Promoter and 3′-Untranslated-Region Haplotypes in the Vitamin D Receptor Gene Predispose to Osteoporotic Fracture: The Rotterdam Study  Yue Fang, Joyce B.J. van Meurs, Arnold d'Alesio, Mila Jhamai, Hongyan Zhao, Fernando Rivadeneira, Albert Hofman, Johannes P.T. van Leeuwen, Frédéric Jehan, Huibert A.P. Pols, André G. Uitterlinden  The American Journal of Human Genetics  Volume 77, Issue 5, Pages 807-823 (November 2005) DOI: 10.1086/497438 Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 1 Genomic structure and LD map of the human VDR gene. a, Physical organization of the 12q12 area containing VDR, mostly based on the Celera database (47032–47145 kb at chromosome 12q12). The arrows for each gene indicate the transcription direction, and distances are in kb. b, Genomic structure of the human VDR gene. Black bars indicate the coding exons of VDR, and the gray bars indicate 5′ exons and the 3′ UTR. c, Sequenced areas and positions of the 62 variations. Gray bars in the 3′-UTR indicate destabilizing elements (DE 1, 2, and 3 [Durrin et al. 1999]). The sequence-variation numbers refer to those given in table 3. d, Haplotype map of VDR in whites, Asians, and African Americans, based on SNPs with an MAF ≥5% in each of the different ethnic populations. Common haplotype alleles in each block with a frequency >3% are presented below the blocks. SNPs and alleles in red indicate htSNPs. Fracture-risk haplotype alleles are underlined. The correspondence is shown for whites to haplotypes in block 5 of the BsmI-ApaI-TaqI haplotype alleles we defined elsewhere (Uitterlinden et al. 1996). The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 1 Genomic structure and LD map of the human VDR gene. a, Physical organization of the 12q12 area containing VDR, mostly based on the Celera database (47032–47145 kb at chromosome 12q12). The arrows for each gene indicate the transcription direction, and distances are in kb. b, Genomic structure of the human VDR gene. Black bars indicate the coding exons of VDR, and the gray bars indicate 5′ exons and the 3′ UTR. c, Sequenced areas and positions of the 62 variations. Gray bars in the 3′-UTR indicate destabilizing elements (DE 1, 2, and 3 [Durrin et al. 1999]). The sequence-variation numbers refer to those given in table 3. d, Haplotype map of VDR in whites, Asians, and African Americans, based on SNPs with an MAF ≥5% in each of the different ethnic populations. Common haplotype alleles in each block with a frequency >3% are presented below the blocks. SNPs and alleles in red indicate htSNPs. Fracture-risk haplotype alleles are underlined. The correspondence is shown for whites to haplotypes in block 5 of the BsmI-ApaI-TaqI haplotype alleles we defined elsewhere (Uitterlinden et al. 1996). The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 2 Conservation of the human and mouse genomic VDR gene sequence. The Y-axis is the homology rate between human and mouse; the X-axis is the physical distance on the human VDR gene. All exons are indicated in purple, the 3′ UTR in light green, and the conserved noncoding region in red. The small black bars on the top of each frame indicate the polymorphisms we observed by resequencing, and the gray arrow on top indicates the transcription direction of VDR. The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 3 LD structure of VDR in whites. a, Blocks with pairwise D′>0.8 are numbered 1–5. The analyzed SNPs (table 1) include 39 VDR SNPs, 5 SNPs in the COL2A1 and VDR intergenic region (“IGR VDR & COL2A1”), and 3 SNPs in the VDR and HDAC7A intergenic region (“IGR HDAC7A & VDR”). SNP IDs correspond to those in figure 1 and tables 1 and 3. The red boxes on the X- and Y-axes indicate the high-LD blocks used to define haplotype alleles. The physical organization of VDR is represented with vertical lines on the Y-axis (see also fig. 1fig. 1). b, Aligned LD analyses from different sources and estimated consensus LD structure of VDR. The total number of SNPs analyzed in each study is indicated in parentheses. Thick lines indicate haplotype blocks, with the number of analyzed SNPs below the line and the name of the block above the line. The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 4 LD maps of VDR in different ethnic groups. a, LD map of 33 SNPs in 107 Asians (214 chromosomes). b, LD map of 41 SNPs in 58 African Americans (116 chromosomes). The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 4 LD maps of VDR in different ethnic groups. a, LD map of 33 SNPs in 107 Asians (214 chromosomes). b, LD map of 41 SNPs in 58 African Americans (116 chromosomes). The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 5 HR for clinical fracture, by VDR genotypes based on haplotype alleles in five haplotype blocks (1–5) and the FokI RFLP. The HR point estimates and the surrounding 95% CIs are represented with colored squares and lines. The HR for one copy of the test allele versus no copy is in blue; the HR for two copies of the allele versus no copy is in red. The logarithmic HR is plotted for the common haplotype alleles (frequency >3%) in all haplotype blocks (see fig. 1d for whites) and the FokI RFLP. The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 6 EMSA of 1a-A−1012G for GATA protein. a, GATA binding assay using Caco2 cell-line nuclear extract. The binding of GATA to the 1a−1012A site was analyzed in competition experiments for Caco2 nuclear extract by use of the well-characterized GATA sites of the EpoR and Lactase gene promoters (Zon et al. 1991; Fang et al. 2001). These experiments showed similar binding characteristics