Perthes disease is one of the most common forms of pediatric femoral head osteonecrosis with an unknown etiology. Coagulation factors were the first genetic factors suspected to have a role in the pathogenesis of this disease, but studies showed inconsistent results. It is described that inflammation is present during early stages of Perthes disease, but its genetic aspect has not been studied extensively. Little is known regarding the status of apoptotic factors during the repair process that leads to the occurrence of hip deformity in patients. Therefore, the aim of this study was to analyze major mediators involved in coagulation, inflammation, and apoptotic processes as possible causative factors of Perthes disease. The study cohort consisted of 37 patients. Gene variants of TNF-α, FV, FII, and MTHFR genes were determined by PCR-RFLP, while IL-3 and PAI-1 were genotyped by direct sequencing. The expression level of Bax, Bcl-2, Bcl2L12, Fas and FasL was analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) technique. Our results showed a significantly increased level of expression of pro-apoptotic factor Bax along with significantly higher Bax/Bcl-2 ratio in the patient group.
Conclusion: The results presented indicate that apoptosis could be one of the factors contributing to the lack of balanced bone remodeling process in Perthes patients.
What is Known:
•The etiology of Perthes disease is unknown. The role of genetic factors involved in the coagulation process has been studied, showing inconsistent results so far.
•Genetic factors involved in inflammation and apoptotic processes that could contribute to development of hip deformity have not been studied extensively.
What is New:
•Our results show significantly increased level of expression of the pro-apoptotic factor Bax as well as significantly higher Bax/Bcl-2 ratios in patient group, indicating that apoptosis could be one of the factors contributing to the lack of a balanced bone remodeling process in Perthes patients.
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Bcl-2-associated X protein
B cell lymphoma 2
Factor V Leiden
Glyceraldehyde 3-phosphate dehydrogenase
Plasminogen activator inhibitor-1
Peripheral blood mononuclear cells
Tumor necrosis factor alpha
Baluchamy S, Sankar N, Navaraj A, Moran E, Thimmapaya B (2007) Relationship between E1A binding to cellular proteins, c-myc activation and S-phase induction. Oncogene 26:781–787
Boss JH, Misselevich I (2003) Osteonecrosis of the femoral head of laboratory animals: the lessons learned from a comparative study of osteonecrosis in man and experimental animals. Vet Pathol 40:345–354
Calder JD, Buttery L, Revell PA, Pearse M, Polak JM (2004) Apoptosis—a significant cause of bone cell death in osteonecrosis of the femoral head. J Bone Joint Surg Br 86:1209–1213
Chang MK, Raggatt LJ, Alexander KA, Kuliwaba JS, Fazzalari NL, Schroder K, Maylin ER, Ripoll VM, Hume DA, Pettit AR (2008) Osteal tissue macrophages are intercalated throughout human and mouse bone lining tissues and regulate osteoblast function in vitro and in vivo. J Immunol 181:1232–1244
Chu X, Dong C, Lei R, Sun L, Wang Z, Dong Y, Shen M, Wang Y, Wang B, Zhang K, Yang L, Li Y, Yuan W, Wang Y, Song H, Jin L, Xiong M, Huang W (2009) Polymorphisms in the interleukin 3 gene show strong association with susceptibility to Graves' disease in Chinese population. Genes Immun 10:260–266
Clark JP, Munson KW, Gu JW, Lamparska-Kupsik K, Chan KG, Yoshida JS, Kawachi MH, Crocitto LE, Wilson TG, Feng Z, Smith SS (2008) Performance of a single assay for both type III and type VI TMPRSS2:ERG fusions in noninvasive prediction of prostate biopsy outcome. Clin Chem 54:2007–2017
Dillman JR RJH (2009) MRI of Legg-Calve-Perthes disease. Am J Roentgenol 193:1394–1407
