Functional & Integrative Genomics

, Volume 10, Issue 1, pp 63–72

Genes influencing spinal bone mineral density in inbred F344, LEW, COP, and DA rats

  • Imranul Alam
  • Qiwei Sun
  • Daniel L. Koller
  • Lixiang Liu
  • Yunlong Liu
  • Howard J. Edenberg
  • Tatiana Foroud
  • Charles H. Turner
Original Paper


Previously, we identified the regions of chromosomes 10q12–q31 and 15p16–q21 harbor quantitative trait loci (QTLs) for lumbar volumetric bone mineral density (vBMD) in female F2 rats derived from Fischer 344 (F344) × Lewis (LEW) and Copenhagen 2331 (COP) × Dark Agouti (DA) crosses. The purpose of this study is to identify the candidate genes within these QTL regions contributing to the variation in lumbar vBMD. RNA was extracted from bone tissue of F344, LEW, COP, and DA rats. Microarray analysis was performed using Affymetrix Rat Genome 230 2.0 Arrays. Genes differentially expressed among the rat strains were then ranked based on the strength of the correlation with lumbar vBMD in F2 animals derived from these rats. Quantitative PCR (qPCR) analysis was performed to confirm the prioritized candidate genes. A total of 285 genes were differentially expressed among all strains of rats with a false discovery rate less than 10%. Among these genes, 18 candidate genes were prioritized based on their strong correlation (r2 > 0.90) with lumbar vBMD. Of these, 14 genes (Akap1, Asgr2, Esd, Fam101b, Irf1, Lcp1, Ltc4s, Mdp-1, Pdhb, Plxdc1, Rabep1, Rhot1, Slc2a4, Xpo4) were confirmed by qPCR. We identified several novel candidate genes influencing spinal vBMD in rats.


Lumbar vBMD Gene expression Microarray QTLs Osteoporotic fracture 


  1. Aitman TJ, Glazier AM, Wallace CA, Cooper LD, Norsworthy PJ, Wahid FN, Al-Majali KM, Trembling PM, Mann CJ, Shoulders CC, Graf D, St Lezin E, Kurtz TW, Kren V, Pravenec M, Ibrahimi A, Abumrad NA, Stanton LW, Scott J (1999) Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats. Nature Genet 21:76–83CrossRefPubMedGoogle Scholar
  2. Alam I, Sun Q, Liu L, Koller DL, Liu L, Edenberg HJ, Econs MJ, Foroud T, Turner CH (2008) Genomic expression analysis of rat chromosome 4 for skeletal traits at femoral neck. Physiol Genomics 35:191–196CrossRefPubMedGoogle Scholar
  3. Arden NK, Baker J, Hogg C, Bann K, Spector TD (1996) The heritability of bone mineral density, ultrasound of the calcaneus and hip axis length: a study of postmenopausal twins. J Bone Miner Res 11:530–534CrossRefPubMedGoogle Scholar
  4. Beamer WG, Shultz KL, Donahue LR, Churchill GA, Sen S, Wergedal JR, Baylink DJ, Rosen CJ (2001) Quantitative trait loci for femoral and lumbar vertebral bone mineral density in C57BL/6J and C3H/HeJ inbred strains of mice. J Bone Miner Res 16:1195–1206CrossRefPubMedGoogle Scholar
  5. Benes H, Weinstein RS, Zheng W, Thaden JJ, Jilka RL, Manolagas SC, Shmookler Reis RJ (2000) Chromosomal mapping of osteopenia-associated quantitative trait loci using closely related mouse strains. J Bone Miner Res 15:626–633CrossRefPubMedGoogle Scholar
  6. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc (B) 57:289–290Google Scholar
  7. Calvano SE, Xiao W, Richards DR, Felciano RM, Baker HV, Cho RJ, Chen RO, Brownstein BH, Cobb JP, Tschoeke SK, Miller-Graziano C, Moldawer LL, Mindrinos MN, Davis RW, Tompkins RG, Lowry SF (2005) A network-based analysis of systemic inflammation in humans. Nature 437:1032–1037CrossRefPubMedGoogle Scholar
  8. Chen HB, Shen J, Ip YT, Xu L (2006) Identification of phosphatases for Smad in the BMP/DPP pathway. Genes Dev 20:648–653CrossRefPubMedGoogle Scholar
