- Email: [email protected]
Monocyte Chemotactic Protein-1 -2518 Gene Polymorphism and Susceptibility to Spinal Tuberculosis
Archives of Medical Research 45 (2014) 183e187
ORIGINAL ARTICLE
Monocyte Chemotactic Protein-1 -2518 Gene Polymorphism and Susceptibility to Spinal Tuberculosis Qile Gao,a Qingbiao Du,b Hongqi Zhang,a Chaofeng Guo,a Shijin Lu,a Ang Deng,a Mingxing Tang,a Shaohua Liu,a Yuxiang Wang,a Jia Huang,a and Qiang Guoa a Department of Spine Surgery, Xiangya Hospital, Central South University, Hunan, China Clinical Laboratory, Secondary Affiliated Hospital, Fujian Medical University, Fujian, China
b
Received for publication July 10, 2013; accepted December 13, 2013 (ARCMED-D-13-00370).
Background and Aims. Monocyte chemotactic protein-1 (MCP-1) gene polymorphisms play important roles in regulating immunological reactions and may be associated with pulmonary tuberculosis. However, the relationship between the MCP-1 -2518 gene polymorphism and susceptibility to spinal tuberculosis remains unknown. We undertook this study to investigate the relationships between MCP-1 promoter 2518 genotype frequency and allele polymorphisms and susceptibility to spinal tuberculosis in a Chinese Han population. Methods. Patients with spinal tuberculosis and healthy volunteers were enrolled between December 2004 and December 2010. MCP-1 -2518 polymorphisms in both groups were detected using polymerase chain reaction and DNA sequencing. MCP-1 genotype was analyzed in all patients. Differences in genotype frequencies between groups were compared using c2 tests. Results. A total of 208 patients with spinal tuberculosis and 210 healthy volunteers were included. The distribution frequencies of MCP-1 -2518 GG, GA and AA genotypes were 36.1, 50.9 and 13.0%, respectively, in the case group and 25.2, 53.8 and 21.0%, respectively, in the control group ( p !0.05). MCP-1 -2518 GG genotype was significantly associated with the onset of spinal tuberculosis (OR 5 2.306, 95% CI 5 1.273e4.178). The G and A allele frequencies were 61.5% and 38.5%, respectively, in the case group, and 52.1% and 47.9% in the control group ( p !0.05), the allele ‘‘G’’ of MCP-1 -2518 showed an association with an increased risk for spinal tuberculosis: OR 5 1.777, 95% CI 5 1.053e2999, p 5 0.03 in the dominant model; OR 5 1.67, 95% CI 5 1.097e2.544, p 5 0.016 in the recessive model. Conclusions. The MCP-1 -2518 GG genotype and presence of the G allele may be associated with susceptibility to spinal tuberculosis in the Chinese Han population. Ó 2014 IMSS. Published by Elsevier Inc. Key Words: Monocyte chemotactic protein-1, Gene polymorphism, Spinal tuberculosis, Susceptibility.
Introduction Spinal tuberculosis is associated with high morbidity and a high disability rate. An increase in the incidence of multidrug-resistant strains of Mycobacterium tuberculosis indicates that the treatment needs of patients with spinal
Address reprint requests to: Hongqi Zhang, MD, Central South University, Xiangya Hospital, Spine Surgery, *87 Xiang Ya Road, Changsha, Hunan 410008, China; Phone: þ86073189753001; E-mail: [email protected] or [email protected]
tuberculosis have changed. A thorough understanding of the individual factors influencing the host antituberculosis immunological reaction is therefore needed to help prevent and treat spinal tuberculosis. Monocyte chemotactic protein-1 (MCP-1) is a monocyte and macrophage chemotactic factor that induces and activates monocytes. MCP-1 can increase the free oxygen concentration in monocyte fluid to enhance the killing effects of monocytes against bacteria (1). Moreover, MCP-1 can mediate cell migration and phagocytosis and other adhesion-dependent functions by regulating cell surface adhesion molecule
0188-4409/$ - see front matter. Copyright Ó 2014 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2013.12.007
184
Gao et al./ Archives of Medical Research 45 (2014) 183e187
expression and cytokine production (2). MCP-1 can restrict the in vivo spreading of M. tuberculosis and regulate secondary immunological reactions and is thus closely related to the occurrence and progression of tuberculosis (3). A previous study demonstrated that the MCP-1 -2518A/G polymorphism was associated with increased susceptibility to pulmonary tuberculosis in the Chinese Han population (4); however, the correlation between the gene polymorphism and spinal tuberculosis remains poorly understood. The present study therefore aimed to clarify the relationship between MCP-1 -2518 polymorphisms and the incidence of spinal tuberculosis in the Chinese Han population.
