Phenotypic Dissection of Bone Mineral Density Reveals Skeletal Site Specificity and Facilitates the Identification of Novel Loci in the Genetic Regulation of Bone Mass Attainment
[['Bone mineral density ( BMD ) at the femoral neck and lumbar spine [as measured by dual-energy X-ray absorptiometry , ( DXA ) ] , represents the primary diagnostic marker for osteoporosis as it serves as a good predictor of bone strength and fracture risk in adults [1] .', 'Bone strength and fracture risk are influenced by:', 'i ) bone acquisition in childhood , adolescence and young adulthood', 'ii ) the subsequent maintenance of bone mass over the life course and', 'iii ) the progressive loss of bone in later life [2] , [3] .', 'Large-scale genome-wide association studies ( GWAS ) using adult-BMD measured at the femoral neck ( FN ) and lumbar spine ( LS ) have successfully identified variants in 56 loci explaining 4–5% of the phenotypic variance in adult-BMD [4]–[6] .', 'However , it is possible that the genetic variants influencing bone acquisition are different from the ones involved in bone maintenance and bone loss across the life course .', 'Consequently , GWAS using paediatric-BMD measurements have recently been performed with the goal of identifying novel genetic variants primarily associated with bone acquisition , whilst limiting the noise introduced by bone maintenance and bone loss [7] .', 'This approach has resulted in the successful identification of novel BMD associated variants in the WNT16 [7] and Osterix ( SP7 ) loci [8] and it is highly likely that more variants will be discovered as the sample size of these paediatric studies increases .', 'In growing children , changes in bone area create artefacts influencing the reproducibility , comparability and interpretation of DXA measurements .', 'For this reason , regions of interest ( ROI ) containing larger bone areas [i . e . total-body , ( TB ) ] , which are less prone to these artefacts , are preferred for paediatric evaluations of bone health [9] .', 'The skull region is generally excluded from TB-DXA scans as its relative contribution to bone mass is proportionally larger with respect to the rest of the body in children , and its inclusion has been shown to make diagnostic interpretation difficult [10] .', 'However , from a locus discovery perspective , it may be advantageous to partition TB-DXA further into different regions , such as the upper and lower limbs and the skull .', 'This is important if genetic heterogeneity exists in terms of loci differentially affecting BMD at different skeletal sites , or whose effect is greater at some locations than in others .', 'Considering that environmental factors ( i . e . mechanical loading ) influence skeletal sites differently , analysis of skull-BMD may be particularly informative and even provide greater power to identify genetic variants .', 'This is the case given that the skull is less influenced by mechanical loading than appendicular and other axial sites .', 'Further , the skull is frequently affected in monogenic conditions involving the skeleton .', 'For example , craniofacial abnormalities such as thickening of the cranium and skull base are cardinal features of van Buchems disease , Sclerosteosis and other sclerosing bone dysplasias [11] , [12] .', 'In the current study we examined whether genetic factors influence bone mass accrual in a site-specific manner , by performing regional analysis of TB-DXA scans , focussing on the total-body less head ( TBLH ) , lower limb ( LL ) , upper limb ( UL ) , and skull ( SK ) regions .', 'Using genome-wide complex trait analysis ( GCTA ) on participants from the Avon Longitudinal Study of Parents and their Children ( ALSPAC ) , we assessed the proportion of BMD variance explained by common genetic variants , across each sub-region and additionally determined the shared genetic and residual correlation between each sub-region .', 'Subsequently , we performed a genome-wide association ( GWA ) meta-analysis of BMD at each skeletal site in the ALSPAC and Generation R studies and went on to identify factors , which preferentially influence one or more skeletal regions .'], ['Univariate GCTA analysis revealed that common genotyped variants explained a greater proportion of the variance in SK-BMD ( vg\u200a=\u200a0 . 51 , SE\u200a=\u200a0 . 07 , P\u200a=\u200a2 . 0×10−13 ) than LL- ( vg\u200a=\u200a0 . 40 , SE\u200a=\u200a0 . 07 , P\u200a=\u200a8 . 0×10−9 ) or UL- ( vg\u200a=\u200a0 . 39 , SE\u200a=\u200a0 . 07 , P\u200a=\u200a2 . 