An Integration of Genome-Wide Association Study and Gene Expression Profiling to Prioritize the Discovery of Novel Susceptibility Loci for Osteoporosis-Related Traits
[['The feasibility of carrying out genome-wide association studies ( GWAS ) has led to the rapid progression of the field of complex-disease genetics over the past few years .', 'Although the GWAS approach has been successful in identifying novel candidate genes leading to new discovery of pathways that are involved in the pathophysiology of diseases , the genetic variants identified so far only explain a small proportion of the heritability for complex traits [1] .', 'Due to the modest genetic effect size and inadequate power to overcome the heterogeneity of genetic effects in meta-analysis , true association signals may not be revealed based on a stringent genome-wide significance threshold alone [2] .', 'In addition , the majority of the GWAS have not provided much information beyond statistical signals to understand the genetic architecture for those usually novel genes that have not been studied for a particular trait/disease before .', 'Thus , the necessity of incorporating additional information when studying the GWAS has become apparent .', "Expression profiling with gene signatures of cellular models have been used to characterize gene's involvement in bone metabolism and disease processes .", 'One such approach is parathyroid hormone ( PTH ) stimulated osteoclastogenesis and osteoblast maturation for osteoblastogenesis [3] .', 'PTH indirectly stimulated osteoclastogenesis via its receptors on osteoblasts , which then signal to osteoclast precursors to stimulate osteoclastogenesis .', 'Impaired osteoblastic differentiation reduces bone formation and causes severe osteoporosis in animals [4] .', 'The TNFRSF11B/OPG gene , a well-known candidate gene for osteoporosis , is involved in osteoclastogenesis through the regulation of PTH [5] .', 'Compared to GWAS-identified candidate genes that do not show differential expression in these cellular models , genes like TNFRSF11B/OPG with differential expression are more likely to be involved in skeletal metabolism and thus more likely to be truly associated with osteoporosis .', 'Given that the majority of the reported genome-wide significant SNPs are in the intergenic or noncoding regions [6] , it is not clear which SNP/gene might be implicated as a causal SNP/gene .', 'Since intergenic or noncoding SNPs do not appear to affect protein sequence , it is likely that these SNPs either are in linkage disequilibrium with the causal variants or located within the transcription regulation elements of nearby genes .', 'The relative quantification of gene transcripts may act as intermediate phenotypes between genetic loci and the clinical phenotypes .', 'Expression quantitative trait loci ( eQTL ) analysis in specific tissues is a valuable tool to identify potentially causal SNPs [7]–[10] .', 'By integration of genetic variants , transcriptome , and phenotypic data , investigators have the potential to provide much-needed support to prioritize the candidate susceptibility genes identified from GWAS for further validation [11]–[13] .', 'Previously , we conducted a pilot GWAS for osteoporosis-related phenotypes in a small subset of the Framingham study participants [14] .', 'Osteoporosis is a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture .', 'The heterogeneity of osteoporosis has both an environmental and genetic basis .', 'Although bone mineral density ( BMD ) is frequently used in the diagnosis and prognosis of osteoporosis [15] , a growing body of evidence indicates that femoral geometry also contributes importantly to hip fracture risk [16] , [17] .', 'Both BMD and hip geometry are strongly heritable , with heritability estimates between 50% and 85% [18] .', 'In an attempt to identify genes that are involved in the regulation of bone health related phenotypes , genetic linkage analyses [19] , [20] , candidate gene association studies [21] and recent GWAS [22]–[27] have been used to implicate several loci and candidate genes , such as OPG/RANK/RANKL [22]–[24] , [28] , LRP5 [22] , [23] , [29] , LRP4 [23] , ESR1 [23] , [30] , VDR [31] , and SP7 [24] , [25] .', 'However , the majority of genes that contribute to genetic susceptibility to osteoporosis remain to be elucidated .', 'Seeking to extend these initial observations , in the current study , we first performed a large-scale GWAS analysis for BMD and hip geometry in 2 , 038 women and 1 , 531 men from the Framingham Osteoporosis Study using 550 , 000 SNPs , and then replicated the top findings in 5 , 595 women and 2 , 126 men from