Subjects and Methods Participants The case group comprised new patients with spinal tuberculosis admitted to our hospital between December 2004 and December 2010. The inclusion criteria were spinal tuberculosis infection confirmed by preoperative imaging and serological examinations, preoperative lung imaging showing no obvious signs of pulmonary tuberculosis infection, exclusion of other extrapulmonary tuberculosis, pathological examination of surgical sections showing tuberculosis infection; and etiological examination positive for acidfast staining or M. tuberculosis in exudate. The control group comprised healthy Han individuals who underwent physical examinations at our hospital between December 2004 and December 2010 and in whom pulmonary tuberculosis, spinal tuberculosis, and other extrapulmonary tuberculosis were excluded. Cases and controls with any of the following conditions were excluded from the study: cultures of multidrugresistant M. tuberculosis, diseases affecting the immune system (infection, trauma, tumor); diseases affecting MCP-1 expression (coronary artery disease, psoriasis), autoimmune disease, tuberculosis of other organs (or history of this type of disease), genetic disease, undergoing spinal surgery, or free of bacillus Calmette-Guerin (BCG) vaccination. The study was approved by the Ethics Committee of Central South University, and informed consent was obtained from all participants. Genomic DNA Extraction Two milliliters of fasting peripheral venous blood was harvested in the morning and placed in acid citrate dextrose tubes. White blood cell genomic DNA was extracted using a whole-blood genomic DNA quick extraction kit (Takara, Dalian, China). DNA concentrations were determined using an ultraviolet spectrophotometer. DNA was diluted to 100 ng/mL with double distilled water and cryopreserved at 70 C. DNA was detected in batches after all samples were collected.
Polymerase Chain Reaction (PCR) Primers were synthesized as described previously (5): forward primer: 5’-TTCTCTCACGCCAGCAC-3’; reverse primer: 5’-TGACTTGGCCTTTGCATATATC-3’. The amplified fragment was 163 bp. PCR reaction conditions were prenaturation at 95 C for 5 min, 94 C for 1 min, 62 C for 30 sec, 72 C for 1 min for 30 cycles in total, followed by extension at 72 C for 10 min and annealing at 4 C. PCR Product Sequencing If the amount and specificity of the products were as expected, they were treated with shrimp alkaline phosphatase and exonuclease on ice and then incubated at 37 C for 90 min and 80 C for 15 min to deactivate the enzymes. In the event that the amount and specificity of products were not satisfactory (e.g., if there were confounding bands), the PCR products were retrieved and purified as follows (50 mL PCR amplification system as example). The target fragment was retrieved under a VitaLight lamp, placed at room temperature for 5 h, ground using a large tip, mixed with 600 mL eluting buffer, mixed overnight on a shaking table at 37 C, and centrifuged at 16,200 g at 4 C for 30 min. The supernatant was transferred to a new Eppendorf tube, mixed with 500 mL absolute alcohol at -20 C for 4 h, and centrifuged at 16,200 g at 4 C for 10 min. White precipitate was eluted with 70% alcohol, centrifuged at 540 g at 4 C for 15 min, dried at room temperature for 5 min, dissolved in 10 mL PCR water for 2 h and then sequenced on an ABI3100 sequencer. Sequencing was performed by Shanghai Majorbio Pharm (Shanghai, China). Statistical Analysis Data were analyzed using SPSS v.13.0 software. The general conditions of the two groups were compared using t tests and c2 tests. The genotype frequency of the control group was calculated to confirm Hardy-Weinberg equilibrium. c2 tests were used to compare differences in genotype frequencies in each group followed by intergroup comparisons of each genotype. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. The results of t tests were expressed as mean SD; p !0.05 was considered statistically significant.
Results Comparison of General Conditions Sequencing results were obtained for 208 patients and 210 controls. There were no significant differences in age (t 5 0.682, p 5 0.496) or gender distribution (c2 5 0.778, p 5 0.378; Table 1) between groups.
