Single nucleotide polymorphisms in bone turnover-related genes in Koreans: ethnic differences in linkage disequilibrium and haplotype

  • Kyung-Seon Kim1Email author,

    Affiliated with

    • Ghi-Su Kim2, 3Email author,

      Affiliated with

      • Joo-Yeon Hwang1,

        Affiliated with

        • Hye-Ja Lee1,

          Affiliated with

          • Mi-Hyun Park1,

            Affiliated with

            • Kwang-joong Kim1,

              Affiliated with

              • Jongsun Jung1,

                Affiliated with

                • Hyo-Soung Cha1,

                  Affiliated with

                  • Hyoung Doo Shin4,

                    Affiliated with

                    • Jong-Ho Kang5,

                      Affiliated with

                      • Eui Kyun Park2, 6,

                        Affiliated with

                        • Tae-Ho Kim2,

                          Affiliated with

                          • Jung-Min Hong2,

                            Affiliated with

                            • Jung-Min Koh2, 3,

                              Affiliated with

                              • Bermseok Oh1,

                                Affiliated with

                                • Kuchan Kimm1,

                                  Affiliated with

                                  • Shin-Yoon Kim2, 7Email author and

                                    Affiliated with

                                    • Jong-Young Lee1Email author

                                      Affiliated with

                                      BMC Medical Genetics20078:70

                                      DOI: 10.1186/1471-2350-8-70

                                      Received: 29 September 2006

                                      Accepted: 26 November 2007

                                      Published: 26 November 2007

                                      Abstract

                                      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.

                                      Methods

                                      We resequenced all the exons, splice junctions and promoter regions of candidate osteoporosis genes using 24 unrelated Korean individuals. Using the common SNPs from our study and the HapMap database, a statistical analysis of deviation in heterozygosity depicted.

                                      Results

                                      We identified 942 variants, including 888 SNPs, 43 insertion/deletion polymorphisms, and 11 microsatellite markers. Of the SNPs, 557 (63%) had been previously identified and 331 (37%) were newly discovered in the Korean population. When compared SNPs in the Korean population with those in HapMap database, 1% (or less) of SNPs in the Japanese and Chinese subpopulations and 20% of those in Caucasian and African subpopulations were significantly differentiated from the Hardy-Weinberg expectations. In addition, an analysis of the genetic diversity showed that there 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 sub-populations 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) [38]. 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 [1214]. 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 [1214]. 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.

                                      Methods

                                      Subjects and candidate genes

                                      The study population consists of 24 unrelated Korean individuals, 11 men and 13 women, who were recruited from Ansan and Ansung area. The men were aged between 41 and 65 years (mean ± SD: 57.8 ± 8.5 years) and the women were aged between 41 and 62 years (mean ± SD: 52.6 ± 6.9 years). They were used for SNP screening and immortalized B lymphocyte cell line generation (cell line IDs GRB2015717, GRB2014744, GRB2014719, GRB2014754, GRB2015301, GRB2014712, GRB2012585, GRB2012949, GRB2012816, GRB2013123, GRB2012811, GRB2012998, GRB2015263, GRB2014890, GRB2014112, GRB2014896, GRB2014197, GRB2010947, GRB2021291, GRB2021404, GRB2021105, GRB2022466, GRB2026940, GRB2021302). Informed consent was obtained from all of the subjects, and this study was approved by the Institutional Review Board of the Korea National Institute of Health. Candidate osteoporosis genes were selected based on their function in bone/chondrocyte formation or bone resorption according to reports in the literature. We included the following genes of interest: those that promote or inhibit bone/chondrocyte formation; those that promote or inhibit bone resorption; and those involved in adipocyte differentiation. Genes of interest that promote bone/chondrocyte formation are as follows: FGFs [15], SOX5,6,9 [15], BMPs [16], LGALS3 [17], LGALS1 [18], DLX5 [16, 19], MSX2 [19], SP7 [19], CBFB [20], TGFBI [21], MSX1 [22], BGLAP [23], SPP1 [24], IBSP [24], IL1RN [25], CTNNB1 [16, 26], WNTs [26], TCF4 [27], OMD [28], VEGFs [29], DMP1 [30], IL13 [31], AR [32], CYP17A1 [32, 33] and CYP19A1 [32, 33]. Genes of interest that inhibit bone/chondrocyte formation are as follows: PTHrP/PTHR1 [15, 19], NPY2R [19], PPARG [19], TWIST1 [16, 24], DKK1 [26], PTH [19, 34], AHSG [35], PPP3CA [36], WIF1 [37], MEPE [38] and IL10 [39, 40]. Genes of interest that promote bone resorption are as follows: PTHrP/PTHR1 [15, 19], PTH [19, 34], PTGS2 [26], IL4 [34], IL6ST [41], CTSK [42], H+ATPase [42], ITGA1 [42, 43], NFKB [42, 44], CALCR [44], CLCN7 [44], FOS [44, 45], FOSB [42, 45], FOSL2 [46, 47], ITGAV [42, 44], CSK [42], TRAF6 [42, 44], MITF [44], CCR1 [48], NFATC1 [49], JDP2 [50], IL15 [51], PTK2B [52], CASR [53], SEMA7A [54], PTGER4 [55] and PLXNA2 [12]. Genes of interest that inhibit bone resorption are as follows: IL13 [31], AR [32], IL3 [56], ZNF675 [57], GPX1 [58] and CAT [59]. In additional, a decrease in bone volume that occurs with age and in osteoporosis is accompanied by an increase in adipose tissue in the bone marrow, suggesting a dysregulation of the mesenchymal stem cell differentiation pathway in favour of adipogenesis. Therefore, we also included the following adipocyte differentiation genes: PPARs [19], CEBPB [19, 47] and DBI [60].

