International Journal of Genomics
Volume 2014 (2014), Article ID 920491, 5 pages
http://dx.doi.org/10.1155/2014/920491

Research Article

Lack of Association of the Polymorphisms IL-17A (−197G/A) and IL-17F (+7488A/G) with Multibacillary Leprosy in Mexican Patients

Mónica Escamilla-Tilch,1,2 Iris Estrada-García,¹ Julio Granados,³ Roberto Arenas-Guzmán,⁴ Rosalio Ramos-Payan,⁵ Thalía Gabriela Pérez-Suárez,² Ma. Isabel Salazar,¹ Riky Luis Pérez-Lucas,³ Sergio Estrada-Parra,¹ and Nora Magdalena Torres-Carrillo²

¹Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, 11340 México, DF, Mexico
²Instituto Nacional de Geriatría, Periférico Sur No. 2767, Col. San Jerónimo Lídice, Del. Magdalena Contreras, 10200 México, DF, Mexico
³Departamento de Trasplantes, División de Inmunogenética, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, 14000 México, DF, Mexico
⁴Sección de Micología, Hospital General Dr. Manuel Gea González, 14080 México, DF, Mexico
⁵Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, 80010 Culiacán, SIN, Mexico

Received 18 August 2014; Accepted 19 October 2014; Published 6 November 2014

Academic Editor: Elena Pasyukova

Copyright © 2014 Mónica Escamilla-Tilch et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Background. Leprosy is a chronic infectious disease caused by the intracellular acid-fast bacilli Mycobacterium leprae; it has been determined that genetic factors of the host play an important role in the disease susceptibility. Thus, in this case-control study, we evaluated the possible association between the IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene SNPs and susceptibility to leprosy disease in Mexican population. Methods. Seventy-five leprosy patients and sixty-nine control subjects were included. Both SNPs were genotyped with the polymerase chain reaction-restriction fragment length polymorphism technique. Results. We found nonsignificant differences in genotype and allele frequencies related to IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene SNPs in MB as well as subclinical forms of leprosy disease versus healthy individuals. Conclusions. Since the sample size is not large enough, it is difficult to sustain an association of susceptibility to leprosy with genotypes or allele frequencies of IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780), suggesting that IL-17 polymorphisms have no significant role in the genetic susceptibility to development of this disease in the Mexican Mestizo population.

1. Introduction

Leprosy is a human chronic infectious disease caused by the intracellular acid-fast bacilli Mycobacterium leprae. It affects the skin and peripheral nerves of susceptible individuals, causing irreversible impairment of nerve function and consequent chronic disability [1]. Despite the global coverage of multidrug therapy promoted by the World Health Organization, 215 new cases of leprosy were reported in Mexico until 2012, with a prevalence of 0.480 cases per every 100,000 inhabitants. In addition, the proportion of multibacillary (MB) cases has increased in some Mexican population associated with a declining incidence [2, 3].

The clinical spectrum of leprosy includes two poles, the lepromatous (LL) and tuberculoid (TT), with several intermediate (I) or dimorphic forms, including borderline tuberculoid (BT), mid-borderline (BB), and borderline lepromatous (BL); furthermore, in the WHO classification standards, the BB, BL, and LL individuals belong to multibacillary (MB) patients, whereas the BT and TT individuals go to paucibacillary (PB) patients. Immunologically, the LL skin lesions are distinguished by predominance of CD8⁺ T cells and it is characterized by a Th2 T-cell immune response, with an antibody complex formation that is unable to contain the infection. By contrast, the TT skin lesions are characterized by a predominance of CD4⁺ T cells; they present a Th1 T-cell cytokine reaction with a vigorous T-cell response that restricts the M. leprae growth [4–6].

On the other hand, this balance between the Th1 (TT) and Th2 (LL) response can be influenced by a third participant, the Th17 cells, which are known to produce a proinflammatory cytokine called interleukin-17 (IL-17) [7]. IL-17 is a newly described cytokine that bonds the adaptive and innate immune systems. So far, six IL-17 family ligands (IL-17A-F) and five receptors (IL-17RA-RD and SEF) have been identified. IL-17A and IL-17F are the members of the IL-17 cytokine family responsible for the pathogenic activity of the Th17 cells; this cytokines family induce multiple proinflammatory mediators, including chemokines, cytokines, and metalloproteinases, from epithelial and fibroblast cells [8].