Monocyte Chemotactic Protein-1 -2518 Gene Polymorphism and Susceptibility to Spinal Tuberculosis Table 1. Age and gender of patients with spinal tuberculosis and controls
185
Table 3. Association analyses of the MCP-1 -2518 genotype and spinal tuberculosis using the dominant model
Group
Age (years)
Male (n)
Female (n)
Control Case
41.35 13.12 40.31 17.79
102 110
108 98
Genotype (n [%]) Group
GG þ GA
AA
p
OR
95% CI
Control Case
166 (79%) 181 (87%)
44 (21.0%) 27 (13.0%)
0.03
1.777
1.053e2.999
Hardy-Weinberg Equilibrium Test
The incidence and progression of tuberculosis is multifactorial, and host predisposing genes play important roles together with pathogen-related factors, host immune status, and socioeconomic and environmental factors (6,7). MCP-1
gene polymorphisms can reduce the ability to resist M. tuberculosis infection and thus increase the risk of tuberculosis. Flores-Villanueva et al. reported that the odds of developing tuberculosis were 2.3- and 5.4-fold higher in Mexican carriers of the MCP-1 genotypes GA and GG compared with AA homozygotes, and 2.8- and 6.9-fold higher in Korean carriers of the GA and GG MCP-1 genotypes compared with AA homozygotes. This was identified as the genetic factor with the strongest influence on adult tuberculosis (8). Another study that focused on Tunisian patients showed that the risk of pulmonary tuberculosis or extrapulmonary tuberculosis was 3.1-fold higher in patients carrying the MCP-1 -2518 GG genotype compared with the MCP-1 -2518GA genotype (9). A meta-analysis showed that the risk of tuberculosis was increased 1.79-fold in an Asian population carrying the MCP-1 -2518 GG genotype (10), whereas another meta-analysis showed that the odds of developing tuberculosis increased 1.51-fold in carriers of the MCP-1 -2518 G allele (4). Gene changes in the MCP-1 promoter region can increase MCP-1 gene transcription and serum MCP-1 levels. Feng et al. reported that the MCP-1-2518 G allele was in linkage disequilibrium with the MCP-1 þ900 C allele, and that MCP-1 þ900 C could alter MCP-1 protein expression, resulting in susceptibility to tuberculosis (11). The MCP-1 -2518 G allele increased binding of the TALE protein family members PREP1 and PBX2 to the MCP-1 promoter, resulting in increased transcriptional activation of MCP-1 and increased serum MCP-1 levels (12). High levels of serum MCP-1 can inhibit interleukin-12 expression and selectively inhibit helper T cell immunological reaction type I, leading to reduced resistance to M. tuberculosis (1). In addition, overexpression of MCP-1 suppresses interferon-g production resulting in reduced granuloma formation, loss of receptor sensitivity to the ligand, and a decreased monocyte chemotactic response (13). Serum MCP-1 can also upregulate interleukin-4 transcription,
Table 2. Distribution of MCP-1 -2518 genotypes and alleles in patients with spinal tuberculosis and controls
Table 4. Association analyses of the MCP-1 -2518 genotype and spinal tuberculosis using the recessive model
The distribution frequencies of the GG, GA and AA genotypes are shown in Table 2. The genotype distributions in the control group conformed to the Hardy-Weinberg equilibrium ( p O0.05). Comparison of Genotype and Allele Distribution Frequencies Between groups The MCP-1 -2518 genotype distribution frequency differed significantly between the groups (c2 5 8.066, p 5 0.018). Paired comparisons of genotypes showed that the risk of spinal tuberculosis was higher in individuals with the GG genotype compared with those carrying the AA genotype (c2 5 7.731, p 5 0.005, OR 5 2.306, 95% CI 5 1.273e4.178). However, there were no significant differences between the GG and GA genotypes (c2 5 3.363, p 5 0.067) or between the AA and GA genotypes (c2 5 2.324, p 5 0.127). The MCP-1 -2518 allele distribution frequencies differed significantly between groups (c2 5 7.52, p 5 0.006). Association of MCP-1 -2518 Gene Polymorphisms with Spinal Tuberculosis Tables 3 and 4 shows the association of the SNPs with spinal tuberculosis. The allele ‘‘G’’ of MCP-1 -2518 showed an association with an increased risk for spinal tuberculosis: OR 5 1.777, 95% CI 5 1.053e2999, p 5 0.03 in the dominant model; OR 5 1.67, 95% CI 5 1.097e2.544, p 5 0.016 in the recessive model.
