Characteristics for the 20 kb Downstream and Upstream Regions around the Jagged 1 Gene
(A) The fine scale recombination rate (right scale) across the studied region (HapMap rel22_B36); and the −log₁₀ (p) values (left scale) plot for single marker association results of BMD at lumbar spine (LS, diamond dots) and femoral neck (FN, circle dots). The colors of dots are coded according to the degree of linkage disequilibrium (LD) with SNP rs2273061 identified originally in the genome-wide association discovery stage (r² ≥ 0.8 red; 0.5 ≤ r² < 0.8 orange; 0.2 ≤ r² < 0.5 yellow; r² < 0.2 white). The solid horizontal line at the bottom represents the JAG1 gene region (NCBI_B36) and its vertical bars represent exons.
(B) LD plot for the studied region based on the r² statistic. Blocks connecting SNP pairs are shaded based on the LD strength between SNPs by using the disequilibrium coefficient r², which are ranged from 0 (white) to 1.0 (black).

JAG1 Gene Expression in Subjects with Different rs2273061 Genotypes
(A and B) JAG1 mRNA expression normalized to GAPDH by reverse transcription PCR (RT-PCR) in human bone-derived cells (HBDCs, A) and peripheral blood mononuclear cells (PBMCs, B) from individual subjects with GG, GA, and AA genotype of rs2273061.
(C) The relative quantity of JAG1 mRNA expression normalized to ribosomal RNA by quantitative real-time PCR (q-PCR) in PBMCs from individuals with different rs2273061 genotypes (n = 20 per genotype, results are mean ± SD).
HBDCs and PBMCs from healthy subjects were isolated as described previously. 46,47 Total RNA was extracted from HBDCs and PBMCs with Trizol reagent according to the manufacturer's instruction (Invitrogen, CA). 0.5 μg RNA was used for cDNA synthesis by M-MLV Reverse Transcriptase according to the manufacturer's protocol (Invitrogen, CA). (A) and (B) showed JAG1 gene expression with RT-PCR with primers (5′-TCGAGTTGGAGATCCTGTCC-3′) and (5′-GGGTGTGGGATGCACTTATC-3′) spanning exon 2 to 4 with annealing temperature at 59°C. Results were normalized to GAPDH gene expression with primers (5′-GCCTCCTGCACCACCACC-3′) and (5′-CCGTTCAGCTCAGGGATGA-3′). (C) showed q-PCR results with Taqman one-step PCR master mix kit with gene-specific target primers and 3-FAM probes of JAG1 gene (ABI gene expression assay ID number Hs00164982_m1). Each sample was simultaneously quantified with eukaryotic 18S primers and target probes (ABI gene expression assay 4319413E). Amplification was performed in an ABI Prism 7000 sequence detector, with standard settings of 40 cycles with an annealing temperature of 60°C. All samples were assayed in duplicates. Results were expressed as fold-change of JAG1 gene expression of GG and GA genotypes relative to AA genotype by comparative Ct method (−2^ΔΔCT). q-PCR in PBMCs revealed the GG genotype group had approximately 30-fold higher JAG1 mRNA expression levels than the AA genotype group (p = 0.037, t test).

Quantile-Quantile Plots of Discovery Test Statistics for the Genome-wide Associations of Single Nucleotide Polymorphisms with Bone Mineral Density
y axis is the observed –log₁₀ (p) values. x axis is the expected –log₁₀ (p) values under the null distribution of no association for studied genome-wide SNPs, which would be expected to follow the solid black line. (A) BMD at lumbar spine (LS); (B) BMD at femoral neck (FN). The green and gray dots are p values without and with correction of the factor for chi-square statistics of the genomic control method (λ = 1.007 for LS BMD and λ = 1.003 for FN BMD), respectively.

Electrophoretic Mobility Shift and Competition Assays with c-Myc Recombinant Protein and Allelic Variants of SNP rs2273061 in JAG1
The allele A probe, corresponding to JAG1 intron 3 sequences centering rs2273061 (underlined in the following sequences) was prepared by annealing of the Biotin-labeled oligonucleotide 5′-GACAACCTGTTACCACTTATTTACCTTCTTTA-3′ with the complementary sequence 5′-TAAAGAAGGTAAATAAGTGGTAACAGGTTGTC-3′; the allele G probe, prepared by annealing the Biotin-labeled oligonucleotide 5′-GACAACCTGTTACCACTTGTTTACCTTCTTTA-3′ with the complementary sequences 5′-TAAAGAAGGTAAACAAGTGGTAACAGGTTGTC-3′. Electrophoretic mobility shift assay (EMSA) was conducted with a commercial kit (Panomics, Fremont, CA). 1 ng of Biotin-labeled probe was incubated with 0.36 μg of c-Myc recombinant protein for 30 min at 15°C in a 10 μl reaction volume containing 2 μl 5 × binding buffer and 1 μg poly d(I-C). For competition reactions, the unlabeled probe was used at 660-fold molar excess of the labeled probe. After incubation, the samples were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel with 0.5 × TBE buffer. DNA-protein complexes were electroblotted to Pall Biodyne B nylon membrane (Pall Corp., Pensacola, FL) and visualized by exposure to Chemiluminescent Detection Film (Agfa, Shanghai, China).
Lanes: 1, labeled A probe; 2, labeled A probe + c-Myc; 3, labeled A probe + c-Myc + unlabeled A probe; 4, labeled G probe; 5, labeled G probe + c-Myc; 6, labeled G probe + c-Myc + unlabeled G probe. Binding was not observed with oligonucleotide containing A allele (lane 2) but was present with oligonucleotide containing the G allele (lane 5). Binding to the G allele resulted in a complex that was specifically competed by unlabeled mutant probe containing the G allele (lane 6).