  9. Cooper C, Atkinson EJ, Jacobsen SJ, O'Fallon WM, Melton LJ 3rd (1993) Population-based study of survival after osteoporotic fractures. Am J Epidemiol 137:1001–1005PubMedGoogle Scholar
  10. Deng HW, Xu FH, Huang QY, Shen H, Deng H, Conway T, Liu YJ, Liu YZ, Li JL, Zhang HT, Davies KM, Recker RR (2002) A whole-genome linkage scan suggests several genomic regions potentially containing quantitative trait loci for osteoporosis. J Clin Endocrinol Metab 87:5151–5159CrossRefPubMedGoogle Scholar
  11. Devoto M, Shimoya K, Caminis J, Ott J, Tnenhouse A, Whyte MP, Sereda L, Hall S, Considine E, Williams CJ (1998) First-stage autosomal genome screen in extended pedigrees suggests genes predisposing to low bone mineral density on chromosomes 1p, 2p and 4q. Euro J Human Genet 6:151–157CrossRefGoogle Scholar
  12. Duncan EL, Browon MA, Sinsheimer J, Bell J, Carr AJ, Wordsworth BP, Wass JA (1999) Suggestive linkage of the parathyroid receptor type 1 to osteoporosis. J Bone Miner Res 14:1993–1999CrossRefPubMedGoogle Scholar
  13. Farber CR, van Nas A, Ghazalpour A, Aten JE, Doss S, Sos B, Schadt EE, Ingram-Drake L, Davis RC, Horvath S, Smith DJ, Drake TA, Lusis AJ (2009) An integrative genetics approach to identify candidate genes regulating BMD: combining linkage, gene expression, and association. J Bone Miner Res 24:105–116CrossRefPubMedGoogle Scholar
  14. Fuchs B, Mahlum E, Halder C, Maran A, Yaszemski M, Bode B, Bolander M, Sarkar G (2007) High expression of tumor endothelial marker 7 is associated with metastasis and poor survival of patients with osteogenic sarcoma. Gene 399:137–143CrossRefPubMedGoogle Scholar
  15. Garnero P, Arden NK, Griffiths G, Delmas PD, Spector TD (1996) Genetic influence on bone turnover in postmenopausal twins. J Clin Endocrinol Metab 81:140–146CrossRefPubMedGoogle Scholar
  16. Giustina A, Mazziotti G, Canalis E (2008) Growth hormone, insulin-like growth factors, and the skeleton. Endocr Rev 29:535–559CrossRefPubMedGoogle Scholar
  17. Hsu YH, Xu X, Terwedow HA, Niu T, Hong X, Wu D, Wang L, Brain JD, Bouxsein ML, Cummings SR, Rosen CJ, Xu X (2007) Large-scale genome-wide linkage analysis for loci linked to BMD at different skeletal sites in extreme selected sibships. J Bone Miner Res 22:184–194CrossRefPubMedGoogle Scholar
  18. Huang C, Ross PD, Wasnich RD (1996) Vertebral fracture and other predictors of physical impairment and health care utilization. Arch Intern Med 156:2469–2475CrossRefPubMedGoogle Scholar
  19. Ioannidis JP, Ng MY, Sham PC, Zintzaras E, Lewis CM, Deng HW, Econs MJ, Karasik D, Devoto M, Kammerer CM, Spector T, Andrew T, Cupples LA, Duncan EL, Foroud T, Kiel DP, Koller D, Langdahl B, Mitchell BD, Peacock M, Recker R, Shen H, Sol-Church K, Spotila LD, Uitterlinden AG, Wilson SG, Kung AW, Ralston SH (2007) Meta-analysis of genome-wide scans provides evidence for sex- and site-specific regulation of bone mass. J Bone Miner Res 22:173–183CrossRefPubMedGoogle Scholar
  20. Johnson ML, Gong G, Kimberling W, Reckér SM, Kimmel DB, Recker RB (1997) Linkage of a gene causing high bone mass to human chromosome 11 (11q12-13). Am J Hum Genet 60:1326–1332CrossRefPubMedGoogle Scholar
  21. Kado DM, Browner WS, Palermo L, Nevitt MC, Genant HK, Cummings SR (1999) Vertebral fractures and mortality in older women: a prospective study. Study of osteoporotic fractures research group. Arch Intern Med 159:1215–1220CrossRefPubMedGoogle Scholar
  22. Kanis JA, Melton LJ, Christiansen C, Johnston CC, Khaltaev N (1994) The diagnosis of osteoporosis. J Bone Miner Res 9:1137–1141CrossRefPubMedGoogle Scholar