Based on the biological relevance of IL-17A and IL-17F cytokines, in this study we analyzed two single-nucleotide polymorphisms (SNPs) within the IL-17A and IL-17F genes including the G-197A (rs227593) and A7488G (His161Arg, rs763780), respectively, which have been identified to be associated with several diseases [9]. Therefore, taking into consideration that the genetic background of patients influences the immune response to acquire the infection and consequently provides different clinical manifestations [10], the aim of this study was to analyze the association between the IL-17A and IL-17F genes SNPs with susceptibility or resistance to develop leprosy in Mexican patients.

2. Materials and Methods

2.1. Subjects

In this study, we recruited a total of 75 patients with leprosy (41% female and 59% male, mean age of years) which were classified according to the International Criteria established by Ridley and Jopling [4]. The patients were residents from the states of Sinaloa (33.3%), San Luis Potosí (25.3%), Puebla (17.4%), Distrito Federal (9.3%), Guanajuato (8%), and Yucatán (6.7%). Sixty-two patients were classified as LL, 9 as dimorphic (D), 2 as TT, and 2 as I. All leprosy cases were MB, with exception of 4 PB. As a control group, we included 69 healthy subjects (55.5% female and 44.5% male, mean age of years), unrelated to the patients and matched by ethnicity. Ethnically, patients and controls were classified as Mestizos, who are defined as those individuals born in Mexico that have a Spanish last name, with Mexican ancestors at least back to the third generation. Mestizos are the result of 500 years of admixture between Spaniards, Amerindians, and Africans, and they currently represent most of the Mexican population (>90%) [11, 12]. The informed written consent was obtained from all subjects before enrollment to the study, according to the ethical guidelines of 2008 Declaration of Helsinki.

2.2. Genotyping

For genotyping, venous blood samples of leprosy patients and controls were collected into EDTA-containing tubes, and the genomic DNA (gDNA) was extracted from peripheral blood leukocytes according to Miller’s salting-out method [13]. The IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene polymorphisms were analyzed by the polymerase chain reaction-restriction fragment length polymorphism system (PCR-RFLP) which was slightly modified from the two previously described methods [14]. The PCR amplification was performed in a total volume of 30 μL, containing 100 ng of gDNA, 1 μM of each oligonucleotide, 0.2 mM of each dNTPs, 2 mM of MgCl₂, 0.5 U/μL of Taq DNA polymerase, and supplied buffer enzyme 1X (Invitrogen Life Technologies, Carlsbad, CA, USA). The amplification conditions were 95°C for 3 min, 35 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 30 s, and a final extension of 72°C for 3 min for IL-17A G-197A and for IL-17F A7488G they were 95°C for 3 min, 35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a final extension of 72°C for 3 min. The PCR products were digested at 37°C for 2 h with 1 U of XagI and NIaIII restriction enzymes (New England BioLabs, Beverly, MA, USA) for the identification of the IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene polymorphisms, respectively. Finally, the digested PCR products were electrophoresed on 3% agarose gels ethidium bromide stained for the identification of the genotypes both polymorphisms.

2.3. Statistical Analysis

Frequencies of genotypes and alleles, Hardy-Weinberg Equilibrium (HWE), and OR with a 95% CI were estimated using the Chi-square test (χ ²) (Epi Info statistical software 3.3.2, Atlanta Georgia). Comparison data were evaluated by χ ² test or Fisher’s exact test when applicable. The P values were adjusted with Bonferroni correction for multiple testing ().

3. Results

We investigated the possible association with leprosy in 144 Mexican Mestizo individuals (75 leprosy patients and 69 healthy controls) who were genotyped for the IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene polymorphisms. For this purpose and taking into account that the majority of cases of leprosy in Mexico are MB [3, 15], we only considered the MB leprosy and control group for this analysis. The distribution of genotypic and allelic frequencies of IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene polymorphisms in patients with leprosy and healthy controls is shown in Table 1. Both SNPs were in HWE in the control group (; data not shown). The distribution of genotypic and allelic frequencies between MB leprosy patients and control group did not show significant differences in IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene polymorphisms (). This lack of association was confirmed when we performed the analysis in the clinical subtypes (LL and D) of MB leprosy, where we did not find significant differences (; Table 2).

Table 1: Allelic and gene frequencies of polymorphisms in IL-17A and IL-17F genes in patients with multibacillary leprosy and healthy controls.