two independent cohorts of Caucasian individuals .', 'We then prioritized the genome-wide association findings by utilizing publicly available experiments relevant to the skeletal system in cellular or whole animal models , and provided supportive biological information for future functional validation of their involvement in bone metabolism .', 'The expression experiments included ( 1 ) gene signatures of a mouse embryo expression atlas and mouse cellular models of osteoblastogenesis and PTH- stimulated osteoblasts; ( 2 ) eQTL analysis in human primary osteoblasts , lymphocytes and liver tissues; and ( 3 ) likelihood-based causality model selection ( LCMS ) by integrating genetic variants , gene expression profiling , and skeletal phenotypes in inbred mice to identify candidate genes causally related to bone phenotypes .', 'An overview of the study design is provided in Figure 1 .'], ['Significant differences of BMD and geometry indices were found between men and women in the Framingham Study with p-values <0 . 001 ( Table S1 ) .', 'Quantile-quantile plots of observed p-values for single SNP association tests under additive genetic effect models are shown in Figure 2 .', 'Except for the tail ( likely comprising true associations ) , the distributions of observed p-values did not deviate from the null distribution , which rules out systematic bias due to bad genotyping or population substructure in our study samples .', 'The estimated genome control λGC for each phenotype ranged from 0 . 99 to 1 . 02 .', 'The regression coefficients analyzed with and without adjusting for the PCs are highly correlated ( r\u200a=\u200a0 . 95–0 . 98 ) .', 'Thus , we do not expect these principal components to influence our results substantially .', 'SNPs associated with each phenotype at p-values <10−6 are listed in Table S2 .', 'For women , the most significant association was found with neck width ( NW ) for SNP rs16965654 ( MAF\u200a=\u200a0 . 01 ) located 13Kb away from the 5′ upstream region of the WD repeat and SOCS box-containing 1 ( WSB1 ) gene on chromosome 17q11 . 1 ( p\u200a=\u200a4 . 15×10−8 ) .', 'For men , the most significant association was found with neck-shaft angle ( NSA ) for SNP rs11573709 ( MAF\u200a=\u200a0 . 23 ) located in intron 7 of the RAD23 homolog B ( RAD23B ) gene on 9q31 . 2 ( p\u200a=\u200a2 . 37×10−7 ) .', 'We also performed association tests by combining men and women together .', 'The most significant association was found with NW for SNP rs16965654 ( p\u200a=\u200a6 . 89×10−10 ) .', 'All genotyped SNPs ( n\u200a=\u200a431–593 for sex-specific phenotypes ) with association test p-values <10−3 in Stage I were examined for replication in the Rotterdam Study ( both men and women ) and TwinsUK Study ( women only ) .', 'We performed meta-analyses by combining results from the Framingham Study and Rotterdam Study in men and all three cohorts in women .', 'P-values <4 . 3×10−7 from meta-analyses are considered as genome-wide significant associations ( See statistical methods section for details ) .', 'We listed the most significant SNP on each chromosome locus with meta-analysis p-values <10−6 in Table 1 .', 'The most significant association for men was found with NSA for SNP rs2278729 located in the intron 4 of TBC1D8 on chromosome 2q11 . 2 ( p\u200a=\u200a1 . 48×10−7 ) .', 'SNP rs7227401 located in intron 4 of OSBPL1A ( 18q11 . 2 ) was found to be strongly associated with NW ( p\u200a=\u200a4 . 22×10−7 ) in men .', 'The most significant association for women from meta-analysis was found with LS BMD for SNP rs2062375 located in the intergenic region of TNFRSF11B and COLEC10 genes on chromosome 8q24 . 12 ( p\u200a=\u200a2 . 68×10−11 ) .', 'SNP rs494453 located in the intron 2 of RAP1A on chromosome 1p13 . 2 was also strongly associated with NW ( p\u200a=\u200a2 . 80×10−7 ) .', 'The association became more significant for SNP rs494453 when combining women and men together ( p\u200a=\u200a3 . 6×10−8 ) .', 'None of the above associated SNPs are exonic coding SNPs .', 'For SNPs listed in Table 1 , no significant heterogeneity across studies was found and the p-values ( as well as regression coefficients ) were not changed with or without adjustment of body weight .', 'The quality scores of imputed SNPs in Table 1 were >0 . 98 ( IMPUTE confidence score ) for the TwinsUK Study and >0 . 84 ( MACH variance ratio ) for the Rotterdam Study .', 'Cis-eQTLs were analyzed for eight candidate genes located within 500 kb in four genome-wide significant loci ( Table 2 ) .', 'All eight candidate genes were expressed in bone tissue estimated by either expressed sequence tag ( EST ) in the CGAP EST cDNA library ( Figure S1 ) or human primary osteoblast samples ( Table 2 ) .', 'However , since transcripts were not presented on expression arrays , expression of TBC1D8 was not available in human primary osteoblast samples .', 'P-values <0 . 