Carolina Medina-Gomez

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, Pu200a=u200a6.2×10−9). 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, Pu200a=u200a2.3×10−12, and −0.16 SD per G allele, Pu200a=u200a1.2×10−15, 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 (ORu200a=u200a1.33, Pu200a=u200a7.3×10−9), with genome-wide suggestive signals from the two missense variants in WNT16 (rs2908004: ORu200a=u200a1.22, Pu200a=u200a4.9×10−6 and rs2707466: ORu200a=u200a1.22, Pu200a=u200a7.2×10−6). 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.

Methylation of bone SOST, its mRNA, and serum sclerostin levels correlate strongly with fracture risk in postmenopausal women

Inhibition of sclerostin, a glycoprotein secreted by osteocytes, offers a new therapeutic paradigm for treatment of osteoporosis (OP) through its critical role as Wnt/catenin signaling regulator. This study describes the epigenetic regulation of SOST expression in bone biopsies of postmenopausal women. We correlated serum sclerostin to bone mineral density (BMD), fractures, and bone remodeling parameters, and related these findings to epigenetic and genetic disease mechanisms. Serum sclerostin and bone remodeling biomarkers were measured in two postmenopausal groups: healthy (BMD T‐scoreu2009>u2009–1) and established OP (BMD T‐scoreu2009<u2009–2.5, with at least one low‐energy fracture). Bone specimens were used to analyze SOST mRNAs, single nucleotide polymorphisms (SNPs), and DNA methylation changes. The SOST gene promoter region showed increased CpG methylation in OP patients (nu2009=u20094) compared to age and body mass index (BMI) balanced controls (nu2009=u20094) (80.5% versus 63.2%, pu2009=u20090.0001) with replication in independent cohorts (nu2009=u200927 and nu2009=u200936, respectively). Serum sclerostin and bone SOST mRNA expression correlated positively with age‐adjusted and BMI‐adjusted total hip BMD (ru2009=u20090.47 and ru2009=u20090.43, respectively; both pu2009<u20090.0005), and inversely to serum bone turnover markers. Five SNPs, one of which replicates in an independent population‐based genomewide association study (GWAS), showed association with serum sclerostin or SOST mRNA levels under an additive model (pu2009=u20090.0016 to 0.0079). Genetic and epigenetic changes in SOST influence its bone mRNA expression and serum sclerostin levels in postmenopausal women. The observations suggest that increased SOST promoter methylation seen in OP is a compensatory counteracting mechanism, which lowers serum sclerostin concentrations and reduces inhibition of Wnt signaling in an attempt to promote bone formation.

Challenges in conducting genome-wide association studies in highly admixed multi-ethnic populations: the Generation R Study

Genome-wide association studies (GWAS) have been successful in identifying loci associated with a wide range of complex human traits and diseases. Up to now, the majority of GWAS have focused on European populations. However, the inclusion of other ethnic groups as well as admixed populations in GWAS studies is rapidly rising following the pressing need to extrapolate findings to non-European populations and to increase statistical power. In this paper, we describe the methodological steps surrounding genetic data generation, quality control, study design and analytical procedures needed to run GWAS in the multiethnic and highly admixed Generation R Study, a large prospective birth cohort in Rotterdam, the Netherlands. Furthermore, we highlight a number of practical considerations and alternatives pertinent to the quality control and analysis of admixed GWAS data.

Assessment of Gene-by-Sex Interaction Effect on Bone Mineral Density

Sexual dimorphism in various bone phenotypes, including bone mineral density (BMD), is widely observed; however, the extent to which genes explain these sex differences is unclear. To identify variants with different effects by sex, we examined gene‐by‐sex autosomal interactions genome‐wide, and performed expression quantitative trait loci (eQTL) analysis and bioinformatics network analysis. We conducted an autosomal genome‐wide meta‐analysis of gene‐by‐sex interaction on lumbar spine (LS) and femoral neck (FN) BMD in 25,353 individuals from 8 cohorts. In a second stage, we followed up the 12 top single‐nucleotide polymorphisms (SNPs; pu2009<u20091u2009×u200910−5) in an additional set of 24,763 individuals. Gene‐by‐sex interaction and sex‐specific effects were examined in these 12 SNPs. We detected one novel genome‐wide significant interaction associated with LS‐BMD at the Chr3p26.1‐p25.1 locus, near the GRM7 gene (male effectu2009=u20090.02 and pu2009=u20093.0u2009×u200910−5; female effectu2009=u2009−0.007 and pu2009=u20093.3u2009×u200910−2), and 11 suggestive loci associated with either FN‐ or LS‐BMD in discovery cohorts. However, there was no evidence for genome‐wide significant (pu2009<u20095u2009×u200910−8) gene‐by‐sex interaction in the joint analysis of discovery and replication cohorts. Despite the large collaborative effort, no genome‐wide significant evidence for gene‐by‐sex interaction was found to influence BMD variation in this screen of autosomal markers. If they exist, gene‐by‐sex interactions for BMD probably have weak effects, accounting for less than 0.08% of the variation in these traits per implicated SNP.

