- Email: [email protected]
O polymorphisms and Mycobacterium tuberculosis infection in populations from the Brazilian Amazon region
Human Immunology 74 (2013) 82–84
Contents lists available at SciVerse ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Rapid Communication
No evidence of association between MBL2A/O polymorphisms and Mycobacterium tuberculosis infection in populations from the Brazilian Amazon region Mauro S. Araújo a, Ednelza S. Graça a, Vânia N. Azevedo b, Izaura Cayres-Vallinoto b, Luiz Fernando A. Machado b, Marluisa O.G. Ishak b, Ricardo Ishak b, Antonio C.R. Vallinoto b,⇑ a b
University Hospital João de Barros Barreto – HUJBB, Federal University of Para, Brazil Virus Laboratory, Institute for Biological Sciences, Federal University of Para, Brazil
a r t i c l e
i n f o
Article history: Received 18 May 2012 Accepted 10 September 2012 Available online 19 September 2012
a b s t r a c t The present study investigated the prevalence of the polymorphisms in the exon 1 of the MBL2 gene in patients with tuberculosis at a hospital in northern Brazil, which is a regional reference for the treatment of the disease. The study group was composed of 167 patients with tuberculosis, 34 of which had the extra-pulmonary form of the disease, while the other 133 had the pulmonary type. The control group consists of 159 healthy individuals. Samples of DNA extracted from leucocytes were submitted to Polymerase Chain Reaction for the amplification of a 120-bp segment of exon 1 of the MBL2 gene. The distribution of allele and genotype frequencies varied little among the different groups, and it was not possible to establish any clear association between the variants of the MBL2 gene and the susceptibility to or clinical profile of tuberculosis infections in the population analyzed. Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The mannose-binding lectin (MBL) is an acute phase serum protein of the collectin family [1,2]. It is produced primarily in the liver, although some leucocytes may also be able to synthesize it [3,4]. The principal biological function of MBL is opsonization, given that its collagenous structures are binding sites for the collectin receptors present in the phagocytes, which act directly as opsonin [5–7]. The MBL is capable of recognizing specific carbohydrate residues, primarily mannose, fucose, and N-acetylglucosamine [4–6,8,9], through which the MBL binds to pathogens or cell surfaces [10]. Variations in the serum concentrations of MBL are attributed to mutations in exon 1 (MBL2⁄B – rs1800450, MBL2⁄C – rs1800451 and MBL2⁄D – rs5030737) and the promoter region ( 550 (H/L) – rs11003125 and 221 (Y/X) – rs7096206) of the MBL2 gene [7,11]. This results in defects in the polymerization of MBL, which leads to a functional deficiency, as well as affecting the expression level of the protein [12,13]. In an in vitro study, Bonar et al. [14] showed that the phagocytosis of Mycobacterium tuberculosis in humans is intensified by the presence of MBL, which suggests that this lectin may facilitate the assimilation of this bacterium into the phagocyte in vivo, and thus ⇑ Corresponding author. E-mail address: [email protected] (A.C.R. Vallinoto).
contribute to infection. Such phagocytosis may be mediated by the recognition of the lipoarabinomannan present on the surface of M. tuberculosis. A study in Africa indicated that susceptibility to tuberculosis may increase in individuals with low serum levels of this protein as a result of their being homozygous for the mutations of the MBL2 gene [15]. However, an increased probability of tuberculosis infection has also been related to elevated serum levels of MBL, which might be disadvantageous in the case of infection by mycobacteria [16]. Given these conflicting results on the influence of MBL on infection by M. tuberculosis, the present study investigated the prevalence of mutations in exon 1 of the MBL2 gene in patients with tuberculosis attending a hospital in northern Brazil, which is a regional reference for the treatment of the disease. 2. Materials and methods 2.1. Subjects The study group was made up of 167 tuberculosis (TB) patients (92 males and 75 females) who were seronegative for HIV-1, and had been diagnosed according to the CDC (Center for Disease Control and Prevention) criteria and treated at the João de Barros Barreto University Hospital in the metropolitan region of Belém in 2006 and 2007. Infection by M. tuberculosis was confirmed in these patients by positive smears of the phlegm and/or bronchial fluid,
0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2012.09.001
83
M.S. Araújo et al. / Human Immunology 74 (2013) 82–84
