Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Lack of influence of GTP cyclohydrolase gene (GCH1) variations on pain sensitivity in humans

Lack of influence of GTP cyclohydrolase gene (GCH1) variations on pain sensitivity in humans Objectives: To assess the effect of variations in GTP cyclohydrolase gene (GCH1) on pain sensitivity in humans. Methods: Thermal and cold pain sensitivity were evaluated in a cohort of 735 healthy volunteers. Among this cohort, the clinical pain responses of 221 subjects after the surgical removal of impacted third molars were evaluated. Genotyping was done for 38 single nucleotide polymorphisms (SNPs) whose heterozygosity > 0.2 in GCH1. Influence of the genetic variations including SNPs and haplotypes on pain sensitivity were analyzed. Results: Minor allele frequencies and linkage disequilibrium show significant differences in European Americans, African Americans, Hispanic Americans and Asian Americans. Association analyses in European Americans do not replicate the previously reported important influence of GCH1 variations on pain sensitivity. Conclusion: Considering population stratification, previously reported associations between GCH1 genetic variations and pain sensitivity appear weak or negligible in this well characterized model of pain. pain protective haplotype associated with the risk of Background The role of 6(R)-t-erythro-5,6,7,8-tetrahydrobiopterin developing persistent pain syndromes, which could be a (BH4) in pain is suggested by the up-regulation of two of useful tool to assess an individual's risk potential for the three enzymes in the synthesis cascade of BH4 in the chronic pain [2]. Based on this reported role of GCH in dorsal root ganglion following sciatic nerve injury [1]. pain both in animals and humans, we investigated its con- GTP cyclohydrolase (GCH) catalyzes the rate limiting tribution to genetic inter-individual variation in clinical step, and sepiapterin reductase catalyzes the final conver- pain sensitivity and analgesic responses. We have investi- sion of 6-pyruvoyltetrahydropterin to the BH4. It was gated the association between pain responses to experi- recently reported that single nucleotide polymorphisms mental and clinical painful stimuli and genetic variations (SNPs) in the gene encoding GCH (GCH1) alter both including SNPs and haplotypes of GCH1. responses in healthy humans to noxious stimuli and the susceptibility of patients to development of neuropathic and inflammatory pain [2]. The authors also suggest a Page 1 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Further haploblock analysis was performed with Euro- Results The minor allelic frequencies of each SNP in the GCH1 are pean Americans only due to the small number of other shown in Table 1. ethnic populations. We found 3 haploblocks from GCH1 (4 SNPs spanning ~17.4 kb, 5 SNPs spanning ~3.7 kb and We next determined what haplotypes are formed by these 29 SNPs spanning ~51.0 kb respectively) based on the SNPs. The total number of haploblocks found were 3 in confidence interval method. European Americans (average minimum span = 24.0 kb) with 12 tag SNPs, 5 haploblocks with 21 tag SNPs in Afri- In the GCH1 haploblock 1, a total of 5 haplotypes were can Americans (average minimum span = 5.3 kb), 3 hap- detected for the 4 SNPs, with the most frequent haplotype loblocks with 14 tag SNPs in Hispanic Ameicans (average (55.4%) composed of A_G_T_C. The 3 frequent haplo- minimum span = 22.7 kb) and 4 haploblocks with 10 tag types (> 5%) were 98% of total haplotypes. In the GCH1 SNPs in Asian Americans (average minimum span = 16.1 haploblock 2, a total of 7 haplotypes were detected for the kb). Position of those SNPs and haploblocks within the 5 chosen SNPs, with the most frequent haplotype loci of GCH1 are shown in Figure 1, 2, 3 and 4. Their D' (55.0%) composed of A_A_T_C_C. Four of the 7 haplo- matrices in each ethnic with confidence intervals and r types were frequent (> 5%), and these 4 haplotypes were population show large differences (see Additional file 1). 99% of the total haplotypes. In the GCH1 haploblock 3, a Table 1: SNPs genotyped in GCH1 Order SNP ID Location from transcription site Nucleotide variation Rarer allele frequency 1 rs8008858 -9,686 A/T 0.28 2 rs8007267 -9,462 G/A 0.28 3 rs2878172 -4,141 T/C 0.44 4 rs8017210 7,694 C/T 0.21 5 rs3783642 9,327 A/G 0.48 6 rs3783641 9,391 A/T 0.21 7 rs7147201 10,653 T/C 0.27 8 rs7147286 10,865 C/T 0.42 9 rs17128052 13,005 C/G 0.18 10 rs998259 14,499 G/A 0.14 11 rs10498471 15,813 C/T 0.22 12 rs8020798 16,162 G/A 0.21 13 rs8004018 18,834 T/C 0.25 14 rs8004445 18,864 C/A 0.25 15 rs3783639 20,864 A/G 0.20 16 rs3783638 21,157 C/T 0.26 17 rs3783637 21,412 G/A 0.18 18 rs10133650 23,257 G/C 0.47 19 rs12147422 25,515 A/G 0.25 20 rs17128050 25,651 A/G 0.17 21 rs7492600 32,655 C/A 0.26 22 rs2183081 32,779 T/C 0.47 23 rs8010282 39,275 T/C 0.21 24 rs9671455 40,450 G/C 0.26 25 rs9671371 40,895 G/A 0.33 26 rs2878169 43,537 C/A 0.06 27 rs12589758 43,668 T/A 0.22 28 rs12587434 43,947 A/C 0.23 29 rs7155309 46,679 C/T 0.27 30 rs2878168 48,745 A/G 0.22 31 rs4411417 48,967 A/G 0.22 32 rs10133662 53,260 T/C 0.39 33 rs10131232 53,622 C/T 0.40 34 rs752688 57,961 G/A 0.23 35 rs841 59,038 C/T 0.23 36 rs7142517 62,726 G/T 0.28 37 rs10483639 63,073 C/G 0.26 38 rs2057369 65,469 C/T 0.22 Page 2 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Li Figure 1 nkage disequilibrium of GCH1 in European Americans (Haploview results with confidence interval method) Linkage disequilibrium of GCH1 in European Americans (Haploview results with confidence interval method). total of 17 haplotypes were detected for the 29 SNPs, with Discussion the most frequent haplotype (28.7%) and 5 frequent hap- We could not repeat any of the finding reported for lotypes were 88% of the total haplotypes. In the whole humans by Tegeder et al. First, the linkage disequilibrium GCH1, 12 SNPs were tag SNPs European American popu- (LD) between SNPs and patterns of haplotype blocks in lation. Among 25 existing combinations of those 12 tag our sample are not consistent with their previous finding SNPs, the most frequent combination occupies 28.3% reporting one single block for GCH1. This inconsistency while 5 frequent (> 5%) combinations of tag SNPs are may be caused by the samples evaluated and the atypical 87% of the total tag SNPs combinations (Table 2). interpretation of their data. The University of North Caro- lina and University of Florida cohorts of Tegeder et al. cite Considering the relatively dominating effect of gender on previous publications[3,4] with mixed population