Biological constraints on GWAS SNPs at suggestive significance thresholds reveal additional BMI loci.

To uncover novel significant association signals (p<5×10-8), genome-wide association studies (GWAS) requires increasingly larger sample sizes to overcome statistical correction for multiple testing. As an alternative, we aimed to identify associations among suggestive signals (5 × 10-8≤p<5×10-4) in increasingly powered GWAS efforts using chromatin accessibility and direct contact with gene promoters as biological constraints. We conducted retrospective analyses of three GIANT BMI GWAS efforts using ATAC-seq and promoter-focused Capture C data from human adipocytes and embryonic stem cell (ESC)-derived hypothalamic-like neurons. This approach, with its extremely low false-positive rate, identified 15 loci at p<5×10-5 in the 2010 GWAS, of which 13 achieved genome-wide significance by 2018, including at NAV1, MTIF3, and ADCY3. Eighty percent of constrained 2015 loci achieved genome-wide significance in 2018. We observed similar results in waist-to-hip ratio analyses. In conclusion, biological constraints on sub-significant GWAS signals can reveal potentially true-positive loci for further investigation in existing data sets without increasing sample size.

A Meta-Analysis of the Transferability of Bone Mineral Density Genetic Loci Associations From European to African Ancestry Populations.

Genetic studies of bone mineral density (BMD) largely have been conducted in European populations. We therefore conducted a meta-analysis of six independent African ancestry cohorts to determine whether previously reported BMD loci identified in European populations were transferable to African ancestry populations. We included nearly 5000 individuals with both genetic data and assessments of BMD. Genotype imputation was conducted using the 1000G reference panel. We assessed single-nucleotide polymorphism (SNP) associations with femoral neck and lumbar spine BMD in each cohort separately, then combined results in fixed effects (or random effects if study heterogeneity was high, I2 index >60) inverse variance weighted meta-analyses. In secondary analyses, we conducted locus-based analyses of rare variants using SKAT-O. Mean age ranged from 12 to 68 years. One cohort included only men and another cohort included only women; the proportion of women in the other four cohorts ranged from 52% to 63%. Of 56 BMD loci tested, one locus, 6q25 (C6orf97, p = 8.87 × 10-4 ), was associated with lumbar spine BMD and two loci, 7q21 (SLC25A13, p = 2.84 × 10-4 ) and 7q31 (WNT16, p = 2.96 × 10-5 ), were associated with femoral neck BMD. Effects were in the same direction as previously reported in European ancestry studies and met a Bonferroni-adjusted p value threshold, the criteria for transferability to African ancestry populations. We also found associations that met locus-specific Bonferroni-adjusted p value thresholds in 11q13 (LRP5, p < 2.23 × 10-4 ), 11q14 (DCDC5, p < 5.35 × 10-5 ), and 17p13 (SMG6, p < 6.78 × 10-5 ) that were not tagged by European ancestry index SNPs. Rare single-nucleotide variants in AKAP11 (p = 2.32 × 10-2 ), MBL2 (p = 4.09 × 10-2 ), MEPE (p = 3.15 × 10-2 ), SLC25A13 (p = 3.03 × 10-2 ), STARD3NL (p = 3.35 × 10-2 ), and TNFRSF11A (p = 3.18 × 10-3 ) were also associated with BMD. The majority of known BMD loci were not transferable. Larger genetic studies of BMD in African ancestry populations will be needed to overcome limitations in statistical power and to identify both other loci that are transferable across populations and novel population-specific variants. © 2020 American Society for Bone and Mineral Research (ASBMR).

Genetically Determined Later Puberty Impacts Lowered Bone Mineral Density in Childhood and Adulthood.