0×10−8 ) BMD .', 'Higher phenotypic correlations were observed when comparing LL- and UL-BMD than with SK-BMD ( Table 1 ) .', 'Similarly , bivariate GCTA analysis indicated that the strongest genetic correlation was between BMD at the two appendicular sites , whereas the genetic correlations involving SK-BMD were more moderate .', 'The residual correlation between the different sites was in general smaller than the genetic correlation , and was higher for BMD between the appendicular sites than for comparisons involving the skull ( Table 1 ) .', 'Highly similar magnitudes and patterns of residual correlations were obtained for a sensitivity analysis in which BMD at all skeletal sites was corrected for age , gender , weight and height ( Table S1 ) .', 'Genome-wide association meta-analyses were performed on TBLH- , LL- , UL- and SK-BMD , using regional BMD data derived from ∼9 , 395 TB-DXA scans .', 'Detailed population characteristics of the ALSPAC and Generation R cohorts are summarised in Tables S2 and S3 .', 'Summary statistics from each GWAS ( after meta-analysis ) indicated that negligible systematic inflation of test statistics was observed ( META λGC\u200a=\u200a1 . 01–1 . 03 ) .', 'In contrast , a marked deviation from the null was observed in the tail of the distribution amongst the lowest observed P-values of the meta-association analyses ( Figure S1 ) .', 'SNPs in thirteen published BMD-associated loci exceeded the genome-wide significance ( GWS ) threshold for association ( P≤5×10−8 , Table 2 ) .', 'They included variants which mapped close to , or within: WNT4 ( 1p36 . 12 ) , WNT16/FAM3C/CPED1 ( 7q31 . 31 ) for all skeletal sites measured , EYA4 ( 6q23 . 2 ) , COLEC10/TNFRS11B ( 8q24 . 12 ) , LIN7C/LGR4 ( 11p14 . 1 ) , PPP6R3/LRP5 ( 11q13 . 2 ) and TNFRSF11A ( 18q21 . 33 ) for SK-BMD , CENPW/RSPO3 ( 6q22 . 32 ) for UL- and SK-BMD , TNFSF11 ( 13q14 . 11 ) and GALNT3 ( 2q24 . 3 ) for UL- and TBLH-BMD .', 'In addition , variants proximal to or within FUBP3 ( 9q34 . 11 ) and KLHDC5/PTHLH ( 12p11 . 22 ) were associated with TBLH- and LL-BMD .', 'Furthermore , a novel signal ( top SNP rs754388 , 14q32 . 12 ) , located within Ras and Rab interactor 3 ( RIN3 ) achieved genome-wide significance after meta-analysis of LL-BMD ( β\u200a=\u200a0 . 13 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a1 . 4×10−10 , Figure 1-I , Table 2 ) and TBLH-BMD ( β\u200a=\u200a0 . 12 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a3 . 0×10−9 , Table 2 and Figure S2 ) .', 'The full list of all genome-wide significant SNPs and regional association plots for each locus and skeletal site are presented in Supplementary Tables S4 , S5 , S6 , S7 and Figures S2 , S3 , S4 , S5 .', 'A followup of 66 independent SNPs at 58 loci , previously associated with BMD [4] , [13] , indicated that 31 loci showed nominal evidence of association ( P<0 . 05 ) with TBLH-BMD , 28 with LL-BMD , 26 with UL-BMD and 26 with SK-BMD ( versus an expectation of 3 . 3 . per phenotype ) ( Table S8 ) .', 'A similar distribution of associations was also observed when a more conservative threshold considering multiple hypothesis testing was adopted that took into account the fact that 66 variants and four phenotypes had been tested ( i . e . α<1 . 9×10−4 ) .', 'Using this threshold nine variants showed evidence of association with TBLH-BMD , seven with LL-BMD , six with UL-BMD and 10 with SK-BMD ( versus an expectation of 0 . 1 per phenotype ) .', 'We note that in all cases where nominal significance was reached , the direction of effect was consistent with previous studies .', 'To ensure that our results were robust to the possible effects of population stratification and our choice of covariates , we performed sensitivity analyses where we either restricted our analysis to white European individuals only , or adjusted for the same set of covariates across all analyses ( i . e . age , gender , height , and weight ) .', 'Similar effect sizes and patterns of association were observed for the top SNPs when adjusting BMD measures of all four regions for age , gender , height and weight ( Model 1a , Table S9 ) and when limiting the GWAS meta-analysis to individuals of European ancestry ( Model 1b , Table S9 ) .', 'In both sensitivity analyses , no additional loci reached the threshold of genome-wide association ( Figure S6 and S7 ) .', 'We assessed the presence of novel secondary association signals at loci that contained genome-wide associated variants .', 'Meta-analysis of conditional association analyses resulted in the attenuation of the majority of our top association signals ( Table S10 , Figures S2 , S3 , S4 , S5 ) , indicating that these loci were not independent from signals previously reported by other BMD GWAS .', 'However , the top signal for SK-BMD ( rs2130604 , β\u200a=\u200a0 . 