Djordjevic V, Stankovic M, Brankovic-Sreckovic V, Rakicevic L, Damnjanovic T, Antonijevic N, Radojkovic D (2012) Prothrombotic genetic risk factors in stroke: a possible different role in pediatric and adult patients. Clin Appl Thromb Hemost 18:658–661
Edwards CJ, Williams E (2010) The role of interleukin-6 in rheumatoid arthritis-associated osteoporosis. Osteoporos Int 21:1287–1293
Feldmann M, Brennan FM, Maini RN (1996) Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14:397–440
Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, Woo P (1998) The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 102:1369–1376
Floros KV, Thomadaki H, Florou D, Talieri M, Scorilas A (2006) Alterations in mRNA expression of apoptosis-related genes BCL2, BAX, FAS, caspase-3, and the novel member BCL2L12 after treatment of human leukemic cell line HL60 with the antineoplastic agent etoposide. Ann N Y Acad Sci 1090:89–97
Jeong MC, Navani A, Oksenberg JR (1998) Limited allelic polymorphism in the human interleukin-3 gene. Mol Cell Probes 12:49–53
Joseph B, Willoughby R (2010) Perthes' disease: a review of contributions from the Asia-pacific region. Malays Orthop J 4:1–8
Kim H, Cho C, Cho Y, Cho S, Yoon K, Kim K (2011) Significant associations of PAI-1 genetic polymorphisms with osteonecrosis of the femoral head. BMC Musculoskelet Disord 12:160
Kim HK (2010) Legg-Calve-Perthes disease. J Am Acad Orthop Surg 18:676–686
Kim HK (2012) Pathophysiology and new strategies for the treatment of Legg-Calve-Perthes disease. J Bone Joint Surg Am 94:659–669
Kim HK, Herring JA (2011) Pathophysiology, classifications, and natural history of Perthes disease. Orthop Clin North Am 42:285–295, v
Kim HK, Morgan-Bagley S, Kostenuik P (2006) RANKL inhibition: a novel strategy to decrease femoral head deformity after ischemic osteonecrosis. J Bone Miner Res 21:1946–1954
Lacey DC, Simmons PJ, Graves SE, Hamilton JA (2009) Proinflammatory cytokines inhibit osteogenic differentiation from stem cells: implications for bone repair during inflammation. Osteoarthritis Cartilage 17:735–742
Loder RT, Skopelja EN (2011) The epidemiology and demographics of Legg-Calve-Perthes' disease. ISRN Orthop 2011:504393
Merkel KD, Erdmann JM, McHugh KP, Abu-Amer Y, Ross FP, Teitelbaum SL (1999) Tumor necrosis factor-alpha mediates orthopedic implant osteolysis. Am J Pathol 154:203–210
Nagase Y, Iwasawa M, Akiyama T, Kadono Y, Nakamura M, Oshima Y, Yasui T, Matsumoto T, Hirose J, Nakamura H, Miyamoto T, Bouillet P, Nakamura K, Tanaka S (2009) Anti-apoptotic molecule Bcl-2 regulates the differentiation, activation, and survival of both osteoblasts and osteoclasts. J Biol Chem 284:36659–36669
Nair SP, Meghji S, Wilson M, Reddi K, White P, Henderson B (1996) Bacterially induced bone destruction: mechanisms and misconceptions. Infect Immun 64:2371–2380
Nanes MS (2003) Tumor necrosis factor-alpha: molecular and cellular mechanisms in skeletal pathology. Gene 321:1–15
Nelitz M, Lippacher S, Krauspe R, Reichel H (2009) Perthes disease: current principles of diagnosis and treatment. Dtsch Arztebl Int 106:517–523
Oltvai ZN, Milliman CL, Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–619
Papageorgiou SG, Kontos CK, Pappa V, Thomadaki H, Kontsioti F, Dervenoulas J, Papageorgiou E, Economopoulos T, Scorilas A (2011) The novel member of the BCL2 gene family, BCL2L12, is substantially elevated in chronic lymphocytic leukemia patients, supporting its value as a significant biomarker. Oncologist 16:1280–1291
Park BL, Kim LH, Choi YH, Lee JH, Rhim T, Lee YM, Uh ST, Park HS, Choi BW, Hong SJ, Park CS, Shin HD (2004) Interleukin 3 (IL3) polymorphisms associated with decreased risk of asthma and atopy. J Hum Genet 49:517–527
Perry DC (2013) Unravelling the enigma of Perthes disease. Ann R Coll Surg Engl 95:311–316
Perry DC, Machin DM, Pope D, Bruce CE, Dangerfield P, Platt MJ, Hall AJ (2012) Racial and geographic factors in the incidence of Legg-Calve-Perthes' disease: a systematic review. Am J Epidemiol 175:159–166
