Abstract
Osteoporosis is a serious public health issue, which is mostly characterized by low bone mineral density (BMD). To search for additional genetic susceptibility loci underlying BMD variation, an effective strategy is to focus on testing of specific variants with high potential of functional effects. Single nucleotide polymorphisms (SNPs) that introduce or disrupt CpG dinucleotides (CpG-SNPs) may alter DNA methylation levels and thus represent strong candidate functional variants. Here, we performed a targeted GWAS for 63,627 potential functional CpG-SNPs that may affect DNA methylation in bone-related cells, in five independent cohorts (n = 5905). By meta-analysis, 9 CpG-SNPs achieved a genome-wide significance level (p < 7.86 × 10−7) for association with lumbar spine BMD and additional 15 CpG-SNPs showed suggestive significant (p < 5.00 × 10−5) association, of which 2 novel SNPs rs7231498 (NFATC1) and rs7455028 (ESR1) also reached a genome-wide significance level in the joint analysis. Several identified CpG-SNPs were mapped to genes that have not been reported for association with BMD in previous GWAS, such as NEK3 and NFATC1 genes, highlighting the enhanced power of targeted association analysis for identification of novel associations that were missed by traditional GWAS. Interestingly, several genomic regions, such as NEK3 and LRP5 regions, contained multiple significant/suggestive CpG-SNPs for lumbar spine BMD, suggesting that multiple neighboring CpG-SNPs may synergistically mediate the DNA methylation level and gene expression pattern of target genes. Furthermore, functional annotation analyses suggested a strong regulatory potential of the identified BMD-associated CpG-SNPs and a significant enrichment in biological processes associated with protein localization and protein signal transduction. Our results provided novel insights into the genetic basis of BMD variation and highlighted the close connections between genetic and epigenetic mechanisms of complex disease.
Highlights
Osteoporosis is a complex disease mainly characterized by low bone mineral density (BMD) and microarchitectural deterioration of bone tissue, which results in an increased risk of bone fragility and susceptibility to fracture [1]
The discovery dataset incorporated a total of 5905 subjects from five Genome-wide association studies (GWAS), of which three studies were “in-house” studies: (1) Omaha Osteoporosis Study (Caucasian ancestry, n = 987), (2) Kansas City Osteoporosis Study (Caucasian ancestry, n = 2250), and (3) China Osteoporosis Study (Han Chinese ancestry, n = 1547), and two studies were “external” studies obtained from the Database on Genotypes and Phenotypes: (1) Women’s Health Initiative Observational Study African-American Substudy (African ancestry, n = 712) and (2) Women’s Health Initiative Observational Study Hispanic Substudy (Hispanic ancestry, n = 409)
Over 50% of these potential functional CpG-Single nucleotide polymorphisms (SNPs) were mapped to introns, we observed a significant enrichment of potential functional CpG-SNPs in 5′/3′-UTR regions and underrepresentation in intergenic regions (Supplementary Figure 1), when comparing to the overall profile of CpG-SNPs in the human genome
Summary
Osteoporosis is a complex disease mainly characterized by low bone mineral density (BMD) and microarchitectural deterioration of bone tissue, which results in an increased risk of bone fragility and susceptibility to fracture [1]. Genome-wide association studies (GWAS) and metaanalyses of these studies have successfully identified over 250 genetic loci associated with BMDs at different skeletal sites [5,6,7,8,9,10,11]. These loci explained approximately 12% of BMD variation [11] and the specific functional variants at these loci were generally unknown. To search for additional genetic loci and to enhance our understanding of the biological mechanisms underlying BMD variation, one effective strategy is to focus on testing of specific variants with high potential of functional effects, such as exonic/nonsynonymous variants [5, 12] or variants that may potentially
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