followed by specific culture, tuberculin skin test, X-ray, and positive biopsy for M. tuberculosis. These tests were run in the hospital’s Clinical Pathology Laboratory. The clinical data of the patients were obtained from their medical records, which were provided by the hospital’s Medical Records Department. These records contained the patients’ full history of medical, clinical, and laboratorial examinations and tests. For analysis, the patients were divided into two subgroups, according to their clinical diagnosis. One group was made up of 34 patients with extra-pulmonary tuberculosis, while the second included all the 133 patients with the pulmonary form of the disease. The control group consisted of 159 health workers of both sexes (58 males and 98 females) that did not have active tuberculosis and were seronegative for HIV-1, and had regular (halfyearly) medical check-ups, including X-ray of the chest, sputum smear, haemogram, VDRL, urine routine, fasting plasma glucose and tuberculin skin test. These individuals were chosen as the control group because of the high risk of exposure to tuberculosis patients during their professional activities. 2.2. Ethics All the individuals were briefed about the study and signed an informed consent form before sample collection. The present study was approved by the Human Ethics Committee of the Institute for Health Sciences, Federal University of Para, under the protocol n ° 034/06 CEP-ICS/UFPA. 2.3. Sample collection Blood samples were collected from the members of both groups in vacuum tubes containing EDTA as an anticoagulant. The cells and plasma were separated and frozen at 20 °C in the Virology Laboratory of the Biological Sciences Institute of the Federal University of Pará, where the molecular analyses of the genetic polymorphism were carried out. 2.4. Molecular analysis The genomic DNA was extracted from the leucocytes using a Puregene kit from Gentra Systemas, Inc. (USA) and subjected to the Polymerase Chain Reaction (PCR) for the amplification of 120 bps of exon 1 of the Mbl2 gene. The amplifications were based on the protocol described by Vallinoto et al. [17].
2.5. Statistical analysis The allele and genotype frequencies were calculated by direct counting. The frequencies recorded in both groups were compared with those expected under Hardy-Weinberg equilibrium and tested using Chi-square, Test G and Fisher’s test, which was run in Tools For Population Genetic Analyses – TFPGA 1.3v [18] and BioEstat 5.3v [19]. 3. Results Six different genotypes (AA, AB, AC, AD, BC, and BD) were recorded in both patient and control groups (Table 1), although there was no significant difference (p = 0.8610) in the distribution of frequencies in the groups. The comparative analysis of the genotype frequencies considered only AA, AO, and OO, where MBL2⁄A is the normal allele, and MBL2⁄O represents any of the other three variants (MBL2⁄B, ⁄C or ⁄D). The AA genotype returned the highest frequency in both groups, with a frequency of 0.611 in the patients and 0.635 in the control group (Table 1). The AO genotype had a frequency of 0.371 among TB patients and 0.352 in the control group, while OO was 0.018 in the former and 0.012 in the latter group. The statistical comparison of expected and observed genotype frequencies indicated that both groups were in Hardy-Weinberg equilibrium (p > 0.05). Comparing the two subgroups of tuberculosis patients, the extra-pulmonary group presented a slightly higher frequency of the MBL2⁄O genotypes (Table 1), although the difference was not significant (p = 0.3121). The analysis of the allele frequencies indicated a predominance of the MBL2⁄A allele in both patient subgroups and the control group. There was no significant difference in the distribution of the MBL2⁄O variant among the different groups (p = 0.0504). 4. Discussion To date, relatively few studies have investigated the impact of a deficiency of MBL on the susceptibility or resistance of individuals to tuberculosis [16,20–22], and the available data are contradictory in relation to the role of this protein in this susceptibility. While Alagarasu et al. [21] concluded that a deficiency of MBL was associated with an increase in susceptibility to tuberculosis, for example, other studies [22,23] indicated that it was associated with
Table 1 Distribution of genotype and allele frequencies of the Mbl2 gene in tuberculosis (TB) patient group and subgroups, and the control group.
a b
Genetic profile
TB patients n (%)
Control n (%)
Genotype AA AO AB AC AD OO BC BD Total
102 (61.1) 62 (37.1) 50 (29.9) 4 (2.4) 8 (4.8) 3 (1.8) 2 (1.2) 1 (0.6) 167 (100)
101 (63.5) 56 (35.2) 48 (30.2) 2 (1.2) 6 (3.8) 2 (1.3) 1 (0.63) 1 (0.63) 159 (100)
Alleles A O B C D Total
266 (79.6) 68 (20.4) 53 (15.9) 6 (1.8) 9 (2.7) 334 (100)
157 (72.3) 60 (27.7) 50 (23.4) 3 (1.4) 7 (3.2) 217 (100)
Test G. Fisher’s test.