005 estimated by false discovery rate ( FDR ) were considered as significant .', 'SNP rs494453 was found to be significantly associated with transcript levels of the RAP1A gene .', 'Allele C of rs494453 is in LD with allele A of rs3767595 ( haplotype ) .', 'The haplotype CA was associated with lower expression of RAP1A , but higher NW ( stronger bone structure ) in women .', 'We also performed eQTL analyses in human lymphocytes and liver tissue .', 'Expression level of the RAP1A gene was not available for either lymphocytes or liver tissue .', 'SNPs on chromosome 2q11 . 2 ( TBC1D8 and RLP31 ) and 8q24 . 12 ( TNFRSF11B ) loci were associated with gene expressions in lymphocytes ( Table 2 ) .', 'The most significant eSNP was found for SNP rs2278729 ( chromosome 2q11 . 2 ) with TBC1D8 expression in lymphocytes ( p\u200a=\u200a2 . 58×10−10 ) and liver tissue ( p<10−16 , Figure S3 ) .', 'Allele A of rs2278729 was associated with smaller NSA in men and also with lower expression of TBC1D8 transcript .', 'The same allele A was also associated with lower RPL31 expression in lymphocytes and was marginally significant in osteoblasts .', 'Consistency between the direction of effect on transcript levels in lymphocytes and LS BMD was observed for TNFRSF11B at the chromosome 8q24 . 12 locus , which confirmed a previous report that increased TNFRSF11B expression levels have been shown to inhibit bone resorption [32] .', 'A previous study also demonstrated that alleles associated with decreased BMD were associated with differential allelic expression of the TNFRSF11B in lymphocytes [22] .', 'However , we did not observe associations of genome-wide significant SNPs in/near the TNFRSF11B gene region with TNFRSF11B expression levels in human primary osteoblasts , possible due to lack of power .', 'We investigated the candidate genes corresponding to the genome-wide significant SNPs in 4 chromosomal regions by looking at reported gene functions ( including biological processes , canonical pathways and organism processes in human and mouse ) , microRNA targets and gene-related human diseases ( Table S3 ) .', 'Except for the TNFRSF11B gene , there were few additional data regarding the potential biological significance of other candidate genes being involved in skeletal development and bone remodeling; therefore , we performed additional analyses on expression profiles in animal experiments ( Table 3 ) .', 'In experiment 1 , we found that PTH negatively regulated expressions of OSBPL1A and TNFRSF11B .', 'RPL31 , IMPACT and RAP1A genes were expressed in PTH stimulated osteoblasts , but not regulated by PTH .', 'TBC1D8 were not expressed in PTH stimulated osteoblasts .', 'In experiment 2 , we analyzed the differential expression of candidates during osteoblast maturation .', 'As a quality control measure , we looked at a number of known osteoblast markers , including runt-related transcription factor 2 ( Runx2 ) , collagen type 1 , alpha 1 ( Col1a1 ) , collagen type 1 , alpha 2 ( Col1a2 ) , osteocalcin , osteopontin and osteonectin .', 'The expected expression patterns ( differential expression during maturation ) were observed in all cases .', 'We observed that the expression of OSBPL1A , IMPACT and COLEC10 was significantly different across a time course ( Day 4 , 5 , 6 , 8 , 16 , 25 and 30 post-differentiation ) of osteoblast development ( p<0 . 0083 ) .', 'In the third experiment using the LCMS algorithm in the B6XC3H F2 intercross mice , we found that OSBPL1A , IMPACT , RAP1A and COLEC10 genes were predicted to be causally linked with bone phenotypes ( detailed phenotypes listed in Table S4 ) based on the evidence of significantly pleiotropic effects on trait QTL and eQTL .', 'A total of 109 suggestive genome-wide associated regions/genes ( most significant SNP with meta-analysis 4 . 