Kate L. Holliday

Genetic Determinants of Serum Testosterone Concentrations in Men

Testosterone concentrations in men are associated with cardiovascular morbidity, osteoporosis, and mortality and are affected by age, smoking, and obesity. Because of serum testosterones high heritability, we performed a meta-analysis of genome-wide association data in 8,938 men from seven cohorts and followed up the genome-wide significant findings in one in silico (n = 871) and two de novo replication cohorts (n = 4,620) to identify genetic loci significantly associated with serum testosterone concentration in men. All these loci were also associated with low serum testosterone concentration defined as <300 ng/dl. Two single-nucleotide polymorphisms at the sex hormone-binding globulin (SHBG) locus (17p13-p12) were identified as independently associated with serum testosterone concentration (rs12150660, p = 1.2×10−41 and rs6258, p = 2.3×10−22). Subjects with ≥3 risk alleles of these variants had 6.5-fold higher risk of having low serum testosterone than subjects with no risk allele. The rs5934505 polymorphism near FAM9B on the X chromosome was also associated with testosterone concentrations (p = 5.6×10−16). The rs6258 polymorphism in exon 4 of SHBG affected SHBGs affinity for binding testosterone and the measured free testosterone fraction (p<0.01). Genetic variants in the SHBG locus and on the X chromosome are associated with a substantial variation in testosterone concentrations and increased risk of low testosterone. rs6258 is the first reported SHBG polymorphism, which affects testosterone binding to SHBG and the free testosterone fraction and could therefore influence the calculation of free testosterone using law-of-mass-action equation.

Obesity is a risk factor for musculoskeletal pain in adolescents: findings from a population-based cohort.

Summary Obesity is associated with the occurrence and severity of several musculoskeletal pain phenotypes, including knee pain and chronic regional pain in adolescents. Abstract Obesity is a risk factor for fibromyalgia in adults, but whether a similar relationship exists in children is uncertain. This study examined whether obesity is associated with reporting of musculoskeletal pain, including chronic regional pain (CRP) and chronic widespread pain (CWP), in adolescents, in a population‐based setting. A pain questionnaire was administered to offspring of the Avon Longitudinal Study of Parents and Children at age 17, asking about site, duration, and pain intensity, from which participants with different types of musculoskeletal pain were identified. Relationships between obesity and pain were examined by calculating odds ratios stratified by gender and adjusted for socioeconomic status as reflected by level of maternal education. A total of 3376 participants (1424 boys) with complete data were identified, mean age 17.8; 44.7% of participants reported any pain within the last month lasting 1 day or longer; 16.3% reported lower back pain, 9.6% shoulder pain, 9.4% upper back pain, 8.9% neck pain, 8.7% knee pain, 6.8% ankle/foot pain, 4.7% CRP, and 4.3% CWP; 7.0% of participants were obese. Obesity was associated with increased odds of any pain (odds ratio [OR] 1.33, P = .04), CRP (OR 2.04, P = .005), and knee pain (OR 1.87, P = .001), but not CWP (OR 1.10, P = .5). Compared with non obese participants, those with any pain, knee pain, and CRP reported more severe average pain (P < .01). Obese adolescents were more likely to report musculoskeletal pain, including knee pain and CRP. Moreover, obese adolescents with knee pain and CRP had relatively high pain scores, suggesting a more severe phenotype with worse prognosis.

Genome-wide association study meta-analysis of chronic widespread pain: evidence for involvement of the 5p15.2 region