MB patients
CS
OR (95% CI)
value

(%)
(%)

Genotype

IL-17A −197

GG
38 (56)
29 (42)
—
—

GA
30 (44)
40 (58)
0.57 (0.27–1.19)
2.11

AA
0 (0)
0 (0)

GA + AA
30 (44)
40 (58)
0.57 (0.27–1.19)
2.11

IL-17F +7488

AA
58 (82)
52 (76)
—
—

AG
13 (18)
16 (24)
0.73 (0.30–1.7)
0.58

GG
0 (0)
0 (0)

AG + GG
13 (18)
16 (24)
0.73 (0.30–1.7)
0.58

Allele

IL-17A −197

G
106 (78)
98 (71)
—
—

A
30 (22)
40 (29)
0.96 (0.39–1.24)
0.24

IL-17F +7488

A
129 (91)
120 (88)
—
—

G
13 (9)
16 (12)
0.76 (0.33–1.74)
0.60

The values are presented as frequency in percentage (%) and number () of genotypes or alleles. The frequencies comparison between groups was analyzed by Chi-square test. Statistical significance was at Yates corrected. OR, odds ratio; CI, confidence interval; , number of individuals; MB, multibacillary leprosy; CS, healthy controls.

Table 2: Allelic and gene frequencies of polymorphisms in IL-17A and IL-17F genes in subclinical forms (LL and D) and healthy controls.

D patients 
(%)
CS 
(%)
OR 
(95% CI)
a value
LL patients 
(%)
OR 
(95% CI)
b value
OR 
(95% CI)
c value

Genotype

IL-17A −197

GG
7 (88)
29 (42)
—
—
31 (52)
—
—
—
—

GA
1 (12)
40 (58)
0.10 (0–0.93)
0.038
29 (48)
1.47 (0.69–3.14)
0.358
0.15 (0.01–1.38)
0.123

AA
0 (0)
0 (0)

0 (0)

GA + AA
1 (12)
40 (58)
0.10 (0–0.93)
0.038
29 (48)
1.47 (0.69–3.14)
0.358
0.15 (0.01–1.38)
0.123

IL-17F +7488

AA
7 (78)
52 (76)
—
—
51 (82)
—
—
—
—

AG
2 (22)
16 (24)
1.08 (0.17–8.38)
0.739
11 (18)
1.43 (0.56–3.67)
0.551
1.32 (0.16–8.64)
0.891

GG
0 (0)
0 (0)

0 (0)

AG + GG
2 (22)
16 (24)
1.08 (0.17–8.38)

11 (18)
1.43 (0.56–3.67)
0.551
1.32 (0.16–8.64)
0.891

Allele

IL-17A −197

G
15 (94)
98 (71)
     6.12  (0.80–1228)
0.039
91 (76)
     0.21  (0.01–1.63)
0.192
     1.28  (0.71–2.32)
0.464

A
1 (6)
40 (29)
29 (24)

IL-17F +7488

A
16 (89)
120 (88)
     1.07  (0.20–7.39)
0.757
113 (91)
     1.28  (0.0–7.08)
0.897
     1.37  (0.57–3.32)
0.575

G
2 (11)
16 (12)
11 (9)

The values are presented as frequency in percentage (%) and number () of genotypes or alleles. The frequencies comparison between groups was analyzed by Chi-square test. Statistical significance was at Yates corrected; OR, odds ratio; CI, confidence interval; , number of individuals; MB, multibacillary leprosy; CS, healthy controls; D, dimorphic; LL, lepromatous leprosy. aD patients versus CS; bLL versus CS; cD versus LL.

4. Discussion

Despite global coverage of multidrug therapy promoted by the World Health Organization the leprosy remains as a public health problem [2]. Nowadays, we know that certain individuals who are exposed to the M. leprae develop leprosy, suggesting that genetic background is relevant in the development of it, since it determines the control of the immune response against this infection [16]. In this study we aimed to identify the genotypic and allelic frequencies of IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene SNPs in the context of leprosy to identify if there was any association with susceptibility to leprosy in the Mexican Mestizo population. We did not find an association between both IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene SNPs and MB leprosy or their clinical subtypes, so our study provides evidence of lack of association with susceptibility in leprosy patients; moreover, the genotype and alleles frequencies have not been reported previously in Mexican population.

