Vitamin D receptor gene (VDR) transcripts in bone, cartilage, muscles and blood and microarray analysis of vitamin D responsive genes expression in paravertebral muscles of juvenile and adolescent idiopathic scoliosis patients

Roman Nowak, Justyna Szota, Urszula Mazurek, Roman Nowak, Justyna Szota, Urszula Mazurek

Abstract

Background: VDR may be considered as a candidate gene potentially related to idiopathic scoliosis susceptibility and natural history. Transcriptional profile of VDR mRNA isoforms might be changed in the structural tissues of the scoliotic spine and potentially influence the expression of VDR responsive genes. The purpose of the study was to determine differences in mRNA abundance of VDR isoforms in bone, cartilage and paravertebral muscles between tissues from curve concavity and convexity, between JIS and AIS and to identify VDR responsive genes differentiating juvenile and adolescent idiopathic scoliosis in paravertebral muscles.

Methods: In a group of 29 patients with JIS and AIS, specimens of bone, cartilage, paravertebral muscles were harvested at the both sides of the curve apex together with peripheral blood samples. Extracted total RNA served as a matrix for VDRs and VDRl mRNA quantification by QRT PCR. Subsequent microarray analysis of paravertebral muscular tissue samples was performed with HG U133A chips (Affymetrix). Quantitative data were compared by a nonparametric Mann Whitney U test. Microarray results were analyzed with GeneSpring 11GX application. Matrix plot of normalized log-intensities visualized the degree of differentiation between muscular tissue transcriptomes of JIS and AIS group. Fold Change Analysis with cutoff of Fold Change ≥2 identified differentially expressed VDR responsive genes in paravertebral muscles of JIS and AIS.

Results: No significant differences in transcript abundance of VDR isoforms between tissues of the curve concavity and convexity were found. Statistically significant difference between JIS and AIS group in mRNA abundance of VDRl isoform was found in paravertebral muscles of curve concavity. Higher degree of muscular transcriptome differentiation between curve concavity and convexity was visualized in JIS group. In paravertebral muscles Tob2 and MED13 were selected as genes differentially expressed in JIS and AIS group.

Conclusions: In Idiopathic Scolioses transcriptional activity and alternative splicing of VDR mRNA in osseous, cartilaginous, and paravertebral muscular tissues are tissue specific and equal on both sides of the curve. The number of mRNA copies of VDRl izoform in concave paravertebral muscles might be one of the factors differentiating JIS and AIS. In paravertebral muscles Tob2 and Med13 genes differentiate Adolescent and Juvenile type of Idiopathic Scoliosis.

Figures

Figure 1
Figure 1
Spinal and rib cage CT scan at the apex of the curve. Measurement of the vertebral rotation angle about the longitudinal axis relative to the sagittal plane RAsag and Rib Hump index RHi defined as the ratio (H-D) / W.
Figure 2
Figure 2
Results of electrophoresis of QRT-PCR products on 6% polyacrylamid gel.
Figure 3
Figure 3
Dissociation curves indicating number of nucleic acids amplification products and Tm of amplimers: amplimer GAPDH (Tm = 80,1°C), amplimer VDR (Tm = 82,2°C), amplimer VDRs (Tm = 83,1°C), amplimer β-actin (Tm = 86,2°C).
Figure 4
Figure 4
Comparison of fluorescence intensity values of 12 paravertebral muscle HGU 133 A (Affymetrix) oligonucleotide microarray chips (M1, M2 – adequately muscular tissue from curve concavity and convexity). Every box-and-whisker plot represents 22283 mRNA probes fluorescence intensity values normalized with RMA algorithm. All signals based to median (black horizontal lines), height of the rectangle (blue) determines the value of interquartile range; outliers marked in red.
Figure 5
Figure 5
Matrix plot illustrating the degree of differentiation between the transcriptomes of muscular tissue in dependence of the side of the curve and the age of scoliosis onset. Red spots-up regulated genes, blue spots-down regulated genes. A1, A2- adequately muscular tissue samples from curve concavity and convexity in group A-Juvenile Idiopathic Scoliosis; B1, B2- adequately muscular tissue samples from curve concavity and convexity in group B-Adolescent Idiopathic Scoliosis.
Figure 6
Figure 6
Scatter plot comparing normalized intensities of 75 mRNA probes of VDR responsive genes between group A –JIS and B-AIS in muscular tissue specimens from the concave (M1) and convex side of the curve (M2). Each rectangle represents the normalized expression of an individual gene within both mRNA populations. Axes of the scatter plot represent the log scale of the normalized fluorescence intensity value in group A and B. The middle dashed line indicates values that represent a ratio of 1 (similar expression in group A and B). The outer lines represent a ratio of 2,0 (upper line; 2-fold greater expression in group B compared with group A) and 0,5 (lower line; 2-fold greater expression in group A compared with group B).