11 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a3 . 3×10−11 ) , mapping near RSPO3 , but closest to CENPW ( previously known as C6orf173 , 6q22 . 32 , Figure 2A-I , Table 2 ) was only marginally attenuated after conditional analysis ( rs2130604 , β\u200a=\u200a0 . 10 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a7 . 1×10−9 , Figure 2A-II , Table S10 ) .', 'This suggests that rs2130604 is largely independent from the previously reported signal at RSPO3 ( rs13204965 , 6q22 . 32 ) , which was identified in a GWAS of individuals with extremely high or low BMD at the hip [12] and later replicated in the second GEnetic Factors for OSteoporosis Consortium ( GEFOS-II ) BMD meta-analysis [4] .', 'This observation is further supported by low estimates of LD ( r2\u200a=\u200a0 . 14 ) between rs2130604 and rs13204965 .', 'Furthermore , the secondary signal ( after conditional analysis ) reached the estimated significance threshold of association after multiple testing correction ( i . e . P≤7 . 2×10−5 ) .', 'Interestingly , after conditioning rs4418209 ( another SNP in the same locus ) on the published BMD-associated SNP rs13204965 , we observed a marked increase in its evidence of association [ ( β\u200a=\u200a0 . 07 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a1 . 1×10−6 ) before and ( β\u200a=\u200a0 . 09 , SE\u200a=\u200a0 . 01 , P\u200a=\u200a7 . 9×10−10 ) after] , ( Figure 2A-II , Table S10 ) .', 'The rs4418209 variant maps closest to CENPW ( 6q22 . 32 ) and is in moderate LD with the secondary independent signal ( rs2130604 , r2\u200a=\u200a0 . 43 ) and in low LD with the published RSPO3 SNP ( rs13204965 , r2\u200a=\u200a0 . 12 ) .', 'Whilst no other SNPs reached the threshold for declaring genome-wide significance ( P<5×10−8 ) , variants from three loci still yielded suggestive evidence for association ( P<1×10−5 ) after conditional analyses ( Table S10 and Figures S2 , S3 , S4 , S5 ) .', 'They included:', 'i ) KLHDC5/PTHLH ( rs4420311 , 12p11 . 22 ) associated with TBLH- ( β\u200a=\u200a0 . 08 , SE\u200a=\u200a0 . 016 , P\u200a=\u200a7 . 6×10−7 ) and LL-BMD ( β\u200a=\u200a0 . 08 , SE\u200a=\u200a0 . 016 , P\u200a=\u200a1 . 9×10−6 ) ,', 'ii ) TNFSF11 ( rs17536328 and rs2148072 , 13q14 . 11 ) associated with TBLH- ( β\u200a=\u200a0 . 08 , SE\u200a=\u200a0 . 015 , P\u200a=\u200a5 . 6×10−7 ) and UL-BMD ( β\u200a=\u200a0 . 07 , SE\u200a=\u200a0 . 015 , P\u200a=\u200a2 . 1×10−6 ) respectively and', 'iii ) LIN7C/LGR4 [rs10160456 , 11p14 . 1 , ( β\u200a=\u200a0 . 07 , SE\u200a=\u200a0 . 015 , P\u200a=\u200a7 . 8×10−6 ) ] with SK-BMD .', 'After conditional analysis , the secondary signal at LIN7C/LGR4 ( i . e . rs10160456 ) mapped closest to CCDC34 and not to LIN7C , the gene closest to the primary signal .', 'All these three loci might represent novel secondary signals as the residual signal reached the predicted locus specific threshold of association after multiple testing correction ( Table S10 ) .', 'However , we cannot exclude that both associations ( i . e . the primary and secondary signals ) could potentially arise from their association with one or more causal variants , which could occur , on the same haplotype background .', 'For example , one such BMD-associated rare variant has recently been identified in LGR4 in Icelandic populations although this mutation appears specific to this population and therefore is unlikely to account for the LIN7C/LGR4 signal we observe [14] .', 'The standardized per allele effect sizes ( β ) of all the top BMD-associated SNPs were compared across three ( SK- , UL- , and LL ) BMD regions to determine if they preferentially influenced one or more skeletal sites ( Table 3 , Figure S8 , S9 , S10 , S11 ) .', 'Effect sizes of the following variants: rs2130604 ( CENPW/RSPO3 , 6q22 . 32 ) , rs3012465 ( EYA4 , 6q23 . 2 ) , rs2450083 ( COLEC10/TNFRS11B , 8q24 . 12 ) , rs10835187 ( LIN7C/LGR4 , 11p14 . 1 ) and rs884205 ( TNFRSF11A , 18q21 . 33 ) appeared to be largest for SK-BMD when compared to UL- and LL-BMD ( Figure S8 ) .', 'Furthermore , differences in the magnitude of the effect were evident when comparing independent genetic variants that occurred in close proximity within a locus , as shown at the CENPW/RSPO3 ( 6q22 . 32 ) and WNT16/FAM3C/CPED1 ( 7q31 . 31 ) loci .', 'Specifically , the independent signal ( rs2130604 , CENPW/RSPO3 , 6q22 . 32 ) associated with SK-BMD [β\u200a=\u200a0 . 11 ( CI95: 0 . 08 , 0 . 15 ) P\u200a=\u200a3 . 3×10−11] , was not strongly related to LL-BMD [β\u200a=\u200a0 . 