Later puberty associates with lower areal bone mineral density (aBMD), and both are risk factors for osteoporosis. However, the association between puberty timing-associated genetic variants and aBMD during development, and the causal relationship between puberty timing and aBMD, remain uncharacterized. We constructed sex-specific polygenic risk scores (GRS) consisting of 333 genetic variants associated with later puberty in European-descent children in the Bone Mineral Density in Childhood Study (BMDCS), consisting of a longitudinal cohort with up to seven assessments (n = 933) and a cross-sectional cohort (n = 486). These GRS were tested for associations with age- and sex-specific aBMD Z-scores at the lumbar spine (LS), femoral neck (FN), total hip, and distal radius, accounting for clinical covariates using sex-stratified linear mixed models. The causal relationship between puberty timing and aBMD was tested in the BMDCS and in publicly available adult data (GEFOS consortium) using two-sample Mendelian randomization (MR). The puberty-delaying GRS was associated with later puberty and lower LS-aBMD in the BMDCS in both sexes (combined beta ± SE = -0.078 ± 0.024; p = 0.0010). In the MR framework, the puberty-delaying genetic instrument also supported a causal association with lower LS-aBMD and FN-aBMD in adults of both sexes. Our results suggest that pubertal timing is causal for diminished aBMD in a skeletal site- and sex-specific manner that tracks throughout life, potentially impacting later risk for osteoporosis, which should be tested in future studies. © 2017 American Society for Bone and Mineral Research.

Multidimensional Bone Density Phenotyping Reveals New Insights Into Genetic Regulation of the Pediatric Skeleton.

Osteoporosis is a complex disease with developmental origins. It is therefore important to understand the genetic contribution to pediatric areal bone mineral density (aBMD). Individual skeletal site phenotyping has been primarily used to identify pediatric aBMD loci. However, this approach is limited because there is a degree of aBMD discordance across skeletal sites. We therefore applied a novel multidimensional phenotyping approach to further understand the genetic regulation of pediatric aBMD. Our sample comprised a prospective, longitudinal cohort of 1293 children of European ancestry (52% female; up to seven annual measurements). Principal components analysis was applied to dual-energy X-ray absorptiometry-derived aBMD Z-scores for total hip, femoral neck, spine, and distal radius to generate multidimensional aBMD phenotypes (ie, principal component scores). We tested the association between a genetic score (percentage of bone lowering alleles at 63 loci) and each principal component. We also performed a genomewide association study (GWAS) using the multiethnic baseline data (n = 1885) to identify novel loci associated with these principal components. The first component (PC1) reflected a concordant phenotypic model of the skeleton (eg, higher loading score = higher BMD across all sites). In contrast, PC2 was discordant for distal radius versus spine and hip aBMD, and PC3 was discordant for spine versus distal radius and hip aBMD. The genetic score was associated with PC1 (beta = -0.05, p = 3.9 × 10-10 ), but was not associated with discordant PC2 or PC3. Our GWAS discovered variation near CPED1 that associated with PC2 (rs67991850, p = 2.5 × 10-11 ) and near RAB11FIP5 (rs58649746, p = 4.8 × 10-9 ) that associated with PC3. In conclusion, an established bone fragility genetic summary score was associated with a concordant skeletal phenotype, but not discordant skeletal phenotypes. Novel associations were observed for the discordant multidimensional skeletal phenotypes that provide new biological insights into the developing skeleton. © 2017 American Society for Bone and Mineral Research.

Implicating candidate genes at GWAS signals by leveraging topologically associating domains.

Genome-wide association studies (GWAS) have contributed significantly to the understanding of complex disease genetics. However, GWAS only report association signals and do not necessarily identify culprit genes. As most signals occur in non-coding regions of the genome, it is often challenging to assign genomic variants to the underlying causal mechanism(s). Topologically associating domains (TADs) are primarily cell-type-independent genomic regions that define interactome boundaries and can aid in the designation of limits within which an association most likely impacts gene function. We describe and validate a computational method that uses the genic content of TADs to prioritize candidate genes. Our method, called 'TAD_Pathways', performs a Gene Ontology (GO) analysis over genes that reside within TAD boundaries corresponding to GWAS signals for a given trait or disease. Applying our pipeline to the bone mineral density (BMD) GWAS catalog, we identify 'Skeletal System Development' (Benjamini-Hochberg adjusted P=1.02x10-5) as the top-ranked pathway. In many cases, our method implicated a gene other than the nearest gene. Our molecular experiments describe a novel example: ACP2, implicated near the canonical 'ARHGAP1' locus. We found ACP2 to be an important regulator of osteoblast metabolism, whereas ARHGAP1 was not supported. Our results via BMD, for example, demonstrate how basic principles of three-dimensional genome organization can define biologically informed association windows.