  23. Karasik D, Myers RH, Cupples LA, Hannan MT, Gagnon DR, Herbert A, Kiel DP (2002) Genome screen for quantitative trait loci contributing to normal variation in bone mineral density: the Framingham Study. J Bone Miner Res 17:1718–1727CrossRefPubMedGoogle Scholar
  24. Karp CL, Grupe A, Schadt E, Ewart SL, Keane-Moore M, Cuomo PJ, Köhl J, Wahl L, Kuperman D, Germer S, Aud D, Peltz G, Wills-Karp M (2000) Identification of complement factor 5 as a susceptibility locus for experimental allergic asthma. Nat Immunol 1:221–226CrossRefPubMedGoogle Scholar
  25. Kaufman JM, Ostertag A, Saint-Pierre A, Cohen-Solal M, Boland A, Van Pottelbergh I, Toye K, de Vernejoul MC, Martinez M (2008) Genome-wide linkage screen of bone mineral density (BMD) in European pedigrees ascertained through a male relative with low BMD values: evidence for quantitative trait loci on 17q21–23, 11q12–13, 13q12–14, and 22q11. J Clin Endocrinol Metab 93:3755–3762CrossRefPubMedGoogle Scholar
  26. Koller DL, Econs MJ, Morin PA, Christian JC, Hui SL, Parry P, Curran ME, Rodriguez LA, Conneally PM, Joslyn G, Peacock M, Johnston CC, Foroud T (2000) Genome screen for QTLs contributing to normal variation in bone mineral density and osteoporosis. J Clin Endocrinol Metab 85:3116–3120CrossRefPubMedGoogle Scholar
  27. Koller DL, Alam I, Sun Q, Liu L, Fishburn T, Carr LG, Econs MJ, Foroud T, Turner CH (2005) Genome screen for bone mineral density phenotypes in Fischer 344 and Lewis rats. Mammalian Genome 16:578–586CrossRefPubMedGoogle Scholar
  28. Koller DL, Liu L, Alam I, Sun Q, Econs MJ, Foroud T, Turner CH (2008) Linkage screen for BMD phenotypes in male and female COP and DA rat strains. J Bone Miner Res 23:1382–1388CrossRefPubMedGoogle Scholar
  29. Koseki T, Gao Y, Okahashi N, Murase Y, Tsujisawa T, Sato T, Yamato K, Nishihara T (2002) Role of TGF-beta family in osteoclastogenesis induced by RANKL. Cell Signal 14:31–36CrossRefPubMedGoogle Scholar
  30. Leidig-Bruckner G, Minne HW, Schlaich C, Wagner G, Scheidt-Nave C, Bruckner T, Gebest HJ, Ziegler R (1997) Clinical grading of spinal osteoporosis: quality of life components and spinal deformity in women with chronic low back pain and women with vertebral osteoporosis. J Bone Miner Res 12:663–675CrossRefPubMedGoogle Scholar
  31. Le Stunff C, Rotwein P (1998) Growth hormone stimulates interferon regulatory factor-1 gene expression in the liver. Endocrinology 139:859–866CrossRefPubMedGoogle Scholar
  32. Li X, Liu H, Qin L, Tamasi J, Bergenstock M, Shapses S, Feyen JH, Notterman DA, Partridge NC (2007) Determination of dual effects of parathyroid hormone on skeletal gene expression in vivo by microarray and network analysis. J Biol Chem 282:33086–33097CrossRefPubMedGoogle Scholar
  33. Lips P, Cooper C, Agnusdei D, Caulin F, Egger P, Johnell O, Kanis JA, Kellingray S, Leplege A, Liberman UA, McCloskey E, Minne H, Reeve J, Reginster JY, Scholz M, Todd C, de Vernejoul MC, Wiklund I (1999) Quality of life in patients with vertebral fractures: validation of the Quality of Life Questionnaire of the European Foundation for Osteoporosis (QUALEFFO). Working Party for Quality of Life of the European Foundation for Osteoporosis. Osteoporos Int 10:150–160CrossRefPubMedGoogle Scholar
  34. Lomaga MA, Yeh WC, Sarosi I, Duncan GS, Furlonger C, Ho A, Morony S, Capparelli C, Van G, Kaufman S, van der Heiden A, Itie A, Wakeham A, Khoo W, Sasaki T, Cao Z, Penninger JM, Paige CJ, Lacey DL, Dunstan CR, Boyle WJ, Goeddel DV, Mak TW (1999) TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev 13:1015–1024CrossRefPubMedGoogle Scholar
