Genome-wide association and follow-up replication studies identified ADAMTS18 and TGFBR3 as bone mass candidate genes in different ethnic groups

Abstract

To identify and validate genes associated with bone mineral density (BMD), which is a prominent osteoporosis risk factor, we tested 379,319 SNPs in 1000 unrelated white U.S. subjects for associations with BMD. For replication, we genotyped the most significant SNPs in 593 white U.S. families (1972 subjects), a Chinese hip fracture (HF) sample (350 cases, 350 controls), a Chinese BMD sample (2955 subjects), and a Tobago cohort of African ancestry (908 males). Publicly available Framingham genome-wide association study (GWAS) data (2953 whites) were also used for in silico replication. The GWAS detected two BMD candidate genes, ADAMTS18 (ADAM metallopeptidase with thrombospondin type 1 motif, 18) and TGFBR3 (transforming growth factor, beta receptor III). Replication studies verified the significant findings by GWAS. We also detected significant associations with hip fracture for ADAMTS18 SNPs in the Chinese HF sample. Meta-analyses supported the significant associations of ADAMTS18 and TGFBR3 with BMD (p values: 2.56 x 10(-5) to 2.13 x 10(-8); total sample size: n = 5925 to 9828). Electrophoretic mobility shift assay suggested that the minor allele of one significant ADAMTS18 SNP might promote binding of the TEL2 factor, which may repress ADAMTS18 expression. The data from NCBI GEO expression profiles also showed that ADAMTS18 and TGFBR3 genes were differentially expressed in subjects with normal skeletal fracture versus subjects with nonunion skeletal fracture. Overall, the evidence supports that ADAMTS18 and TGFBR3 might underlie BMD determination in the major human ethnic groups.

Figure 1

Genome-wide Association Results for BMD in the White U.S. GWAS Sample (A) Genome-wide association results for hip BMD in the female sample by the single-SNP approach. (B) Genome-wide association results for spine BMD in the total sample by the 5-SNP-size sliding-window approach.

Figure 2

Association Signals in the ADAMTS18 Gene in 16q23 (A) Genomic locations of chromosome 16q23 genes between 75873929 and 76026512 bp. (B) The negative log₁₀ p values from the female sample are plotted for genotyped markers in the ADAMTS18 region. (C) Pairwise r² plot for the genotype data of ADAMTS18 in this study. The intensity of shading is proportional to r². The x axis represents physical positions. (D) Enlarged picture of the significant region in ADAMTS18. The numbers on the x axis are the SNP IDs corresponding to those in Table S1.

Figure 3

Association Signals in the TGFBR3 Gene on the Chromosome Region 1p22 (A) Genomic locations of chromosome 1p22 genes between 91900000 and 92150000 bp. (B) The negative log₁₀ p values from the total sample are plotted for genotyped markers in the TGFBR3 region. (C) Pairwise r² plot for the genotype data of TGFBR3 in this study. The intensity of shading is proportional to r². The x axis represents physical positions. (D) Enlarged picture of the significant region in TGFBR3. The numbers on the x axis are the SNP IDs corresponding to those in Table S2.

Figure 4

Average Raw BMD Values for Groups Stratified by Different Alleles at rs11864477 in ADAMTS18 and at rs17131547 in TGFBR3 Error bars denote standard error.

Figure 5

Quantile-Quantile Plots for Hip BMD and Spine BMD Associations (A) Quantile-quantile plots for hip BMD associations in the GWAS female sample. (B) Quantile-quantile plots for spine BMD associations in the GWAS total sample. Axes represent the following information: y axis, observed −log₁₀(p) values; x axis, p values expected under the null distribution for the GWAS SNPs.

Figure 6

Electrophoretic Mobility Shift, Supershift, and Competition Assays with E.coli BL21 Nuclear Cell Extract and Allelic Variants of SNP rs16945612 in ADAMTS18 The 25 bp oligonucleotides containing both allelic variants of SNP rs16945612, representing native and mutated TEL-2b binding sites, were assayed with increasing (0–5.7 μg) amounts of nuclear extract of E.coli BL21 cells. Binding was not observed with oligonucleotide containing native allele T (lanes 1–3) but was present with oligonucleotide containing the mutant allele C (lanes 5–11). Binding to the mutant C allele resulted in a complex that was specifically competed by 125 × excess of unlabeled mutant TEL-2b binding-site probe containing the C allele (lane 10) but not by the same amount of unrelated oligonucleotide (lane 11). Rabbit polyclonal antibody against TEL2 (α-TEL2) revealed a much weakened band due to supershifting of the band (lane 8), whereas unrelated serum did not produce the supershift (lane 9).