Genetics of Pain

Transgenic Knockout Mice and Other Mutants

By far the most common bottom-up approach to pain genetics is the creation and testing of transgenic knockout mice. These are genetically engineered null mutants in which a single gene is effectively ablated via homologous recombination of embryonic stem cells with a transgenic targeting vector (see ). Such knockout mice are compared with “wild-type” and heterozygous littermates, and if the genetic “lesion” results in an aberrant behavioral and/or biochemical pain phenotype (e.g., altered sensitivity on an algesiometric assay), this demonstration provides evidence, subject to certain caveats (see ), that the gene is required for normal expression of the trait. A large number of knockout mice have been tested for nociceptive and analgesic sensitivity; we maintain a web database of findings from such experiments called the Pain Genes Database ( ). As of this writing, the pain-related phenotypes of 322 mutants have been documented (see http://paingeneticslab.ca/ 4105/06_02_pain_genetics_database.asp). A comprehensive review of transgenic studies of pain is beyond the scope of this chapter, however, and findings using this technique will appear elsewhere in this volume. To give the reader a sense, Table 10-1 lists simply the number of genes currently demonstrated to affect pain, hypersensitivity after inflammatory and neuropathic injury, and morphine analgesia via the transgenic technique. It should also be noted that spontaneously occurring mutations (of coat color genes and genes causing obvious phenotypic abnormalities) have been and continue to be studied for their relevance to pain (see ; also see for review). Great sums of money have been spent on the deliberate induction, via chemical mutagenesis, of random point mutations combined with systematic screening for phenotypic alterations. Three public mutagenesis projects ( , , ) included a nociceptive assay (hot plate test), but data describing only one pain-relevant mutant were ever published ( ). A genome-wide screen of Drosophila mutations recently identified a thermal avoidance gene in flies that appears to play a similar role in humans ( ) (see below).

mRNA Knockdown

In the original version of this bottom-up approach, called antisense knockdown, an oligonucleotide is synthesized to be complementary (i.e., in an antisense orientation) to an mRNA transcript of the gene of interest and injected into a target tissue. By a mechanism involving steric hindrance in ribosomes and/or enzymatic degradation, the antisense oligonucleotide prevents (to some degree, hence “knockdown” instead of “knockout”) successful translation of the native mRNA into protein ( ). Although the antisense approach has several major advantages over knockout mice (see ), it is technically difficult and has been used fairly sparsely in pain research. When successful, however, it has provided impressive demonstrations of the role of particular genes in pain (e.g., , , ).

More recently, mRNA knockdown in vitro and in vivo has been achieved with the use of small interfering RNA (siRNA; see , ), which provides much greater translational block efficiency. The role of any number of genes in pain has been demonstrated via in vivo knockdown using siRNA, known as RNA interference ( ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ).

Selective Breeding

Selective breeding is the oldest of the top-down approaches and has been performed in agriculture and animal husbandry for millennia. Also known as artificial selection, the technique as applied scientifically involves two-way breeding of a genetically heterogeneous stock of animals based on their response in regard to a trait of interest (see ). Only extreme responders are used to beget the next generation of offspring, and the resultant “high” and “low” lines will start to diverge phenotypically for all heritable traits. The great advantage of the technique is that all trait-relevant genes will eventually become fixed in alternate allelic states in the two lines such that very robust phenotypic differences are demonstrated. Of greatest relevance to pain genetics are three selective breeding projects: the HA/LA rat lines selected for high and low autotomy after nerve transection ( ), the HA/LA mouse lines selected for high and low analgesia after forced swim stress ( ), and the HAR/LAR mouse lines selected for high and low analgesic response to levorphanol (and in later generations, morphine) ( ). Much has been learned from these lines (see ); however, it remains difficult to identify the fixed genes, and this approach is rarely used today.