References

    1. Kouwenhoven JW, Castelein RM. The pathogenesis of Adolescent Idiopathic Scoliosis. Review of the literature. Spine. 2008;33:2898–2908.
    1. Lowe TG, Edgar M, Marguiles JY, Miller NH, Raso J, Reinker KA, Rivard C. Current concepts review. Etiology of idiopathic scoliosis: current trends in research. J Bone and Joint Surg. 2000;82-A(8):1157–1168.
    1. Burwell RG, Dangerfield PH, Freeman BJ. Concepts on the pathogenesis of adolescent idiopathic scoliosis. Bone growth and mass, vertebral column, spinal cord, brain, skull, extra-spinal left-right length asymmetries, disproportions and molecular pathogenesis. Stud Health Technol Inform. 2008;135:3–52.
    1. Cheng JC, Tang NLS, Yeung H, Miller N. Genetic association of complex traits. Clin Ortop and Rel Res. 2007;462:36–44.
    1. Hermus JP, Rhijn W, Ooij A. Non-genetic expression of adolescent idiopathic scoliosis: a case report and review of the literature. Eur Spine J. 2007;16:S338–S341.
    1. Willner S. A study of growth in girls with adolescent idiopathic structural scoliosis. Clin Ortop Rel Res. 1974;101:129–135.
    1. Ylikowski M. Height of girls with adolescent idiopathic scoliosis. Eur Spine J. 2003;12:288–291.
    1. Cheung CSK, Lee WTK, Tse YK, Tang SP, Lee KM, Guo X, Qin L, Cheng JC. Abnormal peri-pubertal anthropometric measurements and growth pattern in adolescent idiopathic scoliosis: a study of 598 patients. Spine. 2003;28:2152–2157.
    1. Hung VWY, Qin L, Cheung TP, Lam FRC, Ng BKW, Tse YK, Guo X, Lee KM, Cheng JCY. Osteopenia: a new prognostic factor of curve progression in adolescent idiopathic scoliosis. J Bone Joint Surg. 2005;87-A:2709–2716.
    1. Lee WTK, Cheung CSK, Tse YK, Guo X, Qin L, Lam TP, Ng BKW, Cheng JCY. Association of osteopenia with curve severity in adolescent idiopathic scoliosis: a study of 919 girls. Osteoporos Int. 2005;16:1924–1932.
    1. Grundberg E, Brandstrom H, Ribom FE, Ljunggren O, Mallmin H, Kindmark A. Genetic variation in the human vitamin D receptor is associated with muscle strength, fat mass and body weight in Swedish women. Eur J Endocrin. 2004;150:323–328.
    1. Inoue M, Minami S, Nakata Y, Takaso M, Otsuka Y, Kitahara H, Isobe K, Kotani T, Maruta T, Moriya H. Prediction of curve progression in idiopathic scoliosis from gene polymorphic analysis. Stud Health Technol Inform. 2002;91:90–96.
    1. Kitagawa I, Ikumi I, Kitagawa Y, Kawase Y, Nagaya T, Tokudome S. Advanced onset of menarche and higher bone mineral density depending on vitamin D receptor gene polymorphism. Eur J Endocrinol. 1998;139:522–527.
    1. Xiong DH, Xu FH, Liu PY, Shen H, Long JR, Elze L, Recker RR, Deng HW. Vitamin D receptor gene polymorphisms are linked to, and associated with adult height. J Med Genet. 2005;42:228–234.
    1. Xia CW, Qiu Y, Sun X, Qiu XS, Wang SF, Zhu ZZ, Zhu F. Vitamin D receptor gene polymorphism in female adolescent idiopathic scoliosis patients. Zhonghua Yi Xe Za Zhi. 2007;87:1465–1469. abstr.
    1. Suh KT, Eun I, Lee JS. Polymorphism in vitamin D receptor is associated with bone mineral density in patients with adolescent idiopathic scoliosis. Eur Spine J. 2010;19:1545–1550.
    1. Sunn KL, Cock TA, Crofts LA, Eisman JA, Gardiner EM. Novel N-terminal variant of human VDR. Mol Endocrinol. 2001;15:1599–1609.
    1. Bouillon R, Carmeliet G, Verlinden L, Etten E, Verstuyf A, Luderer HF, Lieben L, Mathieu C, Demay M. Vitamin D and human health: Lessons from vitamin D receptor null mice. Endocr Rev. 2008;29:726–776.