                                      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].
                                      Table 1

                                      Gene information for candidate osteoporosis genes

                                      Gene Symbol

                                      Gene Name

                                      Locus ID

                                      NM_#

                                      Genomic Size

                                      Exon #

                                      BGLAP

                                      Bone gamma-carboxyglutamate (gla) protein (osteocalcin)

                                      1q25-q31

                                      NM_000711

                                      263037

                                      8

                                      CALCR

                                      Calcitonin receptor

                                      7q21.3

                                      NM_001742

                                      149952

                                      13

                                      IL6ST

                                      Interleukin 6 signal transducer (gp130, oncostatin M receptor)

                                      5q11

                                      NM_002184

                                      54069

                                      17

                                      LGALS3

                                      Lectin, galactoside-binding, soluble, 3 (galectin 3)

                                      14q21-q22

                                      NM_002306

                                      16124

                                      6

                                      PPARG

                                      Peroxisome proliferative activated receptor, gamma

                                      3p25

                                      NM_005037

                                      146417

                                      7

                                      PTH

                                      Parathyroid hormone

                                      11p15.3-p15.1

                                      NM_000315

                                      3966

                                      3

                                      SP7

                                      Sp7 transcription factor(osterix)

                                      12q13.13

                                      NM_152860

                                      9176

                                      2

                                      TGFBI

                                      Transforming growth factor, beta-induced, 68 kDa

                                      5q31

                                      NM_000358

                                      34810

                                      17

                                      AR

                                      Androgen receptor

                                      Xq11.2-q12

                                      NM_000044

                                      180246

                                      8

                                      BMP7

                                      Bone morphogenetic protein 7

                                      20q13

                                      NM_001719

                                      95747

                                      7

                                      AHSG

                                      Alpha 2 HS-glycoprotein

                                      3q27

                                      NM_001622

                                      8219

                                      7

                                      BMP2

                                      Bone morphogenetic protein 2

                                      20p12

                                      NM_001200

                                      10563

                                      3

                                      BMP4

                                      Bone morphogenetic protein 4

                                      14q22-q23

                                      NM_001202

                                      7156

                                      4

                                      BMP6

                                      Bone morphogenetic protein 6

                                      6p24-p23

                                      NM_001718

                                      154718

                                      7

                                      CBFB

                                      Core-binding factor, beta subunit

                                      16q22.1

                                      NM_022845

                                      71907

                                      6

                                      CTSK

                                      Cathepsin K

                                      1q21

                                      NM_000396

                                      12126

                                      8

                                      DLX5

                                      Distal-less homeobox 5

                                      7q22

                                      NM_005221

                                      4436

                                      3

                                      IBSP

                                      Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II)

                                      4q21-q25

                                      NM_004967

                                      12373

                                      7

                                      IL1RN

                                      Interleukin 1 receptor antagonist

                                      2q14.2

                                      NM_000577

                                      16123

                                      5

                                      LGALS1

                                      Lectin, galactoside-binding, soluble, 1 (galectin 1)

                                      22q13.1

                                      NM_002305

                                      4165

                                      4

                                      MSX1

                                      Msh homeobox homolog 1

                                      4p16.3-p16.1

                                      NM_002448

                                      4053

                                      3

                                      MSX2

                                      Msh homeobox homolog 2

                                      5q34-q35

                                      NM_002449

                                      6300

                                      2

                                      PTHLH

                                      Parathyroid hormone-like hormone

                                      12p12.1-p11.2

                                      NM_002820

                                      9663

                                      4

                                      PTHR1

                                      Parathyroid hormone receptor 1

                                      3p22-p21.1

                                      NM_000316

                                      26052

                                      16

                                      RUNX1

                                      Runt-related transcription factor 1

                                      21q22.3

                                      NM_001754

                                      261497

                                      8

                                      SPP1

                                      Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I)

