This study represents the first published GWAS for hip BS, an important risk factor for HF. Through this study we identified a novel gene, PLCL1 that had four SNPs associated with hip BS at, or approaching, the genome-wide significance level in our female subjects. The gene's association with hip BS was replicated in an independent UK sample despite it using a different platform and selection of SNPs. Two SNPs in the UK sample, that are surrounded by the four interesting SNPs identified in our GWAS, were associated with hip BS with p values less than 0.01 (). Imputation analyses of our GWAS and the UK samples showed a significant overlap of association signals in intron 5 of the PLCL1 gene and immediately downstream from that gene (). In particular, according to the imputation analyses, a narrow window of <6 kb in intron 5 contained four SNPs that achieved p values less than 1×10−5 (including two of genome-wide significance) in our GWAS sample, and four SNPs that achieved p values less than 0.01 in the UK sample, ( and ). Combining p values of the imputed SNPs in our GWAS and the UK samples, we found that eight SNPs of the PLCL1 gene achieved combined p values less than 10−5, five additional SNPs achieved combined p values less than 10−4, and fifteen more SNPs achieved combined p values less than 0.01 ().
Importantly, we also demonstrated the relevance of PLCL1
to HF. A SNP of this gene, rs3771362, that is only ~0.6 kb away from the most significant SNP identified in our GWAS (rs7595412), achieved a p
value of 7.66×10−3
and an odds ratio of 0.26 (95% CI: 0.09–0.75) for association with HF in an independent Chinese sample. PLCL1
encodes an inositol 1,4,5-trisphosphate (IP3) binding protein that can inhibit IP3 mediated calcium signaling 
, an important pathway that regulates the response of bone cells to mechanical signals 
. Overall, our association findings, together with PLCL1
's potential functional relevance to bone mechanical sensing, provide strong evidence for the gene's importance for hip BS variation and the pathogenesis of HF.
In our GWAS sample, the PLCL1
gene's association with hip BS was limited only to women, suggesting female-specificity of this gene's importance to osteoporosis. The female-specificity of the gene cannot be determined in the UK replication cohort since the cohort contains only women but no men 
. However, analysis results in the Chinese HF cohort seem to support the female-specificity of the gene as the gene was associated with HF in females but not in males (data not shown). Our findings are consistent with previous studies suggesting that genetic control of bone parameters may be gender-specific 
In this study, replication of the PLCL1 gene's association with hip BS in the UK sample was achieved “gene-wise” rather than “SNP-wise”. The most significant SNPs of the gene detected/imputed in our GWAS sample did not achieve nominally significant p values in the UK sample (), although the use of different genotyping platforms may be partly responsible. However, significant replication signals did show up in the UK sample in many other SNPs of the PLCL1 gene, which are in neighborhood of those significant SNPs found in the GWAS sample, according to both the experimental data ( and ) and the imputation data ( and & ).
BS can be measured as bone volume (cm3
), bone area (cm2
), or bone length (or diameter), and each of these measurements is legitimate and reflects various properties of bone. Areal BS measured by DXA, as adopted in this study, is a useful and reliable bone phenotype. Compared with other types of BS measurements, areal BS is relatively precise and involves less exposure to radiation during measurement 
, enhancing its safety and feasibility for large-scale clinical investigation. Areal BS is also highly correlated with bone strength 
and osteoporotic fractures 
, and this correlation is largely independent from BMD 
. Consequently, genetic study of BS may provide a unique perspective to osteoporosis research, because current research is largely dominated by studies of BMD.
Population stratification and/or ethnic admixture can be an important source of spurious association in genetic association studies. These factors are unlikely to exist in our samples, however, and are therefore unlikely to interfere with our association results. The study cohort for our GWAS came from an apparently homogenous US mid-west white population, living in Omaha, Nebraska and its surrounding areas. The relative homogeneity of this population is largely due to the predominance of Caucasians as the major ethnic group in this area for many generations. According to the recent 2000 census, ethnic minorities made up only less than 3% of the entire population in the State of Nebraska. In addition, we found that the allelic frequencies of the significant SNPs in our GWAS sample are very similar to those reported in the typical and representative Caucasian samples used in the HapMap CEU (). Furthermore, using Structure 2.2 
, we thoroughly analyzed study subjects used in our GWAS in order to detect potential sub-populations in the sample. In the analyses, all subjects tightly clustered together as a single group, suggesting no population substructure in our sample (Appendix S2
). Calculated through the genomic control method 
, the measure for population stratification (λ) for our GWAS sample, was 1.007, suggesting essentially no stratification. Using the same approaches, we analyzed the UK replication sample and our Chinese HF sample and achieved similar results. For the above reasons, the association results, as detected in our study, are not likely to be plagued by spurious associations due to population admixture/stratification. Moreover the wide generalizability of the PLCL1
gene's association makes this unlikely
In summary, we identified a novel gene, PLCL1
, associated with hip BS through a GWAS. Our success in replicating the association of this gene with hip BS in a UK sample and the relevance of this gene to HF in a Chinese cohort, combined with the gene's functional implication in mechanical sensing by bone cells, makes it a strong candidate for regulation of hip BS and a potential key factor in the pathogenesis of HF. Apparent female specificity for the gene's association with hip BS supports gender-specific genetic basis of osteoporosis as suggested by previous studies 
and provides new insights into our understanding of differential HF risk between females and males. As a future direction of research, we will perform fine-mapping of the PLCL1
gene in an enlarged independent Caucasian cohort to validate the gene's importance to hip BS and HF, including replicating the two SNPs that showed association with hip BS in the UK cohort (). In addition, focused molecular functional studies will be pursued to clarify the mechanisms by which this gene regulates hip BS variation and contributes to HF risk. The gene may also be related to bone growth in general – which also merits further work.