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Genetic Studies of Pain
Pain is a phenomenon that affects every person, but in the case of chronic pain disorders, pain can be
debilitating. There are two components of what is commonly thought of as pain-a sensory experience related
to tissue damage and an emotional response elicited by this damage. The latter varies greatly across individuals
and has many contributing components. The aim of this research was to identify to what degree genetics plays a
role in our perception of pain. We did this by gathering genotypic information at several loci per gene in the form
of single nucleotide polymorphisms (SNPs) in genes known or suspected to be involved in the pain pathway.
The genes we studied were the genes coding for melanocortin-1 receptor (MC1R), the mu-opioid receptor
(OPRM1), the kappa-opioid receptor (OPRK1) and the enzyme catecholamine-O-methyltransferase
(COMT). Genotyping was done via direct sequencing or restriction enzyme digest. We then compared
these genotypes with information from tests on subjects' pain tolerances under normal conditions and
under analgesia to see if there was any correlation to the candidate genes. Also, we looked at several populations
of patients with chronic pain disorders such as irritable bowel syndrome, fibromyalgia and post-injury
chronic shoulder pain to see if there was an association between these disorders and the genes studied. We
found there to be a significant sex x genotype association with ischemic pain in MC1R. No other associations
were statistically significant; however, several trends were noted and further testing with a larger sample size
may prove significant.
Pain is the primary reason for seeking healthcare6, and its treatment costs total around $125 billion annually.16
What is interesting about pain in humans is that is has two components-a sensory input and an
emotional experience. The sensory input is known as nociception, the detection of a tissue-damaging or
potentially tissue-damaging stimulus. "Pain" is the emotional experience that is often involved with
nociception, although the two are not indefinitely linked. The International Association for the Study of Pain
defines pain as "an unpleasant sensory and emotional experience associated with actual or potential tissue
damage, or described in terms of such damage" (www.iasp-pain.org/termsp.html#pain). Just as each of us
can experience happiness or rage in unique way, so can we experience pain in varying degree. As with anything
in human physiology, there are both environmental components and genetic factors in a phenotype. My research
has been part of a large collaboration of specialists to investigate the latter.
Our aim was to examine genes thought to be involved in the pain pathway and investigate how natural
variation among humans in these genes contributed to pain tolerances. Natural variation is found in single
nucleotide polymorphisms, or SNPs, which are single base-pair changes in the genome that may or may not result
in an amino acid change in the translated protein product. Many SNPs are neutral, but others may affect
gene regulation, such as those in promoter regions. Over 4 million SNPs have been documented in the
human genome thus far. These SNP genotypes can be compared with a person's pain measures to determine
if different allelic variations are associated with pain perception or analgesic effect.
The first candidate gene I examined is the melanocortin-1 receptor gene (MC1R). Quantitative trait locus
(QTL) mapping led the collaborators of this project to suspect MC1R to be involved in kappa-opioid analgesia
in female mice.10 Also, previous studies showed that MC1R was expressed in the ventral periaqueductal grey
(PAG) and in glial cells involved in the pain pathway.17,18 Among other roles, MC1R is also responsible for
the regulation of hair and skin pigmentation, and allelic variants can lead to red-headedness.11 The SNP variants
we examined were the amino acid substitutions R151C, R160W, and D294H-all causing red-headedness.13
Other polymorphisms that cause a loss of function of MC1R were investigated including V60L, V92M, and R163W.14,15
Another way humans can modulate pain is through our endogenous opioid system, which involves substances such
as endorphins, enkephalins and dynorphins. There are three types of cell surface receptors on neurons, called
opioid receptors for their ability to bind exogenous opiates such as heroin and opium. The first of these is the
mu opioid receptor. Allelic variations in this receptor have been linked to addiction susceptibility.7 For one of the
SNPs we looked at, A118G (causing N40D amino acid substitution), the binding affinity of B endorphin is
increased three-fold with the G allele.8 We also screened for the rare C17T SNP of OPRM1 to see if it had
any correlation to pain in our shoulder pain investigation (see below), since we were able to genotype it at the
same time as A118G.
Another opioid receptor gene, delta opioid receptor (OPRD1), has been linked to heredity of pain sensitivity in
both mice9 and humans.10 For this study we examined the T80G SNP of OPRD1 which is the most frequent SNP
with a G allele frequency of 9%.8 This SNP causes a phenylalanine at codon 27 to be changed to a cysteine.