                                      4q21-q25

                                      NM_000582

                                      7761

                                      6

                                      TWIST1

                                      Twist homolog 1 (acrocephalosyndactyly 3; Saethre-Chotzen syndrome)

                                      7p21.2

                                      NM_000474

                                      2203

                                      2

                                      CEBPB

                                      CCAAT/enhancer binding protein (C/EBP), beta

                                      20q13.1

                                      NM_005194

                                      1837

                                      1

                                      CYP17A1

                                      Cytochrome P450, family 17, subfamily A, polypeptide 1

                                      10q24.3

                                      NM_000102

                                      6885

                                      8

                                      CYP19A1

                                      Cytochrome P450, family 19, subfamily A, polypeptide 1

                                      15q21.1

                                      NM_000103

                                      129125

                                      10

                                      IL10

                                      Interleukin 10

                                      1q31-q32

                                      NM_000572

                                      4892

                                      5

                                      IL4

                                      Interleukin 4

                                      5q31.1

                                      NM_000589

                                      5996

                                      4

                                      NFKB1

                                      Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105)

                                      4q24

                                      NM_003998

                                      115989

                                      24

                                      VEGF

                                      Vascular endothelial growth factor

                                      6p12

                                      NM_003376

                                      14391

                                      8

                                      NPY2R

                                      Neuropeptide Y receptor Y2

                                      4q31

                                      NM_000910

                                      8447

                                      2

                                      FGF2

                                      Fibroblast growth factor 2

                                      4q26-q27

                                      NM_002006

                                      71528

                                      3

                                      FOS

                                      V-fos FBJ murine osteosarcoma viral oncogene homolog

                                      14q24.3

                                      NM_005252

                                      3383

                                      4

                                      FOSB

                                      FBJ murine osteosarcoma viral oncogene homolog B

                                      19q13.32

                                      NM_006732

                                      7184

                                      4

                                      SOX5

                                      SRY (sex determining region Y)-box 5

                                      12p12.1

                                      NM_152989

                                      1030149

                                      18

                                      SOX6

                                      SRY (sex determining region Y)-box 6

                                      11p15.3

                                      NM_033326

                                      506124

                                      16

                                      SOX9

                                      SRY (sex determining region Y)-box 9

                                      17q24.3-q25.1

                                      NM_000346

                                      5401

                                      3

                                      PTGER4

                                      Prostaglandin E receptor 4 (subtype EP4)

                                      5p13.1

                                      NM_000958

                                      13804

                                      3

                                      CSK

                                      C-src tyrosine kinase

                                      15q23-q25

                                      NM_004383

                                      20790

                                      13

                                      FGF23

                                      Fibroblast growth factor 23

                                      12p13.3

                                      NM_020638

                                      11502

                                      3

                                      FOSL2

                                      Fos-like antigen 2

                                      2p23-p22

                                      NM_005253

                                      21736

                                      4

                                      REL

                                      V-rel reticuloendotheliosis viral oncogene homolog (avian)

                                      2p13-p12

                                      NM_002908

                                      41427

                                      11

                                      RELA

                                      V-rel reticuloendotheliosis viral oncogene homolog A, p65 (avian)

                                      11q13

                                      NM_021975

                                      8559

                                      11

                                      RELB

                                      V-rel reticuloendotheliosis viral oncogene homolog B (avian)

                                      19q13.32

                                      NM_006509

                                      36741

                                      11

                                      NFKB2

                                      Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100)

                                      10q24

                                      NM_002502

                                      6805

                                      23

                                      ITGAV

                                      Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)

                                      2q31-q32

                                      NM_002210

                                      90828

                                      30

                                      JDP2

                                      Jun dimerization protein 2

                                      14q24.3

                                      NM_130469

                                      38320

                                      4

                                      NFATC1

                                      Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1

                                      18q23

                                      NM_172390

                                      72406

                                      8

                                      PPP3CA

                                      Protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform (calcineurin A alpha)

                                      4q24

                                      NM_000944

                                      323767

                                      14

                                      CASR

                                      Calcium sensing receptor

                                      3q21.1

                                      NM_000388

                                      102813

                                      7

                                      ZNF675

                                      Zinc finger protein 675

                                      19p12

                                      AB_209601

                                      33969

                                      4

                                      TRAF6

                                      TNF receptor associated factor 6

                                      11p12

                                      NM_004620

                                      21100

                                      7

                                      CLCN7

                                      Chloride channel 7

                                      16p13.3

                                      NM_001287

                                      29668

                                      25

                                      DBI

                                      Diazepam binding inhibitor (acyl-Coenzyme A binding protein)