02 ( CI95: −0 . 02 , 0 . 05 ) , P\u200a=\u200a0 . 28] , or UL-BMD [β\u200a=\u200a0 . 04 , ( CI95: 0 . 01 , 0 . 07 ) , P\u200a=\u200a0 . 02] ( Table 3 , Figure 2A-III ) .', 'In contrast , a neighbouring SNP ( rs1262476 ) primarily associated with UL-BMD appeared to influence BMD across all skeletal sites ( Table 3 , Figure 2B-III ) .', 'Differential patterns of association between SNPs at neighbouring positions were also observed at the WNT16 locus ( Table 3 , Figure 3A–C ) .', 'Effect sizes were largest for UL-BMD at rs2908004 ( WNT16 , 7q31 . 31 , Table 3 , Figure 3A-II ) when compared to SK- and LL-BMD .', 'Interestingly , as compared to LL-BMD , we observed consistently larger effect sizes for rs13223036 and rs798943 ( CPED1 , previously known as C7orf58 ) for SK- and UL-BMD , ( Table 3 , Figure 3B-II and 3C-II ) .', "To formally determine whether the standardized regression coefficients of each of the above-mentioned variants truly differed across the skeletal sites , we fitted a multivariate normal likelihood model to the raw data in ALSPAC and Generation R ( see Methods ) , and then meta-analysed the results using Fisher's method .", 'Using a conservative threshold ( i . e . α\u200a=\u200a5×10−8 ) , we observed robust evidence indicating that i . e . rs13223036 and rs798943 , located at CPED1 exerted strong effects on UL and SK-BMD , when compared to LL-BMD [P\u200a=\u200a2 . 01×10−37 and P\u200a=\u200a4 . 44×10−36 ( Table3 ) ] , whereas the variant rs2908004 ( WNT16 ) was strongly related to UL-BMD in comparison to BMD at the other sites ( P\u200a=\u200a2 . 31×10−14 ) .', 'Several variants at other loci were also suggestive of some degree of skeletal site specificity including EYA4 and LIN7C , although they did not formally meet the criteria for statistical significance ( Table 3 , Figures S8 , S9 , S10 , S11 ) .', 'To elucidate if any of the novel primary and/or secondary signals , identified during the course of this study , were nominally associated with BMD in adults , we performed a lookup of these variants in the publicly available results of the GEFOS-II meta-analysis of hip and spine BMD ( Table S11 ) [4] .', 'The novel RIN3 variant ( rs754388 ) was not associated with femoral neck ( PFN\u200a=\u200a0 . 87 ) and lumbar spine BMD ( PLS\u200a=\u200a0 . 42 ) .', 'The G allele of the EYA4 variant ( rs3012465 ) , associated with increased SK-BMD ( β\u200a=\u200a0 . 13 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a8 . 3×10−17 ) , but surprisingly showed nominal association with decreased BMD at both the hip ( P\u200a=\u200a7 . 1×10−3 ) and spine ( P\u200a=\u200a0 . 04 ) .', 'A followup of this variant in a recent published GWAS of 4061 premenopausal women aged 20 to 45 revealed no evidence of association with FN-BMD ( P\u200a=\u200a0 . 73 ) [15] .', 'A lookup of the secondary independent SNPs revealed no evidence of a relationship between the TBLH- and LL-BMD-associated KLHDC5/PTHLH variant ( rs4420311 ) and associations with hip or spine BMD ( PFN\u200a=\u200a0 . 33 and PLS\u200a=\u200a0 . 45 ) in GEFOS-II .', 'Similarly no evidence of association was detected for the SK-BMD-associated variant at CENPW/RSPO3 ( rs2130604: PFN\u200a=\u200a0 . 98 and PLS\u200a=\u200a0 . 40 ) .', 'Interestingly , the T allele of the CENPW/RSPO3 variant ( rs4418209 ) , which was associated with increased SK-BMD ( β\u200a=\u200a0 . 07 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a1 . 1×10−6 ) , appeared to be nominally associated with decreased hip BMD ( P\u200a=\u200a5 . 0×10−3 ) but not spine BMD ( P\u200a=\u200a0 . 34 ) .', 'Further inspection revealed that the T allele of rs4418209 was nominally associated with decreased BMD at the TBLH ( β\u200a=\u200a−0 . 03 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a1 . 7×10−2 ) , LL ( β\u200a=\u200a−0 . 04 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a6 . 5×10−3 ) and UL ( β\u200a=\u200a−0 . 04 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a8 . 2×10−3 ) .', 'The T allele of rs17536328 located within TNFSF11 , associated with increased TBLH-BMD , showed nominal evidence of association with increased hip ( P\u200a=\u200a0 . 04 ) but not spine BMD ( P\u200a=\u200a0 . 87 ) .', 'In contrast , the G allele of an independent TNFSF11 variant ( rs2148072 ) associated with increased UL-BMD was associated with decreased spine BMD ( P\u200a=\u200a0 . 05 ) .', 'In addition , the C allele LIN7C/LGR4 variant ( rs10160456 ) associated with increased SK-BMD showed weak evidence of association with increased hip ( P\u200a=\u200a0 . 06 ) and spine ( P\u200a=\u200a0 . 