The last of the opioid receptors is the kappa opioid receptor and its gene OPRK1, which has been associated with
sex differences in analgesia.4 I examined only the G36T SNP, a silent substitution and one of the most frequent
SNPs. It has not yet been studied in the context of pain or analgesia, and at the time this study began, there were
no known OPRK1 SNPs that caused coding region changes. However, this safeguard that allows for
single substitutions to be silent (and thus protects against high mutation rates) becomes considerably less
reliable with subsequent substitutions. More than one SNP in a gene can result in the production of a different
amino acid and phenotype.
In addition to the study of these SNPs and their relation to normal subject pain testing, we used the data to
conduct several association studies with chronic pain disorders. By comparing allelic ratios of certain sets
of individuals to those of a comparable healthy population, it is possible to identify potential susceptibility
genes. Association studies are becoming increasingly popular as a method to identify genes involved with
the development of complex disorders. Some successes with this method have been locating the genetic
changes contributing to Type 1 Diabetes12 and macular degeneration.3
We gathered the above genotyping data on several sets of patients. One set included patients with Irritable
Bowel Syndrome (IBS), a chronic pain syndrome that involves the viscera. Dr. Nicholas Verne studied these
patients. The other subset involved patients who suffer from fibromyalgia, a debilitating chronic pain disorder.
Dr. Roland Staud collected these patients. Both disorders have specific diagnostic criteria. We had approximately
90 samples from each of these two conditions.
We also investigated polymorphisms in patients ages 18-85 currently seeking treatment for a shoulder injury
(rotator cuff) at the University of Florida Department of Orthopedics Research and Sports Medicine Center to see
if there was an altered allele frequency in this population as compared to the normal population. The
clinical measures included rating of pain before and after analgesia administration (including in the normal
shoulder), and before and after shoulder surgery followed by analgesia. One new gene involved in this study is
the catecholamine-O-methyltransferase gene (COMT). COMT has been associated with
myogenous temporomandibular joint disorder (TMD) and accounted for approximately 11% of pain variability in
test subjects of a recent study.2 The SNPs I investigated in this population were OPRM1 C17T and A118G,
OPRD1 T80G and rs678849, and COMT rs4633 and rs4818. As described below, a healthy subject pain
project provided the normal data for comparison to the chronic pain populations, for case-control types of studies.
MATERIALS AND METHODS
All PCR primers were designed using primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.
cgi) and PCR conditions were optimized using a PCR thermal cycler that ran a temperature gradient. Hotmaster
Taq was used with supplied buffer in concentrations as directed by the manufacturer's protocol. PCR products
were visualized by gel electrophoresis on 1.2% agarose gels to check quality and quantity of each subject's
PCR product. Sequences were run on an ABI Prism Sequencer. Those sequences were then analyzed using
Gene Codes Corporation Sequencher version 4.5 software. Some of the genotyping was performed
by pyrosequencing at the UF Center for Pharmacogenomics.
Dr. Roger Fillingim performed pain testing in healthy humans in the UF Clinical Research Center,
including standardized tests for ischemic and thermal pain tolerance. Patients were exposed to various levels
of painful stimuli and self-reported the experienced pain both unanesthetized and with analgesia such as
pentazocine. In addition, some subjects were submitted to pressure pain testing. The latter data were used
for analysis of association with OPRM1 genetic variation.
Most SNPs were examined via sequencing, however, a few sites were genotyped by restriction enzyme digest,
using enzymes from New England Biolabs. The enzymes allowed detection of the two alleles individually. The
MC1R D294H polymorphism was digested with the alpha-TaqI restriction enzyme at conditions according to
the manufacturer's protocol. Alpha-TaqI "cuts" at the "G" allele and not the C allele. The OPRK G36T
polymorphism was digested with PsPOMI enzyme also under the manufacturer's conditions and cut at the "G"
allele. COMT 4633 was digested with BsaAI making a cut again at the "G" allele. Digest products were submitted
to electrophoresis on 8% native polyacrylamide gels and visualized after ethidium bromide staining. I then read
the genotypes for each subject-homozygous cut allele, heterozygous or homozygous uncut allele.
Associations of variations in pain perception and genotype were analyzed for statistical significance using
ANOVA. Differences in genotype and allele frequencies between healthy populations and those with chronic pain
were tested for significance by chi-square tests. I contributed substantial genotyping data to all of the
studies described below.
Figure 1. An example of genotyping via restriction enzyme digest for PCR product of COMT rs4633
with enzyme BsaAI on 8% polyacrylamide gel. Highest band represents the "uncut" C allele while
lower two bands are the result of the "cut" T allele. Homozygotes consists of only upper band (CC)
or lower bands (TT). Heterozygotes are represented by those with both bands (CT).