Background and objectives Chronic widespread pain (CWP) is a common disorder affecting ∼10% of the general population and has an estimated heritability of 48–52%. In the first large-scale genome-wide association study (GWAS) meta-analysis, we aimed to identify common genetic variants associated with CWP. Methods We conducted a GWAS meta-analysis in 1308 female CWP cases and 5791 controls of European descent, and replicated the effects of the genetic variants with suggestive evidence for association in 1480 CWP cases and 7989 controls. Subsequently, we studied gene expression levels of the nearest genes in two chronic inflammatory pain mouse models, and examined 92 genetic variants previously described associated with pain. Results The minor C-allele of rs13361160 on chromosome 5p15.2, located upstream of chaperonin-containing-TCP1-complex-5 gene (CCT5) and downstream of FAM173B, was found to be associated with a 30% higher risk of CWP (minor allele frequency=43%; OR=1.30, 95% CI 1.19 to 1.42, p=1.2×10−8). Combined with the replication, we observed a slightly attenuated OR of 1.17 (95% CI 1.10 to 1.24, p=4.7×10−7) with moderate heterogeneity (I2=28.4%). However, in a sensitivity analysis that only allowed studies with joint-specific pain, the combined association was genome-wide significant (OR=1.23, 95% CI 1.14 to 1.32, p=3.4×10−8, I2=0%). Expression levels of Cct5 and Fam173b in mice with inflammatory pain were higher in the lumbar spinal cord, not in the lumbar dorsal root ganglions, compared to mice without pain. None of the 92 genetic variants previously described were significantly associated with pain (p>7.7×10−4). Conclusions We identified a common genetic variant on chromosome 5p15.2 associated with joint-specific CWP in humans. This work suggests that CCT5 and FAM173B are promising targets in the regulation of pain.

Genetic variation in the hypothalamic–pituitary–adrenal stress axis influences susceptibility to musculoskeletal pain: results from the EPIFUND study

Objectives To determine if genetic variation in genes in the hypothalamic–pituitary–adrenal (HPA) axis, the primary stress response system, influences susceptibility to developing musculoskeletal pain. Methods Pain and comorbidity data was collected at three time points in a prospective population-based cohort study. Pairwise tagging single nucleotide polymorphisms (SNPs) were selected and genotyped for seven genes. Genetic association analysis was carried out using zero-inflated negative binomial regression to test for association between SNPs and the maximum number of pain sites across the three time points in participants reporting pain, reported as proportional changes with 95% CIs. SNPs were also tested for association with chronic widespread pain (CWP) using logistic regression reporting odds ratios and 95% CI. Results A total of 75 SNPs were successfully genotyped in 994 participants including 164 cases with persistent CWP and 172 pain-free controls. Multiple SNPs in SERPINA6 were associated with the maximum number of pain sites; for example, each copy of the T allele of rs941601 was associated with having 16% (proportional change=1.16, 95% CI 1.04 to 1.28, p=0.006) more pain sites compared to participants with the CC genotype. SERPINA6 gene SNPs were also associated with CWP. Significant associations between the maximum number of pain sites and SNPs in the CRHBP and POMC genes were also observed and a SNP in MC2R was also associated with CWP. Associations between SNPs and comorbidity of poor sleep quality and depression explained some of the associations observed. Conclusions Genetic variation in HPA axis genes was associated with musculoskeletal pain; however, some of the associations were explained by comorbidities. Replication of these findings is required in independent cohorts.

Genetic variation in neuroendocrine genes associates with somatic symptoms in the general population: Results from the EPIFUND study

Objective Functional somatic syndromes commonly occur together, share a genetic component and are associated with numerous somatic symptoms. This study aimed to determine if genetic variation in two neuroendocrine systems, the serotoninergic system and the hypothalamic-pituitary-adrenal (HPA) axis, was associated with the number of reported somatic symptoms. Methods This population-based cohort study (Epidemiology of Functional Disorders) recruited participants from three primary care registers in the northwest of England. Somatic symptoms, anxiety, depression, and pain were assessed using the Somatic Symptoms Checklist, Hospital Anxiety and Depression scales, and body manikins, respectively, via a postal questionnaire. Tag Single Nucleotide Polymorphisms (SNPs) (r2>0.8) were selected for serotoninergic system genes (TPH2, SLC6A4 and HTR2A) and HPA axis genes (CRH, CRHR1, CRHBP, MC2R, POMC, NR3C1, and SERPINA6) and genotyped using Sequenom technology. Negative binomial regression was used to test for association between SNPs and the number of somatic symptoms. Stepwise-regression was used to identify independent effects and adjustments were made for anxiety, depression, and pain. Results A total of 967 subjects were successfully genotyped for 143 (87%) SNPs. Multiple SNP associations with the number of somatic symptoms were observed in HTR2A and SERPINA6 as well as two SNPs in TPH2. Stepwise regression identified two effects in HTR2A and a single effect in TPH2 which were independent of anxiety, depression, and pain. A single effect was also identified in SERPINA6 but was no longer significant when adjusted for pain. Conclusion This study finds association of SNPs in HTR2A, SERPINA6, and TPH2 with somatic symptoms implicating them as potentially important in the shared genetic component to functional somatic syndromes, although replication is required.