    1. Figueiredo UM, James JIP. Juvenile idiopathic scoliosis. J Bone Joint Surg. 1981;63-B:61–66.
    1. James JIP. Idiopathic scoliosis. The prognosis, diagnosis and operative indications related to curve patterns and age of onset. J Bone Joint Surg. 1954;36-B:36–49.
    1. Tolo VT, Gillespie R. The characteristics of juvenile idiopathic scoliosis and results of its treatment. J Bone Joint Surg. 1978;60-B:181–188.
    1. Robinson CM, McMaster MJ. Juvenile idiopathic scoliosis. Curve patterns and prognosis in one hundred and nine patients. J Bone Joint Surg. 1996;78-A:1140–1148.
    1. Weinstein SL, Dolan LA, Spratt KF, Peterson KK, Spoonamore MJ, Ponseti IV. Health and function of patients with untreated idiopathic scoliosis. A 50-year natural history study. JAMA. 2003;289:559–567.
    1. Lenke LG, Betz RR, Harms J, Bridwell KH, Clements DH, Lowe TG, Blanke K. Adolescent idiopathic scoliosis. A new classification to determine extent of spinal arthrodesis. J Bone Joint Surg. 2001;83-A(8):1169–1181.
    1. Scoliosis Research Society. SRS Revised Glossary of Terms. 2002. .
    1. Aaro S, Dahlborn M. Estimation of vertebral rotation and the spinal and rib cage deformity in scoliosis by computer tomography. Spine. 1981;6:460–467.
    1. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003;4:249–264.
    1. Affymetrix. .
    1. Sutton AA, MacDonald PN, Vitamin D. More than a”Bone-a-Fide” hormone. Mol Endocrinol. 2003;17:777–791.
    1. Raggio CL. Sexual dimorphism in adolescent idiopathic scoliosis. Orthop Clin North Am. 2006;37:555–558.
    1. Karasik D, Ferrari SL. Contribution of gender-specific factors to osteoporosis risk. Ann Hum Genet. 2008;72:696–714.
    1. Tosi LL, Boyan BD, Boskey AL. Does sex matter in musculoskeletal health? The influence of sex and gender on musculoskeltal health. J Bone Joint Surg. 2005;87A:1631–1647.
    1. Arfai K, Pitukcheewanont PD, Goran MI, Tavare CJ, Heller L, Gilsanz V. Bone, muscle, and fat: sex related differences in prepubertal children. Radiology. 2002;224:338–344.
    1. Wang WJ, Yeung HY, Chu WC, Tang NL, Lee KM, Qiu Y, Burwell RG, Cheng JC. Top theories for the etiopathogenesis of adolescent idiopathic scoliosis. J Pediatr Ortop. 2011;31:S14–S27.
    1. Bagnall KM. Using a synthesis of the research literature related to the aetiology of adolescent idiopathic scoliosis to provide ideas on future directions for success. Scoliosis. 2008;3:5. .
    1. Veldhuizen AG, Wever DJ, Webb PJ. The aetiology of idiopathic scoliosis: biomechanical and neuromuscular factors. Eur Spine J. 2000;9:178–184.
    1. Stokes IAF. Analysis and simulation of progressive adolescent scoliosis by biomechanical growth. Eur Spine J. 2007;16:1621–1628.
    1. Perdriolle R, Becchetti S, Vidal J, Lopez P. Mechanical process and growth cartilages. Essential factors in the progression of scoliosis. Spine. 1993;18:343–349.
    1. Stokes IAF, Burwell RG, Dangerfield PH. Biomechanical spinal growth modulation and progressive adolescent scoliosis-a test of the ‘vicious cycle’ pathogenetic hypothesis: summary of an electronic focus group debate of the IBSE. Scoliosis. 2006. .
    1. Villemure I, Aubin CE, Dansereau J, Labelle H. Biomechanical simulations of thye spine deformation process in adolescent idiopathic scoliosis from different pathogenesis hypotheses. Spine. 2004;13:83–90.
    1. Shea KG, Ford T, Bloebaum RD, D’Astous J, King H. A comparison of the microarchitectural bone adaptations of the concave and convex thoracic spinal facets in idiopathic scoliosis. J Bone Joint Surg. 2004;86-A:1000–1006.