This observation differs with a study in the North India Cohort, where the IL-17F A7488G (His161Arg, rs763780) gene SNP is associated with susceptibility to leprosy; likewise, they found that AG genotypes confer a decreased risk of acquiring leprosy in the North Indian Cohort and observed that this SNP also influences the clinical phenotypes in leprosy disease [17], and to our knowledge, this is the first study that reported association of this polymorphism with leprosy susceptibility. To date, there are some studies related to IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene SNPs and risk of tuberculosis (TB), gastric cancer, rheumatoid arthritis (RA), intestinal bowel disease (IBD), Crohn’s disease (CD), ulcerative colitis (UC), and others [14]. In this previous studies, patients from a Chinese Han population who had the genotype AG/AA of IL-17F A7488G (His161Arg, rs763780) gene SNP were more susceptible to acquiring tuberculosis, compared to the GG genotype [18], whereas in other studies of the China population it was verified that the IL-17F A7488G (His161Arg, rs763780) gene SNP was associated with risk of gastric cancer and GA genotype was associated with the clinic-pathological features and not with the risk of gastric cancer itself. However, in the case of the IL-17A G-197A (rs227593) gene SNP, this polymorphism was not associated with the risk of gastric cancer [14]. Moreover, in Iranian population it was observed that the IL-17A G-197A (rs227593) gene SNP was significantly associated with the risk of gastric cancer, since patients who had the homozygous AA were 2.9 times more likely to develop disease; furthermore, the presence of a single A allele increases the risk of gastric cancer up to 1.7-fold. This association was only observed in early stage gastric adenocarcinomas and was not linked to H. pylori infection [19]. On the other hand, the close association of IL-17A G-197A (rs227593) gene SNP with susceptibility to the development of osteoarthritis (OA) in the Korea population has been described; however, they did not find relationship between the IL-17F A7488G (His161Arg, rs763780) gene SNP and OA susceptibility [20]. With respect to IBD which includes CD and UC, it was reported in German population that the IL-17F A7488G (His161Arg, rs763780) gene SNP is not considered as a marker of susceptibility for IBD per se but is associated with increased disease activity [21], whereas in Chinese population it was demonstrated that the IL-17F A7488G (His161Arg, rs763780) gene SNP has no effect on susceptibility to DC but is associated with protection to UC [22]. The discrepancy in the association of IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene SNPs could be due to the type of pathology studied but mainly to the frequency at which the polymorphic allele in each population is presented, which reflects the genetic heterogeneity of the different population, suggesting that the low frequency or absence of the polymorphic alleles may confer an advantage of decreasing risk of susceptibility. Moreover, environmental and socioeconomic factors may influence the development of different pathologies. In conclusion, we identify lack of association of IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene SNPs with susceptibility to leprosy in Mexican population.

Conflict of Interests

The authors declare that they have no conflict of interests related to the publication of this paper.

Acknowledgments

The authors thank Dr. G. Sanchez-Schmitz (Division of Infectious Diseases, Boston Children’s Hospital and Harvard Medical School, Harvard University) for his critical reading, and they also greatly appreciate the participation in this study of Jesús Lázaro Lopez-Vazquez, laboratory assistant. This study was supported by grants from the National Council of Science and Technology-CONACYT (nos. 106152, 79589, and 160883).

References

A. Gulia, I. Fried, and C. Massone, “New insights in the pathogenesis and genetics of leprosy,” F1000 Medicine Reports, vol. 2, no. 1, article 30, 2010.

World Health Organization, “Global burden of leprosy at the beginning of the year 2012,” Weekly Epidemiological Record, vol. 87, pp. 317–328, 2012.

M. R. Larrea, M. C. Carreño, and P. E. M. Fine, “Patterns and trends of leprosy in Mexico: 1989–2009,” Leprosy Review, vol. 83, no. 2, pp. 184–194, 2012.

D. S. Ridley and W. H. Jopling, “Classification of leprosy according to immunity. A five-group system,” International Journal of Leprosy and Other Mycobacterial Diseases, vol. 34, no. 3, pp. 255–273, 1966.

R. R. Jacobson and J. L. Krahenbuhl, “Leprosy,” The Lancet, vol. 353, no. 9153, pp. 655–660, 1999.

R. L. Modlin, “Th1-Th2 paradigm: insights from leprosy,” Journal of Investigative Dermatology, vol. 102, no. 6, pp. 828–832, 1994.