Genetic Variation in the RANKL/RANK/OPG Signaling Pathway Is Associated With Bone Turnover and Bone Mineral Density in Men

The aim of this study was to determine if single‐nucleotide polymorphisms (SNPs) in RANKL, RANK, and OPG influence bone turnover and bone mineral density (BMD) in men. Pairwise tag SNPs (r2 ≥ 0.8) were selected for RANKL, RANK, and OPG and their 10‐kb flanking regions. Selected tag SNPs plus five SNPs near RANKL and OPG, associated with BMD in published genome‐wide association studies (GWAS), were genotyped in 2653 men aged 40 to 79 years of age recruited for participation in a population‐based study of male aging, the European Male Ageing Study (EMAS). N‐terminal propeptide of type I procollagen (PINP) and C‐terminal cross‐linked telopeptide of type I collagen (CTX‐I) serum levels were measured in all men. BMD at the calcaneus was estimated by quantitative ultrasound (QUS) in all men. Lumbar spine and total‐hip areal BMD (BMDa) was measured by dual‐energy X‐ray absorptiometry (DXA) in a subsample of 620 men. Multiple OPG, RANK, and RANKL SNPs were associated with bone turnover markers. We also identified a number of SNPs associated with BMD, including rs2073618 in OPG and rs9594759 near RANKL. The minor allele of rs2073618 (C) was associated with higher levels of both PINP (β = 1.83, p = .004) and CTX‐I (β = 17.59, p = 4.74 × 10−4), and lower lumbar spine BMDa (β = −0.02, p = .026). The minor allele of rs9594759 (C) was associated with lower PINP (β = −1.84, p = .003) and CTX‐I (β = −27.02, p = 6.06 × 10−8) and higher ultrasound BMD at the calcaneus (β = 0.01, p = .037). Our findings suggest that genetic variation in the RANKL/RANK/OPG signaling pathway influences bone turnover and BMD in European men.

Association of HTR2A polymorphisms with chronic widespread pain and the extent of musculoskeletal pain: Results from two population-based cohorts

OBJECTIVE The aim of the current study was to investigate whether genetic variation in genes across the serotoninergic system is associated with chronic widespread pain (CWP) and the number of pain sites reported. METHODS A discovery cohort, with pain data at 3 time points, was used to investigate genetic associations with 2 phenotypes: 1) CWP (at ≥ 2 time points; n = 164) compared with pain-free controls (at 3 time points; n = 172), and 2) the maximum number of pain sites reported at any 1 of the 3 time points (range of sites 0-29; n = 989). A cohort of 2,285 men for whom a DNA sample and pain data were available (including 203 CWP cases and 929 controls) was used for validation. Pairwise tagging (r(2) > 0.8) single-nucleotide polymorphisms (SNPs) were genotyped. Logistic and zero-inflated negative binomial regression analyses were used to test for SNP associations with CWP and the number of pain sites, respectively. RESULTS SNPs in HTR2A were associated with both pain phenotypes in the discovery cohort, and a number of these SNP associations were replicated in the validation cohort, some of which were attenuated after adjustment for depression. There was an increased likelihood of having CWP in subjects with 1 or 2 copies of the T allele of rs12584920 (odds ratio [OR] 1.64, 95% confidence interval [95% CI] 1.01-2.60 [P = 0.03] in the discovery cohort, and OR 1.46, 95% CI 1.07-2.00 [P = 0.018] in the validation cohort). A similar association was observed between rs17289394 and the maximum number of pain sites reported in both cohorts. Results from a meta-analysis of the data from the 2 cohorts further strengthened these findings. CONCLUSION The findings of this study support the role of HTR2A in the genetic predisposition to musculoskeletal pain.