Discussion
Genotype (n [%]) Group
GG
GA
Allele (n [%]) AA
G
A
Control 53 (25.2%) 113 (53.8%) 44 (21.0%) 219 (52.1%) 201 (47.9%) Case 75 (36.1%) 106 (50.9%) 27 (13.0%) 256 (61.5%) 160 (38.5%)
Genotype (n [%]) Group
GG
AA þ GA
p
OR
95% CI
Control Case
53 (25.2%) 75 (36.1%)
157 (74.8%) 133 (63.9%)
0.016
1.670
1.097e2.544
186
Gao et al./ Archives of Medical Research 45 (2014) 183e187
induce the transformation of Th0 cells into Th2 cells to upregulate the Th2 immunological reaction, and inhibit the Th1 immunological reaction (14). All the above processes can increase an individual’s susceptibility to tuberculosis and increase the risk of M. tuberculosis infection dissemination in vivo. To date, Chinese studies have focused mainly on patents with pulmonary tuberculosis, and little is known about susceptibility to spinal tuberculosis. Schlesinger et al. found that only 33e50% of spinal tuberculosis patients also had pulmonary tuberculosis, whereas most exhibited no obvious pulmonary focus or history of pulmonary tuberculosis (15). This suggests that the factors influencing the incidence and progression of spinal tuberculosis differ from those associated with pulmonary tuberculosis. Previous studies have shown that spinal tuberculosis has independent predisposing genotypes (16), and the predisposing genotypes differed between pulmonary tuberculosis and tuberculosis of the bones and joints (17). The internal environment and host immunity are important factors in the pathogenesis of spinal tuberculosis, and host genes may thus play a more important role in spinal tuberculosis compared with pulmonary tuberculosis. The present study detected significant differences in MCP-1 -2518 genotype distributions between the control and case groups. The risk of developing spinal tuberculosis was increased 2.33-fold in carriers of the GG genotype. Carriers of the allele ‘‘G’’ of MCP-1-2518 also showed an association with an increased risk for spinal tuberculosis. Those indicating that the MCP-1 -2518 GG genotype and G allele may correlate with susceptibility to spinal tuberculosis in the Chinese Han population. To the best of our knowledge, no previous study has reported on the correlation between MCP-1 gene polymorphisms and susceptibility to spinal tuberculosis. However, Motsinger et al. reported that MCP-1 -2518 gene polymorphism was not associated with extrapulmonary tuberculosis (18). Several factors may account for this apparent discrepancy. Motsinger et al. recruited black subjects, and recent meta-analyses demonstrated that the MCP-1 -2518 gene polymorphism was not significantly correlated with susceptibility to tuberculosis in the African population (4,10). In addition, the sample size in the previous study was small, and only 24 patients were included, possibly leading to statistical errors. In terms of disease location, the study of Motsinger et al. included only four cases of bone tuberculosis, and other tuberculous lesions were located in the scrofula, pleura, pericardium and intestine. As the pathogenic mechanisms of tuberculosis differ among various tissues and organs are different, the susceptibility factors might thus also be expected to differ. Spinal tuberculosis results from multiple factors including environmental, social and genetic factors, and its susceptibility is thus regulated by multiple genes. We demonstrated that the MCP-1 -2518 GG genotype is one genetic factor that can increase the risk of spinal
tuberculosis. Further multicenter, large-scale genetic screening studies are needed to identify other susceptibility genes. The development of genetic diagnosis techniques and the study of predisposing genes may provide new strategies for early diagnosis and individualized therapy for spinal tuberculosis, as well as to the development of potential vaccines.
Acknowledgments The study was supported by the Fundamental Research Funds for the Central Universities, No. 2012QNZT101, China, and The National Natural Science Fund, No. 81301576, China.
References 1. Deshmane SL, Kremlev S, Amini S, et al. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res 2009;29: 313e326. 2. Nio Y, Yamauchi T, Iwabu M, et al. Monocyte chemoattractant protein-1 (MCP-1) deficiency enhances alternatively activated M2 macrophages and ameliorates insulin resistance and fatty liver in lipoatrophic diabetic A-ZIP transgenic mice. Diabetologia 2012;55: 3350e3358. 3. Hussain R, Ansari A, Talat N, et al. CCL2/MCP-I genotype-phenotype relationship in latent tuberculosis infection. PLOS ONE 2011;6: e25803. 4. Feng WX, Flores-Villanueva PO, Mokrousov I, et al. CCL2-2518 (A/ G) polymorphisms and tuberculosis susceptibility: a meta-analysis. Int J Tuberc Lung Dis 2012;16:150e156. 5. Thye T, Nejentsev S, Intemann CD, et al. MCP-1 promoter variant -362C associated with protection from pulmonary tuberculosis in Ghana, West Africa. Hum Mol Genet 2009;18:381e388. 6. Allen AR, Minozzi G, Glass EJ, et al. Bovine tuberculosis: the genetic basis of host susceptibility. Proc Biol Sci 2010;277:2737e2745. 7. Vannberg FO, Chapman SJ, Hill AV. Human genetic susceptibility to intracellular pathogens. Immunol Rev 2011;240:105e116. 8. Flores-Villanueva PO, Ruiz-Morales JA, Song CH, et al. A functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with increased susceptibility to pulmonary tuberculosis. J Exp Med 2005;202:1649e1658. 9. Ben-Selma W, Harizi H, Boukadida J. MCP-1 -2518 A/G functional polymorphism is associated with increased susceptibility to active pulmonary tuberculosis in Tunisian patients. Mol Biol Rep 2011;38: 5413e5419. 10. Zhang Y, Zhang J, Zeng L, et al. The -2518A/G polymorphism in the MCP-1 gene and tuberculosis risk: a meta-analysis. PLOS ONE 2012; 7:e38918. 11. Feng WX, Mokrousov I, Wang BB, et al. Tag SNP polymorphism of CCL2 and its role in clinical tuberculosis in Han Chinese pediatric population. PLOS ONE 2011;6:e14652. 12. Page SH, Wright EK Jr, Gama L, et al. Regulation of CCL2 expression by an upstream TALE homeodomain protein-binding site that synergizes with the site created by the A-2578G SNP. PLOS ONE 2011; 6:e22052. 13. Charo IF, Ransohoff RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med 2006;354: 610e621. 14. Mendez A, Hernandez-Pando R, Contreras S, et al. CCL2, CCL18 and sIL-4R in renal, meningeal and pulmonary TB: a 2 year study of patients and contacts. Tuberculosis (Edinb) 2011;91:140e145.