Healthy, IBS and Fibromyalgia
For MC1R in healthy subjects (145), all significant differences in pain and response to analgesia were seen only
in females but not males. In addition, males reported only modest analgesic response to pentazocine where
this effect was much more marked in females with multiple minor alleles. This sex x genotype effect was
significant (p < 0.05) for ischemic pain and approached significance in thermal pain (p = 0.056).
Although OPRD1 and OPRK1 ANOVAs failed to reveal any significant correlations to pain thresholds in
our populations, a recent study has implicated OPRD1 in pain perception. In addition, no significant differences
were found in our association study with FMS or IBS compared to the healthy population. However,
additional subjects will be gathered to be sure there is not an error due to sample size.
Shoulder Pain Study
In our genotyping of 22 shoulder pain patients, in collaboration with Dr. Steven George, I found no
significant differences between the allelic frequencies found in the healthy population and those being treated
for chronic shoulder pain.
A listing of each locus's allele frequency in both healthy and chronic shoulder pain populations. Results of chi-square tests for
significant difference are also shown.
Allele Frequency COMT rs4633 OPRM1 C17T OPRM1 A118G OPRD1 T80G OPRD1 rs678849
fr(C) = 0.5 fr(C) = 0.97 fr(A) = 0.90 fr(T)= 0.885 fr(T) = 0.508
fr(T) = 0.5 fr(T) = 0.03 fr(G) = 0.10 fr(G)= 0.106 fr(C) = 0.492
fr(C) = 0.386 fr(C) = 0.95 fr(A) = 0.977 fr(T)= 0.810 fr(T) = 0.368
fr(T) = 0.613 fr(T) = 0.045 fr(G) = 0.023 fr(G)= 0.190 fr(C) = 0.631
p-value 0.132 0.548 0.085 0.078 0.128
However, several of the polymorphisms approached significance and showed trends toward deviating from the
normal frequencies. For example, the OPRD1 T80G SNP has a minor allele (G) frequency of 0.106. In our patient
set we found a frequency of 0.190 although this was not enough (p = 0.078) to reach significance by a chi-
square test. The rs678849 SNP of OPRD1 also showed deviation from the healthy allele frequencies but fell short
of significance (p = 0.128). The two COMT SNPs are a part of a haploblock, and they reveal the genotype at
two other locations relevant to pain sensitivity. Thus, both genotypes are needed for a thorough investigation of
this gene to test its relationship to shoulder pain. The genotyping for rs4633 has been completed with a
restriction enzyme test, but rs4818 has been problematic, which may indicate an error in the SNP entry in
the National Center for Biolnformatics.
The findings with MC1R are interesting since they show a direct correlation with previous mouse studies and
show female-specific mediation of pain and analgesia. They also demonstrate the multipartite nature of
pain regulation, which has implications in anesthesia and medicine in general. These data also fit with
anecdotal reports about redheaded females needing less analgesia/anesthesia to obtain the same effect as
women with other colored hair, or males. This provides an insight into the mechanism involved in
women's experience of pain and analgesia.
Even the negative results are not discouraging. Given the complexity of pain and relatively small effect of each
gene in a complex trait such as this, any trends are worth pursuing. For instance, the results may become
significant if more modalities of pain were tested or different techniques used. Another limitation may be that
we tested too few SNPs and having more would increase our power at each candidate gene. Also, as with
any statistically dependent study, more subjects would result in more powerful statistical analysis and allow for
more decisive conclusions to be drawn.
This last stumbling block is especially important in the shoulder pain dataset since a maximum of 22 patients
were genotyped in this pilot study. However, it is exciting that even among this small set we see potential trends.
It is particularly interesting that both OPRD1 polymorphisms showed a similar trend.
The next step is to investigate whether these two polymorphisms are in linkage disequilibrium. If so, their
genotypes can be combined into one more-informative genotype consisting of haplotypes, which will give the
study more power. If they are not, we could genotype more polymorphisms within the two haploblocks to
improve informative value.
1. Bond, C., K. S. LaForge, M. Tian, D. Melia, S. Zhang, L. Borg, J. Gong, J. Schluger, J. A. Strong, S. M. Leal, J.
A. Tischfield, M. J. Kreek and L. Yu (1998). "Single nucleotide polymorphism in the human mu opioid receptor
gene alters betaendorphin
binding and activity: possible implications for opiate addiction." Proc Natl Acad Sci USA 95(16): 9608-13.
2. Diatchenko, L., Slade, G.D., Nackley, A.G., Bhalang, K., Sigurdsson, A., Belfer, I., Goldman, D., Xu, K., Shabalina,
S.A., Shagin, D., Max, M.B., S.S. Makarov and W. Maixner (2005). "Genetic basis for individual variations in
pain perception and the development of a chronic pain condition." Hum. Mol. Gen. 14(1):135-143.