Recent Advances in the Understanding of Genetic Susceptibility to Chronic Pain and Somatic Symptoms

Regional (e.g., low back) and widespread chronic pain disorders are common in the general population and are known to be heritable. Recent research suggests that genetic factors increase the risk of developing chronic pain independent of the site of pain. Candidate gene studies have been conducted on key pathways to elucidate susceptibility genes that are likely to be involved in both the sensory and affective components of pain. Findings have been largely equivocal, predominantly due to small sample size, but larger studies of pain in general population samples are being conducted. Interesting candidate genes from animal models and monogenic pain disorders are beginning to emerge. Recent advances in genetics research have yet to make an impact in the pain field but provide considerable scope for future research efforts.

No evidence for a role of the catechol-O-methyltransferase pain sensitivity haplotypes in chronic widespread pain

Objectives The aim of this study was to investigate the association between the catechol-O-methyltransferase (COMT) ‘pain sensitivity’ haplotypes and chronic widespread pain (CWP) in two distinct cohorts. Methods Cases of CWP and controls free of pain were selected from two population-based studies: the Epidemiology of Functional Disorders study (EPIFUND) (UK) and the European Male Ageing Study (European). The number of cases and controls were 164 and 172, and 204 and 935, respectively. Identical American College of Rheumatology criteria were used in both studies to ascertain CWP status. The EPIFUND study had three time points and cases were classified as subjects with CWP at two or three time points and controls as those free of pain at all three time points. Four single nucleotide polymorphisms (SNP): rs6269, rs4633, rs4818 and rs4680 (V158M) were genotyped using Sequenom technology. Allele and genotype frequencies were compared and haplotype analysis was conducted using PLINK software. Results No differences in allele or genotype frequencies for any of the four SNP were observed between cases and controls for either cohort. Haplotype analysis also showed no difference in the frequency of haplotypes between cases and controls. Conclusions There was no evidence of association between the COMT ‘pain sensitivity’ haplotypes and CWP in two population-based cohorts.

HTR2A polymorphisms are associated with chronic widespread pain and the extent of musculoskeletal pain: Results from two population based cohorts.

OBJECTIVE The aim of the current study was to investigate whether genetic variation in genes across the serotoninergic system is associated with chronic widespread pain (CWP) and the number of pain sites reported. METHODS A discovery cohort, with pain data at 3 time points, was used to investigate genetic associations with 2 phenotypes: 1) CWP (at ≥ 2 time points; n = 164) compared with pain-free controls (at 3 time points; n = 172), and 2) the maximum number of pain sites reported at any 1 of the 3 time points (range of sites 0-29; n = 989). A cohort of 2,285 men for whom a DNA sample and pain data were available (including 203 CWP cases and 929 controls) was used for validation. Pairwise tagging (r(2) > 0.8) single-nucleotide polymorphisms (SNPs) were genotyped. Logistic and zero-inflated negative binomial regression analyses were used to test for SNP associations with CWP and the number of pain sites, respectively. RESULTS SNPs in HTR2A were associated with both pain phenotypes in the discovery cohort, and a number of these SNP associations were replicated in the validation cohort, some of which were attenuated after adjustment for depression. There was an increased likelihood of having CWP in subjects with 1 or 2 copies of the T allele of rs12584920 (odds ratio [OR] 1.64, 95% confidence interval [95% CI] 1.01-2.60 [P = 0.03] in the discovery cohort, and OR 1.46, 95% CI 1.07-2.00 [P = 0.018] in the validation cohort). A similar association was observed between rs17289394 and the maximum number of pain sites reported in both cohorts. Results from a meta-analysis of the data from the 2 cohorts further strengthened these findings. CONCLUSION The findings of this study support the role of HTR2A in the genetic predisposition to musculoskeletal pain.