  35. Lynch MP, Capparelli C, Stein JL, Stein GS, Lian JB (1998) Apoptosis during bone-like tissue development in vitro. J Cell Biochem 68:31–49CrossRefPubMedGoogle Scholar
  36. Macdonald KK, Cheung CY, Anseth KS (2007) Cellular delivery of TGFbeta1 promotes osteoinductive signalling for bone regeneration. J Tissue Eng Regen Med 1:314–317CrossRefPubMedGoogle Scholar
  37. Magnusson MK, Meade KE, Brown KE, Arthur DC, Krueger LA, Barrett AJ, Dunbar CE (2001) Rabaptin-5 is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia. Blood 98:2518–2525CrossRefPubMedGoogle Scholar
  38. Maor G, Karnieli E (1999) The insulin-sensitive glucose transporter (GLUT4) is involved in early bone growth in control and diabetic mice, but is regulated through the insulin-like growth factor I receptor. Endocrinology 140:1841–1851CrossRefPubMedGoogle Scholar
  39. McClintick JN, Edenberg HJ (2006) Effects of filtering by present call on analysis of microarray experiments. BMC Bioinformatics 7:49CrossRefPubMedGoogle Scholar
  40. Mengus G, Fadloun A, Kobi D, Thibault C, Perletti L, Michel I, Davidson I (2005) TAF4 inactivation in embryonic fibroblasts activates TGFbeta signalling and autocrine growth. EMBO J 24:2753–2767CrossRefPubMedGoogle Scholar
  41. Nakamura I, Jimi E (2006) Regulation of osteoclast differentiation and function by interleukin-1. Vitam Horm 74:357–3570CrossRefPubMedGoogle Scholar
  42. Olney RC (2003) Regulation of bone mass by growth hormone. Med Pediatr Oncol 41:228–234CrossRefPubMedGoogle Scholar
  43. Peacock M, Turner CH, Econs MJ, Foroud T (2002) Genetics of osteoporosis. Endocr Rev 23:378–383CrossRefGoogle Scholar
  44. Polzer K, Joosten L, Gasser J, Distler JH, Ruiz G, Baum W, Redlich K, Bobacz K, Smolen JS, van den Berg W, Schett G, Zwerina J (2009) IL-1 is essential for systemic inflammatory bone loss. Ann Rheum Dis (in press). PMID: 19196726Google Scholar
  45. Ralston SH (2005) Genetic determinants of osteoporosis. Curr Opin Rheumatol 17:475–479CrossRefPubMedGoogle Scholar
  46. Ray NF, Chan JK, Thamer M, Melton LJ (1997) Medical expenditures for the treatment of osteoporotic fractures in the United States in 1995: report from the National Osteoporosis Foundation. J Bone Miner Res 12:24–35CrossRefPubMedGoogle Scholar
  47. Riddick CA, Serio KJ, Hodulik CR, Ring WL, Regan MS, Bigby TD (1999) TGF-beta increases leukotriene C4 synthase expression in the monocyte-like cell line, THP-1. J Immunol 162:1101–1107PubMedGoogle Scholar
  48. Rozzo SJ, Allard JD, Choubey D, Vyse TJ, Izui S, Peltz G, Kotzin BL (2001) Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus. Immunity 15:435–443CrossRefPubMedGoogle Scholar
  49. Schneider HG, Michelangeli VP, Frampton RJ, Grogan JL, Ikeda K, Martin TJ, Findlay DM (1992) Transforming growth factor-beta modulates receptor binding of calciotropic hormones and G protein-mediated adenylate cyclase responses in osteoblast-like cells. Endocrinology 131:1383–1389CrossRefPubMedGoogle Scholar
  50. Shen H, Zhang YY, Long JR, Xu FH, Liu YZ, Xiao P, Zhao LJ, Xiong DH, Liu YJ, Dvornyk V, Rocha-Sanchez S, Liu PY, Li JL, Conway T, Davies KM, Recker RR, Deng HW (2004) A genome-wide linkage scan for bone mineral density in an extended sample: evidence for linkage on 11q23 and Xq27. J Med Genet 41:743–751CrossRefPubMedGoogle Scholar
  51. Smyth GK (2004) Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:1–26Google Scholar