    1. Zhu F, Qiu Y, Yeung HY, Lee KM, Cheng CJ. Trabecular bone micro-architecture and bone mineral density in adolescent idiopathic and congenital scoliosis. Orthop Surg. 2009;1:78–83.
    1. Cheng JC, Guo X. Osteopenia. in adolescent idiopathic scoliosis. A primary problem or secondary to the spinal deformity? Spine. 1997;22:1716–1721.
    1. Cheng JC, Guo X, Sher AH. Persistent osteopenia in adolescent idiopathic scoliosis. A longitudinal follow up study. Spine. 1999;24:1218–1222.
    1. Cheng JC, Tang SP, Guo X. Osteopenia in adolescent idiopathic scoliosis: a histomorphometric study. Spine. 2001;26:E19–E23.
    1. Cheung CSK, Lee WTK, Tse YK, Lee KM, Guo X, Qin L, Cheng JCY. Generalized osteopenia in adolescent idiopathic scoliosis – association with abnormal pubertal growth, bone turnover and calcium intake? Spine. 2006;31:330–338.
    1. Sadat-Ali M, Al-Othman A, Bubshait D, Al-Dakheel D. Does scoliosis causes low bone mass? A comparative study between siblings. Eur Spine J. 2008;17:944–947.
    1. Park WW, Suh KT, Kim JI, Kim S, Lee JS. Decreased osteogenic differentiation of mesenchymal stem cells and reduced bone mineral density in patients with adolescent idiopathic scoliosis. Eur Spine J. 2009;18:1920–1926.
    1. Seremak-Mrozikiewicz A, Drews K, Mrozikiewicz P, Bartkowiak-Wieczorek J, Marcinkowska M, Wawrzyniak A, Słomski R, Kalak R, Czerny B, Horst-Sikorska W. Correlation of vitamin D receptor gene (VDR) polymorphism with osteoporotic changes in Polish postmenopausal women. Neuro Endocrinol Lett. 2009;30:540–546.
    1. Colin E, Uitterlinden A, Meurs J, Bergink A, Van de Klift M, Fang Y, Arp P, Hofman A, Van Leeuwen J, Pols H. Interaction between vitamin D receptor genotype and estrogen receptor genotype influences vertebral fracture risk. J Clin Endocrinol Metab. 2003;88:3777–3784.
    1. Cholewicki J, Panjabi MM, Khachatryan A. Stabilizing function of trunk flexor-extensor muscles around a neutral spine posture. Spine. 1997;22:2207–2212.
    1. Masi AT, Dorsch JL, Cholewicki J. Are adolescent idiopathica scoliosis and ankylosing spondylitis counteropposing conditions? A hypothesis on biomechanical contributions predisposing to these spinal disorders. Clin Exp Rheumatol. 2003;21:573–580.
    1. Mannion A, Meier M, Grob D, Muntener M. Paraspinal muscle fibre type alterations associated with scoliosis: an old problem revisited with new evidence. Eur Spine J. 1998;7:289–293.
    1. Gonzalez-Reimers E, Duran-Castellon MC, Lopez-Lirola A, Santolaria-Fernandez F, Abreu-Gonzalez P, Alvisa-Negrin J, Sanchez-Perez MJ. Alcoholic myopathy: vitamin d deficiency is related to muscle fibre atrophy in a murine model. Alcohol Alcohol. 2010;45:223–230.
    1. Kindsfater K, Lowe T, Lawellin D, Weinstein D, Akmakjian J. Levels of platelet calmodulin for the prediction of progression and severity of adolescent idiopathic scoliosis. J Bone Joint Surg. 1994;76-A:1186–1192.
    1. Lowe T, Burwell R, Dangerfield P. Platelet calmodulin levels in adolescent idiopathic scoliosis (AIS): can they predict curve progression and severity? Summary of an electronic focus group debate of the IBSE. Eur Spine J. 2004;13:257–265.
    1. Acaroglu E, Akel I, Alanay A, Yazici M, Marcucio R. Comparison of the melatonin and calmodulin in paravertebral muscle and platelets of patients with or without Adolescent Idiopathic Scoliosis. Spine. 2009;34:E659–E663.
