Bmc Medical Genetics Single Nucleotide Polymorphisms in Bone Turnover-related Genes in Koreans: Ethnic Differences in Linkage Disequilibrium and Haplotype

Background: Osteoporosis is defined as the loss of bone mineral density that leads to bone fragility with aging. Population-based case-control studies have identified polymorphisms in many candidate genes that have been associated with bone mass maintenance or osteoporotic fracture. To investigate single nucleotide polymorphisms (SNPs) that are associated with osteoporosis, we examined the genetic variation among Koreans by analyzing 81 genes according to their function in bone formation and resorption during bone remodeling.

were no significant differences among Korean, Han Chinese and Japanese populations, but African and Caucasian populations were significantly differentiated in selected genes. Nevertheless, in the detailed analysis of genetic properties, the LD and Haplotype block patterns among the five subpopulations were substantially different from one another.

Conclusion:
Through the resequencing of 81 osteoporosis candidate genes, 118 unknown SNPs with a minor allele frequency (MAF) > 0.05 were discovered in the Korean population. In addition, using the common SNPs between our study and HapMap, an analysis of genetic diversity and deviation in heterozygosity was performed and the polymorphisms of the above genes among the five populations were substantially differentiated from one another. Further studies of osteoporosis could utilize the polymorphisms identified in our data since they may have important implications for the selection of highly informative SNPs for future association studies.

Background
Bone is continuously remodeled in vertebrates through coordinated phases of bone formation and resorption in order to maintain bone volume and phosphorus and calcium homeostasis [1]. Bone remodeling by direct contact with bone cells or by the release of soluble effectors is also altered by other cell protagonists present in the bone microenvironment such as monocytes/macrophages, lymphocytes, and endothelial cells [2]. In the disease state, the loss of bone homeostasis is potentially associated with changes in the numerous cellular protagonists that are responsible for the interactions between bone tissue, the immune system, and the vascular compartment. The study of bone homeostasis can therefore be utilized to elicit a better understanding of the pathologies associated with bone diseases such as osteoporosis [2]. Bone mass also has a very strong genetic determination: Twin and family studies showed that genetic factor could cause 50 to 90% of variance in bone mineral density (BMD) [3][4][5][6][7][8]. In addition, both the calcium-sensing receptor (CASR) and the interleukin 6 (IL-6) are important candidate genes for osteoporosis as well as in bone and mineral metabolism. These genes may have effects on BMD variation in Chinese nuclear families [9]. Determining SNPs for bone remodeling-related genes is becoming a more feasible and efficient tool for analyzing the processes associated with osteoporosis. However, an investigation of the distribution of SNPs within human populations is laborious and costly, mainly due to the necessity of testing large numbers of individuals and SNPs. Some SNPs for bone remodeling genes have already been reported; however, there are significant differences in allele frequency distributions among population groups, indicating that the populations exhibit genetic heterogeneity with respect to the incidence of these SNPs. Moreover, racial differences in the prevalence of certain alleles could account for a certain proportion of bone disease trait variation between different ethnicities [10]. The genetic variability of Asian and Caucasian populations was observed at restriction sites exhibiting polymorphisms of five important candidate genes for BMD: CASR-BsaHI, alpha 2HS-glycoprotein (AHSG)-SacI, estrogen receptor alpha (ESR1)-PvuII and XbaI, vitamin D receptor (VDR)-ApaI and parathyroid hormone (PTH)-BstBI. The results of the statistical analysis between the two populations revealed a significant allelic and genotypic differentiation in polymorphisms associated with osteoporosis. Intra-and inter-population variability implies that the studied pattern of variation at some loci may be affected by various types of natural selection [11]. A case-control approach is normally used to investigate the association of osteoporosis with SNPs in osteoporosis-related genes. A few of the newly discovered candidate genes (PLXNA2, CAT and SEMA7A) in our study were also used in case-control association studies in a Korean population [12][13][14]. These genes were screened in 24 individuals and then were genotyped in 560 postmenopausal women to compare gene and bone properties. Statistical analyses found a genetic linkage of the SNPs and haplotypes from the above genes with a risk of vertebral fracture or with BMD at the lumbar spine and at the femur neck [12][13][14]. Thus, to facilitate further association studies using SNPs of genes involved in osteoporosis, we selected 81 candidate genes involved in bone formation and resorption. We have characterized the genetic variants of these candidate osteoporosis genes, including gene-based haplotype diversity. These SNPs may be useful for genetic association studies that compare the SNP and haplotype information of ethnic groups.