3×10−7< p-value ≤5×10−5 ) were selected based on the criteria that p-values showed nominal association in the Framingham , Rotterdam and TwinsUK studies .', 'Among them , 16 candidate genes were prioritized with results either involving the differential expression in osteoblasts or causally linked ( LCMS algorithm ) with bone phenotypes in mice ( Table 4 ) .', 'Among 16 prioritized candidate genes/loci , PPAP2B , GPR177 , TGFBI , DOCK1 , SOX6 and PDGFD gene expressions were regulated by PTH in osteoblasts .', 'Significant differential expression during osteoblast development was found for GPR177 , TGFBI , SOX6 and CDH2 genes .', 'IRX2 , TGFBI and CDH2 genes showed strong expression in the skeleton compared to 24 other subsets of organ/tissue systems of the mouse embryo .', 'Using the LCMS algorithm in inbred mice , 12 genes were predicted to be causally linked with bone phenotypes ( detailed phenotypes listed in Table S4 ) .', 'All of the prioritized candidate genes are expressed in bone tissues .', '10 genes were found to be expressed in human bone tissue from the CGAP EST cDNA library ( Figure S1 ) and the remained genes ( HECW2 , CASR , MMRN1 , IRX2 , SOX6 and SALL1 ) were found to be expressed in human primary osteoblasts .', 'To test the probability of our candidate genes clustering into a particular biological pathway , we performed a gene set enrichment test on 24 candidate genes ( 20 loci ) from Table 2 and Table 4 .', 'Due to lack of biological or functional annotation , IRX2 and FBXO31 genes were excluded from analyses .', 'We found a significant clustering ( Fisher exact test p\u200a=\u200a1 . 65×10−4; Benjamini-Hochberg multiple testing corrected p-value\u200a=\u200a0 . 03 ) of genes involved in adhesion of cells , including CASR , CDH2 , PPAP2B , RAP1A , TGFBI and TNFRSF11B genes .', 'We also estimated expression abundance by number of expressed sequence tag ( EST ) sequences per 200 , 000 tags in the CGAP EST cDNA library for these 24 candidate genes .', 'Among 48 human tissues and organs , candidate genes were expressed in bone ( 17 candidate genes ) , liver ( 22 candidate genes ) , muscle ( 18 ) and adipose tissue ( 12 ) ( Figure S1 and Figure S2 ) .', 'Expression levels of RAP1A ( p\u200a=\u200a2 . 51×10−4 ) , RPL31 ( p\u200a=\u200a3 . 03×10−7 ) and TNFRSF11B ( p\u200a=\u200a1 . 69×10−3 ) genes showed over-representation in bone ( Figure S1 ) .'], ['In this study we performed sex-specific genome-wide association studies for BMD at the LS and FN skeletal sites as well as geometric indices of the hip in adults from the Framingham Osteoporosis Study and then replicated the top finding in two independent studies .', 'As a result of meta-analyses on 7 , 633 women and 3 , 657 men , we discovered three novel genome-wide significant loci , including chromosome 1p13 . 2 RAP1A locus ( p\u200a=\u200a3 . 62×10−8; NW in men and women combined ) , 2q11 . 2 TBC1D8 locus ( p\u200a=\u200a1 . 48×10−7 , NSA in men ) and 18q11 . 2 OSBPL1A locus ( p\u200a=\u200a4 . 22×10−7 , NW in men ) .', 'We also confirmed TNFRSF11B gene on chromosome 8q24 . 12 to be associated with LS BMD in women only ( p\u200a=\u200a2 . 68×10−11 ) .', 'The RAP1A gene ( chromosome 1p13 . 2 ) was predicted to be causally linked with bone phenotypes in B6xC3H F2 intercross mice .', 'Compared to other tissues , expression levels of RAP1A showed over-representation in human bone tissue .', 'An eSNP ( rs494453 ) located in intron 2 of RAP1A gene was also found to be significantly associated with RAP1A gene expression in human primary osteoblasts .', 'A marginally significant differential expression during osteoblast maturation was also found in our study .', 'RAP1A , a GTPase that mediates calcium signal transduction , has been found to mediate activities of JnK [33] .', 'JnK has been reported to be involved in late stage osteoblast differentiation [34] and apoptosis of osteoblasts [35] .', 'Therefore , variants in the RAP1A gene may change the activities of JnK and then impact osteoblast maturation .', 'Further experiments are necessary to explore the role of the RAP1A gene .', 'Both OSBPL1A and IMPACT genes located in chromosome 18q11 . 2 region were predicted to be causally linked with bone phenotypes in mice .', 'Expressions of both genes were found to be significantly differential during osteoblast maturation .', 'However , only expression of the OSBPL1A gene in osteoblasts was regulated by PTH .', 'No significant eQTL was found in this region .', 'Given the genome-wide significant SNPs were located in the OSBPL1A gene , we still cannot rule out the involvment of the nearby IMPACT gene .', 'In addition , an in vitro study has shown that DDIT3 over-expression enhances osteoblastic differentiation in ST-2 stromal cells , a mechanism that may involve the formation of heterodimers with C/EBP-β and the sensitization of the BMP/Smad signaling pathway [36] .', 'IMPACT protein has found to decrease expression of mouse DDIT3 protein [37]; therefore , IMPACT may negatively regulate bone formation .', 'We estimated the statistical power of our meta-analysis at an α-level of 10−7 .', 'In women , the power was 62–99% and >80% for effect size ( h2 ) equal to 1% and 2% , respectively .', 'In men , the statistical power was 35–75% and >70% for effect size equal to 1% and 2% , respectively .', 'Inadequate statistical power seems to be one of the limitations in our study .', 'Therefore , we prioritized 16 candidate genes/loci out of 109 suggestive genome-wide suggestive candidate genes ( 4 . 