3. Esfandiary, H., U. Chakravarthy, C. Patterson, I. Young and A. E. Hughes (2005). "Association study of
detoxification genes in age related macular degeneration." Br J Ophthalmol 89(4): 470-4.
4. Gear, R. W., C. Miaskowski, N. C. Gordon, S. M. Paul, P. H. Heller and J. D. Levine (1996). "Kappa-opioids
produce significantly greater analgesia in women than in men." Nat Med 2(11): 1248-50
5. Kim, H., J. K. Neubert, A. San Miguel, K. Xu, R. K. Krishnaraju, M. J. Iadarola, D. Goldman and R. A. Dionne
(2004). "Genetic influence on variability in human acute experimental pain sensitivity associated with
gender, ethnicity and
psychological temperament." Pain 109(3): 488-96.
6. Knapp, D. A. and H. Koch (1984). "The management of new pain in office-based ambulatory care:
National Ambulatory Medical Care Survey, 1980 and 1981." Adv Data(97): 1-9.
7. Liu, J. G. and P. L. Prather (2001). "Chronic exposure to mu-opioid agonists produces constitutive activation of
mu-opioid receptors in direct proportion to the efficacy of the agonist used for pretreatment." Mol Pharmacol 60
8. Mayer, P. and V. Hollt (2001). "Allelic and somatic variations in the endogenous opioid system of humans."
Pharmacol Ther 91(3): 167-77.
9. Mogil, J. S., S. P. Richards, L. A. O'Toole, M. L. Helms, S. R. Mitchell, B. Kest and J. K. Belknap (1997).
"Identification of a sex-specific quantitative trait locus mediating nonopioid stress-induced analgesia in female
mice." J Neurosci 17(20): 7995-8002.
10. Mogil, J. S., J. Ritchie, S. B. Smith, K. Strasburg, L. Kaplan, M. R. Wallace, R. R. Romberg, H. Bijl, E. Y. Sarton, R.
B. Fillingim and A. Dahan (2005). "Melanocortin-1 receptor gene variants affect pain and mu-opioid analgesia in mice
and humans." J Med Genet 42(7): 583-7.
11. Mogil, J. S., S. G. Wilson, E. J. Chesler, A. L. Rankin, K. V. Nemmani, W. R. Lariviere, M. K. Groce, M. R. Wallace,
L. Kaplan, R. Staud, T. J. Ness, T. L. Glover, M. Stankova, A. Mayorov, V. J. Hruby, J. E. Grisel and R. B.
Fillingim (2003). "The
melanocortin-1 receptor gene mediates female-specific mechanisms of analgesia in mice and humans." Proc
Natl Acad Sci U S A 100(8): 4867-72.
12. Onengut-Gumuscu, S., K. G. Ewens, R. S. Spielman and P. Concannon (2004). "A functional polymorphism (1858C/
T) in the PTPN22 gene is linked and associated with type I diabetes in multiplex families." Genes Immun 5(8):
13. Rees, J. L., M. Birch-Machin, N. Flanagan, E. Healy, S. Phillips and C. Todd (1999). "Genetic studies of the
human melanocortin-1 receptor." Ann N Y Acad Sci 885:134-42.
14. Schieoth, H. B., S. R. Phillips, R. Rudzish, M. A. Birch-Machin, J. E. Wikberg and J. L. Rees (1999). "Loss of
function mutations of the human melanocortin 1 receptor are common and are associated with red hair."
Biochem Biophys Res Commun 260(2):
15. Scott, M. C., K. Wakamatsu, S. Ito, A. L. Kadekaro, N. Kobayashi, J. Groden, R. Kavanagh, T. Takakuwa, V.
Virador, V. J. Hearing and Z. A. Abdel-Malek (2002). "Human melanocortin 1 receptor variants, receptor function
and melanocyte response to UV radiation." J Cell Sci 115(Pt 11): 2349-55.
16. Turk, D. C., A. Okifuji and D. Kalauokalani (1999). Clinical outcome and economical evaluation of multi-
disciplinary pain centers. Mahwah, NJ, Erlbaum.
17. Wikberg, J. E. (1999). "Melanocortin receptors: perspectives for novel drugs." Eur J Pharmacol 375(1-3): 295-310.
18. Xia, Y., J. E. Wikberg and V. Chhajlani (1995). "Expression of melanocortin 1 receptor in periaqueductal gray
matter." Neuroreport 6(16): 2193-6.
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