                                      2q14.2

                                      NM_020548

                                      5461

                                      5

                                      CTNNB1

                                      Catenin (cadherin-associated protein), beta 1, 88 kDa

                                      3p21

                                      NM_001904

                                      40935

                                      15

                                      TCF4

                                      Transcription factor 4

                                      18q21.2

                                      NM_003199

                                      360475

                                      20

                                      OMD

                                      Osteomodulin

                                      9q22.31

                                      NM_005014

                                      10023

                                      3

                                      DMP1

                                      Dentin matrix acidic phosphoprotein

                                      4q22.1

                                      NM_004407

                                      14049

                                      6

                                      WIF1

                                      WNT inhibitory factor 1

                                      12q14.3

                                      NM_007191

                                      70681

                                      10

                                      MEPE

                                      Matrix extracellular phosphoglycoprotein with ASARM motif (bone)

                                      4q22.1

                                      NM_020203

                                      13807

                                      4

                                      CCR1

                                      Chemokine (C-C motif) receptor 1

                                      3p21.31

                                      NM_001295

                                      6101

                                      2

                                      ATP6V0D2

                                      ATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d2

                                      8q21.3

                                      NM_152565

                                      55316

                                      8

                                      IL15

                                      Interleukin 15

                                      4q31.21

                                      NM_172174

                                      96859

                                      8

                                      VEGFC

                                      Vascular endothelial growth factor C

                                      4q34.3

                                      BC_063685

                                      109205

                                      7

                                      PTK2B

                                      PTK2B protein tyrosine kinase 2 beta

                                      8p21.2

                                      NM_004103

                                      147905

                                      35

                                      WNT9A

                                      Wingless-type MMTV integration site family, member 9A

                                      1q42.13

                                      NM_003395

                                      26903

                                      4

                                      PPARA

                                      Peroxisome proliferative activated receptor, alpha

                                      22q13.31

                                      NM_001001928

                                      93155

                                      8

                                      CAT

                                      Catalase

                                      11p13

                                      NM_001752

                                      33115

                                      13

                                      GPX1

                                      Glutathione peroxidase 1

                                      3p21.3

                                      NM_201397

                                      1181

                                      1

                                      ITGA1

                                      Integrin, alpha 1\

                                      5q11.2

                                      NM_181501

                                      165350

                                      29

                                      MITF

                                      Microphthalmia-associated transcription factor

                                      3p14.2-p14.1

                                      NM_198159

                                      228855

                                      10

                                      PLXNA2

                                      Plexin A2

                                      1q32.2

                                      NM_025179

                                      216898

                                      32

                                      PTGS2

                                      Prostaglandin-endoperoxide synthase 2

                                      1q25.2-q25.3

                                      NM_000963

                                      8588

                                      10

                                      SEMA7A

                                      Semaphorin 7A, GPI membrane anchor

                                      15q22.3-q23

                                      NM_003612

                                      23954

                                      14

                                      DKK1

                                      Dickkopf homolog 1

                                      10q11.2

                                      NM_012242

                                      3063

                                      4

                                      IL3

                                      Interleukin 3 (colony-stimulating factor, multiple)

                                      5q31.1

                                      NM_000588

                                      2550

                                      5

                                      IL13

                                      Interleukin 13

                                      5q31

                                      NM_002188

                                      2937

                                      4

                                      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].

                                      Results

                                      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.
                                      Table 2

                                      Summary of polymorphisms discovered in candidate osteoporosis genes

                                      Gene

                                      IND

                                      MIC

                                      SNP

                                           

                                      Promoter

                                      5'UTR

                                      Syn

                                      Nonsyn

                                      3'UTR

                                      Intron

                                      Intergenic

                                      Total

                                       

                                      aT

                                      bN

                                      T

                                      N

                                      T

                                      N

                                      T

                                      N

                                      T

                                      N

                                      T

                                      N

                                      T

                                      N

                                      T

                                      N

                                      T

                                      N

                                      T

                                      N

                                      BGLAP

                                          

                                      1

                                           

                                      1

                                      1

                                            

                                      2

                                      1

                                      CALCR

                                      3

                                      2

                                          

                                      1

                                         

                                      1

                                       

                                      4

                                      2

                                      3

                                      1

                                        

                                      9

                                      3

                                      IL6ST

                                          

                                      1

                                      1

                                      1

                                      1

                                        

                                      2

                                      1

                                        

                                      5

                                      5

                                        

                                      9

                                      8

                                      LGALS3

                                              

                                      1

                                      1

                                      3

                                      1

                                        

                                      4

                                      1

                                        

                                      8

                                      3

                                      PPARG

                                        

                                      2

                                      2

                                        

                                      1

                                       

                                      1

                                           

                                      3

                                      2

                                        

                                      5

                                      2

                                      PTH

                                              

                                      1

                                         

                                      1

                                      1

                                      1

                                         

                                      3

                                      1

                                      SP7

                                              

                                      1

                                               

                                      1

                                      0

                                      TGFBI

                                              

                                      5

                                      1

                                        

                                      2

                                       