A Genomewide Association Study Identifies Two Sex-Specific Loci, at SPTB and IZUMO3, Influencing Pediatric Bone Mineral Density at Multiple Skeletal Sites.

Failure to achieve optimal bone mineral accretion during childhood and adolescence results in subsequent suboptimal peak bone mass, contributing to osteoporosis risk later in life. To identify novel genetic factors that influence pediatric bone mass at discrete skeletal sites, we performed a sex-stratified genomewide association study of areal bone mineral density (BMD) measured by dual-energy X-ray absorptiometry at the 1/3 distal radius, spine, total hip, and femoral neck in a cohort of 933 healthy European American children. We took forward signals with p < 5 × 10-5 and minor allele frequency (MAF) >5% into an independent cohort of 486 European American children in search of replication. In doing so, we identified five loci that achieved genome wide significance in the combined cohorts (nearest genes: CPED1, IZUMO3, RBFOX1, SPBT, and TBPL2), of which the last four were novel and two were sex-specific (SPTB in females and IZUMO3 in males), with all of them yielding associations that were particularly strong at a specific skeletal site. Annotation of potential regulatory function, expression quantitative trait loci (eQTL) effects and pathway analyses identified several potential target genes at these associated loci. This study highlights the importance of sex-stratified analyses at discrete skeletal sites during the critical period of bone accrual, and identifies novel loci for further functional follow-up to pinpoint key genes and better understand the regulation of bone development in children. © 2017 American Society for Bone and Mineral Research.

Relative Skeletal Maturation and Population Ancestry in Nonobese Children and Adolescents.

More rapid skeletal maturation in African-American (AA) children is recognized and generally attributed to an increased prevalence of obesity. The objective of the present study was to evaluate the effects of population ancestry on relative skeletal maturation in healthy, non-obese children and adolescents, accounting for body composition and sexual maturation. To do this, we leveraged a multiethnic, mixed-longitudinal study with annual assessments for up to 7 years (The Bone Mineral Density in Childhood Study and its ancillary cohort) conducted at five US clinical centers. Participants included 1592 children, skeletally immature (45% females, 19% AA) who were aged 5 to 17 years at study entry. The primary outcome measure was relative skeletal maturation as assessed by hand-wrist radiograph. Additional covariates measured included anthropometrics, body composition by dual-energy X-ray absorptiometry (DXA), and Tanner stage of sexual maturation. Using mixed effects longitudinal models, without covariates, advancement in relative skeletal maturation was noted in self-reported AA girls (∼0.33 years, p < 0.001) and boys (∼0.43 years, p < 0.001). Boys and girls of all ancestry groups showed independent positive associations of height, lean mass, fat mass, and puberty with relative skeletal maturation. The effect of ancestry was attenuated but persistent after accounting for covariates: for girls, 0.19 years (ancestry by self-report, p = 0.02) or 0.29 years (ancestry by admixture, p = 0.004); and for boys, 0.20 years (ancestry by self-report, p = 0.004), or 0.29 years (ancestry by admixture, p = 0.004). In summary, we conclude that advancement in relative skeletal maturation was associated with AA ancestry in healthy, non-obese children, independent of growth, body composition, and puberty. Further research into the mechanisms underlying this observation may provide insights into the regulation of skeletal maturation. © 2016 American Society for Bone and Mineral Research.

Physical Activity Benefits the Skeleton of Children Genetically Predisposed to Lower Bone Density in Adulthood.

