Adrian E Sayers

WNT16 influences bone mineral density, cortical bone thickness, bone strength, and osteoporotic fracture risk.

We aimed to identify genetic variants associated with cortical bone thickness (CBT) and bone mineral density (BMD) by performing two separate genome-wide association study (GWAS) meta-analyses for CBT in 3 cohorts comprising 5,878 European subjects and for BMD in 5 cohorts comprising 5,672 individuals. We then assessed selected single-nucleotide polymorphisms (SNPs) for osteoporotic fracture in 2,023 cases and 3,740 controls. Association with CBT and forearm BMD was tested for ∼2.5 million SNPs in each cohort separately, and results were meta-analyzed using fixed effect meta-analysis. We identified a missense SNP (Thr>Ile; rs2707466) located in the WNT16 gene (7q31), associated with CBT (effect size of −0.11 standard deviations [SD] per C allele, P = 6.2×10−9). This SNP, as well as another nonsynonymous SNP rs2908004 (Gly>Arg), also had genome-wide significant association with forearm BMD (−0.14 SD per C allele, P = 2.3×10−12, and −0.16 SD per G allele, P = 1.2×10−15, respectively). Four genome-wide significant SNPs arising from BMD meta-analysis were tested for association with forearm fracture. SNP rs7776725 in FAM3C, a gene adjacent to WNT16, was associated with a genome-wide significant increased risk of forearm fracture (OR = 1.33, P = 7.3×10−9), with genome-wide suggestive signals from the two missense variants in WNT16 (rs2908004: OR = 1.22, P = 4.9×10−6 and rs2707466: OR = 1.22, P = 7.2×10−6). We next generated a homozygous mouse with targeted disruption of Wnt16. Female Wnt16−/− mice had 27% (P<0.001) thinner cortical bones at the femur midshaft, and bone strength measures were reduced between 43%–61% (6.5×10−13<P<5.9×10−4) at both femur and tibia, compared with their wild-type littermates. Natural variation in humans and targeted disruption in mice demonstrate that WNT16 is an important determinant of CBT, BMD, bone strength, and risk of fracture.

DNA Methylation Patterns in Cord Blood DNA and Body Size in Childhood

Background Epigenetic markings acquired in early life may have phenotypic consequences later in development through their role in transcriptional regulation with relevance to the developmental origins of diseases including obesity. The goal of this study was to investigate whether DNA methylation levels at birth are associated with body size later in childhood. Principal Findings A study design involving two birth cohorts was used to conduct transcription profiling followed by DNA methylation analysis in peripheral blood. Gene expression analysis was undertaken in 24 individuals whose biological samples and clinical data were collected at a mean ± standard deviation (SD) age of 12.35 (0.95) years, the upper and lower tertiles of body mass index (BMI) were compared with a mean (SD) BMI difference of 9.86 (2.37) kg/m2. This generated a panel of differentially expressed genes for DNA methylation analysis which was then undertaken in cord blood DNA in 178 individuals with body composition data prospectively collected at a mean (SD) age of 9.83 (0.23) years. Twenty-nine differentially expressed genes (>1.2-fold and p<10−4) were analysed to determine DNA methylation levels at 1–3 sites per gene. Five genes were unmethylated and DNA methylation in the remaining 24 genes was analysed using linear regression with bootstrapping. Methylation in 9 of the 24 (37.5%) genes studied was associated with at least one index of body composition (BMI, fat mass, lean mass, height) at age 9 years, although only one of these associations remained after correction for multiple testing (ALPL with height, p Corrected = 0.017). Conclusions DNA methylation patterns in cord blood show some association with altered gene expression, body size and composition in childhood. The observed relationship is correlative and despite suggestion of a mechanistic epigenetic link between in utero life and later phenotype, further investigation is required to establish causality.