BMD Loci Contribute to Ethnic And Developmental Differences in Skeletal Fragility Across Populations: Assessment of Evolutionary Selection Pressures

Bone mineral density (BMD) is a highly heritable trait used both for the diagnosis of osteoporosis in adults and to assess bone health in children. Ethnic differences in BMD have been documented, with markedly higher levels in individuals of African descent, which partially explain disparity in osteoporosis risk across populations. To date, 63 independent genetic variants have been associated with BMD in adults of Northern-European ancestry. Here, we demonstrate that at least 61 of these variants are predictive of BMD early in life by studying their compound effect within two multiethnic pediatric cohorts. Furthermore, we show that within these cohorts and across populations worldwide the frequency of those alleles associated with increased BMD is systematically elevated in individuals of Sub-Saharan African ancestry. The amount of differentiation in the BMD genetic scores among Sub-Saharan and non-Sub-Saharan populations together with neutrality tests, suggest that these allelic differences are compatible with the hypothesis of selective pressures acting on the genetic determinants of BMD. These findings constitute an explorative contribution to the role of selection on ethnic BMD differences and likely a new example of polygenic adaptation acting on a human trait.

Bone Mass and Strength in School Age Children Exhibit Sexual Dimorphism Related to Differences in Lean Mass: The Generation R Study

Bone strength, a key determinant of fracture risk, has been shown to display clear sexual dimorphism after puberty. We sought to determine whether sex differences in bone mass and hip bone geometry as an index of strength exist in school‐age prepubertal children and the degree to which the differences are independent of body size and lean mass. We studied 3514 children whose whole‐body and hip scans were measured using the same densitometer (GE‐Lunar iDXA) at a mean age of 6.2 years. Hip dual‐energy X‐ray absorptiometry (DXA) scans underwent hip structural analyses (HSA) with derivation of bone strength indices. Sex differences in these parameters were assessed by regression models adjusted for age, height, ethnicity, weight, and lean mass fraction (LMF). Whole‐body bone mineral density (BMD) and bone mineral content (BMC) levels were 1.3% and 4.3% higher in girls after adjustment by LMF. Independent of LMF, boys had 1.5% shorter femurs, 1.9% and 2.2% narrower shaft and femoral neck with 1.6% to 3.4% thicker cortices than girls. Consequent with this geometry configuration, girls observed 6.6% higher stresses in the medial femoral neck than boys. When considering LMF, the sexual differences on the derived bone strength indices were attenuated, suggesting that differences in muscle loads may reflect an innate disadvantage in bone strength in girls, as consequence of their lower muscular acquisition. In summary, we show that bone sexual dimorphism is already present at 6 years of age, with boys having stronger bones than girls, the relation of which is influenced by body composition and likely attributable to differential adaptation to mechanical loading. Our results support the view that early life interventions (ie, increased physical activity) targeted during the pre‐ and peripubertal stages may be of high importance, particularly in girls, because before puberty onset, muscle mass is strongly associated with bone density and geometry in children.

Genome-wide association study identifies nine novel loci for 2D:4D finger ratio, a putative retrospective biomarker of testosterone exposure in utero

Abstract The ratio of the length of the index finger to that of the ring finger (2D:4D) is sexually dimorphic and is commonly used as a non-invasive biomarker of prenatal androgen exposure. Most association studies of 2D:4D ratio with a diverse range of sex-specific traits have typically involved small sample sizes and have been difficult to replicate, raising questions around the utility and precise meaning of the measure. In the largest genome-wide association meta-analysis of 2D:4D ratio to date (Nu2009=u200915 661, with replication Nu2009=u200975 821), we identified 11 loci (9 novel) explaining 3.8% of the variance in mean 2D:4D ratio. We also found weak evidence for association (βu2009=u20090.06; Pu2009=u20090.02) between 2D:4D ratio and sensitivity to testosterone [length of the CAG microsatellite repeat in the androgen receptor (AR) gene] in females only. Furthermore, genetic variants associated with (adult) testosterone levels and/or sex hormone-binding globulin were not associated with 2D:4D ratio in our sample. Although we were unable to find strong evidence from our genetic study to support the hypothesis that 2D:4D ratio is a direct biomarker of prenatal exposure to androgens in healthy individuals, our findings do not explicitly exclude this possibility, and pathways involving testosterone may become apparent as the size of the discovery sample increases further. Our findings provide new insight into the underlying biology shaping 2D:4D variation in the general population.