Both genetics and physical activity (PA) contribute to bone mineral density (BMD), but it is unknown if the benefits of physical activity on childhood bone accretion depend on genetic risk. We, therefore, aimed to determine if PA influenced the effect of bone fragility genetic variants on BMD in childhood. Our sample comprised US children of European ancestry enrolled in the Bone Mineral Density in Childhood Study (N = 918, aged 5 to 19 years, and 52.4% female). We used a questionnaire to estimate hours per day spent in total, high-, and low-impact PA. We calculated a BMD genetic score (% BMD lowering alleles) using adult genome-wide association study (GWAS)-implicated BMD variants. We used dual-energy X-ray absorptiometry to estimate femoral neck, total hip, and spine areal-BMD and total body less head (TBLH) bone mineral content (BMC) Z-scores. The BMD genetic score was negatively associated with each bone Z-score (eg, TBLH-BMC: estimate = -0.03, p = 1.3 × 10(-6) ). Total PA was positively associated with bone Z-scores; these associations were driven by time spent in high-impact PA (eg, TBLH-BMC: estimate = 0.05, p = 4.0 × 10(-10) ) and were observed even for children with lower than average bone Z-scores. We found no evidence of PA-adult genetic score interactions (p interaction > 0.05) at any skeletal site, and there was no evidence of PA-genetic score-Tanner stage interactions at any skeletal site (p interaction > 0.05). However, exploratory analyses at the individual variant level revealed that PA statistically interacted with rs2887571 (ERC1/WNT5B) to influence TBLH-BMC in males (p interaction = 7.1 × 10(-5) ), where PA was associated with higher TBLH-BMC Z-score among the BMD-lowering allele carriers (rs2887571 AA homozygotes: estimate = 0.08 [95% CI 0.06, 0.11], p = 2.7 × 10(-9) ). In conclusion, the beneficial effect of PA on bone, especially high-impact PA, applies to the average child and those genetically predisposed to lower adult BMD (based on GWAS-implicated BMD variants). Independent replication of our exploratory individual variant findings is warranted. © 2016 American Society for Bone and Mineral Research.

Rare EN1 Variants and Pediatric Bone Mass.

A recent whole-genome sequencing study in search of variation associated with adult areal bone mineral density (aBMD) identified rare variants near EN1, with markedly large effect sizes, and a common variant near SOX6. To understand the developmental effects of these loci, we sought to determine if they were associated with pediatric dual-energy X-ray absorptiometry-derived aBMD and bone mineral content (BMC) and if the associations were modified by sex. Our sample comprised 733 females and 685 males of European ancestry enrolled in the longitudinal Bone Mineral Density in Childhood Study (up to 7 annual study visits). Sex- and age-specific Z-scores, adjusted for height, were calculated for the total hip, femoral neck, spine, and distal radius. Total body less head (TBLH) BMC Z-scores were also calculated. The previously reported single nucleotide polymorphisms (SNPs) near EN1 and SOX6 were derived from our imputed data set. Linear mixed-effects models were used to test associations between each SNP and bone Z-scores, plus interactions with sex were explored. The rare T allele of lead EN1 SNP rs11692564 was associated with higher aBMD Z-score for total hip (beta = 0.62, p = 9.0 × 10(-4) ) and femoral neck (beta = 0.53, p = 0.010). In sex-stratified analyses, this variant was associated with higher bone Z-scores in females only, with the associations being strongest for total hip (sex interaction p = 1.9 × 10(-4) ; beta females = 0.86, p = 6.6 × 10(-6) ) and femoral neck (sex interaction p = 0.016; beta females = 0.73, p = 0.001). The common G allele of SOX6 SNP rs11024028 was associated with higher aBMD Z-score for total hip (beta = 0.12, p = 0.009), femoral neck (beta = 0.13, p = 0.003), and TBLH-BMC (beta = 0.09, p = 0.007); furthermore, this association strengthened in males in the sex-stratified analyses. Our findings reveal that rare genetic variation near EN1 and common variation near SOX6 operates in childhood and has implications for the lifelong risk of osteoporosis and fracture. The sex differences observed need to be independently replicated. © 2016 American Society for Bone and Mineral Research.