3×10−7<p≤5×10−5 ) based on the expression profiling and the LCMS modeling relevant to the skeletal system .', 'Among 16 prioritized candidate genes/loci , PPAP2B , GPR177 , SOX6 and CDH2 genes have been reported to be involved in Wnt-signaling .', 'CASR , TGFBI and CACNB2 genes are involved in ossification , endochondrial bone formation in cartilage and calcium ion transportation , respectively ( Table S5 ) .', 'CASR knockout mice have demonstrated decreased bone density and abnormal bone mineralization [38] .', 'Variants in GPR177 , SOX6 and CASR genes were associated with LSBMD in women .', 'Variants in GPR177 and SOX6 ( 2 out of 3 above genes ) have been successfully replicated in a large-scale meta-analysis of BMD on 19 , 195 Caucasian subjects ( majority of whom were women ) with association p-values <10−9 [27] , but none of the non-prioritized candidates ( associated with BMD ) did .', 'These results support the concept of our prioritization strategy .', 'Candidate gene/SNP prioritization strategies by gene expression and bioinformatic databases leverage the complexity of the disease phenotypes , which offers some advantages over traditional association studies that rely on strictly p-value driven approaches .', 'A recent study demonstrated that using functional information in published references to identify the key biological relationships between genes was able to predict the success of validation in replication genotyping [39] , which also provides additional evidence for the soundness of using biological functional relevance to prioritize candidate genes from GWAS for future validation .', 'We exploited eSNP/eQTL in multiple human tissues .', 'Given that ( 1 ) disease-related human tissues are often difficult to obtain for research purposes; ( 2 ) eQTL analysis requires a large sample size to reach the statistical power necessary to observe subtle changes in gene expression [40]; and ( 3 ) all of the selected candidate genes were expressed in bone tissues , we believe that performing eQTL in multiple tissues , although not replacing eQTL analysis in bone tissue , does provide complementary information .', 'Genetic control of biological functions may be tissue-specific .', 'Analysis of cis- eQTL in the tissue type directly relevant to the phenotype has been generally shown to be more informative than the same analysis in unrelated tissue types ( such as blood ) .', 'However , studies have found that cis-eQTLs are conserved across tissues , when genes are actively expressed in those tissues [10] , [12] , [13] , [41]–[44] .', 'eQTL analyses in liver , adipose , brain and muscle tissues from the same individual mice suggested that , for a gene exhibiting significant cis-eQTL associations in one tissue , 63–88% ( dependent on tissue types ) of them also exhibit cis-eQTL associations in another tissue [42] .', 'Two recent studies , quantifying allele-specific gene expression in four human cell lines ( lymphoblastoid cell , two primary fibroblasts and primary keratinocytes ) from the same individuals , observed that only 2 . 3–10% of the mRNA-associated SNPs showed tissue-specific cis-expression across these cell lines [43] , [44] .', 'They also found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations , much more so than by specific tissue types or growth conditions [43] .', 'Among the highly heritable transcripts ( within the upper 25th percentile for heritability ) , 70% of expression transcripts that had a significant cis-eQTL in adipose tissue also had a significant cis-eQTL in blood cells [45] .', 'Comparing eQTL in human primary fibroblasts , Epstein-Barr virus-immortalized B cells and T cells revealed that cell-type-shared eQTL tend to have larger effects , higher significance and to cluster tightly around the transcription start site [46] .', 'As for bone tissue , comparing gene expression in 58 human primary osteoblast samples and 57 lymphoblastoid cell samples , despite tissues obtained from different individuals , indicated that overall , there is a large overlap in genes expressed in these two cell types , as well as the associated functional pathways [47] .', '60% of the top 100 eSNP in human lymphoblastoid cells also showed associations in human primary osteoblasts , which indicated that both tissue-independent and dependent eSNP were observed in primary osteoblasts and lymphoblastoid cells [47] .', 'Taken together this evidence