioid receptor would be associated with morphine analgesic magnitude. The candidate gene on mouse chromosome 9 is perhaps more heuristic. Within this region lies the Htr1b gene (46 cM), encoding the serotonin-1B (5-HT_1B) receptor subtype (the mouse analog of the human 5-HT_1Dβ receptor). Based on the hypothesis that Htr1b might represent the QTL for morphine analgesia on chromosome 9, we conducted a series of pharmacological experiments that provided substantial support for the involvement of spinal 5-HT_1B receptors (87). Although data exist indicating a relationship between 5-HT_1B receptors and opioid analgesia (e.g., ref. 92), there is still much confusion regarding the specific role of the 5-HT_1A versus 5-HT_1B subtypes, and until very recently subtype-specific ligands were decidedly lacking (93). Thus, it is unlikely that the studies we conducted would have been conceived of in the absence of the QTL mapping data.

Basal nociceptive sensitivity.

By using similar methodology to that described above, we recently mapped basal thermal nociceptive sensitivity by using the hot-plate test (94). The most promising of six putative linkages in BxD RI strains—the D4Mit71 region (50–70 cM) of mouse chromosome 4—was largely confirmed by using F₂ mice. This QTL displayed evidence of sex specificity, with a combined BxD/F₂ P value of 0.005 for males but only 0.085 for females. The identification of a candidate gene in this region, the Oprd1 gene (65 cM) encoding the mouse δ-opioid receptor type, inspired a simple pharmacological experiment in which B6 and D2 mice of both sexes were administered μ-, κ-, and δ-specific antagonists prior to assessment of hot-plate sensitivity. As predicted by the hypothesis of Oprd1 as a male-specific QTL for this trait, we observed a sex- and strain-dependent pattern of responses, with the δ-specific antagonist, naltrindole, lowering nociceptive latencies in the following order of efficacy: D2 male > B6 male > D2 female > B6 female (94).

Nonopioid SIA.

It has long been known that many forms of analgesia are resistant to antagonism by naloxone, representing the recruitment of non-opioid mechanisms (95). To shed light on the mediation of these powerful but little understood systems, we conducted a QTL mapping experiment of non-opioid SIA resulting from 3-min forced swims in 15°C water (96). Six putative QTLs were identified in the BxD RI phase, of which four were subsequently disconfirmed by using F₂ hybrids. Of the two remaining QTLs, one on chromosome 8 (50–80 cM) was confirmed beyond Lander and Kruglyak’s (85) threshold for significant linkage. This QTL (dubbed Siafq1) exhibited compelling evidence of sex specificity, reaching a combined BxD/F₂ P value of 0.00000012 for females but only 0.038 for males. Female F₂ mice inheriting two copies of the D2 allele at this locus displayed 3-fold more SIA than those inheriting two copies of the B6 allele (96). This finding of a female-specific QTL for SIA is of special interest because we (36, 97) and others (e.g., ref. 98) had previously demonstrated the existence of qualitative sex differences in the neurochemical mediation of this trait.

Sex-Specific Genetic Mediation of Pain and Analgesia

The two findings of sex-specific QTLs described above—a male-specific QTL for baseline thermal nociceptive sensitivity and a female-specific QTL for non-opioid SIA—exemplify a phenomenon we and others have found repeatedly, i.e., sex/genotype interactions of relevance to nociception and its modulation. The discovery of sex-specific QTLs on autosomes, first reported by Melo et al. (99) for alcohol preference, was surprising to many, but now more and more examples are being uncovered. It should be emphasized that the existence of autosomal, sex-specific QTLs does not imply that the sexes possess or express different genes, but rather that different genes are associated with trait variability in each sex. The existence of sex-specific QTLs does, however, imply that males and females possess at least partially independent physiological mechanisms underlying the traits in question.

Sex differences in nociception and analgesia are controversial, but when differences are found, males of a number of species consistently display higher thresholds, tolerance, and analgesic sensitivity (see refs. 100 and 101 for reviews). The inability of some to observe these sex differences has been attributed to estrus cycle variability, test specificity, and experimental parameters (e.g., ref. 102). Recent data from my laboratory suggest that an important factor contributing to variable results in this literature has been overlooked, i.e., genotype of the test subjects. For example, a recent survey of supraspinal morphine analgesia in 11 inbred strains revealed no significant sex differences in morphine analgesic potency in seven of these strains (58). In three strains (AKR, B6, and SWR), males exhibited 3.5- to 7-fold higher sensitivity to intracerebroventricularly administered morphine than their female counterparts. Finally, one strain (CBA) was identified in which females were 5-fold more sensitive to morphine than males. In another, just completed study specifically comparing outbred mouse strains, we found that a large male-vs.-female difference in baseline tail-flick latencies can be seen in SW mice obtained from Simonsen Laboratories (Gilroy, CA), but the analogous sex difference in SW and CD-1 mice from Harlan–Sprague–Dawley is either absent or too small to detect statistically with n = 16–32 (unpublished data). This “vendor effect” between SW mice from two different suppliers is likely due to genetic factors, because both populations have been bred in my vivarium for several generations. Ultimately, then, the failure of some to detect sex differences may simply be due to the fact that in the subject population chosen, there is no sex difference to detect.