Association of maternal vitamin D status during pregnancy with bone-mineral content in offspring: a prospective cohort study

Summary Background Maternal vitamin D status in pregnancy is a suggested determinant of bone-mineral content (BMC) in offspring, but has been assessed in small studies. We investigated this association in a large prospective study. Methods Eligible participants were mother-and-singleton-offspring pairs who had participated in the Avon Longitudinal Study of Parents and Children, and in which the mother had recorded measurements of 25(OH)D concentration in pregnancy and the offspring had undergone dual-energy x-ray absorptiometry at age 9–10 years. 25(OH)D concentrations in pregnancy were assessed per 10·0 nmol/L and classified as sufficient (more than 50·00 nmol/L), insufficient (49·99–27·50 nmol/L), or deficient (lower than 27·50 nmol/L). Associations between maternal serum 25(OH)D concentrations and offspring total body less head (TBLH) and spinal BMC were assessed by trimester. Results 3960 mother-and-offspring pairs, mainly of white European origin, were assessed (TBLH BMC n=3960, spinal BMC n=3196). Mean offspring age was 9·9 years. 2644 (67%) mothers had sufficient, 1096 (28%) insufficient, and 220 (6%) deficient 25(OH)D concentrations in pregnancy, but TBLH and spinal BMC did not differ between offspring of mothers in the lower two groups versus sufficient 25(OH)D concentration. No associations with offspring BMC were found for any trimester, including the third trimester, which is thought to be most relevant (TBLH BMC confounder-adjusted mean difference −0·03 g per 10·0 nmol/L, 95% CI −1·71 to 1·65; spinal BMC 0·04 g per 10·0 nmol/L, 95% CI −0·12 to 0·21). Conclusions We found no relevant association between maternal vitamin D status in pregnancy and offspring BMC in late childhood. Funding UK Medical Research Council, Wellcome Trust, and University of Bristol.

Falling threshold for treatment of borderline elevated thyrotropin levels-balancing benefits and risks: evidence from a large community-based study.

IMPORTANCE Rates of thyroid hormone prescribing in the United States and the United Kingdom have increased substantially. If some of the increase is due to lowering the thyrotropin threshold for treatment, this may result in less benefit and greater harm. OBJECTIVE To define trends in thyrotropin levels at the initiation of levothyroxine sodium therapy and the risk of developing a suppressed thyrotropin level following treatment. DESIGN, SETTING, PARTICIPANTS, AND EXPOSURE: Retrospective cohort study using data from the United Kingdom Clinical Practice Research Datalink. Among 52,298 individuals who received a prescription for levothyroxine between January 1, 2001, and October 30, 2009, we extracted data about the thyrotropin level before levothyroxine therapy initiation, clinical symptoms, and thyrotropin levels up to 5 years after levothyroxine was initiated. We excluded persons who had a history of hyperthyroidism, pituitary disease, or thyroid surgery; those who were taking thyroid-altering medication or if the levothyroxine prescription was related to pregnancy; and those who did not have a thyrotropin level measured within 3 months before the initiation of levothyroxine. MAIN OUTCOMES AND MEASURES The median thyrotropin level at the time of the index levothyroxine prescription, the odds of initiation of levothyroxine therapy at thyrotropin levels of 10.0 mIU/L or less, and the age-stratified odds of developing a low or suppressed thyrotropin level after levothyroxine therapy. RESULTS Between 2001 and 2009, the median thyrotropin level at the initiation of levothyroxine therapy fell from 8.7 to 7.9 mIU/L. The odds ratio for prescribing levothyroxine at thyrotropin levels of 10.0 mIU/L or less in 2009 compared with 2001 (adjusted for changes in population demographics) was 1.30 (95% CI, 1.19-1.42; P < .001). Older individuals and individuals with cardiac risk factors had higher odds of initiation of levothyroxine therapy with a thyrotropin level 10.0 mIU/L or less. At 5 years after levothyroxine initiation, 5.8% of individuals had a thyrotropin level of <0.1 mIU/L. Individuals with depression or tiredness at baseline had increased odds of developing a suppressed thyrotropin level, whereas individuals with cardiac risk factors (eg, atrial fibrillation, diabetes mellitus, hypertension, and raised lipid levels) did not. CONCLUSIONS AND RELEVANCE We observed a trend toward levothyroxine treatment of more marginal degrees of hypothyroidism and a substantial risk of developing a suppressed thyrotropin level following therapy. Large-scale prospective studies are required to assess the risk-benefit ratio of current practice.