groups pain, male and female subjects were analyzed separately and leave open the question whether their present study is for the association between genetic variations (individual using mixed ethnicity groups as well. It has been shown SNPs and combination of haplotypes) and responses to that the ethnic demographics can have profound influ- experimental and clinical painful stimuli. However, we ence on LD between SNPs, haploblock structure, SNP fre- could not find any significant genetic associations quency, and estimated effect size drawn from the data. It between variations of GCH1 including SNPs and haplo- is for these very reasons that haplotype data are reported types and pain sensitivity or analgesic responses in this separately by ethnicity in the HapMap Project database. cohort of European American females and males even without multiple testing corrections. Figure 5 shows an When we analyzed haplotypes from our samples and the example of non-significant association between GCH1 HapMap website within the same region using Haplov- genetic variations and pain sensitivity in European Amer- iew, we obtained different haplotype blocks between each ican females (GCH1 tag SNP combinations and heat and ethnic groups, regardless of the algorithm used (imple- cold pain ratings). mented in Haploview, confidence interval, 4-gamete and Page 3 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Figure 2 Linkage disequilibrium of GCH1 in African Americans (Haploview results with confidence interval method) Linkage disequilibrium of GCH1 in African Americans (Haploview results with confidence interval method). solid spine method). Ethnic differences in the structure of supplementary table table 2 in Tegeder et al. The authors haplotypes are consistent with other reports [5,6]. The sin- seem to interpret their data completely opposite direction. gle large 72 kb block identified by Tegeder et al for GCH1 Unique haplotype patterns including block size, block with 168 Caucasian chronic lumbar root pain subjects is numbers, contributing SNPs, tag SNPs along with LDs not consistent with the HapMap Caucasians (CEU), acquired in each ethnic population in our sample are con- which may reflect a lack of generalizability for the Tegeder sistent with HapMap data and clearly suggest that haplo- et al results. It is unclear how the data from the other type analysis with mixed population be avoided. cohorts is affected by one single large haploblock gener- ated from the back pain study, especially when that one Using a mixed ethnic sample may also induce population large block is not observed in the Phase II data released by stratification. This can cause false associations since the the HapMap project. We found 3 haplotype blocks across pattern of genetic variations as well as pain rating is not GCH1 including its flanking region based on confidence uniform between ethnic populations [7], requiring use of interval method in European Americans and 5 blocks in homogenous population in genetic association studies African Americans with different size and different con- [8]. Considering significant differences of allele frequen- tributing SNPs. Hispanic Americans and Asian Americans cies and pain sensitivities among ethnic populations, it also show unique haplotype blocks and LD values would be more informative to analyze single ethnic origin between each SNPs in GCH1. These results are similar to population only compared to the total mixed sample to HapMap data and the general concept of smaller block determine if the apparent association is due to population size in samples of African ancestry. stratification. Additionally, Tegeder et al proposed the low minor allele Another possible reason of inconsistency is the pain phe- frequencies of a few markers as the cause of the disruption notype. Because responses to different types of painful of the LD. However, almost 0 values of D' were obtained stimuli are genetically dissociable [9], GCH1 genetic vari- between 3 most frequent SNPs (10374 c>t as 29.69%, - ation may affect the responses to a specific type of painful 4289 t>c as 37.42% and 3932 g>t as 35.76% in their stimulus, while not influencing other types of stimuli. minor allele frequencies) in supplementary figure 4 and However, Tegeder et al. used Z score for their analyses for Page 4 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Figure 3 Linkage disequilibrium of GCH1 in Hispanic Americans (Haploview results with confidence interval method) Linkage disequilibrium of GCH1 in Hispanic Americans (Haploview results with confidence interval method). a chimeric phenotype that combines pain threshold and risk, requiring large samples to convincingly identify such pain tolerance, outcome measures at opposite ends of the variants [11]. Given these questions of study design sensory continuum. Use of an artificial outcome measure related to population stratification, haploblock size, and that has been transformed may not be an appropriate disparate pain measures in Tegeder et al with similar sta- physiological representative of pain sensation and could tistical power of our samples, it is more probable that this potentially result in an artificial genetic association. Con- association between GCH1 genetic variations and pain sidering the non-significant association between GCH1 sensitivity are, if any, weak or negligible regardless of their genetic variations and similar thermal pain sensitivity in strong in vitro and animal findings. our sample, the inconsistency cannot be easily explained by the differences in stimulus modalities. Additionally, It is doubtful that a single gene accounting for large por- the Z score is based on the assumption that the variables tion of the variability can explain the polygenic nature of follow normal distribution, which pain as a phenotype pain. In many genetic association studies, true positive does not [10]. Since the Z score is calculated by the mean associations are rare, and most of the "significant" results and standard deviation, it would increase the risk of error rarely represent a true-positive association for which the when samples come from different means and standard genetic effect is accurately estimated [11]. Considering the deviations. Experimental heat pain threshold and toler- overwhelming size of the human genome and relatively ance have been reported that Caucasians have different high risk of potential flaws in study design and analysis of means and standard deviations from African ancestry complicated new methodology [12], replication of the population. association with