Another intriguing sex/genotype interaction is that demonstrated by Rady and Fujimoto (103). As described above, these investigators have determined that heroin analgesia is mediated by μ-opioid receptors in ICR mice of both sexes but by δ-opioid receptors in SW mice of both sexes (39). An analysis of reciprocal (ICR × SW) F₁ hybrid mice revealed that male offspring displayed an ICR-like phenotype and female offspring displayed a SW-like phenotype. That is, in F₁ males, heroin analgesia was blocked by μ-opioid- but not δ-opioid-specific antagonists, whereas the reverse was true for F₁ females (103). This is likely an example of sex-influenced autosomal dominant inheritance (see ref. 104). Further analysis of this phenomenon, if it can be replicated in inbred strains, may help to illuminate the basis of sex/genotype interactions in analgesia. Also potentially enlightening is our ongoing mapping study of supraspinal morphine analgesia in (AKR × CBA) F₂ mice, focusing specifically on the identification of sex-specific QTLs. Male mice of these two strains exhibit equipotent analgesic sensitivity to morphine, whereas the AD₅₀s of female mice differ by a factor of 35 (58).

Allelic Variants of Pain-Related Genes

Once the genes mediating a trait have been positively identified, a crucial task still remains—the identification of alternate alleles of that gene giving rise to the original phenotypic difference between individuals and/or populations. This effort is again rendered more difficult when considering complex genetic traits, because the allelic variants are more likely to be single base pair changes (single-nucleotide polymorphisms) than chromosomal rearrangements or large deletions. Also, whereas the mutations giving rise to disease phenotypes are likely to occur in the coding region of a gene, allelic variation causing subtle changes in gene expression can occur outside (even far outside) the coding region. Nonetheless, some success has been reported.

Opioid Receptor Genes.

The coding and much of the regulatory and intronic regions of human opioid receptor genes have been sequenced (e.g., ref. 105). By using direct sequencing of hundreds of individuals, three separate investigations identified two common variants of the OPRM gene coding for the μ-opioid receptor (106–108), with allele frequencies estimated to be 6.6–11%. An A118G variant (i.e., an A → G substitution in nucleotide 118 of exon 1, resulting in a Asn → Asp change in amino acid residue 40) was found to be present in a lower proportion of opioid-dependent subjects than controls, whereas the C17T variant was more common in opioid-dependent subjects (106, 107). The A118G variant was found not to be associated with susceptibility to alcohol dependence (108). An A118G μ-opioid receptor constructed by using site-directed mutagenesis and stably transfected into cell lines displayed higher binding affinity for β-endorphin than the more common wild-type receptor (106).

An allelic variant (T307C) of the OPRD gene encoding the δ-opioid receptor has also been found (109). Although the amino acid sequence remains unchanged by this substitution, the investigators found that heroin addicts were significantly more likely than controls to possess a CC genotype and less likely to possess a TT genotype. It was concluded that, although by unknown mechanisms, the C allele predisposes to heroin abuse. The direct relevance of any such opioid receptor variants to pain or analgesic sensitivity is as yet unpublished, although this work is no doubt underway in several laboratories.

Cytochrome P450.

One genetic polymorphism of well documented relevance to pain is of the gene coding for the neuronal cytochrome P450IID6 (CYP2D6; sparteine/debrisoquine oxygenase) enzyme (see ref. 110 for review). This enzyme is in fact absent in ≈7–10% of Caucasians, who are thus unable to convert codeine to morphine by O-demethylation (111). Because much evidence indicates that codeine produces analgesic effects by being biotransformed to morphine, these “poor metabolizers” will receive minimal therapeutic benefit from administration of codeine but are generally subject nonetheless to its side effects (112, 113). It has been shown as well that poor metabolizers report increased pain compared with “extensive metabolizers” in the cold pressor test (114). An animal model of this phenomenon exists, with the female Dark Agouti (DA) rat showing a poor metabolizer phenotype (115, 116). This well known example should serve to remind that much individual variability in drug response may be due to polymorphisms related to pharmacokinetics rather than pharmacodynamics.

Future Directions

The construction of high-density genomic maps and the initial priority of the Mouse and Human Genome Projects will greatly facilitate (and already has) the identification of the 50,000–100,000 mammalian genes. Given the high degree of redundancy and pleiotropy known to exist in biological systems, the determination of which genes participate in which physiological mechanisms will remain a daunting task, occupying scientists for decades to come. New, high-throughput genomic technologies (e.g., “gene chips”) may further accelerate the rate of discovery. It is likely that the focus in Homo sapiens will be on pathology, whereas animal models like the mouse will continue to be used to investigate more subtle questions involving the normal range of behavior. Although the use of genetic techniques naturally garners much excitement, it must be borne in mind that even variability in pain pathologies is largely determined by environmental factors. Thus, investigations into the psychosocial determinants of pain tolerance and pain behaviors must continue unabated. Nonetheless, knowledge of the genetic bases of pain-related traits may have important scientific and clinical implications, facilitating both the development of novel analgesic strategies and improved, idiosyncratic treatment of pain using conventional therapies.

ABBREVIATIONS

RI

recombinant inbred

SIA

stress-induced analgesia

CIP

congenital insensitivity to pain

cM

centimorgan

QTL

quantitative trait locus

5-HT

serotonin