p
Pulmonary TB n (%)
Extra-pulmonary TB n (%)
p
0.8610a
84 (63.2) 47 (35.3) 35 (26.3) 4 (3.0) 8 (6.0) 2 (1.5) 1 (0.75) 1 (0.75) 133 (100)
18 (52.9) 15 (44.1) 15 (44.1) – – 1 (3.0) 1 (3.0) – 34 (100)
0.3121a
215 (80,82) 51 (19.2) 37 (14.0) 5 (1.8) 9 (3.4) 266 (100)
51 (75,00) 17 (25.0) 16 (23.5) – 1 (1.5) 68 (100)
0.0606b
0.2052a
0.0504b
0.0831a
84
M.S. Araújo et al. / Human Immunology 74 (2013) 82–84
resistance to the disease. The results of the present study were equally inconclusive, and no association was found between the variant genotypes (OO) that reduce the production of MBL [24] and either susceptibility or resistance to tuberculosis. Similarly inconclusive results were obtained in a study in Denmark [25], which indicated that the mutation at codon 57 (MBL⁄C) may have a protector effect against infection, although no evidence was found of any specific effect related to any other allele. In the present study, the frequency of the MBL⁄C mutation was very low in both patient and control groups, impeding the reliable analysis of any potential effect on infection rates. It is perhaps important to note that most of the studies that have evaluated the role of MBL in the infection by M. tuberculosis have focused on this disease as a co-infection of the human immunodeficiency virus 1, HIV-1 [21,26,27]. In the present study, however, positive HIV-1 infection was a criterion for the exclusion of subjects from both groups. Given this, the apparent associations observed with the polymorphism of the MBL2 gene may have been related to HIV-1 infections [17,24], rather than to the occurrence or absence of M. tuberculosis. The present study also considered the different types of tuberculosis – pulmonary and extra-pulmonary – separately for analysis. However, no difference was found between the two subgroups in relation to a possible association between the polymorphism of exon 1 of the MBL2 gene and the different clinical profiles of the disease. This contrasts with the findings of Selvaraj et al. [28], who suggested an association between the AA genotype and extra-pulmonary tuberculosis in patients infected with HIV-1. Cosar et al. [22] also observed a significant reduction in the frequency of the AB genotype and the MBL2⁄B allele in patients with extra-pulmonary tuberculosis, although there was no significant difference between the pulmonary patients and the healthy control group. Additionally, Singla et al. [29] found the mutant allele MBL2⁄B confers a protective role against TB in the study population. The variation found in the available literature may be related to a series of factors, such as the ethnic origin of the study population, an inadequate control group, small sample size, and incomplete genotyping. In addition, immunity to M. tuberculosis is an extremely complex phenomenon affected by a wide variety of factors and circumstances, not all of which are well understood [20,30]. This emphasizes the importance of further studies in regions where the disease is endemic, such as the Amazon basin, for a more conclusive analysis of the potential role of the genetic profile of hosts in their susceptibility to infection by M. tuberculosis. Acknowledgments This study has been supported in part by PROPESP/FADESP/ UFPA. We thank all subjected enrolled in the present study. References [1] Ezerkowitz RA. Genetic heterogeneity of mannose-binding proteins: the Jekyl and Hyde of innate immunity? Am J Hum Genet 1998;62:6–9. [2] Thiel S, Holmskov U, Hviid L, Laursen SB, Jensenius JC. The concentration of Ctype lectin, mannan-binding protein, in human plasma increases during the acute phase response. Clin Exp Immunol 1992;90:31–5. [3] Ogden CA, Cathelineau A, Hoffmann PR, Bratton D, Ghebrehiwet B, Fadok VA, et al. C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake apoptotic cells. J Exp Med 2001;194:781–95.