independent population samples from studies strictly observing rules of genetic research is critical It is also possible that our results may be false-negative. [13]. Many phenotypes with modest estimated genetic effects may result in false negative due to its underpowered stud- Conclusion ies and probably contribute to inconsistent replication. Considering population stratification, previously There are probably many common variants in the human reported associations between GCH1 genetic variations genome with modest but real effects on common disease Page 5 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Figure 4 Linkage disequilibrium of GCH1 in Asian Americans (Haploview results with confidence interval method) Linkage disequilibrium of GCH1 in Asian Americans (Haploview results with confidence interval method). and pain sensitivity appear weak or negligible in this well as needed to facilitate extraction of the impacted lower characterized model of pain. third molars. SNP genotyping Methods Subjects For genotyping, 50 ml of venous blood from each subject Normal subjects (443 females and 292 males) were eval- was collected. DNA isolation was performed with the uated following informed consent under a human Puregene™ DNA isolation kit (Gentra Systems Inc., Min- research protocol approved by the IRB. Demographics neapolis, Minnesota, USA) following manufacturer's and characteristics of the cohort were previously described instructions. [14]. Briefly, subjects were not experiencing any clinical pain as symptomatic patients were referred elsewhere at For SNP genotyping, Assays-on-Demand or Assays-by- the time of initial screening. Self reported ethnicity in the Design SNP Genotyping Products (Applied Biosystems, sample was 50.1% European American, 22.8% African Foster City, California, USA) were used. Each well con- American, 10.6% Hispanic, and 14.1% Asian American in tained 2.5 μl of Taqman universal master mix, 0.25 μl of composition. For the genotype linkage, we excluded indi- genotyping assay mix and 2.25 μl of DNAse free water. viduals with mixed race parentage (2.3%). Polymerase chain reaction (PCR) was performed under the following conditions: 95°C, 10 min followed by 40 Among 735 subjects, 221 patients underwent standard- cycles of 92°C, 15 seconds and 60°C, 1 minute in a Per- ized surgery by the same oral surgeon removing third kin-Elmer™ 9700 thermocycler (Perkin-Elmer Inc., Bos- molar teeth that included at least one bony impacted ton, Massachusetts, USA). Following PCR, fluorescence of mandibular third molar [15]. After receiving pre-medica- each well was measured using the ABI Prism 7900 tion with intravenous midazolam (4.9 ± 0.2 mg) and local Sequence Detection System (Applied Biosystems, Foster anesthesia with 2% lidocaine (250.6 ± 43.0 mg) with City, California, USA). From the genomic sequences epinephrine 1:100,000, a mucoperiosteal flap was raised including their flanking regions, 38 SNPs (heterozygosity and retracted, bone removed, and the teeth were sectioned > 0.2, average distance = 2.0 kb) from GCH1 (genomic Page 6 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Table 2: Frequent haploblocks of GCH1 in European Americans Haplotype Frequency Haploblock 1 A_G_T_C 0.55 A_G_C_C 0.25 T_A_C_T 0.17 total 0.98 Haploblock 2 A_A_T_C_C 0.55 G_T_C_T_G 0.18 G_A_T_T_C 0.17 G_A_T_C_C 0.09 total 0.99 Haploblock 3 G_C_G_T_C_A_C_G_G_A_A_C_T_T_G_G_C_T_A_C_A_A_T_C_G_C_G_C_C 0.29 G_C_A_T_C_G_C_G_C_A_A_C_C_T_C_A_C_A_C_T_G_G_C_T_A_T_G_G_T 0.19 G_T_G_C_A_A_T_A_C_G_G_A_C_C_G_G_C_T_A_C_A_A_T_C_G_C_T_C_C 0.15 G_C_G_T_C_A_C_G_G_A_A_C_T_T_G_A_A_T_A_C_A_A_C_T_G_C_G_C_C 0.06 A_C_G_T_C_A_C_G_G_A_A_C_T_T_G_G_C_T_A_C_A_A_T_C_G_C_T_C_C 0.20 total 0.88 Tag SNPs A_T_C_A_A_C_G_C_G_G_G_G 0.28 A_T_C_A_A_C_A_C_G_G_G_T 0.19 T_C_T_G_T_T_G_C_A_C_A_G 0.16 A_C_C_G_A_T_G_T_G_C_G_T 0.15 A_C_C_G_A_C_G_C_G_G_A_G 0.08 total 0.87 size = 75 kbs including flanking region) were screened. tain constant pressure with the skin. The probe was self- Detailed information of genotyped SNPs is in Table 1. applied at six different sites on the volar forearm within an area of ~40 × 100 mm. Four iterations were completed for Genotype discrimination was performed using Taqman each temperature by moving the probe from spot to spot Sequence Detector version 2.1 software. Samples that to eliminate the possibility of sensitization or tissue dam- failed to amplify were not included in the final analysis. age. The order of each temperature was pre-determined for each trial, but the order was quasi-random to prevent Experimental pain sensitivity measurements subjects from anticipating each subsequent stimulus. Sub- We measured pain sensitivity in response to experimental jects were blinded with regard to the temperature of the painful thermal stimuli and cold stimuli with separate vis- stimulus. ual analogue scale (VAS) ratings for pain intensity. Indi- viduals were trained to use a sliding VAS by rating a visual Clinical Pain measurement gray scale. This procedure also provides an indication of Clinically induced pain was recorded with a paper and each subject's comprehension of the rating process using pencil form of a 100 mm VAS. After the extraction of the a VAS [16]. For cold stimuli, we recorded cold pain inten- impacted third molars, pain was recorded every 20 min- sity (CPI) VAS ratings every 30 seconds following submer- utes by VAS until subjects requested analgesic medication sion of the subject's hand up to the wrist into an insulated as the local anesthesia was eliminated and post-operative bucket filled with iced-water (2–4°C). We instructed sub- pain onset occurred. Ketorolac tromethamine (Toradol) jects to keep their hand submerged while clenching and was administered intravenously at the recommended unclenching repeatedly to prevent local warming in the dose (30 mg) and pain was recorded by VAS again at 15 water until the pain reached an "unbearable level" or 180 minutes interval for 180 minutes. The maximum post- seconds, whichever occurred soonest. The temperature of operative pain rating, onset time of post-operative pain, iced water was maintained by ice cubes separated from the the analgesic onset time after medication and pain relief subject's hand by a wire mesh. Subjects rated CPI at 30 at 180 minutes after medication were used as measures of seconds, if they withdrew their hand prior to 30 seconds, clinical pain. CPI was rated at their cold withdrawal time (CWT). Data analysis Individuals rated heat pain intensity (HPI) using a VAS To resolve phase unknown genotypes and estimate popu- following application of 35, 43, 44, 