    1. Drittani L, de Boland A, Boland R. Stimulation of calmodulin synthesis in proliferating myoblasts by 1,25-dihydroxy-vitamin D3. Mol Cell Endocrinol. 1990;74:143–153.
    1. Wise CA, Gao X, Shoemaker S, Gordon D, Herring JA. Understanding genetic factors in idiopathic scoliosis, a complex disease of childhood. Current Genomics. 2008;9:51–59.
    1. Lenke LG, Dobbs MB. Management of juvenile idiopathic scoliosis. J Bone Joint Surg. 2007;89-A:S55–S63.
    1. Charles YP, Daures J, Rosa V, Dimeglio A. Progression risk of idiopathic juvenile scoliosis during pubertal growth. Spine. 2006;31:1933–1942.
    1. Weinstein SL. Natural history. Spine. 1999;24:2592–2600.
    1. Ward K, Ogilvie J, Argyle V, Nelson L, Meade M, Braun J, Chettier R. Polygenic inheritance of adolescent idiopathic scoliosis: a study of extended families in Utah. Am J Med Genet A. 2010;152A:1178–1188.
    1. Ogilvie JW. Update on prognostic genetic testing in Adolescent Idiopathic Scoliosis (AIS) J Pediatr Orthop. 2011;31:S46–S48.
    1. Jia S, Meng A. Tob genes in development and homeostasis. Dev Dyn. 2007;236:913–921.
    1. Yuan J, Cao J, Tang Z, Wang N, Li K. Molecular characterization of Tob1 in muscle development in pigs. Int J Mol Sci. 2011;12:4315–4326.
    1. Ajima R, Akiyama T, Usui M, Yoneda M, Yoshida Y, Nakamura T, Minowa O, Noda M, Tanaka S, Noda T, Yamamoto T. Osteoporotic bone formation in mice lacking tob2; involvment of Tob2 in RANK ligand expression and osteoclasts differentiation. FEBS Lett. 2008;582:1313–1318.
    1. Sartori R, Milan G, Patron M, Mammucari C, Blaauw B, Abraham R, Sandri M. Smad2 and 3 transcription factors control muscle mass in adulthood. Am J Physiol Cell Physiol. 2009;296:C1248–C1257.
    1. Bubnoff A, Cho W. Intracellular BMP signaling regulation in vertebrates: pathway or network? Develop Biol. 2001;239:1–14.
    1. Carrera I, Janody F, Leeds N, Duveau F, Treisman J. Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13. PNAS. 2008;105:6644–6649.
    1. Knuesel M, Meyer K, Bernecky C, Taatjes D. The human CDK8 subcomplex is a molecular switch that controls Mediator coactivators function. Genes Dev. 2009;23:439–451.
    1. Kim Y, Lis J. Interactions between subunits of Drosophila Mediator and activator protein. Trends Biochem Sci. 2005;30:245–249.
    1. Conaway R, Conaway J. Function and regulation of the Mediator complex. Curr Op Genet Develop. 2011;21:225–230.
    1. Conaway R, Sato S, Tomomori-Sato C, Yao T, Conaway J. The mammalian Mediator complex and its role in transcriptional regulation. Trends Biochem Sci. 2005;30:250–255.
    1. Oda Y, Chalkley R, Burlingame A, Bikle D. The transcriptional coactivators DRIP/Mediator complex is involved in vitamin D receptor function and regulates keratinocyte proliferation and differentiation. J Invest Dermatol. 2010;130:2377–2388.
    1. Chen Z, Tang NL, Cao X, Yi L, Cheng JC, Qiu Y. Promoter polymorphism of matrillin-1 gene predisposes to adolescent idiopathic scoliosis in Chinese population. Eur Hum Genet. 2009;17:525–532.
    1. Yeung H, Tang N, Lee K, Nq B, Hung V, Kwok R, Guo X, Cheng J. Genetic association study of insulin-like growth factor-1 (IGF-1) gene with curve severity and osteopenia in adolescent idiopathic scoliosis. Stud Health Technol Inform. 2006;123:18–24.
    1. Inoue M, Minami S, Nakata Y, Kitahara H, Otsuka Y, Isobe K, Takaso M, Tokunaga M, Nishikawa S, Maruta T, Moriya H. Association between estrogen receptor gene polymorphisms and curve severity of idiopathic scoliosis. Spine. 2002;27(21):2357–2362.

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