[4] Kilpatrick DC, Delahooke TES, Koch C. Mannan-binding lectin and hepatitis C infection. Clin Exp Immunol 2003;132:92–5. [5] Holmskov U, Malhotra R, Sim RB, Jensenius JC. Collectins: collagenous C-type lectins of the innate immune defense system. Immunol Today 1994;15:67–74. [6] Summerfield JA, Ryder S, Sumiya M. Mannose binding protein gene mutations associated with unusual and severe infections in adults. Lancet 1995;345:886–9. [7] Turner MW. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol Today 1996;17:532–40. [8] Ghiran I, Barbashov SF, Klickstein LB, Tas SW, Jensenius JC, Nicholson-Weller A. Complement receptor 1/CD35 is a receptor for mannan binding lectin. J Exp Med 2000;192:1797–807. [9] Jack DL, Lee ME, Turner MW. Anti-microbial activities of mannose-binding lectin. Biochem Soc Trans 2003;31:753–7. [10] Dommett RM, Klein N, Turner MW. Mannose-binding lectin in innate immunity: past, present and future. Tissue Antigens 2006;68:193–209. [11] Garred P, Thiel S, Madsen HO, Ryder LP, Jensenius JC, Svejgaard A. Gene frequency and partial protein characterization of an allelic variant of mannan binding protein associated with low serum concentrations. Clin Exp Immunol 1992;90:517–21. [12] Presanis JS, Kojima M, Sim RB. Biochemistry and genetics of mannan-binding lectin (MBL). Biochem Soc Trans 2003;31:748–52. [13] Turner MW. The role of mannose-binding lectin in health and disease. Mol Immunol 2003;40:423–9. [14] Bonar A, Chmiela M, Rudnicka W, Rózalska B. Mannose-binding lectin enhances the attachment and phagocytosis of mycobacteria in vitro. Arch Immunol Ther Exp 2005;53:437–41. [15] Sumiya M, Super M, Tabona P, Levinsky RJ, Arai T, Turner MW, et al. Molecular basis of opsonic defect in immunodeficient children. Lancet 1991;337:1569–70. [16] Bonar A, Chmiela M, Rozalska B. Level of mannose-binding lectin (MBL) in patients with tuberculosis. Pneumonol Pol 2004;72:201–5. [17] Vallinoto AC, Menezes MRC, Alves AE, Machado LF, Azevedo VN, Souza LL, et al. Mannose-binding lectin gene polymorphism and its impact on human immunodeficiency virus 1 infection. Mol Immunol 2006;43:1358–62. [18] Miller MP. Tools for population genetic analyses – TFPGA, 1.3. Flagstaff, AZ: Department of Biological Sciences, Northern Arizona University, UFA; 1997. [19] Ayres M, Ayres Jr, Ayres Dl, Santos AS. BioEstat 5.3v: aplicação estatísticas nas áreas das ciências biológicas e médicas. Belém, Brasil: Sociedade Civil Mamirauá; 2011 [p. 193]. [20] Berrington WR, Hawn TR. Mycobacterium tuberculosis, macrophages, and the innate immune response: does common variation matter? Immunol Rev 2007:167–86. [21] Alagarasu K, Selvaraj P, Swaminathan S, Raghavan S, Narendran G, Narayanan PR. Mannose binding lectin gene variants and susceptibility to tuberculosis in HIV-1 infected patients of South India. Tuberculosis 2007;87:535–43. [22] Cosar H, Ozkinay F, Onay H, Bayram N, Bakiler AR, Anil M, et al. Low levels of mannose-binding lectin confers protection against tuberculosis in Turkish children. Eur J Clin Microbiol Infect Dis 2009;27:1165–9. [23] Hoal-Van Helden EG, Epstein J, Victor TC. Mannose-binding protein B allele confers protection against tuberculous meningitis. Pediatr Res 1999;45:459–64. [24] Garred P, Madsen HO, Balslev U, Hofmann B, Pedersen C, Gerstoft J, et al. Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-binding lectin. Lancet 1997;349:236–40. [25] Soborg C, Madsen HO, Andersen AB, Lillebaek T, Kok-Jensen A, Garred P. Mannose-binding lectin polymorphismsin in clinical tuberculosis. J Infect Dis 2003;188:777–82. [26] Mombo LE, Lu CY, Ossari S, Bedjabaga I, Sica L, Krishnamoorthy R, et al. Mannose-binding lectin alleles in sub-Saharan Africans and relation with susceptibility to infections. Genes Immun 2003;4:362–7. [27] Garcia-Laorden MI, Pena MJ, Caminero JA, Garcia-Saavedra A, Campos-Herrero MI, Caballero A, et al. Influence of mannose-binding lectin on HIV infection and tuberculosis in a Western-European population. Mol Immunol 2006;43:2143–50. [28] Selvaraj P, Jawahar MS, Rajeswari DN, Alagarasu K, Vidyarani M, Narayanan PR. Role of mannose binding lectin gene variants on its protein levels and macrophage phagocytosis with live Mycobacterium tuberculosis in pulmonary tuberculosis. FEMS Immunol Med Microbiol 2006;2006(46):433–7. [29] Singla N, Gupta D, Joshi A, Batra N, Singh J, Birbian N. Association of mannosebinding lectin gene polymorphism with tuberculosis susceptibility and sputum conversion time. Int J Immunogenet 2012;39:10–4. http:// dx.doi.org/10.1111/j.1744-313X.2011.01047.x. [30] Rosemberg J. Mecanismo imunitário da tubeerculose síntese e atualização. Boletim de Epidemiologia Sanitária 2001;1:35–57.