45, 46, 47, and 49°C lation frequencies in unrelated individuals we employed thermal stimuli for 5 seconds. The thermode probe area PHASE method, a probability based Bayesian algorithm. was 1 cm in diameter and mounted in a housing to main- Haploblocks based on confidence interval rule [5] were Page 7 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Non s Figure 5 ignificant association between pain sensitivity and GCH1 tag SNP combinations in European American females Non significant association between pain sensitivity and GCH1 tag SNP combinations in European American females. tag SNP combination A:A_T_C_A_A_C_G_C_G_G_G_G. B: A_T_C_A_A_C_A_C_G_G_G_T. C: T_C_T_G_T_T_G_C_A_C_A_G. D: A_C_C_G_A_T_G_T_G_C_G_T. E: A_C_C_G_A_C_G_C_G_G_A_G. generated by Haploview version 3.11. To quantify linkage Authors' contributions disequilibrium (LD), widely used D' and r were calcu- HK carried out the molecular genetic studies, participated lated. Both measures are built on the basic pairwise-dise- in the DNA extraction, genotyping, statistical analysis and quilibrium coefficient, D, which is the difference between drafted the manuscript. RD conceived of the study and the probability of observing two marker alleles on the participated in its design and coordination and helped to same haplotype and observing them independently in the draft the manuscript. All authors read and approved the population. A value of 0 implies independence, whereas final manuscript. 1.0 means complete co-transfer. Additional material Due to the smaller sample size when subdivided into eth- nic groups, second association analyses with experimental Additional File 1 and clinical pain was done only in European Americans. D' with confidence intervals and r matrices. D' with confidence intervals For the associations between haplotypes, individual SNPs and r matrices between SNPs in European Americans, African Ameri- and cold/heat pain sensitivity and/or clinical pain cans, Hispanic Americans and Asian Americans. responses, statistical evaluation was performed with anal- Click here for file [http://www.biomedcentral.com/content/supplementary/1744- ysis of variance (ANOVA) models. Groups of frequency 8069-3-6-S1.txt] less than 5% were excluded for the association analyses. Competing interests The author(s) declare that they have no competing inter- ests. Page 8 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Acknowledgements We thank Yin Yao, PhD, Johns Hopkins University, Bloomberg School of Public Health, for her statistical consultation. This study was supported by the Division of Intramural Research, National Institute of Nursing Research, National Institutes of Health, Bethesda, MD 20892, USA. References 1. Costigan M, Befort K, Karchewski L, Griffin RS, D'Urso D, Allchorne A, Sitarski J, Mannion JW, Pratt RE, Woolf CJ: Replicate high-den- sity rat genome oligonucleotide microarrays reveal hun- dreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci 2002, 3:16. 2. Tegeder I, Costigan M, Griffin RS, Abele A, Belfer I, Schmidt H, Ehnert C, Nejim J, Marian C, Scholz J, Wu T, Allchorne A, Diatchenko L, Bin- shtok AM, Goldman D, Adolph J, Sama S, Atlas SJ, Carlezon WA, Parsegian A, Lotsch J, Fillingim RB, Maixner W, Geisslinger G, Max MB, Woolf CJ: GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence. Nat Med 2006, 12:1269-1277. 3. Diatchenko L, Slade GD, Nackley AG, Bhalang K, Sigurdsson A, Belfer I, Goldman D, Xu K, Shabalina SA, Shagin D, Max MB, Makarov SS, Maixner W: Genetic basis for individual variations in pain per- ception and the development of a chronic pain condition. Hum Mol Genet 2005, 14:135-143. 4. Hastie BA, Riley JL 3rd, Robinson ME, Glover T, Campbell CM, Staud R, Fillingim RB: Cluster analysis of multiple experimental pain modalities. Pain 2005, 116:227-237. 5. Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, Higgins J, DeFelice M, Lochner A, Faggart M, Liu-Cordero SN, Rotimi C, Adeyemo A, Cooper R, Ward R, Lander ES, Daly MJ, Altshuler D: The structure of haplotype blocks in the human genome. Sci- ence 2002, 296:2225-2229. 6. Fullerton SM, Bartoszewicz A, Ybazeta G, Horikawa Y, Bell GI, Kidd KK, Cox NJ, Hudson RR, Di Rienzo A: Geographic and haplotype structure of candidate type 2 diabetes susceptibility variants at the calpain-10 locus. Am J Hum Genet 2002, 70:1096-1106. 7. Strachan T, Read AP: Human molecular genetics. 3rd edition. London and New York, Garland Science; 2004. 8. Healy DG: Case-control studies in the genomic era: a clini- cian's guide. Lancet Neurol 2006, 5:701-707. 9. Lariviere WR, Wilson SG, Laughlin TM, Kokayeff A, West EE, Adhikari SM, Wan Y, Mogil JS: Heritability of nociception. III. Genetic relationships among commonly used assays of noci- ception and hypersensitivity. Pain 2002, 97:75-86. 10. Kim H, Neubert JK, San Miguel A, Xu K, Krishnaraju RK, Iadarola M, Goldman D, Dionne R: Genetic influence on variability in human pain sensitivity associated with gender, ethnicity and psychological temperament. Pain 2004, 109:488-496. 11. Lohmueller KE, Pearce CL, Pike M, Lander ES, Hirschhorn JN: Meta- analysis of genetic association studies supports a contribu- tion of common variants to susceptibility to common dis- ease. Nat Genet 2003, 33:177-182. 12. Dupuy A, Simon RM: Critical review of published microarray studies for cancer outcome and guidelines on statistical anal- ysis and reporting. J Natl Cancer Inst 2007, 99:147-157. 13. Newton-Cheh C, Hirschhorn JN: Genetic association studies of complex traits: design and analysis issues. Mutat Res 2005, 573:54-69. 14. Kim H, Mittal DP, Iadarola MJ, Dionne RA: Genetic predictors for Publish with Bio Med Central and every acute experimental cold and heat pain sensitivity in humans. scientist can read your work free of charge J Med Genet 2006, 43:e40. 15. Kim H, Lee H, Rowan J, Brahim J, Dionne RA: Genetic polymor- "BioMed Central will be the most significant development for phisms in monoamine neurotransmitter systems show only disseminating the results of biomedical researc h in our lifetime." weak association with acute post-surgical pain in humans. Sir Paul Nurse, Cancer Research UK Mol Pain 2006, 2:24. 16. Rosier EM, Iadarola MJ, Coghill RC: Reproducibility of pain meas- Your research papers will be: urement and pain perception. Pain 2002, 98:205-216. available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Pain Springer Journals

Lack of influence of GTP cyclohydrolase gene (GCH1) variations on pain sensitivity in humans

Kim, Hyungsuk; Dionne, Raymond
Molecular Pain , Volume 3 (1) – Mar 7, 2007
Download PDF
9 pages

Loading next page...