  52. Sozzi G, Miozzo M, Di Palma S, Minelli A, Calderone C, Danesino C, Pastorino U, Pierotti MA, Della Porta G (1990) Involvement of the region 13q14 in a patient with adamantinoma of the long bones. Hum Genet 85:513–515CrossRefPubMedGoogle Scholar
  53. Stone KL, Seeley DG, Lui LY, Cauley JA, Ensrud K, Browner WS, Nevitt MC, Cummings SR (2003) BMD at multiple sites and risk of fracture of multiple types: long-term results from the Study of Osteoporotic Fractures. J Bone Miner Res 18:1947–1954CrossRefPubMedGoogle Scholar
  54. Styrkarsdottir U, Cazier JB, Kong A, Rolfsson O, Larsen H, Bjarnadottir E, Johannsdottir VD, Sigurdardottir MS, Bagger Y, Christiansen C, Reynisdottir I, Grant SF, Jonasson K, Frigge ML, Gulcher JR, Sigurdsson G, Stefansson K (2003) Linkage of osteoporosis to chromosome 20p12 and association to BMP2. PLoS Biol 1:E69CrossRefPubMedGoogle Scholar
  55. Styrkarsdottir U, Halldorsson BV, Gretarsdottir S, Gudbjartsson DF, Walters GB, Ingvarsson T, Jonsdottir T, Saemundsdottir J, Center JR, Nguyen TV, Bagger Y, Gulcher JR, Eisman JA, Christiansen C, Sigurdsson G, Kong A, Thorsteinsdottir U, Stefansson K (2008) Multiple genetic loci for bone mineral density and fractures. N Engl J Med 358:2355–2365CrossRefPubMedGoogle Scholar
  56. Swanton E, Bishop N, Woodman P (1999) Human rabaptin-5 is selectively cleaved by caspase-3 during apoptosis. J Biol Chem 274:37583–37590CrossRefPubMedGoogle Scholar
  57. Tsai JA, Rong H, Torring O, Matsushita H, Bucht E (2000) Interleukin-1beta upregulates PTHrP-mRNA expression and protein production and decreases TGF-beta in normal human osteoblast-like cells. Calcif Tissue Int 66:363–369CrossRefPubMedGoogle Scholar
  58. Turner CH, Roeder RK, Wieczorek A, Foroud T, Liu G, Peacock M (2001) Variability in skeletal mass, structure, and biomechanical properties among inbred strains of rats. J Bone Miner Res 16:1532–1539CrossRefPubMedGoogle Scholar
  59. Wada Y, Sato M, Niimi M, Tamaki M, Ishida T, Takahara J (1995) Inhibitory effects of interleukin-1 on growth hormone secretion in conscious male rats. Endocrinology 136:3936–3941CrossRefPubMedGoogle Scholar
  60. Wang J, Zhou J, Bondy CA (1999) Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy. FASEB J 13:1985–1990PubMedGoogle Scholar
  61. Wilson SG, Reed PW, Bansal A, Chiano M, Lindersson M, Langdown M, Prince RL, Thompson D, Thompson E, Bailey M, Kleyn PW, Sambrook P, Shi MM, Spector TD (2003) Comparison of genome screens for two independent cohorts provides replication of suggestive linkage of bone mineral density to 3p21 and 1p36. Am J Hum Genet 72:144–155CrossRefPubMedGoogle Scholar
  62. Zhou H, Choong PC, Chou ST, Kartsogiannis V, Martin TJ, Ng KW (1995) Transforming growth factor beta 1 stimulates bone formation and resorption in an in-vivo model in rabbits. Bone 17:443S–448SCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Imranul Alam
    • 1
  • Qiwei Sun
    • 1
  • Daniel L. Koller
    • 2
  • Lixiang Liu
    • 2
  • Yunlong Liu
    • 3
  • Howard J. Edenberg
    • 4
  • Tatiana Foroud
    • 2
  • Charles H. Turner
    • 1
  1. 1.Department of Biomedical EngineeringIndiana University Purdue University IndianapolisIndianapolisUSA
  2. 2.Medical and Molecular GeneticsIndiana University Purdue University Indianapolis (IUPUI)IndianapolisUSA
  3. 3.Medicine, Indiana University Purdue University Indianapolis (IUPUI)IndianapolisUSA
  4. 4.Biochemistry and Molecular BiologyIndiana University Purdue University Indianapolis (IUPUI)IndianapolisUSA

Personalised recommendations