suggests that if genes are expressed across tissues , their allele-specific expression can be preserved and highly correlated across tissues .', 'Thus , the expression of a gene in liver or other non-bone tissues may not directly cause a change in bone; however , it is possible that its allele-specific expression in liver is highly correlated with allele-specific expression in bone .', "Because of these correlations it is possible that a gene's expression in adipose or liver can serve as surrogate markers to study the eQTL; however , the real causal relationship would be occurring in bone .", "It is important to note that a lack of evidence from mining publicly available gene expression experiments does not necessarily exclude a gene's involvment in skeletal metabolism , given that ( 1 ) experimental models such as osteoblastogenesis or early skeletal development , do not represent all relevant processes related to osteoporosis; ( 2 ) variation in a gene leading to disease may affect protein function but not expression; and ( 3 ) absence of association between a transcript and disease-associated SNP may be due to limited statistical power or under different environmental conditions .", 'An inherent limitation in most of the cell line in-vitro gene expression profiling experiments , such as our PTH treated osteoblasts or Epstein-Barr virus-immortalized lymphoblastoid cell lines used to perform eQTL analysis in most of the GWAS , is that the expression profiling of the cultured cells may be varying from actual expression within in-vivo cells [10] .', 'An additonal challenge of using available experimental data is that most of the studies performed gene profing using commercialized “genome-wide” chips , which usually have a fixed number of genes and often do not include all set of genes of given interest .', 'Therefore , prioritization of candidate genes will be biased towards well-studied genes .', 'Few published GWAS have addressed the potential sex-difference in genetic risks of diseases .', 'BMD and hip geometry for men and women are known to differ , as does the prevalence of osteoporotic fractures [48] .', 'Gender differences in the heritability of osteoporosis-related phenotypes have been reported ( reviewed in [49] ) .', 'In the current study , few overlapping associated SNPs between men and women were found , which may be expected based on epidemiological and clinical data and may also be due to lack of power .', 'Sex-specific associations may be due to lifestyle and environmental variation between men and women .', 'However , it also indicates that common genetic effects for both genders may be relatively rare and therefore , larger sample sizes of men and women is needed to detect their existence .', 'Another limitation is that we are unable to distinguish the gender-specific differential expressions , since gene expression is measured in a pooled mixture of osteoblasts from males and females , although , differentiated expression between sexes is actually less likely to occur in-vitro .', 'In summary , our study identified three novel genome-wide significant loci and prioritized 16 genome-wide suggestive candidate genes for BMD and hip geometry traits .', 'Beyond generating a list of top associated SNPs by statistical signals , we highlighted the efficiency of our approach to reasonably prioritize association findings by utilizing publicly available expression profiling relevant to the skeletal system in cellular or whole animal models; and to provide supportive biological information for future functional validation of their involvements in bone metabolism .', 'Resequencing of these loci is needed to determine the causal variants and genes , along with experimental functional studies to establish their precise mechanism linked to bone health related phenotypes .'], ['Joint analysis for results from both discovery and replication stages almost always results in greater power than analyzing discovery and replication stages separately [61] .', 'We selected SNPs with association test p-values less than 10−3 from Stage I discovery GWAS , and replicated them using meta-analysis by combining results from the Framingham Study and two independent population-based cohorts including the Rotterdam Study and the TwinsUK Study .', 'Since both the Rotterdam and TwinsUK studies performed whole-genome genotyping using different platforms ( Illumina platforms ) , SNP imputation was performed .', 'Fixed effect meta-analyses were then used to estimate combined p-values .', 'We conducted expression quantitative trait locus ( eQTL ) analysis to evaluate whether the genome-wide significant SNPs for each locus also influence transcript levels of nearby genes as a cis-effect regulator ( eSNP ) in human primary osteoblasts , lymphocytes and liver tissue .', 'In each locus , we selected nearby genes in which the genome-wide significant SNP was located within 500 Kb in the 5′ upstream of candidate genes with the assumption that SNPs are located in ( or in LD with the variants located in ) regulation elements of candidate genes .', 'Expression experiments in primary osteoblasts , lymphocytes and liver tissues were conducted in three different study samples .', 'For un-genotyped SNPs , imputed SNPs ( MACH variance ratio >0 . 