 
/lp/springer-journals/lack-of-influence-of-gtp-cyclohydrolase-gene-gch1-variations-on-pain-Th24dHBrlw

References (19)

Publisher
Springer Journals
Copyright
Copyright © 2007 by Kim and Dionne; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
ISSN
1744-8069
eISSN
1744-8069
DOI
10.1186/1744-8069-3-6
pmid
17343757
Publisher site
See Article on Publisher Site

Abstract

Objectives: To assess the effect of variations in GTP cyclohydrolase gene (GCH1) on pain sensitivity in humans. Methods: Thermal and cold pain sensitivity were evaluated in a cohort of 735 healthy volunteers. Among this cohort, the clinical pain responses of 221 subjects after the surgical removal of impacted third molars were evaluated. Genotyping was done for 38 single nucleotide polymorphisms (SNPs) whose heterozygosity > 0.2 in GCH1. Influence of the genetic variations including SNPs and haplotypes on pain sensitivity were analyzed. Results: Minor allele frequencies and linkage disequilibrium show significant differences in European Americans, African Americans, Hispanic Americans and Asian Americans. Association analyses in European Americans do not replicate the previously reported important influence of GCH1 variations on pain sensitivity. Conclusion: Considering population stratification, previously reported associations between GCH1 genetic variations and pain sensitivity appear weak or negligible in this well characterized model of pain. pain protective haplotype associated with the risk of Background The role of 6(R)-t-erythro-5,6,7,8-tetrahydrobiopterin developing persistent pain syndromes, which could be a (BH4) in pain is suggested by the up-regulation of two of useful tool to assess an individual's risk potential for the three enzymes in the synthesis cascade of BH4 in the chronic pain [2]. Based on this reported role of GCH in dorsal root ganglion following sciatic nerve injury [1]. pain both in animals and humans, we investigated its con- GTP cyclohydrolase (GCH) catalyzes the rate limiting tribution to genetic inter-individual variation in clinical step, and sepiapterin reductase catalyzes the final conver- pain sensitivity and analgesic responses. We have investi- sion of 6-pyruvoyltetrahydropterin to the BH4. It was gated the association between pain responses to experi- recently reported that single nucleotide polymorphisms mental and clinical painful stimuli and genetic variations (SNPs) in the gene encoding GCH (GCH1) alter both including SNPs and haplotypes of GCH1. responses in healthy humans to noxious stimuli and the susceptibility of patients to development of neuropathic and inflammatory pain [2]. The authors also suggest a Page 1 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Further haploblock analysis was performed with Euro- Results The minor allelic frequencies of each SNP in the GCH1 are pean Americans only due to the small number of other shown in Table 1. ethnic populations. We found 3 haploblocks from GCH1 (4 SNPs spanning ~17.4 kb, 5 SNPs spanning ~3.7 kb and We next determined what haplotypes are formed by these 29 SNPs spanning ~51.0 kb respectively) based on the SNPs. The total number of haploblocks found were 3 in confidence interval method. European Americans (average minimum span = 24.0 kb) with 12 tag SNPs, 5 haploblocks with 21 tag SNPs in Afri- In the GCH1 haploblock 1, a total of 5 haplotypes were can Americans (average minimum span = 5.3 kb), 3 hap- detected for the 4 SNPs, with the most frequent haplotype loblocks with 14 tag SNPs in Hispanic Ameicans (average (55.4%) composed of A_G_T_C. The 3 frequent haplo- minimum span = 22.7 kb) and 4 haploblocks with 10 tag types (> 5%) were 98% of total haplotypes. In the GCH1 SNPs in Asian Americans (average minimum span = 16.1 haploblock 2, a total of 7 haplotypes were detected for the kb). Position of those SNPs and haploblocks within the 5 chosen SNPs, with the most frequent haplotype loci of GCH1 are shown in Figure 1, 2, 3 and 4. Their D' (55.0%) composed of A_A_T_C_C. Four of the 7 haplo- matrices in each ethnic with confidence intervals and r types were frequent (> 5%), and these 4 haplotypes were population show large differences (see Additional file 1). 99% of the total haplotypes. In the GCH1 haploblock 3, a Table 1: SNPs genotyped in GCH1 Order SNP ID Location from transcription site Nucleotide variation Rarer allele frequency 1 rs8008858 -9,686 A/T 0.28 2 rs8007267 -9,462 G/A 0.28 3 rs2878172 -4,141 T/C 0.44 4 rs8017210 7,694 C/T 0.21 5 rs3783642 9,327 A/G 0.48 6 rs3783641 9,391 A/T 0.21 7 rs7147201 10,653 T/C 0.27 8 rs7147286 10,865 C/T 0.42 9 rs17128052 13,005 C/G 0.18 10 rs998259 14,499 G/A 0.14 11 rs10498471 15,813 C/T 0.22 12 rs8020798 16,162 G/A 0.21 13 rs8004018 18,834 T/C 0.25 14 rs8004445 18,864 C/A 0.25 15 rs3783639 20,864 A/G 0.20 16 rs3783638 21,157 C/T 0.26 17 rs3783637 21,412 G/A 0.18 18 rs10133650 23,257 G/C 0.47 19 rs12147422 25,515 A/G 0.25 20 rs17128050 25,651 A/G 0.17 21 rs7492600 32,655 C/A 0.26 22 rs2183081 32,779 T/C 0.47 23 rs8010282 39,275 T/C 0.21 24 rs9671455 40,450 G/C 0.26 25 rs9671371 40,895 G/A 0.33 26 rs2878169 43,537 C/A 0.06 27 rs12589758 43,668 T/A 0.22 28 rs12587434 43,947 A/C 0.23 29 rs7155309 46,679 C/T 0.27 30 rs2878168 48,745 A/G 0.22 31 rs4411417 48,967 A/G 0.22 32 rs10133662 53,260 T/C 0.39 33 rs10131232 53,622 C/T 0.40 34 rs752688 57,961 G/A 0.23 35 rs841 59,038 C/T 0.23 36 rs7142517 62,726 G/T 0.28 37 rs10483639 63,073 C/G 0.26 38 rs2057369 65,469 C/T 0.22 Page 2 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Li Figure 1 nkage disequilibrium of GCH1 in European Americans (Haploview results with confidence interval method) Linkage disequilibrium of GCH1 in European Americans (Haploview results with confidence interval method). total of 17 haplotypes were detected for the 29 SNPs, with Discussion the most frequent haplotype (28.7%) and 5 frequent hap- We could not repeat any of the finding reported for lotypes were 88% of the total haplotypes. In the whole humans by Tegeder et al. First, the linkage disequilibrium GCH1, 12 SNPs were tag SNPs European American popu- (LD) between SNPs and patterns of haplotype blocks in lation. Among 25 existing combinations of those 12 tag our sample are not consistent with their previous finding SNPs, the most frequent combination occupies 28.3% reporting one single block for GCH1. This inconsistency while 5 frequent (> 5%) combinations of tag SNPs are may be caused by the samples evaluated and the atypical 87% of the total tag SNPs combinations (Table 2). interpretation of their data. The University of North Caro- lina and University of Florida cohorts of Tegeder et