Resequencing analysis
To identify SNPs in the 81 candidate osteoporosis genes (Table 1), we resequenced all exons, including the coding region, the 5' UTR and the 3' UTR up to the splice junctions, as well as the promoter regions of approximately 0.5 kb proximal to the transcription start site in genomic DNA samples. For sequencing analysis, genomic DNA information was obtained from GenBank. Polymerase chain reaction (PCR) primers were designed using the Primer 3 program [61]. Genomic DNA was isolated from the 24 immortalized B lymphocyte cell lines of the selected subjects. PCR products were sequenced using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 3730 automated sequencer (Applied Biosystems). SNPs were detected by multiple alignments of the sequences using the Phred/ Phrap/Consed package [62,63] and polyphred [64]. All data for the SNPs discovered in the Korean samples have been deposited in the KSNP database [65].

Statistical analysis
The HapMap database [66] was used to compare the Korean population with other populations. To measure the genetic differentiation between populations, Wright's F ST (the classic measure of population divergence) was calculated from the genotypic data. Haplotypes were suggested using the Partition Ligation-Expectation Maximization (PL-EM) algorithm [67]. We used the KSNP database to analyze LD and haplotype blocks and for tagging the detected SNPs. We defined LD blocks according to the method of LD-based blocking with bootstrapping [68], and haplotype tagging of selected SNPs was accomplished using the Entropy method [69].

Identification of SNPs in candidate osteoporosis genes in the Korean population
We directly sequenced 81 candidate osteoporosis genes including all exons, their intron boundaries, and ~1.5 kb of the 5' flanking region. We identified 942 variants, including 888 SNPs, 43 insertion/deletion polymorphisms, and 11 microsatellite markers ( Table 2). Of the 888 SNPs, 118 were located in promoter regions, 21 in 5' untranslated regions (UTRs), 157 in coding regions, 435 in introns, 119 in 3' UTRs and 38 in intergenic regions ( Table 2). With regard to the minor allele frequency (MAF), we classified the 888 SNPs into low (MAF < 0.05), intermediate (0.05-0.15), and high (>0.15) frequency classes as described by Cargill et al [70] (Fig. 1A). Of the 888 SNPs, we identified 331 unknown SNPs which were not reported in dbSNP (build 124), and the rest were known (Fig. 1B). Of the 888 SNPs, 401 belonged to the high MAF class, of which 53 (13.2%) were unknown SNPs. In addition, the majority of the low MAF class (70.3%) were also unknown SNPs, suggesting that a large portion of newly identified SNPs exist in a recessive model. Overall, about two-third of the SNPs identified in this study are common in the Korean population (MAF > 0.05). When functionally classified, 76% of the nonsynonymous SNPs (cSNP) belonged to the low MAF class whereas only 52.2% of SNPs in the promoter regions belonged to this class (Fig. 1C). In addition, newly identified SNPs with MAF > 0. 15 represented 16% of all the discovered SNPs. In functional aspect, we found some unknown SNPs in the coding region of the genes encoding interleukin 6 signal transducer (IL6ST), the androgen receptor (AR), and the core-binding factor beta subunit (CBFB) which were not reported in dbSNP database. However, there were no SNPs in the coding region of NFKB2 in both our dataset and dbSNP, suggesting that they are functionally and evolutionary highly conserved genes.
It has been reported that the Japanese SNP database (JSNP) was constructed through the gene-based resquencing method of 24 individuals [71]. Therefore, the newly discovered SNPs for candidate genes of osteoporosis from this study were compared with those in the JSNP database. Of 70 SNPs in the exon region (excluding UTR) with MAF > 0.05 in our data, 28 SNPs were common between our study and the JSNP database. The ratio of the common SNPs to all SNPs from our data and those from JSNP for the selected genes was 28/70 and 28/43, respectively.
In order to determine the genetic diversity between subpopulations, both Nei's standard genetic distance and Latter's F ST distance were also calculated [77,78] and listed in Table 3. Overall, both distance measures agreed with each other in terms of the trend, but overall, Nei's distances were lower than those of Latter's. The genetic distance between the KR and either the CHB (0.012) or the JPT (0.013) subpopulations was very close to each other. On the other hand, the genetic distance of the KR population was closer to the YRI population (0.594) than that of the CEU population (0.646) in these SNPs of selected genes. Therefore, the genetic diversity between KR compared with the other populations for the selected genes also agreed with the F ST analysis result.