of the complexes bound to the 1a−1012A site and to the other GATA sites (compare lane 7 to lanes 1 and 4). In addition, the signal found on the 1a−1012A site was eliminated by a 100-fold excess of unlabeled GATA sites of the EpoR and Lactase genes (see lanes 7–10). Conversely, a 100-fold excess of the 1a−1012A site eliminated the binding of GATA to the EpoR and Lactase GATA sites (lane 1 vs. 2 and lane 4 vs. 5). b, GATA binding using HEK293 cell-line nuclear extract. Competition experiments for HEK293 nuclear extract using the −1012 GATA site as a probe revealed that the 1a−1012G variant was unable to compete with the binding of the 1a−1012A variant. The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 7 Relative luciferase activity in HEK293 cells of VDR exon 1a promoter sequences, including two SNPs. a, The three constructs containing the 2-kb 1a promoter sequence with SNPs 1a-G−1521C and 1a-A−1012G. b, β-galactosidase (beta-Gal)–normalized luciferase activity for the three constructs. The block 2–hap1 allele is set at 100% to be the reference group; P values were calculated by independent t test. The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 8 Two SNPs in the 1e promoter region of the human VDR gene, located at a Cdx-2 binding site. a, Alignment of 15 well-characterized Cdx-2 sites from mammalian gene promoters. Base usage is summarized in a table and is compared with DNA sequences surrounding SNPs 1e-C−2090T and 1e-G−1739A. DNA bases involved in SNPs are underlined. b, EMSA experiments performed using Caco-2 cell nuclear extracts. Double-strand oligonucleotides containing the sucrose isomaltase (SIF) Cdx2-binding site as a control or sequences encompassing SNPs 1e-C−2090T and 1e-G−1739A were 32P-labeled and were purified on a 10% polyacrylamide gel. The competition experiments were done with an oligonucleotide containing the SIF element. Gel-shift experiments were performed in the absence (−) or presence (+) of a 100-fold excess of cold SIF probe as a competitor, which resulted in relatively more elimination of these specific complexes for the A allele of 1e-G−1739A and the T allele of 1e-C−2090T than for their allelic counterparts. c, Antibody experiments with monoclonal anti–Cdx-2. EMSA was performed with nuclear extract alone (−) or in the presence of monoclonal anti–Cdx-2 antibody (+). Supershift complexes are identified by an arrow. A clear supershift was observed with SIF, but it had a weak signal with 1e−1739G, and a very low intensity of the complex was seen with 1e−2090. Comparison of the surrounding 1e-C−2090T and 1e-G−1739A sequences with the consensus Cdx-2 sequence (panel a) evidenced the presence of a substitution (T→A) in the 1a−2090 sequence, somehow corrupting the Cdx-2 site, which may explain the lower intensity observed in EMSA for this sequence compared with 1e-G−1739A. The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 8 Two SNPs in the 1e promoter region of the human VDR gene, located at a Cdx-2 binding site. a, Alignment of 15 well-characterized Cdx-2 sites from mammalian gene promoters. Base usage is summarized in a table and is compared with DNA sequences surrounding SNPs 1e-C−2090T and 1e-G−1739A. DNA bases involved in SNPs are underlined. b, EMSA experiments performed using Caco-2 cell nuclear extracts. Double-strand oligonucleotides containing the sucrose isomaltase (SIF) Cdx2-binding site as a control or sequences encompassing SNPs 1e-C−2090T and 1e-G−1739A were 32P-labeled and were purified on a 10% polyacrylamide gel. The competition experiments were done with an oligonucleotide containing the SIF element. Gel-shift experiments were performed in the absence (−) or presence (+) of a 100-fold excess of cold SIF probe as a competitor, which resulted in relatively more elimination of these specific complexes for the A allele of 1e-G−1739A and the T allele of 1e-C−2090T than for their allelic counterparts. c, Antibody experiments with monoclonal anti–Cdx-2. EMSA was performed with nuclear extract alone (−) or in the presence of monoclonal anti–Cdx-2 antibody (+). Supershift complexes are identified by an arrow. A clear supershift was observed with SIF, but it had a weak signal with 1e−1739G, and a very low intensity of the complex was seen with 1e−2090. Comparison of the surrounding 1e-C−2090T and 1e-G−1739A sequences with the consensus Cdx-2 sequence (panel a) evidenced the presence of a substitution (T→A) in the 1a−2090 sequence, somehow corrupting the Cdx-2 site, which may explain the lower intensity observed in EMSA for this sequence compared with 1e-G−1739A. The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions

Figure 9 VDR mRNA expression level and stability analysis by 3′-UTR haplotypes in different cell lines. a, VDR 3′-UTR with sequence variations that distinguish hap1 (corresponding to block 5–hap1) from hap2 (corresponding to block 5–hap2). The SNP variation number (in parentheses) refers to those given in table 3. b, Neomycin-normalized VDR mRNA expression levels (mean ± SD) for VDR 3′-UTR hap1 versus hap2. The level of hap2 is set at 100% to be the reference group; P values were calculated by independent t test; n = number of experiments for each cell line. c, Decay rate of VDR mRNA by hap1 versus hap2, determined in the MG63 cell line. Time point 0 h is defined as 100% mRNA level for both haplotypes; P values for each time point were calculated by independent t test. The American Journal of Human Genetics 2005 77, 807-823DOI: (10.1086/497438) Copyright © 2005 The American Society of Human Genetics Terms and Conditions