al. cite Considering the relatively dominating effect of gender on previous publications[3,4] with mixed population groups pain, male and female subjects were analyzed separately and leave open the question whether their present study is for the association between genetic variations (individual using mixed ethnicity groups as well. It has been shown SNPs and combination of haplotypes) and responses to that the ethnic demographics can have profound influ- experimental and clinical painful stimuli. However, we ence on LD between SNPs, haploblock structure, SNP fre- could not find any significant genetic associations quency, and estimated effect size drawn from the data. It between variations of GCH1 including SNPs and haplo- is for these very reasons that haplotype data are reported types and pain sensitivity or analgesic responses in this separately by ethnicity in the HapMap Project database. cohort of European American females and males even without multiple testing corrections. Figure 5 shows an When we analyzed haplotypes from our samples and the example of non-significant association between GCH1 HapMap website within the same region using Haplov- genetic variations and pain sensitivity in European Amer- iew, we obtained different haplotype blocks between each ican females (GCH1 tag SNP combinations and heat and ethnic groups, regardless of the algorithm used (imple- cold pain ratings). mented in Haploview, confidence interval, 4-gamete and Page 3 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Figure 2 Linkage disequilibrium of GCH1 in African Americans (Haploview results with confidence interval method) Linkage disequilibrium of GCH1 in African Americans (Haploview results with confidence interval method). solid spine method). Ethnic differences in the structure of supplementary table table 2 in Tegeder et al. The authors haplotypes are consistent with other reports [5,6]. The sin- seem to interpret their data completely opposite direction. gle large 72 kb block identified by Tegeder et al for GCH1 Unique haplotype patterns including block size, block with 168 Caucasian chronic lumbar root pain subjects is numbers, contributing SNPs, tag SNPs along with LDs not consistent with the HapMap Caucasians (CEU), acquired in each ethnic population in our sample are con- which may reflect a lack of generalizability for the Tegeder sistent with HapMap data and clearly suggest that haplo- et al results. It is unclear how the data from the other type analysis with mixed population be avoided. cohorts is affected by one single large haploblock gener- ated from the back pain study, especially when that one Using a mixed ethnic sample may also induce population large block is not observed in the Phase II data released by stratification. This can cause false associations since the the HapMap project. We found 3 haplotype blocks across pattern of genetic variations as well as pain rating is not GCH1 including its flanking region based on confidence uniform between ethnic populations [7], requiring use of interval method in European Americans and 5 blocks in homogenous population in genetic association studies African Americans with different size and different con- [8]. Considering significant differences of allele frequen- tributing SNPs. Hispanic Americans and Asian Americans cies and pain sensitivities among ethnic populations, it also show unique haplotype blocks and LD values would be more informative to analyze single ethnic origin between each SNPs in GCH1. These results are similar to population only compared to the total mixed sample to HapMap data and the general concept of smaller block determine if the apparent association is due to population size in samples of African ancestry. stratification. Additionally, Tegeder et al proposed the low minor allele Another possible reason of inconsistency is the pain phe- frequencies of a few markers as the cause of the disruption notype. Because responses to different types of painful of the LD. However, almost 0 values of D' were obtained stimuli are genetically dissociable [9], GCH1 genetic vari- between 3 most frequent SNPs (10374 c>t as 29.69%, - ation may affect the responses to a specific type of painful 4289 t>c as 37.42% and 3932 g>t as 35.76% in their stimulus, while not influencing other types of stimuli. minor allele frequencies) in supplementary figure 4 and However, Tegeder et al. used Z score for their analyses for Page 4 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Figure 3 Linkage disequilibrium of GCH1 in Hispanic Americans (Haploview results with confidence interval method) Linkage disequilibrium of GCH1 in Hispanic Americans (Haploview results with confidence interval method). a chimeric phenotype that combines pain threshold and risk, requiring large samples to convincingly identify such pain tolerance, outcome measures at opposite ends of the variants [11]. Given these questions of study design sensory continuum. Use of an artificial outcome measure related to population stratification, haploblock size, and that has been transformed may not be an appropriate disparate pain measures in Tegeder et al with similar sta- physiological representative of pain sensation and could tistical power of our samples, it is more probable that this potentially result in an artificial genetic association. Con- association between GCH1 genetic variations and pain sidering the non-significant association between GCH1 sensitivity are, if any, weak or negligible regardless of their genetic variations and similar thermal pain sensitivity in strong in vitro and animal findings. our sample, the inconsistency cannot be easily explained by the differences in stimulus modalities. Additionally, It is doubtful that a single gene accounting for large por- the Z score is based on the assumption that the variables tion of the variability can explain the polygenic nature of follow normal distribution, which pain as a phenotype pain. In many genetic association studies, true positive does not [10]. Since the Z score is calculated by the mean associations are rare, and most of the "significant" results and standard deviation, it would increase the risk of error rarely represent a true-positive association for which the when samples come from different means and standard genetic effect is accurately estimated [11]. Considering the deviations. Experimental heat pain threshold and toler- overwhelming size of the human genome and relatively ance have been reported that Caucasians have different high risk of potential flaws in study design and analysis of means and standard deviations from