03 ) BMD .', 'We fine mapped the RIN3 region by imputing common and rare variants using a reference panel from the 1000 Genomes Project and identified a missense variant ( rs117068593 ) that was in strong linkage disequilibrium ( r2\u200a=\u200a0 . 96 ) with the top LL-and TBLH-BMD associated RIN3 variant ( rs754388 ) .', 'The C allele of rs117068593 ( EAF\u200a=\u200a0 . 82 ) was associated with increased BMD of the lower limbs ( β\u200a=\u200a0 . 13 , SE\u200a=\u200a0 . 020 , P\u200a=\u200a5 . 97×10−11 ) and total-body less head ( β\u200a=\u200a0 . 12 , SE\u200a=\u200a0 . 020 , P\u200a=\u200a1 . 87×10−9 ) .', 'A search of the SIFT database [16] revealed that the missense variant could negatively affect RIN3 functioning .', 'This prediction was further supported by a search of the Regulome database [17] , which suggested that the missense variant alters the binding of the following transcription factors: EBF1 , EGR1 , SP1 , NFKB1 and POLR2A , in lymphoblastic cell lines .', 'Evaluation of cis-expression quantitative trait loci ( eQTLs ) from primary human osteoblasts using array-based gene expression suggested that variants located within 1MB of RIN3 ( i . e . including variants tagging SLC24A4 , LGMN , GOLGA5 , CHGA and ITPK1 ) were nominally associated with ITPK1 expression ( P\u200a=\u200a0 . 04 ) .', 'This observation failed to meet the level of significance after correction for multiple testing .', 'Examination of the temporal pattern of gene expression across osteoblastogenesis , using mouse calvarial derived cells , starting with the pre-osteoblast stage , through to mature osteoblasts revealed that Rin3 , Golga5 and Lgmn , and Iptk1 were expressed in this cell type ( Figure S12 ) .', 'In contrast , Slc24a4 and Chga were not expressed at all in the pre- or mature osteoblast , as determined by RNAseq .', 'A further investigation of the expression profiles of the aforementioned genes in human mesenchymal stem cells [ ( hMSCs ) , differentiated into adipocytes and osteoblasts] and peripheral blood monocytes [ ( PBMCs ) differentiated into osteoclasts] indicated the following: SLC24A4 was not expressed in any of these cell lines when differentiated , GOLGA5 had an intermediate expression level in both differentiating hMSCs and PBMCs and LGMN was immediately upregulated upon differentiation into adipocytes ( 8 fold ) , osteoblasts ( 5 fold ) and osteoclasts [ ( 5 fold ) , Figure S13 and S14] .', 'Moreover , we found that the expression of RIN3 was 2-fold downregulated during the proliferative phase of differentiating PBMCs into osteoclasts ( Figure S13 ) .', 'Finally a comparison of expression profiles across the RIN3 region of illiac bone biopsies derived from 39 osteoporotic and 27 healthy postmenopausal donors revealed one transcript ( i . e . 220439_at , originating from RIN3 ) , that demonstrated reduced expression in the osteoporotic group relative to the control group [P\u200a=\u200a2 . 7×10−3 , ( Table S13 ) ] .'], ['This study assessed whether regional analysis of skeletal sites from TB-DXA could be used to estimate the extent to which genetic and environmental factors influence bone mass accrual of differentially loaded skeletal sites ( skull , lower limbs , and upper limbs ) .', 'Point estimates indicated that common SNPs on a commercially available genotyping array , explained a larger proportion of the overall variance of SK-BMD , when compared to BMD measured at the appendicular sites ( i . e . lower and upper limbs ) .', 'These differences potentially reflect differential exposure of each skeletal site to varying environmental stimuli that influence BMD .', 'Specifically the skull , as opposed to appendicular sites , is less influenced by environmental factors , particularly those acting through mechanical loading .', 'To explore this result further , we estimated the residual correlation ( i . e . the proportion of environmental and other sources of variation not tagged by SNPs on the Illumina platform ) across the different skeletal sites and found that whilst the environmental ( and other residual ) factors influencing the appendicular sites were moderately similar to each other , they appeared to be appreciably different from the factors influencing SK-BMD .', 'Taken together , lower vg estimates , coupled with a high residual correlation between the two appendicular sites , may reflect the greater exposure of these sites to loading and muscular stimulation , when compared to the skull .', 'Likewise , estimates of the genetic correlations indicated that the appendicular limbs shared a more similar genetic architecture when compared to the skull , possibly reflecting the