                                      12

                                      4

                                        

                                      19

                                      5

                                      AR

                                              

                                      2

                                      2

                                      5

                                      5

                                      1

                                      1

                                      4

                                      4

                                      1

                                      1

                                      13

                                      13

                                      BMP7

                                                  

                                      1

                                       

                                      7

                                      1

                                        

                                      8

                                      1

                                      AHSG

                                              

                                      2

                                       

                                      2

                                         

                                      2

                                         

                                      6

                                      0

                                      BMP2

                                              

                                      1

                                       

                                      3

                                      1

                                        

                                      1

                                         

                                      5

                                      1

                                      BMP4

                                                

                                      1

                                         

                                      9

                                      5

                                        

                                      10

                                      5

                                      BMP6

                                              

                                      2

                                      1

                                      1

                                      1

                                      2

                                      1

                                      4

                                      1

                                        

                                      9

                                      4

                                      CBFB

                                                

                                      1

                                      1

                                      5

                                      5

                                      4

                                      4

                                        

                                      10

                                      10

                                      CTSK

                                                

                                      1

                                      1

                                        

                                      1

                                      1

                                        

                                      2

                                      2

                                      DLX5

                                                

                                      1

                                      1

                                        

                                      1

                                      1

                                        

                                      2

                                      2

                                      IBSP

                                          

                                      2

                                      2

                                        

                                      2

                                       

                                      5

                                             

                                      9

                                      2

                                      IL1RN

                                          

                                      4

                                      1

                                      3

                                       

                                      1

                                         

                                      1

                                       

                                      24

                                       

                                      1

                                       

                                      34

                                      1

                                      LGALS1

                                          

                                      1

                                               

                                      4

                                      2

                                        

                                      5

                                      2

                                      MSX1

                                        

                                      1

                                                 

                                      2

                                      1

                                        

                                      2

                                      1

                                      MSX2

                                      1

                                      1

                                      1

                                               

                                      2

                                           

                                      2

                                      0

                                      PTHLH

                                      1

                                               

                                      1

                                      1

                                      1

                                       

                                      2

                                      1

                                        

                                      4

                                      2

                                      PTHR1

                                            

                                      1

                                      1

                                      1

                                           

                                      4

                                         

                                      6

                                      1

                                      RUNX1

                                                  

                                      11

                                      10

                                      3

                                      1

                                        

                                      14

                                      11

                                      SPP1

                                          

                                      6

                                      5

                                        

                                      2

                                         

                                      3

                                      1

                                      7

                                      2

                                        

                                      18

                                      8

                                      TWIST1

                                      2

                                      2

                                        

                                      1

                                      1

                                      1

                                                 

                                      2

                                      1

                                      FGF2

                                                  

                                      6

                                      1

                                      2

                                      1

                                      1

                                      1

                                      9

                                      3

                                      FOS

                                      1

                                         

                                      1

                                       

                                      2

                                       

                                      1

                                           

                                      1

                                         

                                      5

                                      0

                                      FOSB

                                          

                                      1

                                      1

                                            

                                      3

                                      1

                                      1

                                         

                                      5

                                      2

                                      CSK

                                              

                                      3

                                      2

                                        

                                      3

                                       

                                      3

                                      3

                                        

                                      9

                                      5

                                      PTGER4

                                          

                                      2

                                      1

                                            

                                      2

                                      2

                                          

                                      4

                                      3

                                      FGF23

                                                

                                      2

                                      1

                                            

                                      2

                                      1

                                      FOSL2

                                                

                                      1

                                      1

                                        

                                      2

                                         

                                      3

                                      1

                                      ITGAV

                                                

                                      1

                                       

                                      3

                                      1

                                      14

                                      7

                                        

                                      18

                                      8

                                      REL

                                                

                                      1

                                      1

                                        

                                      2

                                      1

                                        

                                      3

                                      2

                                      RELA

                                                    

                                      2

                                         

                                      2

                                      0

                                      RELB

                                        

                                      1

                                       

                                      1

                                      1

                                            

                                      1

                                      1

                                      3

                                      1

                                        

                                      5

                                      3

                                      SOX5

                                      1

                                      1

                                            

                                      2

                                      2

                                        

                                      1

                                      1

                                      2

                                         

                                      5

                                      3

                                      SOX6

                                              

                                      2

                                      1

                                          

                                      8

                                      6

                                        

                                      10

                                      7

                                      SOX9

                                                  

                                      2

                                       

                                      1

                                      1

                                        

                                      3

                                      1

                                      IL3

                                                

                                      1

                                             

                                      1

                                      0

                                      IL4

                                          

                                      1

                                           

                                      1

                                      1

                                        

                                      3

                                         

                                      5

                                      1

                                      IL13

                                                

                                      1

                                       

                                      1

                                       

                                      1

                                         