Monocyte Chemotactic Protein-1 -2518 Gene Polymorphism and Susceptibility to Spinal Tuberculosis 15. Schlesinger N, Lardizabal A, Rao J, et al. Tuberculosis of the spine: experience in an inner city hospital. J Clin Rheumatol 2005;11: 17e20. 16. Zhang HQ, Deng A, Guo CF, et al. Association between FokI polymorphism in vitamin D receptor gene and susceptibility to spinal tuberculosis in Chinese Han population. Arch Med Res 2010;41:46e49.
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17. Selvaraj P, Kurian SM, Chandra G, et al. Vitamin D receptor gene variants of BsmI, ApaI, TaqI, and FokI polymorphisms in spinal tuberculosis. Clin Genet 2004;65:73e76. 18. Motsinger A, Antas P, Oki N, et al. Polymorphisms in IL-1beta, vitamin D receptor Fok1, and Toll-like receptor 2 are associated with extrapulmonary tuberculosis. BMC Med Genet 2010;11:37.
ORIGINAL ARTICLE
Monocyte Chemotactic Protein-1 -2518 Gene Polymorphism and Susceptibility to Spinal Tuberculosis Qile Gao,a Qingbiao Du,b Hongqi Zhang,a Chaofeng Guo,a Shijin Lu,a Ang Deng,a Mingxing Tang,a Shaohua Liu,a Yuxiang Wang,a Jia Huang,a and Qiang Guoa a Department of Spine Surgery, Xiangya Hospital, Central South University, Hunan, China Clinical Laboratory, Secondary Affiliated Hospital, Fujian Medical University, Fujian, China
b
Received for publication July 10, 2013; accepted December 13, 2013 (ARCMED-D-13-00370).
Background and Aims. Monocyte chemotactic protein-1 (MCP-1) gene polymorphisms play important roles in regulating immunological reactions and may be associated with pulmonary tuberculosis. However, the relationship between the MCP-1 -2518 gene polymorphism and susceptibility to spinal tuberculosis remains unknown. We undertook this study to investigate the relationships between MCP-1 promoter 2518 genotype frequency and allele polymorphisms and susceptibility to spinal tuberculosis in a Chinese Han population. Methods. Patients with spinal tuberculosis and healthy volunteers were enrolled between December 2004 and December 2010. MCP-1 -2518 polymorphisms in both groups were detected using polymerase chain reaction and DNA sequencing. MCP-1 genotype was analyzed in all patients. Differences in genotype frequencies between groups were compared using c2 tests. Results. A total of 208 patients with spinal tuberculosis and 210 healthy volunteers were included. The distribution frequencies of MCP-1 -2518 GG, GA and AA genotypes were 36.1, 50.9 and 13.0%, respectively, in the case group and 25.2, 53.8 and 21.0%, respectively, in the control group ( p !0.05). MCP-1 -2518 GG genotype was significantly associated with the onset of spinal tuberculosis (OR 5 2.306, 95% CI 5 1.273e4.178). The G and A allele frequencies were 61.5% and 38.5%, respectively, in the case group, and 52.1% and 47.9% in the control group ( p !0.05), the allele ‘‘G’’ of MCP-1 -2518 showed an association with an increased risk for spinal tuberculosis: OR 5 1.777, 95% CI 5 1.053e2999, p 5 0.03 in the dominant model; OR 5 1.67, 95% CI 5 1.097e2.544, p 5 0.016 in the recessive model. Conclusions. The MCP-1 -2518 GG genotype and presence of the G allele may be associated with susceptibility to spinal tuberculosis in the Chinese Han population. Ó 2014 IMSS. Published by Elsevier Inc. Key Words: Monocyte chemotactic protein-1, Gene polymorphism, Spinal tuberculosis, Susceptibility.