composition of bone at each skeletal site and the biological processes that govern their growth and maintenance .', 'For example , appendicular sites consist of broadly equivalent proportions of cortical and trabecular bone .', 'The skull on the other hand is mainly comprised of flat bones , which consist primarily of cortical bone [18] .', 'The developmental processes also differ between long and flat bones , with dermal bones such as the skull vault arising exclusively through intramembranous bone formation , in contrast to long bones , which form through endochondral bone formation involving intermediary formation of cartilage [19] .', 'To further explore the basis for the above-mentioned differences in underlying genetic architecture , GWA meta-analyses of sub-regional TB-DXA data were performed .', 'These analyses helped identifying genetic signals that were associated with one or more skeletal region ( s ) .', 'When comparing the evidence of association for all SNPs ( identified in this effort ) across each skeletal site , our GWA meta-analyses echoed the findings of our GCTA results , supporting the notion that although the underlying genetic architecture influencing BMD appears to be largely similar , it does vary according to skeletal site .', 'The majority of the top SNPs were nominally associated ( P≤0 . 05 ) with BMD across all skeletal sites ( i . e . SNPs at WNT4 , WNT16 , FAM3C , GALNT3 , FUBP3 , KLHDC5/PTHLH , TNSF11 , LIN7C/LGR4 and PPP6R3/LRP5 ) .', 'In contrast , variants near or within CPED1 , COLEC10/TNFRS11B and EYA4 were strongly associated with UL- and SK-BMD , but not LL-BMD .', 'A further variant was identified within TNFRSF11A that appeared to be solely related to SK-BMD .', 'Most notably we observed a novel association between rs754388 ( located within RIN3 ) and LL-/UL-BMD , but not SK-BMD .', 'To the best of our knowledge this is the first GWAS to report an association between RIN3 and BMD .', "It seems likely that this association reflects a true relationship with BMD as the same RIN3 signal ( as determined by conditional analysis ) has previously been associated with an increased risk of Paget's Disease [i . e . rs10498635-C OR: 1 . 44 , 95%-CI ( 1 . 29–1 . 60 ) P\u200a=\u200a3 . ×10−11] [20] .", 'In an attempt to further understand how the genetic variation surrounding RIN3 may influence BMD , we fine mapped RIN3 and identified a missense variant ( rs117068593 ) that was in high LD with our LL-BMD associated SNP .', 'Data mining of SIFT and ENCODE databases suggested a functional role of the missense variant that putatively affects binding of several transcription factors in lymphoblastic cell lines .', 'We further evaluated expression quantitative trait locus ( eQTL ) data from primary human osteoblasts using SNP data from HapMap ( i . e . not including rs117068593 ) and found no substantial evidence that our LL-BMD associated SNPs located at 14q32 . 12 regulated the expression of RIN3 or any of the genes located nearby .', 'However , differential patterns gene expression were detected when comparing RIN3 expression profiles of osteoporotic and healthy individuals .', 'Further , we also observed differential expression during osteoclast differentiation that was not present in osteoblast and adipocyte differentiation processes .', "Collectively , the aforementioned observations appear to be in line with previous findings that suggest that RIN3 could influence osteoclast activity , especially when considering the prior association of RIN3 with Paget's Disease , a disease driven by osteoclast dysfunction and molecular studies that indicate that RIN3 is involved in vesicular trafficking , a process critical for bone resorption [20] , [21] .", 'Further study is however needed to elucidate the precise role of role of RIN3 in bone metabolism .', 'To further understand the preferential associations of some variants with different skeletal sites , we compared the standardized effect sizes of all the genome-wide significant BMD-associated variants , across each skeletal site using a formal multivariate normal likelihood model .', 'Variants at the CPED1 locus were strongly associated with BMD at the skull and upper limb sites , but not with LL-BMD .', 'Similarly variants at WNT16 were more strongly related to UL-BMD , than to BMD at the other sites .', 'Several other SNPs showed evidence for site specificity including variants at the EYA4 and LIN7C loci that were very strongly related to SK-BMD , although these variants did not surpass our conservative criterion for declaring significant heterogeneity , corroboration is needed from