Genetic Risk Scores Implicated in Adult Bone Fragility Associate With Pediatric Bone Density.

Using adult identified bone mineral density (BMD) loci, we calculated genetic risk scores (GRS) to determine if they were associated with changes in BMD during childhood. Longitudinal data from the Bone Mineral Density in Childhood Study were analyzed (N = 798, 54% female, all European ancestry). Participants had up to 6 annual dual energy X-ray scans, from which areal BMD (aBMD) Z-scores for the spine, total hip, and femoral neck were estimated, as well as total body less head bone mineral content (TBLH-BMC) Z-scores. Sixty-three single-nucleotide polymorphisms (SNPs) were genotyped, and the percentage of BMD-lowering alleles carried was calculated (overall adult GRS). Subtype GRS that include SNPs associated with fracture risk, pediatric BMD, WNT signaling, RANK-RANKL-OPG, and mesenchymal stem cell differentiation were also calculated. Linear mixed effects models were used to test associations between each GRS and bone Z-scores, and if any association differed by sex and/or chronological age. The overall adult, fracture, and WNT signaling GRS were associated with lower Z-scores (eg, spine aBMD Z-score: ßadult = -0.04, p = 3.4 × 10(-7) ; ßfracture = -0.02, p = 8.9 × 10(-6) ; ßWNT = -0.01, p = 3.9 × 10(-4) ). The overall adult GRS was more strongly associated with lower Z-scores in females (p-interaction ≤ 0.05 for all sites). The fracture GRS was more strongly associated with lower Z-scores with increasing age (p-interaction ≤ 0.05 for all sites). The WNT GRS associations remained consistent for both sexes and all ages (p-interaction > 0.05 for all sites). The RANK-RANKL-OPG GRS was more strongly associated in females with increasing age (p-interaction < 0.05 for all sites). The mesenchymal stem cell GRS was associated with lower total hip and femoral neck Z-scores, in both boys and girls, across all ages. No associations were observed between the pediatric GRS and bone Z-scores. In conclusion, adult identified BMD loci associated with BMD and BMC in the pediatric setting, especially in females and in loci involved in fracture risk and WNT signaling.

Association of the BANK 1 R61H variant with systemic lupus erythematosus in Americans of European and African ancestry.

Recently an association was demonstrated between the single nucleotide polymorphism (SNP), rs10516487, within the B-cell gene BANK1 and systemic lupus erythematosus (SLE) as a consequence of a genome wide association study of this disease in European and Argentinean populations. In a bid for replication, we examined the effects of the R61H non-synonymous variant with respect to SLE in our genotyped American cohorts of European and African ancestry. Utilizing data from our ongoing genome-wide association study in our cohort of 178 Caucasian SLE cases and 1808 Caucasian population-based controls plus 148 African American (AA) SLE cases and 1894 AA population-based controls we investigated the association of the previously described non-synonymous SNP at the BANK1 locus with the disease in the two ethnicities separately. Using a Fisher's exact test, the minor allele frequency (MAF) of rs10516487 in the Caucasian cases was 22.6% while it was 31.2% in Caucasian controls, yielding a protective odds ratio (OR) of 0.64 (95% CI 0.49-0.85; one-sided p = 7.07 × 10(-4)). Furthermore, the MAF of rs10516487 in the AA cases was 18.7% while it was 23.3% in AA controls, yielding a protective OR of 0.75 (95% CI 0.55-1.034; one-sided p = 0.039). The OR of the BANK1 variant in our study cohorts is highly comparable with that reported previously in a South American/European SLE case-control cohort (OR = 0.72). As such, R61H in the BANK1 gene confers a similar magnitude of SLE protection, not only in European Americans, but also in African Americans.