Inbred Strains and Linkage Mapping

The use of inbred strains for analysis of classic pain genetics is the dominant modern top-down strategy. Inbred strains are produced by repeated brother × sister mating over at least 20 generations, a breeding scheme that eliminates all genetic heterozygosity and renders individual members of the strain isogenic (i.e., clones) to all others, barring the very rare occurrence of new mutations ( ). Many existing inbred mouse strains are the descendents of European and East Asian “fancy mice” bred at the turn of the 20th century by Abbie Lathrop and William Castle, and excellent genealogical records are available in many cases ( ). A comparison of randomly selected inbred strains allows one to survey alleles of trait-relevant genes existing in the European and East Asian “fancy mouse” population of the early 1900s fixed into a homozygous state in every strain but into a different state in phenotypically contrasting strains. That is, each laboratory inbred strain is a different fixed “mosaic” of M. musculus domesticus , M. musculus castaneus , and M. musculus musculus alleles ( ). Inbred strains derived from stocks of Japanese wild mice ( M. musculus molossinus ) may have even greater genetic variability ( ). Because they are isogenic, inbred strains are tremendously useful for establishing the heritability of traits since within-strain variability is by definition of environmental origin and between-strain variability is very probably genetic. An inbred “strain survey” can identify the most robust pair-wise strain differences, and these extreme-responding strains are the ideal progenitors of linkage-mapping populations.

Linkage mapping, which when applied to complex traits is known as quantitative trait locus (QTL) mapping, searches for genomic regions that are co-inherited with trait variability in genetically segregating populations (e.g., F ₂ intercross, backcross, recombinant inbred strains) (see ). As a practical matter, one correlates the phenotypic responses of, say, F ₂ hybrid mice with their inherited genotype at non-coding, polymorphic DNA markers (microsatellites or SNPs), and where such genetic “linkage” is established then infers the existence of a trait-relevant gene or genes in the genomic vicinity of the markers. By contrast to all other strategies in current use, QTL mapping allows one to identify genes containing the very polymorphisms that are the cause of the original strain difference since what is being examined is not gene expression but the DNA sequence itself. The disadvantage of QTL mapping is that identifying the broad genomic region linked to the trait (i.e., the QTL) is far easier than “positional cloning,” or identifying the gene and polymorphism responsible ( ). Positional cloning has historically been attempted by creating advanced mapping populations such as congenic strains ( ), followed by cloning and sequencing.

A very useful advance is haplotype mapping, also known as in silico QTL mapping ( ; also see ). Because DNA variants are not inherited independently but rather in “blocks” known as haplotypes (see later) and since sequencing in multiple inbred mouse strains has already occurred, one can now simply search for statistical associations between strain means and haplotypic patterns. With a sufficiently high number of strains, significant linkages can be evinced, and the mapping resolution is extremely good (generally within the genes themselves or very close to them). Despite many false positives, this technique has been used profitably in mouse pain genetics in recent years.

Gene Expression Profiling with Microarrays

The most recently developed top-down strategy is microarray or “gene chip” expression profiling. The technique, developed by Pat Brown’s laboratory at Stanford ( ) and made widely available by commercial entities such as Affymetrix, Inc., allows quantification of mRNA expression in a massively parallel fashion (see ). Other related techniques, such as differential display polymerase chain reaction ( ) and massively parallel signature sequencing ( ), allow the same sort of analysis. To run a profiling experiment, one isolates the total RNA from at least two contrasting tissue sources (e.g., dorsal root ganglia from a rat in neuropathic pain versus dorsal root ganglia from a control rat), and after a series of reverse transcription, amplification, and labeling steps, hybridizes the samples to a nylon or glass array. In the common Affymetrix version, the glass “chip” contains synthesized oligonucleotides corresponding to many thousands of known and unknown gene transcripts in catalogued locations. The fluorescent probes are excited by a laser and detected with a scanning confocal microscope; the intensity and color of fluorescent labeling at each location correspond to the absolute and relative (between groups) abundance of that particular gene’s mRNA in the tissue.

To date, approximately 30 microarray studies directly focused on chronic pain and using standard assays have been published ( ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ). The results of all these investigations (also see , , ) illustrate the power of and problems with the approach. Depending on the criteria used to establish “significance,” these studies have identified up to hundreds of genes up- or down-regulated by the injury. On the plus side, many of these genes were not previously known to play any role whatsoever in chronic pain, and thus the heuristic potential is immense. Most investigators were surprised, for example, by the large number of “hits” among genes relevant to neuroinflammation ( , ). Various forms of cluster analysis applied to microarray data may identify groups of genes that are co-regulated ( ), an impossible task with other approaches. However, it is difficult to evaluate the relative importance (and cause-and-effect relationships) of these hundreds of genes and also very difficult to determine which of them are specifically related to pain versus the nerve injury itself. It is also difficult (without confirmation by other techniques) to separate true from false positives. We recently performed a meta-analysis of the first 20 published chronic pain–relevant microarray studies ( ).

How Heritable Is Pain in Rats and Mice?