independent studies .', 'Conceivably , differences in the pattern of results across SNPs may have arisen from an artefact of the measurement ( i . e . where sub-regional-specific associations reflect how accurately BMD is measured at each skeletal site ) .', 'However , if the latter were the case , one would expect to observe a consistent pattern of results across all loci ( i . e . the strength of association should be greatest at those sites measured more accurately ) .', 'From our results , this is clearly not the case as evidence of association is sometimes greatest for the skull , whilst for other SNPs evidence is greatest for lower and/or upper limbs .', 'In terms of biological explanations , larger effect sizes of genetic variants that influence SK-BMD possibly reflect their preferential involvement in cortical as opposed to trabecular bone metabolism and/or the involvement of intramembranous ossification vs . endochondral ossification [13] .', 'Certain genetic factors also appeared to influence UL-BMD more strongly than LL-BMD , or vice versa .', 'Since the composition and developmental origin of these two sites is broadly similar , presumably , other explanations are responsible .', 'It is reasonable to think that genetic factors , which we identified , could be acting to alter responses to stimuli that are themselves site-specific .', 'For example , adipose tissue has previously been reported to influence cortical bone of the tibia in preference to the radius [22] .', 'Quantitative SK-BMD measurements have traditionally been ignored by genetic and epidemiological studies as they are thought to be prone to errors such as dental augmentation .', 'Despite these concerns , a study conducted in premenopausal woman found a high correlation between the upper half of the skull ( i . e . cranial vault ) and total skull-BMD ( r2\u200a=\u200a0 . 991 , n\u200a=\u200a91 , Age range 19–30 years ) , with a mean difference of −0 . 004 g/cm2 , suggesting that these two measurements of bone mass are similar [23] .', 'We found that paediatric SK-BMD measures are well suited to GWAS , as indicated by the very low P-values obtained at some of the known BMD associated loci ( 10−17 to 10−28 ) despite our relatively small sample size .', 'This observation may reflect the fact that SK-BMD is considerably less subject to environmental influences , such as those acting through mechanical loading .', 'In addition , genetic variants associated with SK-BMD identified in this study may primarily reflect molecular pathways involved in bone mass accrual and growth , in contrast to variants identified from previous adult scans which may be more strongly related to mechanisms involved in bone maintenance and/or loss .', 'Almost all the loci we have identified in this study ( i . e . with the exception of SNPs in RIN3 and EYA4 ) have been associated with BMD at either the hip or the lumbar spine previously .', "Variants mapping to RIN3 have been implicated in Paget's disease but this is the first time the locus is associated with BMD , and interestingly , the alleles associated with increased BMD are associated with increased risk for the condition .", 'This shows that performing GWAS of BMD at sites other than at the femoral neck ( FN-BMD ) or lumbar spine ( LS-BMD ) can be used to identify loci that exert pleiotropic effects on bone .', 'Potential advantages of examining these additional sites from a locus discovery perspective are that', '( i ) genetic variants may exert stronger effects at these sites than at FN-BMD/LS-BMD , and/or', '( ii ) the genetic effects may be more apparent at these sites because the effect of environmental noise is minimized .', 'For example , the P-values for skull BMD at several loci ( e . g . variants around CPED1 , EYA4 and LIN7C ) are many orders of magnitude stronger than the corresponding P-values for TBLH-BMD ( see Table S8 ) .', 'Likewise variants in LIN7C were first discovered using a GWAS meta-analysis of lumbar spine that was over five times the size of the present study , and even then only just exceeded the threshold for genome-wide significance [4] , whereas in our study a variant at this locus has P<1×10−16 with SK-BMD .', 'Hence , GWAS of BMD at sites such as the skull could be used to efficiently detect clinically relevant loci that might be more difficult to discover in GWAS of the femoral neck and/or lumbar spine .', 'To further illustrate the value of SK-BMD , we draw attention to rs3012465 , a variant proximal to the eyes absent ( EYA4 ) gene and associated with increased SK-BMD .', 'We show that the signal is analogous to that previously associated with increased volumetric cortical BMD of the tibia ( i . e . C allele of rs271170: β\u200a=\u200a0 . 11 , P\u200a=\u200a2 . 7×10−12 ) , based on a GWAS in ALSPAC and other young adult cohorts [13] , suggesting that both findings reflect the relationship of the EYA4 locus with cortical bone .', 'However , a look-up in a separate cortical bone site ( i . e . the femoral neck of the hip ) , from a GWAS in older adults , revealed that the BMD-increasing allele at the EYA4 locus was in fact associated with lower BMD for both rs3012465 and rs271170 [4] .', 'Taken together , 