E. Bettelli, T. Korn, and V. K. Kuchroo, “Th17: the third member of the effector T cell trilogy,” Current Opinion in Immunology, vol. 19, no. 6, pp. 652–657, 2007.

T. Shibata, T. Tahara, I. Hirata, and T. Arisawa, “Genetic polymorphism of interleukin-17A and -17F genes in gastric carcinogenesis,” Human Immunology, vol. 70, no. 7, pp. 547–551, 2009.

L. Southam, O. Heath, K. Chapman, and J. Loughlin, “Association analysis of the interleukin 17 genes IL17A and IL17F as potential osteoarthritis susceptibility loci,” Annals of the Rheumatic Diseases, vol. 65, no. 4, pp. 556–557, 2006.

A. Alter, A. Grant, L. Abel, A. Alcaïs, and E. Schurr, “Leprosy as a genetic disease,” Mammalian Genome, vol. 22, no. 1-2, pp. 19–31, 2011.

H. Rangel-Villalobos, J. F. Muñoz-Valle, A. González-Martín, A. Gorostiza, M. T. Magaña, and L. A. Páez-Riberos, “Genetic admixture, relatedness, and structure patterns among Mexican populations revealed by the Y-chromosome,” American Journal of Physical Anthropology, vol. 135, no. 4, pp. 448–461, 2008.

R. Rubi-Castellanos, M. Anaya-Palafox, E. Mena-Rojas, D. Bautista-España, J. F. Muñoz-Valle, and H. Rangel-Villalobos, “Genetic data of 15 autosomal STRs (Identifiler kit) of three Mexican Mestizo population samples from the States of Jalisco (West), Puebla (Center), and Yucatan (Southeast),” Forensic Science International: Genetics, vol. 3, no. 3, pp. e71–e76, 2009.

S. A. Miller, D. D. Dykes, and H. F. Polesky, “A simple salting out procedure for extracting DNA from human nucleated cells,” Nucleic Acids Research, vol. 16, no. 3, p. 1215, 1988.

X. Wu, Z. Zeng, B. Chen et al., “Association between polymorphisms in interleukin-17A and interleukin-17F genes and risks of gastric cancer,” International Journal of Cancer, vol. 127, no. 1, pp. 86–92, 2010.

M. Escamilla-Tilch, N. M. Torres-Carrillo, R. Ramos Payan, et al., “Genetic polymorphism of HLA-DRB1 antigens in the susceptibility to lepromatous leprosy in Mexicans,” Biomedical Reports, vol. 1, no. 6, pp. 945–949, 2013.

J. M. Blackwell, S. E. Jamieson, and D. Burgner, “HLA and infectious diseases,” Clinical Microbiology Reviews, vol. 22, no. 2, pp. 370–385, 2009.

V. S. Chaitanya, R. S. Jadhav, M. Lavania, et al., “Interleukin-17F single-nucleotide polymorphism (7488T>C) and its association with susceptibility to leprosy,” International Journal of Immunogenetics, vol. 41, no. 2, pp. 131–137, 2014.

R. Peng, J. Yue, M. Han, Y. Zhao, L. Liu, and L. Liang, “The IL-17F sequence variant is associated with susceptibility to tuberculosis,” Gene, vol. 515, no. 1, pp. 229–232, 2013.

A. Rafiei, V. Hosseini, G. Janbabai et al., “Polymorphism in the interleukin-17A promoter contributes to gastric cancer,” World Journal of Gastroenterology, vol. 19, no. 34, pp. 5693–5699, 2013.

L. Han, H. S. Lee, J. H. Yoon et al., “Association of IL-17A and IL-17F single nucleotide polymorphisms with susceptibility to osteoarthritis in a Korean population,” Gene, vol. 533, no. 1, pp. 119–122, 2014.

J. Seiderer, I. Elben, J. Diegelmann et al., “Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease (IBD): upregulated colonic IL-17F expression in active Crohn's disease and analysis of the IL17F p.His161Arg polymorphism in IBD,” Inflammatory Bowel Diseases, vol. 14, no. 4, pp. 437–445, 2008.

B. Chen, Z. Zeng, J. Hou, M. Chen, X. Gao, and P. Hu, “Association of interleukin-17F 7488 single nucleotide polymorphism and inflammatory bowel disease in the Chinese population,” Scandinavian Journal of Gastroenterology, vol. 44, no. 6, pp. 720–726, 2009.