African ancestry complicated new methodology [12], replication of the population. association with independent population samples from studies strictly observing rules of genetic research is critical It is also possible that our results may be false-negative. [13]. Many phenotypes with modest estimated genetic effects may result in false negative due to its underpowered stud- Conclusion ies and probably contribute to inconsistent replication. Considering population stratification, previously There are probably many common variants in the human reported associations between GCH1 genetic variations genome with modest but real effects on common disease Page 5 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Figure 4 Linkage disequilibrium of GCH1 in Asian Americans (Haploview results with confidence interval method) Linkage disequilibrium of GCH1 in Asian Americans (Haploview results with confidence interval method). and pain sensitivity appear weak or negligible in this well as needed to facilitate extraction of the impacted lower characterized model of pain. third molars. SNP genotyping Methods Subjects For genotyping, 50 ml of venous blood from each subject Normal subjects (443 females and 292 males) were eval- was collected. DNA isolation was performed with the uated following informed consent under a human Puregene™ DNA isolation kit (Gentra Systems Inc., Min- research protocol approved by the IRB. Demographics neapolis, Minnesota, USA) following manufacturer's and characteristics of the cohort were previously described instructions. [14]. Briefly, subjects were not experiencing any clinical pain as symptomatic patients were referred elsewhere at For SNP genotyping, Assays-on-Demand or Assays-by- the time of initial screening. Self reported ethnicity in the Design SNP Genotyping Products (Applied Biosystems, sample was 50.1% European American, 22.8% African Foster City, California, USA) were used. Each well con- American, 10.6% Hispanic, and 14.1% Asian American in tained 2.5 μl of Taqman universal master mix, 0.25 μl of composition. For the genotype linkage, we excluded indi- genotyping assay mix and 2.25 μl of DNAse free water. viduals with mixed race parentage (2.3%). Polymerase chain reaction (PCR) was performed under the following conditions: 95°C, 10 min followed by 40 Among 735 subjects, 221 patients underwent standard- cycles of 92°C, 15 seconds and 60°C, 1 minute in a Per- ized surgery by the same oral surgeon removing third kin-Elmer™ 9700 thermocycler (Perkin-Elmer Inc., Bos- molar teeth that included at least one bony impacted ton, Massachusetts, USA). Following PCR, fluorescence of mandibular third molar [15]. After receiving pre-medica- each well was measured using the ABI Prism 7900 tion with intravenous midazolam (4.9 ± 0.2 mg) and local Sequence Detection System (Applied Biosystems, Foster anesthesia with 2% lidocaine (250.6 ± 43.0 mg) with City, California, USA). From the genomic sequences epinephrine 1:100,000, a mucoperiosteal flap was raised including their flanking regions, 38 SNPs (heterozygosity and retracted, bone removed, and the teeth were sectioned > 0.2, average distance = 2.0 kb) from GCH1 (genomic Page 6 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Table 2: Frequent haploblocks of GCH1 in European Americans Haplotype Frequency Haploblock 1 A_G_T_C 0.55 A_G_C_C 0.25 T_A_C_T 0.17 total 0.98 Haploblock 2 A_A_T_C_C 0.55 G_T_C_T_G 0.18 G_A_T_T_C 0.17 G_A_T_C_C 0.09 total 0.99 Haploblock 3 G_C_G_T_C_A_C_G_G_A_A_C_T_T_G_G_C_T_A_C_A_A_T_C_G_C_G_C_C 0.29 G_C_A_T_C_G_C_G_C_A_A_C_C_T_C_A_C_A_C_T_G_G_C_T_A_T_G_G_T 0.19 G_T_G_C_A_A_T_A_C_G_G_A_C_C_G_G_C_T_A_C_A_A_T_C_G_C_T_C_C 0.15 G_C_G_T_C_A_C_G_G_A_A_C_T_T_G_A_A_T_A_C_A_A_C_T_G_C_G_C_C 0.06 A_C_G_T_C_A_C_G_G_A_A_C_T_T_G_G_C_T_A_C_A_A_T_C_G_C_T_C_C 0.20 total 0.88 Tag SNPs A_T_C_A_A_C_G_C_G_G_G_G 0.28 A_T_C_A_A_C_A_C_G_G_G_T 0.19 T_C_T_G_T_T_G_C_A_C_A_G 0.16 A_C_C_G_A_T_G_T_G_C_G_T 0.15 A_C_C_G_A_C_G_C_G_G_A_G 0.08 total 0.87 size = 75 kbs including flanking region) were screened. tain constant pressure with the skin. The probe was self- Detailed information of genotyped SNPs is in Table 1. applied at six different sites on the volar forearm within an area of ~40 × 100 mm. Four iterations were completed for Genotype discrimination was performed using Taqman each temperature by moving the probe from spot to spot Sequence Detector version 2.1 software. Samples that to eliminate the possibility of sensitization or tissue dam- failed to amplify were not included in the final analysis. age. The order of each temperature was pre-determined for each trial, but the order was quasi-random to prevent Experimental pain sensitivity measurements subjects from anticipating each subsequent stimulus. Sub- We measured pain sensitivity in response to experimental jects were blinded with regard to the temperature of the painful thermal stimuli and cold stimuli with separate vis- stimulus. ual analogue scale (VAS) ratings for pain intensity. Indi- viduals were trained to use a sliding VAS by rating a visual Clinical Pain measurement gray scale. This procedure also provides an indication of Clinically induced pain was recorded with a paper and each subject's comprehension of the rating process using pencil form of a 100 mm VAS. After the extraction of the a VAS [16]. For cold stimuli, we recorded cold pain inten- impacted third molars, pain was recorded every 20 min- sity (CPI) VAS ratings every 30 seconds following submer- utes by VAS until subjects requested analgesic medication sion of the subject's hand up to the wrist into an insulated as the local anesthesia was eliminated and post-operative bucket filled with iced-water (2–4°C). We instructed sub- pain onset occurred. Ketorolac tromethamine (Toradol) jects to keep their hand submerged while clenching and was administered intravenously at the recommended unclenching repeatedly to prevent local warming in the dose (30 mg) and pain was recorded by VAS again at 15 water until the pain reached an "unbearable level" or 180 minutes interval for 180 minutes. The maximum post- seconds, whichever occurred soonest. The temperature of operative pain rating, onset time of post-operative pain, iced water was maintained by ice cubes separated from the the analgesic onset time after medication and pain relief subject's hand by a wire mesh. Subjects rated CPI at 30 at 180 minutes after medication were used as measures of seconds, if they withdrew their hand prior to 30 seconds, clinical pain. CPI was rated at their cold withdrawal time (CWT). Data analysis Individuals rated heat pain intensity (HPI) using a VAS To resolve