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Heritability of bone mineral density ( BMD ) varies across skeletal sites , reflecting different relative contributions of genetic and environmental influences .', 'To quantify the degree to which common genetic variants tag and environmental factors influence BMD , at different sites , we estimated the genetic ( rg ) and residual ( re ) correlations between BMD measured at the upper limbs ( UL-BMD ) , lower limbs ( LL-BMD ) and skull ( SK-BMD ) , using total-body DXA scans of ∼4 , 890 participants recruited by the Avon Longitudinal Study of Parents and their Children ( ALSPAC ) .', 'Point estimates of rg indicated that appendicular sites have a greater proportion of shared genetic architecture ( LL-/UL-BMD rg\u200a=\u200a0 . 78 ) between them , than with the skull ( UL-/SK-BMD rg\u200a=\u200a0 . 58 and LL-/SK-BMD rg\u200a=\u200a0 . 43 ) .', 'Likewise , the residual correlation between BMD at appendicular sites ( re\u200a=\u200a0 . 55 ) was higher than the residual correlation between SK-BMD and BMD at appendicular sites ( re\u200a=\u200a0 . 20–0 . 24 ) .', 'To explore the basis for the observed differences in rg and re , genome-wide association meta-analyses were performed ( n∼9 , 395 ) , combining data from ALSPAC and the Generation R Study identifying 15 independent signals from 13 loci associated at genome-wide significant level across different skeletal regions .', 'Results suggested that previously identified BMD-associated variants may exert site-specific effects ( i . e . differ in the strength of their association and magnitude of effect across different skeletal sites ) .', 'In particular , variants at CPED1 exerted a larger influence on SK-BMD and UL-BMD when compared to LL-BMD ( P\u200a=\u200a2 . 01×10−37 ) , whilst variants at WNT16 influenced UL-BMD to a greater degree when compared to SK- and LL-BMD ( P\u200a=\u200a2 . 31×10−14 ) .', "In addition , we report a novel association between RIN3 ( previously associated with Paget's disease ) and LL-BMD ( rs754388: β\u200a=\u200a0 . 13 , SE\u200a=\u200a0 . 02 , P\u200a=\u200a1 . 4×10−10 ) .", 'Our results suggest that BMD at different skeletal sites is under a mixture of shared and specific genetic and environmental influences .', 'Allowing for these differences by performing genome-wide association at different skeletal sites may help uncover new genetic influences on BMD .']
['The heritability of bone mineral density ( BMD ) varies across skeletal sites , reflecting different relative contributions of genetic and environmental influences .', 'To investigate whether the genes underlying bone acquisition act in a site-specific manner , we quantified the shared genetic influences across axial and appendicular skeletal sites by estimating the genetic and residual correlation of BMD at the upper limb , lower limb and the skull .', 'Our results suggest that different skeletal sites as measured by total-body Dual-Energy X-Ray Absorptiometry are to a certain extent under distinct genetic and environmental influences .', 'To further explore the basis for these differences , genome-wide association meta-analyses were performed to identify genetic loci that are preferentially associated with one or more skeletal regions .', 'Variants at 13 loci ( including RIN3 , a novel BMD associated locus ) reached genome-wide significance and several displayed evidence of differential association with BMD across the different skeletal sites in particular CPED1 and WNT16 .', 'Our results suggest that it may be advantageous to decompose the total-body BMD measures and perform GWAS at separate skeletal regions .', 'By allowing for site-specific differences , new genetic variants affecting BMD and future risk of osteoporosis may be uncovered .']
['genome-wide association studies', 'biochemistry', 'rheumatology', 'medicine and health sciences', 'genome analysis', 'genetics', 'biology and life sciences', 'computational biology', 'metabolism', 'bone and mineral metabolism', 'human genetics']