3 ) were used in the lymphocyte expression dataset and surrogate SNPs with LD r2≥0 . 5 were used in primary osteoblasts and liver tissue datasets .']]
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Osteoporosis is a complex disorder and commonly leads to fractures in elderly persons .', 'Genome-wide association studies ( GWAS ) have become an unbiased approach to identify variations in the genome that potentially affect health .', 'However , the genetic variants identified so far only explain a small proportion of the heritability for complex traits .', 'Due to the modest genetic effect size and inadequate power , true association signals may not be revealed based on a stringent genome-wide significance threshold .', 'Here , we take advantage of SNP and transcript arrays and integrate GWAS and expression signature profiling relevant to the skeletal system in cellular and animal models to prioritize the discovery of novel candidate genes for osteoporosis-related traits , including bone mineral density ( BMD ) at the lumbar spine ( LS ) and femoral neck ( FN ) , as well as geometric indices of the hip ( femoral neck-shaft angle , NSA; femoral neck length , NL; and narrow-neck width , NW ) .', 'A two-stage meta-analysis of GWAS from 7 , 633 Caucasian women and 3 , 657 men , revealed three novel loci associated with osteoporosis-related traits , including chromosome 1p13 . 2 ( RAP1A , p\u200a=\u200a3 . 6×10−8 ) , 2q11 . 2 ( TBC1D8 ) , and 18q11 . 2 ( OSBPL1A ) , and confirmed a previously reported region near TNFRSF11B/OPG gene .', 'We also prioritized 16 suggestive genome-wide significant candidate genes based on their potential involvement in skeletal metabolism .', 'Among them , 3 candidate genes were associated with BMD in women .', 'Notably , 2 out of these 3 genes ( GPR177 , p\u200a=\u200a2 . 6×10−13; SOX6 , p\u200a=\u200a6 . 4×10−10 ) associated with BMD in women have been successfully replicated in a large-scale meta-analysis of BMD , but none of the non-prioritized candidates ( associated with BMD ) did .', 'Our results support the concept of our prioritization strategy .', 'In the absence of direct biological support for identified genes , we highlighted the efficiency of subsequent functional characterization using publicly available expression profiling relevant to the skeletal system in cellular or whole animal models to prioritize candidate genes for further functional validation .']
['BMD and hip geometry are two major predictors of osteoporotic fractures , the most severe consequence of osteoporosis in elderly persons .', 'We performed sex-specific genome-wide association studies ( GWAS ) for BMD at the lumbar spine and femor neck skeletal sites as well as hip geometric indices ( NSA , NL , and NW ) in the Framingham Osteoporosis Study and then replicated the top findings in two independent studies .', 'Three novel loci were significant: in women , including chromosome 1p13 . 2 ( RAP1A ) for NW; in men , 2q11 . 2 ( TBC1D8 ) for NSA and 18q11 . 2 ( OSBPL1A ) for NW .', 'We confirmed a previously reported region on 8q24 . 12 ( TNFRSF11B/OPG ) for lumbar spine BMD in women .', 'In addition , we integrated GWAS signals with eQTL in several tissues and publicly available expression signature profiling in cellular and whole-animal models , and prioritized 16 candidate genes/loci based on their potential involvement in skeletal metabolism .', 'Among three prioritized loci ( GPR177 , SOX6 , and CASR genes ) associated with BMD in women , GPR177 and SOX6 have been successfully replicated later in a large-scale meta-analysis , but none of the non-prioritized candidates ( associated with BMD ) did .', 'Our results support the concept of using expression profiling to support the candidacy of suggestive GWAS signals that may contain important genes of interest .']
['genetics and genomics/gene expression', 'genetics and genomics/complex traits', 'genetics and genomics/genetics of disease', 'diabetes and endocrinology/bone and mineral metabolism', 'genetics and genomics/bioinformatics']