Sarcopenia and Its Clinical Correlates in the General Population: The Rotterdam Study: PREVALANCE OF SARCOPENIA IN THE GENERAL POPULATION

Sarcopenia, a complex multifactorial condition, is characterized by loss of muscle mass and function, which increases progressively with age. The existence of different definitions has contributed to the large variation in the prevalence estimates of sarcopenia. We aimed to estimate the prevalence of sarcopenia in the general population using the European Working Group on Sarcopenia in Older People (EWGSOP) proposed definition and compared baseline demographic and clinical characteristics between the nonsarcopenia, presarcopenia, and sarcopenia individuals, with particular emphasis on the overlap with osteoporosis and fracture risk. We studied 5911 subjects at a mean age of 69.2 years (55.8% female) with data on sarcopenia participating in the Rotterdam Study, a prospective population‐based cohort study in Rotterdam, the Netherlands. Presarcopenia was defined as having only low muscle mass, whereas sarcopenia was defined based on the presence of low muscle mass, plus either low muscle strength or low physical performance. The prevalence of presarcopenia and sarcopenia was 5.9% and 4.4%, respectively. Individuals with sarcopenia were older, more often males, smokers, with less optimal dietary intake, and more often disabled with lower physical activity. Although the prevalence of fractures was higher in individuals with low lean mass (presarcopenic [16.6%] and sarcopenic [23.5%]) compared with the no sarcopenic group (15.5%), the differences were not present after correcting for age and sex. There were no statistical differences in the prevalence of chronic diseases, with the exception of a higher prevalence of COPD in presarcopenic (29.1%) and sarcopenic (26.9%) individuals compared with nonsarcopenic (13.4%) individuals. Osteoporotic individuals with (odds ratio [OR]u2009=u20092.59, 95% confidence interval [CI] 1.41–4.45) and without sarcopenia (ORu2009=u20092.75, 95% CI 2.01–3.75) had similar elevated risk of nonvertebral fractures. The presence of sarcopenia appears to be independent of chronic diseases with the exception of COPD and more related to lifestyle factors and disabilities. Sarcopenic individuals in the general population are at no greater risk of fracture than what is determined by their low bone mineral density.

Genome-wide mapping identifies beta-1,4-N-acetyl-galactosaminyl-transferase as a novel determinant of sclerostin levels and bone mineral density

ABSTRACT In bone, sclerostin is mainly osteocyte-derived and plays an important local role in adaptive responses to mechanical loading. Sclerostin is also present at detectable concentrations within the circulation. Our genome wide association study (GWAS) meta-analysis of 10,584 European-descent individuals identified two novel serum sclerostin loci, B4GALNT3 (standard deviation (SD) change in sclerostin per A allele β=0.20, P=4.6x10-49), and GALNT1 (β=0.11 per G allele, P=4.4x10-11), of which the former is a known locus for BMD estimated by heel ultrasound (eBMD). Common variants across the genome explained 16% of the phenotypic variation of serum sclerostin. Mendelian randomization revealed an inverse causal relationship between serum sclerostin and femoral neck BMD and eBMD, and a positive relationship with fracture risk. Colocalization analysis demonstrated common genetic signals within the B4GALNT3 locus for higher sclerostin, lower BMD, and greater B4GALNT3 expression in arterial tissue (Probability>99%). Renal and cortical bone tissue, and osteoblast cultures, were found to express high levels of B4GALNT3, an N-acetylgalactosaminyltransferase which adds a terminal LacdiNAc disaccharide to target glycocoproteins. Together, these findings raise the possibility that sclerostin is a substrate for B4GALNT3, such that its modification leads to higher levels, possibly through greater stability. GALNT1, an enzyme causing mucin-type O-linked glycosylation, may act in a similar capacity. We conclude that genetic variation in glycosylation enzymes represents a novel determinant of BMD and fracture risk, acting via alterations in levels of circulating sclerostin.