Introduction Spinal tuberculosis is associated with high morbidity and a high disability rate. An increase in the incidence of multidrug-resistant strains of Mycobacterium tuberculosis indicates that the treatment needs of patients with spinal
Address reprint requests to: Hongqi Zhang, MD, Central South University, Xiangya Hospital, Spine Surgery, *87 Xiang Ya Road, Changsha, Hunan 410008, China; Phone: þ86073189753001; E-mail: [email protected] or [email protected]
tuberculosis have changed. A thorough understanding of the individual factors influencing the host antituberculosis immunological reaction is therefore needed to help prevent and treat spinal tuberculosis. Monocyte chemotactic protein-1 (MCP-1) is a monocyte and macrophage chemotactic factor that induces and activates monocytes. MCP-1 can increase the free oxygen concentration in monocyte fluid to enhance the killing effects of monocytes against bacteria (1). Moreover, MCP-1 can mediate cell migration and phagocytosis and other adhesion-dependent functions by regulating cell surface adhesion molecule
0188-4409/$ - see front matter. Copyright Ó 2014 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2013.12.007
184
Gao et al./ Archives of Medical Research 45 (2014) 183e187
expression and cytokine production (2). MCP-1 can restrict the in vivo spreading of M. tuberculosis and regulate secondary immunological reactions and is thus closely related to the occurrence and progression of tuberculosis (3). A previous study demonstrated that the MCP-1 -2518A/G polymorphism was associated with increased susceptibility to pulmonary tuberculosis in the Chinese Han population (4); however, the correlation between the gene polymorphism and spinal tuberculosis remains poorly understood. The present study therefore aimed to clarify the relationship between MCP-1 -2518 polymorphisms and the incidence of spinal tuberculosis in the Chinese Han population.
Subjects and Methods Participants The case group comprised new patients with spinal tuberculosis admitted to our hospital between December 2004 and December 2010. The inclusion criteria were spinal tuberculosis infection confirmed by preoperative imaging and serological examinations, preoperative lung imaging showing no obvious signs of pulmonary tuberculosis infection, exclusion of other extrapulmonary tuberculosis, pathological examination of surgical sections showing tuberculosis infection; and etiological examination positive for acidfast staining or M. tuberculosis in exudate. The control group comprised healthy Han individuals who underwent physical examinations at our hospital between December 2004 and December 2010 and in whom pulmonary tuberculosis, spinal tuberculosis, and other extrapulmonary tuberculosis were excluded. Cases and controls with any of the following conditions were excluded from the study: cultures of multidrugresistant M. tuberculosis, diseases affecting the immune system (infection, trauma, tumor); diseases affecting MCP-1 expression (coronary artery disease, psoriasis), autoimmune disease, tuberculosis of other organs (or history of this type of disease), genetic disease, undergoing spinal surgery, or free of bacillus Calmette-Guerin (BCG) vaccination. The study was approved by the Ethics Committee of Central South University, and informed consent was obtained from all participants. Genomic DNA Extraction Two milliliters of fasting peripheral venous blood was harvested in the morning and placed in acid citrate dextrose tubes. White blood cell genomic DNA was extracted using a whole-blood genomic DNA quick extraction kit (Takara, Dalian, China). DNA concentrations were determined using an ultraviolet spectrophotometer. DNA was diluted to 100 ng/mL with double distilled water and cryopreserved at 70 C. DNA was detected in batches after all samples were collected.
Polymerase Chain Reaction (PCR) Primers were synthesized as described previously (5): forward primer: 5’-TTCTCTCACGCCAGCAC-3’; reverse primer: 5’-TGACTTGGCCTTTGCATATATC-3’. The amplified fragment was 163 bp. PCR reaction conditions were prenaturation at 95 C for 5 min, 94 C for 1 min, 62 C for 30 sec, 72 C for 1 min for 30 cycles in total, followed by extension at 72 C for 10 min and annealing at 4 C. PCR Product Sequencing If the amount and specificity of the products were as expected, they were treated with shrimp alkaline phosphatase and exonuclease on ice and then incubated at 37 C for 90 min and 80 C for 15 min to deactivate the enzymes. In the event that the amount and specificity of products were not satisfactory (e.g., if there were confounding bands), the PCR products were retrieved and purified as follows (50 mL PCR amplification system as example). The target fragment was retrieved under a VitaLight lamp, placed at room temperature for 5 h, ground using a large tip, mixed with 600 mL eluting buffer, mixed overnight on a shaking table at 37 C, and centrifuged at 16,200 g at 4 C for 30 min. The supernatant was transferred to a new Eppendorf tube, mixed with 500 mL absolute alcohol at -20 C for 4 h, and centrifuged at 16,200 g at 4 C for 10 min. White precipitate was eluted with 70% alcohol, centrifuged at 540 g at 4 C for 15 min, dried at room temperature for 5 min, dissolved in 10 mL PCR water for 2 h and then sequenced on an ABI3100 sequencer. Sequencing was performed by Shanghai Majorbio Pharm (Shanghai, China). Statistical Analysis Data were analyzed using SPSS v.13.0 software. The general conditions of the two groups were compared using t tests and c2 tests. The genotype frequency of the control group was calculated to confirm Hardy-Weinberg equilibrium. c2 tests were used to compare differences in genotype frequencies in each group followed by intergroup comparisons of each genotype. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. The results of t tests were expressed as mean SD; p !0.05 was considered statistically significant.