Contents lists available at SciVerse ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Rapid Communication
No evidence of association between MBL2A/O polymorphisms and Mycobacterium tuberculosis infection in populations from the Brazilian Amazon region Mauro S. Araújo a, Ednelza S. Graça a, Vânia N. Azevedo b, Izaura Cayres-Vallinoto b, Luiz Fernando A. Machado b, Marluisa O.G. Ishak b, Ricardo Ishak b, Antonio C.R. Vallinoto b,⇑ a b
University Hospital João de Barros Barreto – HUJBB, Federal University of Para, Brazil Virus Laboratory, Institute for Biological Sciences, Federal University of Para, Brazil
a r t i c l e
i n f o
Article history: Received 18 May 2012 Accepted 10 September 2012 Available online 19 September 2012
a b s t r a c t The present study investigated the prevalence of the polymorphisms in the exon 1 of the MBL2 gene in patients with tuberculosis at a hospital in northern Brazil, which is a regional reference for the treatment of the disease. The study group was composed of 167 patients with tuberculosis, 34 of which had the extra-pulmonary form of the disease, while the other 133 had the pulmonary type. The control group consists of 159 healthy individuals. Samples of DNA extracted from leucocytes were submitted to Polymerase Chain Reaction for the amplification of a 120-bp segment of exon 1 of the MBL2 gene. The distribution of allele and genotype frequencies varied little among the different groups, and it was not possible to establish any clear association between the variants of the MBL2 gene and the susceptibility to or clinical profile of tuberculosis infections in the population analyzed. Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The mannose-binding lectin (MBL) is an acute phase serum protein of the collectin family [1,2]. It is produced primarily in the liver, although some leucocytes may also be able to synthesize it [3,4]. The principal biological function of MBL is opsonization, given that its collagenous structures are binding sites for the collectin receptors present in the phagocytes, which act directly as opsonin [5–7]. The MBL is capable of recognizing specific carbohydrate residues, primarily mannose, fucose, and N-acetylglucosamine [4–6,8,9], through which the MBL binds to pathogens or cell surfaces [10]. Variations in the serum concentrations of MBL are attributed to mutations in exon 1 (MBL2⁄B – rs1800450, MBL2⁄C – rs1800451 and MBL2⁄D – rs5030737) and the promoter region ( 550 (H/L) – rs11003125 and 221 (Y/X) – rs7096206) of the MBL2 gene [7,11]. This results in defects in the polymerization of MBL, which leads to a functional deficiency, as well as affecting the expression level of the protein [12,13]. In an in vitro study, Bonar et al. [14] showed that the phagocytosis of Mycobacterium tuberculosis in humans is intensified by the presence of MBL, which suggests that this lectin may facilitate the assimilation of this bacterium into the phagocyte in vivo, and thus ⇑ Corresponding author. E-mail address: [email protected] (A.C.R. Vallinoto).
