Genome-wide copy-number-variation study identified a susceptibility gene, UGT2B17, for osteoporosis

Abstract

Osteoporosis, a highly heritable disease, is characterized mainly by low bone-mineral density (BMD), poor bone geometry, and/or osteoporotic fractures (OF). Copy-number variation (CNV) has been shown to be associated with complex human diseases. The contribution of CNV to osteoporosis has not been determined yet. We conducted case-control genome-wide CNV analyses, using the Affymetrix 500K Array Set, in 700 elderly Chinese individuals comprising 350 cases with homogeneous hip OF and 350 matched controls. We constructed a genomic map containing 727 CNV regions in Chinese individuals. We found that CNV 4q13.2 was strongly associated with OF (p = 2.0 x 10(-4), Bonferroni-corrected p = 0.02, odds ratio = 1.73). Validation experiments using PCR and electrophoresis, as well as real-time PCR, further identified a deletion variant of UGT2B17 in CNV 4q13.2. Importantly, the association between CNV of UGT2B17 and OF was successfully replicated in an independent Chinese sample containing 399 cases with hip OF and 400 controls. We further examined this CNV's relevance to major risk factors for OF (i.e., hip BMD and femoral-neck bone geometry) in both Chinese (689 subjects) and white (1000 subjects) samples and found consistently significant results (p = 5.0 x 10(-4) -0.021). Because UGT2B17 encodes an enzyme catabolizing steroid hormones, we measured the concentrations of serum testosterone and estradiol for 236 young Chinese males and assessed their UGT2B17 copy number. Subjects without UGT2B17 had significantly higher concentrations of testosterone and estradiol. Our findings suggest the important contribution of CNV of UGT2B17 to the pathogenesis of osteoporosis.

Figure 1

Genomic Distribution of 727 CNVRs Identified in Chinese Subjects The approximate locations of all CNVRs are shown by lines. CNVRs observed in only one subject (singleton) are indicated in green, and CNVRs identified in multiple individuals are indicated in blue (<1% frequency) and red (>1% frequency).

Figure 2

Examples of Copy-Number Determination at 4q13.2 by CNAT Analyses with Affymetrix Human Mapping 500K Array Data and Electrophoresis Analyses A: CNV 4q13.2 calculated by CNAT analyses with the 350 unrelated control subjects used as the reference set. AI: Three genotypes of the CNV 4q13.2: four copy numbers (a), three copy numbers (b), and two copy numbers (c). The x axis denotes genomic position. The y axis denotes the log2 ratio, which was calculated by comparison of the allele-intensity values of the test sample with those of the reference set. AII: Electrophoresis analysis for PCR products (with deletion-specific primers) from the three genotypes at the CNV. Results for 4q13.2 inferred to have two copy numbers by CNAT analyses reveal no PCR products, indicating that this sample actually reflects a homozygous deletion. B: CNV 4q13.2 calculated by CNAT with all of the subjects with nonhomozygous deletion used as the updated reference set. BI: Three genotypes of CNV 4q13.2: two copy numbers (a), one copy number, (b) and zero copy numbers (c). BII: On the basis of the UCSC Human Genome Brower, five genes are located in 4q13.2. The heavy arrow above “UGT2B17” means that the gene was found in CNV 4q13.2 with SNPs of rs4260611 and rs4860308 used as boundaries. When the boundaries were extended to SNPs of rs1730872 and rs293430 (the interval between these two SNPs was the maximal potential region for this CNV), four more protein-coding genes, named YTHDC1, TMPRSS11E, TMPRSS11E2, and UGT2B15, were found to be localized within this region (light arrows). CNV was only observed in UGT2B17. BIII: Electrophoresis analysis for PCR products from gene-specific markers C (within exon 1 of UGT2B17), D (within 5′ upstream of UGT2B17), and E (within exon 6 of UGT2B17) and from deletion marker J of UGT2B17 . Subjects with two copy numbers of UGT2B17 had the ability to amplify markers C, D, and E but not marker J (a). Subjects with heterozygous deletion showed the ability to amplify markers C, D, E, and J simultaneously (b). Subjects with homozygous deletion had the ability to amplify only marker J (c). The results revealed that there was a deletion polymorphism in a 150 kb interval spanning the whole UGT2B17 gene.

Figure 3

Comparisons of Hip BMD, CT, and BR Values for CNVs of UGT2B17 in Chinese and White Unrelated Samples (A) Chinese unrelated sample. (B) White unrelated sample. p values were estimated with the use of an ANOVA. For the Chinese unrelated sample, because a low frequency (1.6%) of two copy numbers of the UGT2B17 gene were detected, we combined the subjects with two copy numbers and heterozygous deletions as one group to perform the association analysis. Error bars denote standard error. Abbreviations are as follows: BMD, bone mineral density; CT, cortical thickness; BR, buckling ratio.