Results Comparison of General Conditions Sequencing results were obtained for 208 patients and 210 controls. There were no significant differences in age (t 5 0.682, p 5 0.496) or gender distribution (c2 5 0.778, p 5 0.378; Table 1) between groups.
Monocyte Chemotactic Protein-1 -2518 Gene Polymorphism and Susceptibility to Spinal Tuberculosis Table 1. Age and gender of patients with spinal tuberculosis and controls
185
Table 3. Association analyses of the MCP-1 -2518 genotype and spinal tuberculosis using the dominant model
Group
Age (years)
Male (n)
Female (n)
Control Case
41.35 13.12 40.31 17.79
102 110
108 98
Genotype (n [%]) Group
GG þ GA
AA
p
OR
95% CI
Control Case
166 (79%) 181 (87%)
44 (21.0%) 27 (13.0%)
0.03
1.777
1.053e2.999
Hardy-Weinberg Equilibrium Test
The incidence and progression of tuberculosis is multifactorial, and host predisposing genes play important roles together with pathogen-related factors, host immune status, and socioeconomic and environmental factors (6,7). MCP-1
gene polymorphisms can reduce the ability to resist M. tuberculosis infection and thus increase the risk of tuberculosis. Flores-Villanueva et al. reported that the odds of developing tuberculosis were 2.3- and 5.4-fold higher in Mexican carriers of the MCP-1 genotypes GA and GG compared with AA homozygotes, and 2.8- and 6.9-fold higher in Korean carriers of the GA and GG MCP-1 genotypes compared with AA homozygotes. This was identified as the genetic factor with the strongest influence on adult tuberculosis (8). Another study that focused on Tunisian patients showed that the risk of pulmonary tuberculosis or extrapulmonary tuberculosis was 3.1-fold higher in patients carrying the MCP-1 -2518 GG genotype compared with the MCP-1 -2518GA genotype (9). A meta-analysis showed that the risk of tuberculosis was increased 1.79-fold in an Asian population carrying the MCP-1 -2518 GG genotype (10), whereas another meta-analysis showed that the odds of developing tuberculosis increased 1.51-fold in carriers of the MCP-1 -2518 G allele (4). Gene changes in the MCP-1 promoter region can increase MCP-1 gene transcription and serum MCP-1 levels. Feng et al. reported that the MCP-1-2518 G allele was in linkage disequilibrium with the MCP-1 þ900 C allele, and that MCP-1 þ900 C could alter MCP-1 protein expression, resulting in susceptibility to tuberculosis (11). The MCP-1 -2518 G allele increased binding of the TALE protein family members PREP1 and PBX2 to the MCP-1 promoter, resulting in increased transcriptional activation of MCP-1 and increased serum MCP-1 levels (12). High levels of serum MCP-1 can inhibit interleukin-12 expression and selectively inhibit helper T cell immunological reaction type I, leading to reduced resistance to M. tuberculosis (1). In addition, overexpression of MCP-1 suppresses interferon-g production resulting in reduced granuloma formation, loss of receptor sensitivity to the ligand, and a decreased monocyte chemotactic response (13). Serum MCP-1 can also upregulate interleukin-4 transcription,
Table 2. Distribution of MCP-1 -2518 genotypes and alleles in patients with spinal tuberculosis and controls
Table 4. Association analyses of the MCP-1 -2518 genotype and spinal tuberculosis using the recessive model
The distribution frequencies of the GG, GA and AA genotypes are shown in Table 2. The genotype distributions in the control group conformed to the Hardy-Weinberg equilibrium ( p O0.05). Comparison of Genotype and Allele Distribution Frequencies Between groups The MCP-1 -2518 genotype distribution frequency differed significantly between the groups (c2 5 8.066, p 5 0.018). Paired comparisons of genotypes showed that the risk of spinal tuberculosis was higher in individuals with the GG genotype compared with those carrying the AA genotype (c2 5 7.731, p 5 0.005, OR 5 2.306, 95% CI 5 1.273e4.178). However, there were no significant differences between the GG and GA genotypes (c2 5 3.363, p 5 0.067) or between the AA and GA genotypes (c2 5 2.324, p 5 0.127). The MCP-1 -2518 allele distribution frequencies differed significantly between groups (c2 5 7.52, p 5 0.006). Association of MCP-1 -2518 Gene Polymorphisms with Spinal Tuberculosis Tables 3 and 4 shows the association of the SNPs with spinal tuberculosis. The allele ‘‘G’’ of MCP-1 -2518 showed an association with an increased risk for spinal tuberculosis: OR 5 1.777, 95% CI 5 1.053e2999, p 5 0.03 in the dominant model; OR 5 1.67, 95% CI 5 1.097e2.544, p 5 0.016 in the recessive model.