contribute to infection. Such phagocytosis may be mediated by the recognition of the lipoarabinomannan present on the surface of M. tuberculosis. A study in Africa indicated that susceptibility to tuberculosis may increase in individuals with low serum levels of this protein as a result of their being homozygous for the mutations of the MBL2 gene [15]. However, an increased probability of tuberculosis infection has also been related to elevated serum levels of MBL, which might be disadvantageous in the case of infection by mycobacteria [16]. Given these conflicting results on the influence of MBL on infection by M. tuberculosis, the present study investigated the prevalence of mutations in exon 1 of the MBL2 gene in patients with tuberculosis attending a hospital in northern Brazil, which is a regional reference for the treatment of the disease. 2. Materials and methods 2.1. Subjects The study group was made up of 167 tuberculosis (TB) patients (92 males and 75 females) who were seronegative for HIV-1, and had been diagnosed according to the CDC (Center for Disease Control and Prevention) criteria and treated at the João de Barros Barreto University Hospital in the metropolitan region of Belém in 2006 and 2007. Infection by M. tuberculosis was confirmed in these patients by positive smears of the phlegm and/or bronchial fluid,
0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2012.09.001
83
M.S. Araújo et al. / Human Immunology 74 (2013) 82–84
followed by specific culture, tuberculin skin test, X-ray, and positive biopsy for M. tuberculosis. These tests were run in the hospital’s Clinical Pathology Laboratory. The clinical data of the patients were obtained from their medical records, which were provided by the hospital’s Medical Records Department. These records contained the patients’ full history of medical, clinical, and laboratorial examinations and tests. For analysis, the patients were divided into two subgroups, according to their clinical diagnosis. One group was made up of 34 patients with extra-pulmonary tuberculosis, while the second included all the 133 patients with the pulmonary form of the disease. The control group consisted of 159 health workers of both sexes (58 males and 98 females) that did not have active tuberculosis and were seronegative for HIV-1, and had regular (halfyearly) medical check-ups, including X-ray of the chest, sputum smear, haemogram, VDRL, urine routine, fasting plasma glucose and tuberculin skin test. These individuals were chosen as the control group because of the high risk of exposure to tuberculosis patients during their professional activities. 2.2. Ethics All the individuals were briefed about the study and signed an informed consent form before sample collection. The present study was approved by the Human Ethics Committee of the Institute for Health Sciences, Federal University of Para, under the protocol n ° 034/06 CEP-ICS/UFPA. 2.3. Sample collection Blood samples were collected from the members of both groups in vacuum tubes containing EDTA as an anticoagulant. The cells and plasma were separated and frozen at 20 °C in the Virology Laboratory of the Biological Sciences Institute of the Federal University of Pará, where the molecular analyses of the genetic polymorphism were carried out. 2.4. Molecular analysis The genomic DNA was extracted from the leucocytes using a Puregene kit from Gentra Systemas, Inc. (USA) and subjected to the Polymerase Chain Reaction (PCR) for the amplification of 120 bps of exon 1 of the Mbl2 gene. The amplifications were based on the protocol described by Vallinoto et al. [17].
2.5. Statistical analysis The allele and genotype frequencies were calculated by direct counting. The frequencies recorded in both groups were compared with those expected under Hardy-Weinberg equilibrium and tested using Chi-square, Test G and Fisher’s test, which was run in Tools For Population Genetic Analyses – TFPGA 1.3v [18] and BioEstat 5.3v [19]. 3. Results Six different genotypes (AA, AB, AC, AD, BC, and BD) were recorded in both patient and control groups (Table 1), although there was no significant difference (p = 0.8610) in the distribution of frequencies in the groups. The comparative analysis of the genotype frequencies considered only AA, AO, and OO, where MBL2⁄A is the normal allele, and MBL2⁄O represents any of the other three variants (MBL2⁄B, ⁄C or ⁄D). The AA genotype returned the highest frequency in both groups, with a frequency of 0.611 in the patients and 0.635 in the control group (Table 1). The AO genotype had a frequency of 0.371 among TB patients and 0.352 in the control group, while OO was 0.018 in the former and 0.012 in the latter group. The statistical comparison of expected and observed genotype frequencies indicated that both groups were in Hardy-Weinberg equilibrium (p > 0.05). Comparing the two subgroups of tuberculosis patients, the extra-pulmonary group presented a slightly higher frequency of the MBL2⁄O genotypes (Table 1), although the difference was not significant (p = 0.3121). The analysis of the allele frequencies indicated a predominance of the MBL2⁄A allele in both patient subgroups and the control group. There was no significant difference in the distribution of the MBL2⁄O variant among the different groups (p = 0.0504). 4. Discussion To date, relatively few studies have investigated the impact of a deficiency of MBL on the susceptibility or resistance of individuals to tuberculosis [16,20–22], and the available data are contradictory in relation to the role of this protein in this susceptibility. While Alagarasu et al. [21] concluded that a deficiency of MBL was associated with an increase in susceptibility to tuberculosis, for example, other studies [22,23] indicated that it was associated with
Table 1 Distribution of genotype and allele frequencies of the Mbl2 gene in tuberculosis (TB) patient group and subgroups, and the control group.