                                      3

                                      0

                                      NFKB1

                                      3

                                      3

                                        

                                      3

                                      2

                                      1

                                      1

                                        

                                      2

                                      2

                                        

                                      23

                                      5

                                        

                                      29

                                      10

                                      VEGF

                                      3

                                      3

                                        

                                      1

                                       

                                      2

                                             

                                      10

                                      3

                                      6

                                      1

                                      19

                                      4

                                      IL10

                                      1

                                      1

                                                

                                      3

                                      1

                                      5

                                      2

                                        

                                      8

                                      3

                                      NPY2R

                                      1

                                      1

                                      1

                                      1

                                          

                                      2

                                       

                                      1

                                      1

                                      1

                                      1

                                      10

                                      6

                                        

                                      14

                                      8

                                      CAT

                                          

                                      2

                                       

                                      1

                                       

                                      1

                                       

                                      2

                                      2

                                        

                                      9

                                      2

                                      2

                                       

                                      17

                                      4

                                      CEBPB

                                          

                                      4

                                      2

                                                  

                                      4

                                      2

                                      CYP17A1

                                          

                                      2

                                       

                                      1

                                       

                                      3

                                      1

                                      1

                                      1

                                        

                                      10

                                      1

                                        

                                      17

                                      3

                                      CYP19A1

                                      2

                                      1

                                            

                                      2

                                      1

                                      2

                                      2

                                        

                                      15

                                      4

                                      2

                                       

                                      21

                                      7

                                      GPX1

                                          

                                      1

                                             

                                      1

                                           

                                      2

                                      0

                                      ITGA1

                                      2

                                      2

                                        

                                      5

                                      2

                                        

                                      2

                                       

                                      5

                                      2

                                      13

                                      3

                                      31

                                      3

                                      5

                                      1

                                      61

                                      11

                                      MITF

                                          

                                      4

                                      2

                                          

                                      1

                                      1

                                      5

                                      3

                                      3

                                      2

                                      2

                                      1

                                      15

                                      9

                                      PLXNA2

                                      1

                                             

                                      5

                                      2

                                      8

                                      2

                                        

                                      33

                                      16

                                      1

                                      1

                                      47

                                      21

                                      PTGS2

                                      2

                                         

                                      4

                                       

                                      1

                                       

                                      3

                                       

                                      2

                                       

                                      3

                                       

                                      6

                                       

                                      3

                                       

                                      22

                                      0

                                      SEMA7A

                                          

                                      1

                                      1

                                        

                                      5

                                      1

                                      1

                                      1

                                      2

                                       

                                      6

                                      3

                                        

                                      15

                                      6

                                      DKK1

                                          

                                      1

                                         

                                      1

                                           

                                      2

                                      1

                                        

                                      4

                                      1

                                      CASR

                                      2

                                      2

                                        

                                      2

                                       

                                      1

                                      1

                                      2

                                      2

                                      3

                                      1

                                      2

                                       

                                      5

                                      1

                                        

                                      15

                                      5

                                      CCR1

                                      2

                                      2

                                            

                                      1

                                      1

                                      1

                                      1

                                      5

                                       

                                      10

                                      5

                                      1

                                       

                                      18

                                      7

                                      CLCN7

                                      2

                                      2

                                        

                                      10

                                      5

                                        

                                      2

                                      1

                                        

                                      2

                                      1

                                      12

                                      5

                                      1

                                       

                                      27

                                      12

                                      CTNNB1

                                      1

                                      1

                                        

                                      1

                                             

                                      3

                                      1

                                      3

                                      1

                                        

                                      7

                                      2

                                      DBI

                                          

                                      11

                                      4

                                        

                                      1

                                      1

                                          

                                      4

                                      1

                                      1

                                       

                                      17

                                      6

                                      DMP1

                                      2

                                      2

                                        

                                      6

                                           

                                      2

                                      1

                                      2

                                       

                                      4

                                         

                                      14

                                      1

                                      IL15

                                          

                                      2

                                      1

                                            

                                      4

                                      1

                                      10

                                      2

                                        

                                      16

                                      4

                                      JDP2

                                          

                                      2

                                           

                                      1

                                       

                                      2

                                           

                                      5

                                      0

                                      MEPE

                                              

                                      1

                                       

                                      1

                                      1

                                      1

                                       

                                      9

                                      2

                                        

                                      12

                                      3

                                      NFATC1

                                          

                                      4

                                      1

                                        

                                      3

                                       

                                      1

                                      1

                                      1

                                      1

                                      9

                                      6

                                        

                                      18

                                      9

                                      OMD

                                          

                                      4

                                           

                                      1

                                      1

                                      1

                                      1

                                          

                                      6

                                      2

                                      PPARA

                                      1

                                      1

                                      1

                                      1

                                      2

                                      1

                                        

                                      1

                                      1

                                      2

                                      1

                                          