Discussion
Genotype (n [%]) Group
GG
GA
Allele (n [%]) AA
G
A
Control 53 (25.2%) 113 (53.8%) 44 (21.0%) 219 (52.1%) 201 (47.9%) Case 75 (36.1%) 106 (50.9%) 27 (13.0%) 256 (61.5%) 160 (38.5%)
Genotype (n [%]) Group
GG
AA þ GA
p
OR
95% CI
Control Case
53 (25.2%) 75 (36.1%)
157 (74.8%) 133 (63.9%)
0.016
1.670
1.097e2.544
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Gao et al./ Archives of Medical Research 45 (2014) 183e187
induce the transformation of Th0 cells into Th2 cells to upregulate the Th2 immunological reaction, and inhibit the Th1 immunological reaction (14). All the above processes can increase an individual’s susceptibility to tuberculosis and increase the risk of M. tuberculosis infection dissemination in vivo. To date, Chinese studies have focused mainly on patents with pulmonary tuberculosis, and little is known about susceptibility to spinal tuberculosis. Schlesinger et al. found that only 33e50% of spinal tuberculosis patients also had pulmonary tuberculosis, whereas most exhibited no obvious pulmonary focus or history of pulmonary tuberculosis (15). This suggests that the factors influencing the incidence and progression of spinal tuberculosis differ from those associated with pulmonary tuberculosis. Previous studies have shown that spinal tuberculosis has independent predisposing genotypes (16), and the predisposing genotypes differed between pulmonary tuberculosis and tuberculosis of the bones and joints (17). The internal environment and host immunity are important factors in the pathogenesis of spinal tuberculosis, and host genes may thus play a more important role in spinal tuberculosis compared with pulmonary tuberculosis. The present study detected significant differences in MCP-1 -2518 genotype distributions between the control and case groups. The risk of developing spinal tuberculosis was increased 2.33-fold in carriers of the GG genotype. Carriers of the allele ‘‘G’’ of MCP-1-2518 also showed an association with an increased risk for spinal tuberculosis. Those indicating that the MCP-1 -2518 GG genotype and G allele may correlate with susceptibility to spinal tuberculosis in the Chinese Han population. To the best of our knowledge, no previous study has reported on the correlation between MCP-1 gene polymorphisms and susceptibility to spinal tuberculosis. However, Motsinger et al. reported that MCP-1 -2518 gene polymorphism was not associated with extrapulmonary tuberculosis (18). Several factors may account for this apparent discrepancy. Motsinger et al. recruited black subjects, and recent meta-analyses demonstrated that the MCP-1 -2518 gene polymorphism was not significantly correlated with susceptibility to tuberculosis in the African population (4,10). In addition, the sample size in the previous study was small, and only 24 patients were included, possibly leading to statistical errors. In terms of disease location, the study of Motsinger et al. included only four cases of bone tuberculosis, and other tuberculous lesions were located in the scrofula, pleura, pericardium and intestine. As the pathogenic mechanisms of tuberculosis differ among various tissues and organs are different, the susceptibility factors might thus also be expected to differ. Spinal tuberculosis results from multiple factors including environmental, social and genetic factors, and its susceptibility is thus regulated by multiple genes. We demonstrated that the MCP-1 -2518 GG genotype is one genetic factor that can increase the risk of spinal
tuberculosis. Further multicenter, large-scale genetic screening studies are needed to identify other susceptibility genes. The development of genetic diagnosis techniques and the study of predisposing genes may provide new strategies for early diagnosis and individualized therapy for spinal tuberculosis, as well as to the development of potential vaccines.
Acknowledgments The study was supported by the Fundamental Research Funds for the Central Universities, No. 2012QNZT101, China, and The National Natural Science Fund, No. 81301576, China.
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