a b
Genetic profile
TB patients n (%)
Control n (%)
Genotype AA AO AB AC AD OO BC BD Total
102 (61.1) 62 (37.1) 50 (29.9) 4 (2.4) 8 (4.8) 3 (1.8) 2 (1.2) 1 (0.6) 167 (100)
101 (63.5) 56 (35.2) 48 (30.2) 2 (1.2) 6 (3.8) 2 (1.3) 1 (0.63) 1 (0.63) 159 (100)
Alleles A O B C D Total
266 (79.6) 68 (20.4) 53 (15.9) 6 (1.8) 9 (2.7) 334 (100)
157 (72.3) 60 (27.7) 50 (23.4) 3 (1.4) 7 (3.2) 217 (100)
Test G. Fisher’s test.
p
Pulmonary TB n (%)
Extra-pulmonary TB n (%)
p
0.8610a
84 (63.2) 47 (35.3) 35 (26.3) 4 (3.0) 8 (6.0) 2 (1.5) 1 (0.75) 1 (0.75) 133 (100)
18 (52.9) 15 (44.1) 15 (44.1) – – 1 (3.0) 1 (3.0) – 34 (100)
0.3121a
215 (80,82) 51 (19.2) 37 (14.0) 5 (1.8) 9 (3.4) 266 (100)
51 (75,00) 17 (25.0) 16 (23.5) – 1 (1.5) 68 (100)
0.0606b
0.2052a
0.0504b
0.0831a
84
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resistance to the disease. The results of the present study were equally inconclusive, and no association was found between the variant genotypes (OO) that reduce the production of MBL [24] and either susceptibility or resistance to tuberculosis. Similarly inconclusive results were obtained in a study in Denmark [25], which indicated that the mutation at codon 57 (MBL⁄C) may have a protector effect against infection, although no evidence was found of any specific effect related to any other allele. In the present study, the frequency of the MBL⁄C mutation was very low in both patient and control groups, impeding the reliable analysis of any potential effect on infection rates. It is perhaps important to note that most of the studies that have evaluated the role of MBL in the infection by M. tuberculosis have focused on this disease as a co-infection of the human immunodeficiency virus 1, HIV-1 [21,26,27]. In the present study, however, positive HIV-1 infection was a criterion for the exclusion of subjects from both groups. Given this, the apparent associations observed with the polymorphism of the MBL2 gene may have been related to HIV-1 infections [17,24], rather than to the occurrence or absence of M. tuberculosis. The present study also considered the different types of tuberculosis – pulmonary and extra-pulmonary – separately for analysis. However, no difference was found between the two subgroups in relation to a possible association between the polymorphism of exon 1 of the MBL2 gene and the different clinical profiles of the disease. This contrasts with the findings of Selvaraj et al. [28], who suggested an association between the AA genotype and extra-pulmonary tuberculosis in patients infected with HIV-1. Cosar et al. [22] also observed a significant reduction in the frequency of the AB genotype and the MBL2⁄B allele in patients with extra-pulmonary tuberculosis, although there was no significant difference between the pulmonary patients and the healthy control group. Additionally, Singla et al. [29] found the mutant allele MBL2⁄B confers a protective role against TB in the study population. The variation found in the available literature may be related to a series of factors, such as the ethnic origin of the study population, an inadequate control group, small sample size, and incomplete genotyping. In addition, immunity to M. tuberculosis is an extremely complex phenomenon affected by a wide variety of factors and circumstances, not all of which are well understood [20,30]. This emphasizes the importance of further studies in regions where the disease is endemic, such as the Amazon basin, for a more conclusive analysis of the potential role of the genetic profile of hosts in their susceptibility to infection by M. tuberculosis. Acknowledgments This study has been supported in part by PROPESP/FADESP/ UFPA. We thank all subjected enrolled in the present study. References [1] Ezerkowitz RA. Genetic heterogeneity of mannose-binding proteins: the Jekyl and Hyde of innate immunity? Am J Hum Genet 1998;62:6–9. [2] Thiel S, Holmskov U, Hviid L, Laursen SB, Jensenius JC. The concentration of Ctype lectin, mannan-binding protein, in human plasma increases during the acute phase response. Clin Exp Immunol 1992;90:31–5. [3] Ogden CA, Cathelineau A, Hoffmann PR, Bratton D, Ghebrehiwet B, Fadok VA, et al. C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake apoptotic cells. J Exp Med 2001;194:781–95.
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