Estimated maternal ultraviolet B exposure levels in pregnancy influence skeletal development of the child

CONTEXT Relationships between vitamin D exposure of the mother in pregnancy and skeletal development of the child are poorly understood. OBJECTIVES Our objectives were to establish whether background UVB levels in the third trimester of pregnancy are related to bone mineral content (BMC) of the child, and to examine whether these relationships are explained by effects on height, fat, or lean mass. DESIGN This was a prospective cohort study. SETTING The Avon Longitudinal Study of Parents and Children, a population-based birth cohort, was studied. PARTICIPANTS A total of 6995 boys and girls with a mean age of 9.9 yr was studied. OUTCOME MEASURES Prespecified analyses of relationships between background UVB levels in the third trimester of pregnancy, and total body less head BMC, bone area (BA), bone mineral density, and area-adjusted BMC as measured by dual-energy x-ray absorptiometry scans at 9.9 yr were performed. RESULTS Maternal UVB exposure was positively related to BMC (g) [9.6 (5.3, 13.8)], BA (cm(2)) [8.1 (4.3, 11.9)], and bone mineral density (g/cm(-2)) [0.003 (0.001, 0.004)], but not area-adjusted BMC (g) [0.69 (-0.22, 1.56)], suggesting an effect on bone size. Both height-dependent (cm) [0.18 (0.03, 0.32)] and height-independent (cm(2)) [4.1, (2.0, 6.2)] effects contributed to this association between UVB and BA. Although maternal UVB exposure was also related to lean mass (g) [163 (89, 237)], a positive association between UVB and BA persisted after adjusting for both height and lean mass [2.8 (1.0, 4.6)]. CONCLUSIONS Maternal UVB exposure is related to bone size at age 9.9 yr independently of height and lean mass, suggesting that vitamin D status in pregnancy exerts direct effects on periosteal bone formation in subsequent childhood.

How does body fat influence bone mass in childhood? A Mendelian randomization approach.

Fat mass may be a causal determinant of bone mass, but the evidence is conflicting, possibly reflecting the influence of confounding factors. The recent identification of common genetic variants related to obesity in children provides an opportunity to implement a Mendelian randomization study of obesity and bone outcomes, which is less subject to confounding and several biases than conventional approaches. Genotyping was retrieved for variants of two loci reliably associated with adiposity (the fat mass and obesity‐related gene FTO and that upstream of the MC4R locus) within 7470 children from the Avon Longitudinal Study of Parents and Children (ALSPAC) who had undergone total body DXA scans at a mean of 9.9 yr. Relationships between both fat mass/genotypes and bone measures were assessed in efforts to determine evidence of causality between adiposity and bone mass. In conventional tests of association, both with and without height adjustment, total fat mass was strongly related to total body, spinal, and upper and lower limb BMC (ratio of geometric means [RGM]: 1.118 [95% CI: 1.112, 1.123], 1.110 [95% CI: 1.102, 1.119], 1.101 [95% CI: 1.093, 1.108], 1.146 [95% CI: 1.143, 1.155]; p < 10−10 [adjusted for sex, height, and sitting height]). Equivalent or larger effects were obtained from instrumental variable (IV) regression including the same covariates (1.139 [95% CI: 1.064, 1.220], 1.090 [95% CI: 1.010, 1.177], 1.142 [95% CI: 1.049, 1.243], 1.176 [95% CI: 1.099, 1.257]; p = 0.0002, 0.03, 0.002, and 2.3−6 respectively). Similar results were obtained after adjusting for puberty, when truncal fat mass was used in place of total fat, and when bone area was used instead of bone mass. In analyses where total body BMC adjusted for bone area (BA) was the outcome (reflecting volumetric BMD), linear regression with fat mass showed evidence for association (1.004 [95% CI: 1.002, 1.007], p = 0.0001). IV regression also showed a positive effect (1.031 [95% CI: 1.000, 1.062], p = 0.05). When MC4R and FTO markers were used as instruments for fat mass, similar associations with BMC were seen to those with fat mass as measured by DXA. This suggests that fat mass is on the causal pathway for bone mass in children. In addition, both directly assessed and IV‐assessed relationships between fat mass and volumetric density showed evidence for positive effects, supporting a hypothesis that fat effects on bone mass are not entirely accounted for by association with overall bone size.

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.

Fat mass exerts a greater effect on cortical bone mass in girls than boys.