phase unknown genotypes and estimate popu- following application of 35, 43, 44, 45, 46, 47, and 49°C lation frequencies in unrelated individuals we employed thermal stimuli for 5 seconds. The thermode probe area PHASE method, a probability based Bayesian algorithm. was 1 cm in diameter and mounted in a housing to main- Haploblocks based on confidence interval rule [5] were Page 7 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Non s Figure 5 ignificant association between pain sensitivity and GCH1 tag SNP combinations in European American females Non significant association between pain sensitivity and GCH1 tag SNP combinations in European American females. tag SNP combination A:A_T_C_A_A_C_G_C_G_G_G_G. B: A_T_C_A_A_C_A_C_G_G_G_T. C: T_C_T_G_T_T_G_C_A_C_A_G. D: A_C_C_G_A_T_G_T_G_C_G_T. E: A_C_C_G_A_C_G_C_G_G_A_G. generated by Haploview version 3.11. To quantify linkage Authors' contributions disequilibrium (LD), widely used D' and r were calcu- HK carried out the molecular genetic studies, participated lated. Both measures are built on the basic pairwise-dise- in the DNA extraction, genotyping, statistical analysis and quilibrium coefficient, D, which is the difference between drafted the manuscript. RD conceived of the study and the probability of observing two marker alleles on the participated in its design and coordination and helped to same haplotype and observing them independently in the draft the manuscript. All authors read and approved the population. A value of 0 implies independence, whereas final manuscript. 1.0 means complete co-transfer. Additional material Due to the smaller sample size when subdivided into eth- nic groups, second association analyses with experimental Additional File 1 and clinical pain was done only in European Americans. D' with confidence intervals and r matrices. D' with confidence intervals For the associations between haplotypes, individual SNPs and r matrices between SNPs in European Americans, African Ameri- and cold/heat pain sensitivity and/or clinical pain cans, Hispanic Americans and Asian Americans. responses, statistical evaluation was performed with anal- Click here for file [http://www.biomedcentral.com/content/supplementary/1744- ysis of variance (ANOVA) models. Groups of frequency 8069-3-6-S1.txt] less than 5% were excluded for the association analyses. Competing interests The author(s) declare that they have no competing inter- ests. Page 8 of 9 (page number not for citation purposes) Molecular Pain 2007, 3:6 http://www.molecularpain.com/content/3/1/6 Acknowledgements We thank Yin Yao, PhD, Johns Hopkins University, Bloomberg School of Public Health, for her statistical consultation. This study was supported by the Division of Intramural Research, National Institute of Nursing Research, National Institutes of Health, Bethesda, MD 20892, USA. References 1. Costigan M, Befort K, Karchewski L, Griffin RS, D'Urso D, Allchorne A, Sitarski J, Mannion JW, Pratt RE, Woolf CJ: Replicate high-den- sity rat genome oligonucleotide microarrays reveal hun- dreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci 2002, 3:16. 2. Tegeder I, Costigan M, Griffin RS, Abele A, Belfer I, Schmidt H, Ehnert C, Nejim J, Marian C, Scholz J, Wu T, Allchorne A, Diatchenko L, Bin- shtok AM, Goldman D, Adolph J, Sama S, Atlas SJ, Carlezon WA, Parsegian A, Lotsch J, Fillingim RB, Maixner W, Geisslinger G, Max MB, Woolf CJ: GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence. Nat Med 2006, 12:1269-1277. 3. Diatchenko L, Slade GD, Nackley AG, Bhalang K, Sigurdsson A, Belfer I, Goldman D, Xu K, Shabalina SA, Shagin D, Max MB, Makarov SS, Maixner W: Genetic basis for individual variations in pain per- ception and the development of a chronic pain condition. Hum Mol Genet 2005, 14:135-143. 4. Hastie BA, Riley JL 3rd, Robinson ME, Glover T, Campbell CM, Staud R, Fillingim RB: Cluster analysis of multiple experimental pain modalities. Pain 2005, 116:227-237. 5. Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, Higgins J, DeFelice M, Lochner A, Faggart M, Liu-Cordero SN, Rotimi C, Adeyemo A, Cooper R, Ward R, Lander ES, Daly MJ, Altshuler D: The structure of haplotype blocks in the human genome. Sci- ence 2002, 296:2225-2229. 6. Fullerton SM, Bartoszewicz A, Ybazeta G, Horikawa Y, Bell GI, Kidd KK, Cox NJ, Hudson RR, Di Rienzo A: Geographic and haplotype structure of candidate type 2 diabetes susceptibility variants at the calpain-10 locus. Am J Hum Genet 2002, 70:1096-1106. 7. Strachan T, Read AP: Human molecular genetics. 3rd edition. London and New York, Garland Science; 2004. 8. Healy DG: Case-control studies in the genomic era: a clini- cian's guide. Lancet Neurol 2006, 5:701-707. 9. Lariviere WR, Wilson SG, Laughlin TM, Kokayeff A, West EE, Adhikari SM, Wan Y, Mogil JS: Heritability of nociception. III. Genetic relationships among commonly used assays of noci- ception and hypersensitivity. Pain 2002, 97:75-86. 10. Kim H, Neubert JK, San Miguel A, Xu K, Krishnaraju RK, Iadarola M, Goldman D, Dionne R: Genetic influence on variability in human pain sensitivity associated with gender, ethnicity and psychological temperament. Pain 2004, 109:488-496. 11. Lohmueller KE, Pearce CL, Pike M, Lander ES, Hirschhorn JN: Meta- analysis of genetic association studies supports a contribu- tion of common variants to susceptibility to common dis- ease. Nat Genet 2003, 33:177-182. 12. Dupuy A, Simon RM: Critical review of published microarray studies for cancer outcome and guidelines on statistical anal- ysis and reporting. J Natl Cancer Inst 2007, 99:147-157. 13. Newton-Cheh C, Hirschhorn JN: Genetic association studies of complex traits: design and analysis issues. Mutat Res 2005, 573:54-69. 14. Kim H, Mittal DP, Iadarola MJ, Dionne RA: Genetic predictors for Publish with Bio Med Central and every acute experimental cold and heat pain sensitivity in humans. scientist can read your work free of charge J Med Genet 2006, 43:e40. 15. Kim H, Lee H, Rowan J, Brahim J, Dionne RA: Genetic polymor- "BioMed Central will be the most significant development for phisms in monoamine neurotransmitter systems show only disseminating the results of biomedical researc h in our lifetime." weak association with acute post-surgical pain in humans. Sir Paul Nurse, Cancer Research UK Mol Pain 2006, 2:24. 16. Rosier EM, Iadarola MJ, Coghill RC: Reproducibility of pain meas- Your research papers will be: urement and pain perception. Pain 2002, 98:205-216. available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes)

Journal

Molecular Pain – Springer Journals

Published: Mar 7, 2007

There are no references for this article.