Heritability ( h ² ; see later for further discussion) is the proportion of trait variance attributable to genetic inheritance. Heritability can be estimated in laboratory animals either by using panels of inbred strains or by assessing the response to artificial selection (see ). A large number of studies noting pain-relevant strain differences have compared the thermal nociception–sensitive and opioid analgesia–resistant C57BL/6 mouse strain with the nociception-resistant and opioid analgesia–sensitive DBA/2 strain (see , ). These strains do not represent the extreme responders among commonly available mouse strains, however, and in any case pair-wise comparisons do not provide much power to estimate heritability. In the only systematic inbred strain surveys to date, the heritability of response to 22 common algesiometric assays has been found to range from h ² = 0.30–0.76, with a median value of 0.46 ( , ). This range of heritability is highly similar to that obtained in large human twin studies of experimental pain published in recent years ( , ). Using the same set of strains, the heritability of the efficacy of eight analgesics was found to be slightly lower, h ² = 0.12–0.45, with a median value of 0.29 ( ). The three existing selective breeding studies of relevance to pain (see earlier) have yielded heritability estimates of h ² = 0.30 for autotomy ( ), h ² = 0.32 for levorphanol analgesia ( ), and h ² > 0.32 for swim stress–induced analgesia ( ).

Genetic Correlations

One interesting use of both selected lines and inbred strains involves the investigation of genetic correlations (see ). Most genes are known or thought to be pleiotropic, that is, affecting more than one trait. If selection or chance has fixed the alleles of a gene into a homozygous state, that fixation will probably affect multiple traits. Genetic correlation is demonstrated if lines selected for their performance on one trait are observed to differ on another or if the same distribution of phenotypic responses on two traits is observed among a panel of inbred strains.

As might be expected, the sensitivity of inbred strains and selected lines on certain nociceptive assays is genetically correlated with sensitivity on other nociceptive assays, but not all other nociceptive assays. Inbred mouse strains sensitive to tail flick test nociception are by and large the same mouse strains sensitive to paw withdrawal (Hargreaves’) test nociception. By contrast, a largely different set of strains are sensitive to formalin test nociception ( ). In our analysis of 22 nociceptive assays in a common set of 12 strains, we provided evidence using multivariate analysis of five “clusters” of assays: (1) thermal nociception, (2) ongoing nociception from chemical stimuli, (3) mechanical hypersensitivity, (4) thermal hypersensitivity, and (5) afferent-dependent (featuring initial ongoing nociception) thermal hypersensitivity ( ). Since variability measured on tests within a cluster should be mediated by common genes but variability on tests in different clusters by different genes, we believe that these clusters represent fundamental “types” of pain in the mouse. This is a nice example of how a genetic approach can address a non-genetic question. Species specificity may apply, however, since similar studies using rat strains have yielded somewhat different results ( , ).

Another remarkable genetic correlation pertains to analgesia of different modalities. Such correlation was hinted at by findings in selected lines, for example, when mice selected for high and low stress-induced analgesia were found to be high and low responders, selectively, to levorphanol, morphine, and the κ-opioid agonist U50,488 ( , ). We recently evaluated the analgesic potency of five neurochemically distinct analgesics in 12 inbred strains: morphine; U50,488; clonidine; epibatidine; and the cannabinoid agonist WIN55,212-2 ( ). Although these drugs all activate descending pain-inhibitory mechanisms in the central nervous system, they bind to five distinct molecular sites. Thus, we were surprised to discover a remarkably high degree of genetic correlation among them ( r = 0.33–0.68). This finding has important implications. First, it appears that a “master” analgesia variability gene or set of genes must exist, and it is highly unlikely that this gene is related to the binding site of each drug. If this is true, current attempts to explain variable analgesic drug action (i.e., pharmacogenetics) solely by searching for polymorphisms in the binding site gene will be incomplete. In a follow-up study, a similar genetic correlation was observed between acetylsalicylic acid and indomethacin analgesia, but acetaminophen analgesia appeared to be genetically distinct ( ).

A related genetic correlation is the repeatedly noted correlation between baseline nociceptive sensitivity and subsequent sensitivity to inhibition of the stimulus by analgesics ( , , ). That is, mouse strains initially sensitive to nociception are also resistant to analgesia, and vice versa. This principle works within a number of nociceptive modalities and suggests that the “master” analgesia gene or genes referred to above may in fact be ones with a primary action on nociception, which may subsequently affect analgesia as well. hypothesized that this can be explained in terms of genetic differences in effective stimulus intensity affecting fractional receptor occupancy of the analgesic.