                                      6

                                      5

                                      11

                                      8

                                      PPP3CA

                                      1

                                      1

                                      1

                                      1

                                      2

                                             

                                      4

                                      3

                                      7

                                      5

                                        

                                      13

                                      8

                                      PTK2B

                                          

                                      8

                                      4

                                      3

                                      1

                                      10

                                      3

                                      1

                                       

                                      1

                                       

                                      24

                                      6

                                      3

                                      2

                                      50

                                      16

                                      TCF4

                                      1

                                      1

                                      1

                                      1

                                          

                                      1

                                           

                                      11

                                      7

                                        

                                      12

                                      7

                                      TRAF6

                                      3

                                      3

                                        

                                      3

                                      1

                                          

                                      1

                                      1

                                      1

                                      1

                                          

                                      5

                                      3

                                      VEGFC

                                      1

                                      1

                                        

                                      2

                                      2

                                        

                                      1

                                      1

                                          

                                      3

                                      1

                                        

                                      6

                                      4

                                      WIF1

                                      1

                                      1

                                      1

                                      1

                                      4

                                      4

                                            

                                      1

                                       

                                      3

                                      1

                                        

                                      8

                                      5

                                      WNT9A

                                          

                                      2

                                         

                                      2

                                         

                                      3

                                      1

                                      1

                                      1

                                      1

                                       

                                      9

                                      2

                                      ATP6V0D2

                                      2

                                      1

                                      1

                                      1

                                      3

                                      1

                                      1

                                      1

                                      1

                                      1

                                        

                                      2

                                      1

                                      3

                                       

                                      1

                                      1

                                      11

                                      5

                                      ZNF675

                                              

                                      1

                                      1

                                      1

                                         

                                      2

                                      2

                                        

                                      4

                                      3

                                      Total

                                      43

                                      35

                                      11

                                      8

                                      118

                                      46

                                      21

                                      6

                                      81

                                      27

                                      76

                                      40

                                      119

                                      47

                                      435

                                      151

                                      38

                                      14

                                      888

                                      331

                                      SNPs, single nucleotide polymorphisms; IND, insertion/deletion polymorphism; MIC, microsatellite; UTR, untranslated region. Syn, Synonymous cSNP, indicates there is no change in the amino acid; Nonsyn, Nonsynonymous cSNP, substitutions in coding regions that result in a different amino acid

                                      aT, sum of known SNPs and newly identified SNPs; bN, newly identified SNP in this study

                                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2350-8-70/MediaObjects/12881_2006_Article_264_Fig1_HTML.jpg
                                      Figure 1

                                      Distribution of the SNPs identified in the 81 candidate osteoporosis genes. (A) Classification of the SNPs into minor allele frequency (MAF) classes. (B) Number of known and unknown SNPs. (C) Distribution of SNPs according to location or type. The percentages in (A), (B), and (C) refer to the percentage of SNPs within each MAF class in the given categories.

                                      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.

                                      Deviation in Heterozygosity and Genetic diversity

                                      We used HapMap to compare the allele frequencies of diverse ethnic groups with that of the Korean population [72]. Among the 557 known SNPs detected in this study, 313 were found in HapMap. We thus evaluated genetic differences between Koreans and the diverse populations by measuring the Wright's F ST coefficients using the 313 common SNPs assuming the Hardy-Weinberg principle. F IS is the average deviation in heterozygosity within subpopulations, F ST is the deviation due to subdivision alone, and F IT is the overall deviation in heterozygosity in the total population [73]. The mean values of F IS , F ST and F IT for multiple loci with five subpopulations (KR, CHB, JPT, CEU and YRI) are -0.0121, 0.3366 and 0.3287, respectively, indicating that the SNPs in genes associated with osteoporosis were significantly differentiated among the five subpopulations while the SNPs within the subpopulations were consistent with the Hardy-Weinberg expectations. In addition, the pairwise F ST (s) of KR compared with each of the four subpopulations using the 313 individual SNPs were calculated. The distribution of the pairwise F ST (s) values is plotted in Fig. 2. Interestingly, two distribution patterns were observed that grouped KR-CHB with KR-JPT and KR-CEU with KR-YRI. In addition, the F ST values for KR-CHB and KR-JPT continually decreased to 0.05 whereas those of KR-CEU and KR-YRI continued to 0.2 or more from which point the overall major and minor alleles are reversed, suggesting that there is a large genetic barrier among continental populations. When a threshold (F ST = 0.1 or higher) as the level of significance was applied [74] to our data, 2, 2, 73 and 92 out of 313 SNPs were significantly deviated between KR compared with CHB, JPT, YRI and CEU, respectively. In order to investigate the difference in linkage disequilibrium (LD) patterns between the significantly diverse SNPs in the sub-populations, two highly polymorphic genes (PTK2B and IL1RN) in terms of the number of SNPs per gene were selected and their Haplotype blocks using Haploview [75, 76] were plotted against five subpopulations, KR, CHB, JPT, CEU and YRI, as shown in Fig. 3. Interestingly, all five haplotype blocks for each gene were different from one another. Overall, the largest block was found in the CEU population whereas smaller blocks were found in the two genes of the KR and YRI populations. This result implies that determining genetic properties, such as, LD is a powerful method to elucidate the subtle differences in genetic diversity between sub-populations.
                                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2350-8-70/MediaObjects/12881_2006_Article_264_Fig2_HTML.jpg
                                      Figure 2

                                      Distribution of F ST among the sub-populations.