Pettit AR, Chang MK, Hume DA, Raggatt LJ (2008) Osteal macrophages: a new twist on coupling during bone dynamics. Bone 43:976–982
Scorilas A, Kyriakopoulou L, Yousef GM, Ashworth LK, Kwamie A, Diamandis EP (2001) Molecular cloning, physical mapping, and expression analysis of a novel gene, BCL2L12, encoding a proline-rich protein with a highly conserved BH2 domain of the Bcl-2 family. Genomics 72:217–221
Srzentic S, Spasovski V, Spasovski D, Zivkovic Z, Matanovic D, Bascarevic Z, Terzic-Supic Z, Stojiljkovic M, Karan-Djurasevic T, Stankovic B, Pavlovic S, Nikcevic G, Vukasinovic Z (2014) Association of gene variants in TLR4 and IL-6 genes with Perthes disease. Srp Arh Celok Lek 142:450–456
Vandermeer JS, Kamiya N, Aya-ay J, Garces A, Browne R, Kim HK (2011) Local administration of ibandronate and bone morphogenetic protein-2 after ischemic osteonecrosis of the immature femoral head: a combined therapy that stimulates bone formation and decreases femoral head deformity. J Bone Joint Surg Am 93:905–913
Walsh NC, Reinwald S, Manning CA, Condon KW, Iwata K, Burr DB, Gravallese EM (2009) Osteoblast function is compromised at sites of focal bone erosion in inflammatory arthritis. J Bone Miner Res 24:1572–1585
Woratanarat P, Thaveeratitharm C, Woratanarat T, Angsanuntsukh C, Attia J, Thakkinstian A (2014) Meta-analysis of hypercoagulability genetic polymorphisms in Perthes disease. J Orthop Res 32:1–7
Wu X, McKenna MA, Feng X, Nagy TR, McDonald JM (2003) Osteoclast apoptosis: the role of Fas in vivo and in vitro. Endocrinology 144:5545–5555
Yamada R, Tanaka T, Unoki M, Nagai T, Sawada T, Ohnishi Y, Tsunoda T, Yukioka M, Maeda A, Suzuki K, Tateishi H, Ochi T, Nakamura Y, Yamamoto K (2001) Association between a single-nucleotide polymorphism in the promoter of the human interleukin-3 gene and rheumatoid arthritis in Japanese patients, and maximum-likelihood estimation of combinatorial effect that two genetic loci have on susceptibility to the disease. Am J Hum Genet 68:674–685
Yogesha SD, Khapli SM, Wani MR (2005) Interleukin-3 and granulocyte-macrophage colony-stimulating factor inhibits tumor necrosis factor (TNF)-alpha-induced osteoclast differentiation by down-regulation of expression of TNF receptors 1 and 2. J Biol Chem 280:11759–11769
This work was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia (Grant No. III41004). This study is in memoriam of Prof. Zoran Vukasinovic, who was the research coordinator for the subproject: “Orthopedic rare diseases: molecular basis of Perthes disease.” His enthusiasm and willpower contributed most to the opening of this new research topic in Serbia.
The study was approved by the Ethics Committee of the Institute for Orthopedic Surgery “Banjica,” Belgrade, Serbia (I-86/34; 7-2, 12. 12. 2012.). Written informed consent was obtained for all patients.
Conflict of interest
The authors declare that they have no conflict of interest.
DS, ZB and ZŽ performed the case and control sample collection and clinical management of patients. ZTŠ and DM participated in clinical management of patients and participated in studz design. VDJ provided control sample for coagulation study and participated in data analyzes. SS and VS performed the laboratory work and statistical analysis. SS and GN analyzed data and wrote first draft of the paper. VS and SP designed the study and wrote the final version of the paper. VS take the primary responsibility for the paper.
Sanja Srzentić and Gordana Nikčević contributed equally to this work.
Communicated by Beat Steinmann
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Srzentić, S., Nikčević, G., Spasovski, D. et al. Predictive genetic markers of coagulation, inflammation and apoptosis in Perthes disease—Serbian experience. Eur J Pediatr 174, 1085–1092 (2015). https://doi.org/10.1007/s00431-015-2510-z
- Blood coagulation factors
- Bone remodeling
- Perthes disease