CONTEXT It is unclear whether fat mass (FM) and lean mass (LM) differ in the way they influence cortical bone development in boys and girls. OBJECTIVE The aim of the study was to investigate the contributions of total body FM and LM to parameters related to cortical bone mass and geometry. DESIGN/SETTING We conducted a longitudinal birth cohort study, the Avon Longitudinal Study of Parents and Children. PARTICIPANTS A total of 4005 boys and girls (mean age, 15.5 yr) participated in the study. OUTCOME MEASURES We measured cortical bone mass, cortical bone mineral content (BMC(C)), cortical bone mineral density, periosteal circumference (PC), and endosteal circumference by tibial peripheral quantitative computed tomography. RESULTS LM had a similar positive association with BMC(C) in boys and girls [regression coefficients with 95% confidence interval (CI); P for gender interactions: boys/girls, 0.952 (0.908, 0.997); P = 0.85]. However, the mechanisms by which LM influenced bone mass differed according to gender because LM was positively associated with PC more strongly in girls [boys, 0.579 (0.522, 0.635); girls, 0.799 (0.722, 0.875); P < 0.0001], but was only associated with cortical bone mineral density in boys [boys, 0.443 (0.382, 0.505); girls, 0.014 (-0.070, 0.097); P < 0.0001]. There was a stronger positive association between FM and BMC(C) in girls [boys, 0.227 (0.185, 0.269); girls, 0.355 (0.319, 0.392); P < 0.0001]. This reflected both a greater positive association of FM with PC in girls [boys, 0.213 (0.174, 0.253); girls, 0.312 (0.278, 0.347); P = 0.0002], and a stronger negative association with endosteal circumference(PC) [boys, -0.059 (-0.096, 0.021); girls, -0.181 (-0.215, -0.146); P < 0.0001]. CONCLUSIONS Whereas LM stimulates the accrual of cortical bone mass to a similar extent in boys and girls, FM is a stronger stimulus for accrual of cortical bone mass in girls, reflecting a greater tendency in females for FM to stimulate periosteal growth and suppress endosteal expansion.

Genetic determinants of trabecular and cortical volumetric bone mineral densities and bone microstructure

Most previous genetic epidemiology studies within the field of osteoporosis have focused on the genetics of the complex trait areal bone mineral density (aBMD), not being able to differentiate genetic determinants of cortical volumetric BMD (vBMD), trabecular vBMD, and bone microstructural traits. The objective of this study was to separately identify genetic determinants of these bone traits as analysed by peripheral quantitative computed tomography (pQCT). Separate GWA meta-analyses for cortical and trabecular vBMDs were performed. The cortical vBMD GWA meta-analysis (n = 5,878) followed by replication (n = 1,052) identified genetic variants in four separate loci reaching genome-wide significance (RANKL, rs1021188, p = 3.6×10−14; LOC285735, rs271170, p = 2.7×10−12; OPG, rs7839059, p = 1.2×10−10; and ESR1/C6orf97, rs6909279, p = 1.1×10−9). The trabecular vBMD GWA meta-analysis (n = 2,500) followed by replication (n = 1,022) identified one locus reaching genome-wide significance (FMN2/GREM2, rs9287237, p = 1.9×10−9). High-resolution pQCT analyses, giving information about bone microstructure, were available in a subset of the GOOD cohort (n = 729). rs1021188 was significantly associated with cortical porosity while rs9287237 was significantly associated with trabecular bone fraction. The genetic variant in the FMN2/GREM2 locus was associated with fracture risk in the MrOS Sweden cohort (HR per extra T allele 0.75, 95% confidence interval 0.60–0.93) and GREM2 expression in human osteoblasts. In conclusion, five genetic loci associated with trabecular or cortical vBMD were identified. Two of these (FMN2/GREM2 and LOC285735) are novel bone-related loci, while the other three have previously been reported to be associated with aBMD. The genetic variants associated with cortical and trabecular bone parameters differed, underscoring the complexity of the genetics of bone parameters. We propose that a genetic variant in the RANKL locus influences cortical vBMD, at least partly, via effects on cortical porosity, and that a genetic variant in the FMN2/GREM2 locus influences GREM2 expression in osteoblasts and thereby trabecular number and thickness as well as fracture risk.

Habitual Levels of Vigorous, But Not Moderate or Light, Physical Activity Is Positively Related to Cortical Bone Mass in Adolescents

Vigorous physical activity is positively related to cortical bone mass (BMCC) independent of associations with fat and lean mass.