                                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2350-8-70/MediaObjects/12881_2006_Article_264_Fig3_HTML.jpg
                                      Figure 3

                                      Comparison of LD patterns of PTK2B and IL1RN among the sub-populations.

                                      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.
                                      Table 3

                                      Pairwise genetic distance among five population

                                       

                                      KOR

                                      CHB

                                      JPT

                                      CEU

                                      YRI

                                      KOR

                                       

                                      0.01197

                                      0.01331

                                      0.64578

                                      0.59374

                                      CHB

                                      0.05259

                                       

                                      0.01207

                                      0.68158

                                      0.58691

                                      JPT

                                      0.05775

                                      0.04840

                                       

                                      0.71527

                                      0.60823

                                      CEU

                                      0.85137

                                      0.86316

                                      0.86480

                                       

                                      1.00000

                                      YRI

                                      0.83252

                                      0.82958

                                      0.83475

                                      1.00000

                                       

                                      The upper triangle is for Nei's standard genetic distance [19] and the lower triangle is for Latter's F ST distance [20].

                                      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].

                                      The 888 polymorphisms identified in this study were obtained from 24 unrelated individuals. Three hundred and thirty-one (37.3%) variants were newly identified polymorphisms that were not present in the public database examined, whereas 557 (62.7%) of the polymorphisms found by resequencing were already present in the database. Of the 331 variants that were not reported in the database, 64.4% belonged to the low minor allele frequency group (MAF < 0.05) in Koreans and variants, 35.6% were common SNPs in the Korean population. These common SNPs could be useful for further case-control association studies of osteoporosis in Koreans. We identified new SNPs that had low allele frequencies. This may be due to the fact that previous studies used various factors, such as a mixture of populations, or had a relatively smaller sample size, thereby limiting their ability to discover low allele frequency SNPs. Alternatively, as an ethnically homogeneous population, the Korean samples may have allele frequencies that significantly differ from those from mixed samples. Of the 557 variants that were already present in the dbSNP database, only 16.2% had a minor allele frequency lower than 0.05, 21.4% between 0.05 to 0.15 and 62.5% greater than 0.15. Therefore, our resequencing effort provided experimental validation for more than 460 polymorphisms that were already in the database.

                                      In our study, we measured the LD block structure of the candidate genes, excluding cases of one or two SNPs and uncommon SNPs (MAF < 0.05) in each gene, from the limited sample using normalized D' statistics between all pairwise SNP markers with MAF > 0.05 that satisfied the Hardy-Weinberg's equilibrium (p < 0.05). The LD and haplotype results are shown in the KSNP database [65]. A comparison of the haplotype blocks of two highly polymorphic genes (PTK2B and IL1RN) from the KR population with those from the 4 subpopulations in HapMap, showed diverse block patterns (Fig. 3). Therefore, the LD and haplotype information could be valuable resources for ethnicity comparison, tagging SNPs and recombination signals of the osteoporosis-related genes in future studies.

                                      In this study, the nonsynonymous cSNPs tended to have a larger proportion of low allele frequencies compared 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 sub-populations 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 haplotype patterns showed that all-sub populations were substantially different.

                                      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.

                                      Declarations

                                      Acknowledgements

                                      This work was supported by intramural grants from the Korea National Institute of Health, Korea Center for Disease Control, Republic of Korea (Project No.: 347-6111-211) and a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (Project No.: A010252)

                                      Authors’ Affiliations

                                      (1)
                                      Center for Genome Science, National Institute of Health
                                      (2)
                                      Skeletal Diseases Genome Research Center, Kyungpook National University Hospital
                                      (3)
                                      Division of Endocrinology and Metabolism, University of Ulsan College of Medicine, Asan Medical Center
                                      (4)
                                      Department of Genetic Epidemiology, SNP Genetics, Inc.
                                      (5)
                                      World Meridian Venture Center 10F
                                      (6)
                                      Department of Pathology and Regenerative Medicine, School of Dentistry, Kyungpook National University
                                      (7)
                                      Department of Orthopedic Surgery, Kyungpook National University School of Medicine

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                                      © Kim et al. 2007

                                      This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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