Discussion
In this study, 81 candidate genes of osteoporosis were sequenced to identify common genetic polymorphisms that might alter bone remodeling. In the analysis of differences among ethnic group allele frequencies using the measure of genetic distance, we showed that the Han Chinese and Japanese populations were close to the Korean population. This implies a strong genetic linkage among the Han Chinese, Japanese and Korean populations, which may reflect either a recent common ancestry or high levels of mutual immigration among these groups [79].
In this study, the nonsynonymous cSNPs tended to have a larger proportion of low allele frequencies compared Distribution of the SNPs identified in the 81 candidate osteoporosis genes  with the synonymous cSNPs, the noncoding SNPs, and the promoter SNPs. This trend is consistent with a selection pressure against SNPs that cause amino acid changes [80]. In contrast, the promoter regions, which had a wide range of allele frequencies overall, had more SNPs with high allele frequency compared with the other regions. These results indicate that the promoter variants found in this study might be utilized as genetic determinants for future studies [81]. The several million human SNPs reported in the HapMap international project will likely prove useful for association studies; however SNPs located close to functionally important genes are more valuable as markers than random genomic SNPs. Moreover, SNPs located in the coding or promoter regions have the added benefit of potentially causing the genetic variation that directly contributes to disease. Therefore, additional resequencing efforts are still needed for comprehensive studies of osteoporosis candidate genes across ethnic groups as such data should prove important for future association studies of osteoporosis.

Conclusion
We directly resequenced 81 candidate osteoporosis genes and identified 942 variants including 888 SNPs, 43 insertion/deletion polymorphisms, and 11 microsatellite markers. Of the 888 SNPs, 331 SNPs have not been previously identified and 557 SNPs were already reported in the dbSNP database, of which more than 460 were validated by our resequencing effort.
Statistical analysis of deviation in heterozygosity with the HapMap data depicted that compared with SNPs in Koreans, 1%(or less) of SNPs in Japanese and Chinese and 20% of those in Caucasian and African were significantly differentiated from the Hardy-Weinberg expectations. In addition, the analysis of genetic diversity between Korean and the other four populations showed that the order of the closest neighbor (in terms of genetic distance) is Han Chinese, Japanese, African and Caucasian. In general, we didn't find any significant differences among three subpopulations from KR, CHB and JPT, but these Asian populations, CEU and YRI were significantly different in both the F ST and genetic diversity results in selected genes. Nevertheless, analysis using genetic properties, such as LD and   Overall, through the resequencing of 81 osteoporosis candidate genes, 118 unknown SNPs with MAF > 0.05 were discovered in a Korean population. In addition, our newly discovered SNPs were compared with those in HapMap to elucidate diversity and deviation in heterozygosity, resulting in strong genetic linkages between the Han Chinese, Japanese and Korean populations. This result may reflect either a recent common ancestry or high levels of mutual immigration among these groups. Yet, using a genetic property, such as LD patterns, is a powerful method to elucidate the subtle differences between the Korean, Chinese and Japanese populations. Our results could aid in the design of case-controlled and population stratification studies in the Korean population. The upper triangle is for Nei's standard genetic distance [19] and the lower triangle is for Latter's F ST distance [20].