Address of Corresponding Author

Respiration 2005;72:365-368 (DOI: 10.1159/000086249)

 Outline

• Key Words
• Abstract
• Introduction
• Materials and Methods
• Patients and Controls
• Genotyping Using PCR-RFLP
• Statistical Analysis
• Results
• Baseline Clinical Characteristics of the TB Patients
• Genotype Frequencies of the 5 SNPs in TB Patients and Controls
• Discussion
• Acknowledgements
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Tuberculosis
• Genetic susceptibility
• Interleukin-12 receptor
• Single nucleotide polymorphism
• Koreans

  Abstract

Background: The fact that only 10% of people infected with Mycobacterium tuberculosis develop clinical tuberculosis (TB) suggests the presence of genetic factors in the pathogenesis of TB. To date, a number of single nucleotide polymorphisms (SNPs) in several candidate genes have been proposed as genetic risk factors of TB; however, reports are conflicting. Objectives: We investigated whether SNPs in the interleukin (IL)-12 receptor 1 gene are associated with TB in Koreans. Methods: One hundred and fifteen patients with bacteriologically or pathologically confirmed TB and 151 healthy anonymous blood donors were enrolled. The genotypes of 5 SNPs on IL-12 receptor 1 gene, +705A/G (Q214R), +1158T/C (M365T), +1196G/C (G378R), +1637G/A (A525T) and +1664 C/T (P534S), were determined by PCR-RFLP. Results: No difference was observed between TB patients and controls in terms of the genotype frequencies of the 5 SNPs of the IL-12 receptor 1 gene or of their haplotypes. Conclusions: In view of the finding that these SNPs have been reported to be associated with TB in the Japanese and Moroccan populations, our results may reflect racial differences in genetic susceptibility to TB.

Copyright © 2005 S. Karger AG, Basel

 Introduction

Tuberculosis (TB) is the second most common cause of death due to infectious disease in the world. An estimated 8.3 million new cases of TB occurred in the year 2000, and 1.8 million deaths from TB were reported in the same year [1]. Moreover, the incidence of HIV-associated TB continues to increase [2, 3], and TB caused 11% of all adult AIDS deaths in 1997 [1].

The fact that only 10% of people infected with Mycobacterium tuberculosis develop clinical TB [4] suggests that genetic factors play a role in the pathogenesis of TB. This hypothesis is further supported by the higher concordance rate of TB in monozygotic twins rather than in dizygotic twins [5]. In this context, several polymorphisms in genes associated with immune defense against M. tuberculosis have been tested. Polymorphisms in the following genes: NRAMP1 [6,7,8,9,10], vitamin D receptor [11, 12], interleukin (IL)-1 [13, 14], IL-10 [14] and TNF [15] have been reported to be genetic factors involved in the development of clinical TB. Unfortunately, these associations have proven to be irreproducible in most cases.

IL-12-dependent interferon- production is crucial to the host's defense mechanism against mycobacteria [16, 17]. Moreover, polymorphisms in the coding sequence of the IL-12 receptor gene have been reported to be associated with tuberculosis in the Japanese and Moroccan populations [18, 19], but not with leprosy in Koreans [20]. In this study, we examined whether single nucleotide polymorphisms (SNPs) in IL-12 receptor 1 gene are associated with TB in Koreans.

 Materials and Methods

 Patients and Controls

One hundred and fifteen patients with bacteriologically or pathologically confirmed TB were enrolled. Patients with a positive HIV test or being administered immunosuppressive agents were excluded. A control group comprised of 151 healthy anonymous blood donors was evaluated to determine the distribution of alleles and genotypes in a random control population. All enrollees were Korean. This study was approved by our institutional review board and written informed consent was obtained from all subjects, before blood sampling.

 Genotyping Using PCR-RFLP

Three milliliters of peripheral blood was drawn from each subject into EDTA-containing tubes. DNA was extracted using a Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, Minn., USA) following the manufacturer's protocol. We typed for 5 previously reported SNPs that cause missense mutations in the coding sequence of the IL-12 receptor 1 gene in Koreans [20], namely +705A/G (Q214R, NCBI SNP ID:rs 11575934), +1158T/C (M365T, NCBI SNP ID:rs 375947), +1196G/C (G378R, NCBI SNP ID:rs 401502), +1637G/A (A525T, NCBI SNP ID:rs 11575935) and +1664 C/T (P534S, HGBASE database ID:SNP001745641). For +705A/G genotyping, genomic DNA was amplified using the primer set 5'-ggttaagtgactggtgccaag-3' and 5'-ctcaaaccactggcctcaag-3'. The PCR fragment obtained was restricted with BbvI (New England BioLabs, Beverly, Mass., US) at 37°C for 10 h. For +1158T/C typing (M365T), 5'-aacaaacgccatctgctacc-3' and 5'-caacacctctctgggcctta-3', and Hsp92II (Promega, Madison, Wisc., USA) were used at 37°C. For +1196G/C, 5'-aacaaacgccatctgctacc-3' and 5'-agagtgagaggccacctgag-3', and MspI(Promega, Madison, Wisc., USA) were used at 37°C. For +1637G/A and +1664 C/T, 5'-ggctgtggtagcccagcct-3' and 5'-ggaagcgcagtgcagtgcatg-3' were used and restricted with MaeII (Roche, Mannheim, Germany) at 50°C and BsrI (New England BioLabs) at 65°C, respectively. PCR was carried out in PCR buffer (1.5 mM MgCl₂, 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton^® X-100), 100 µM of each dNTP, 25 µM primers and 1 unit of Taq polymerase (Invitrogen, Carlsbad, Calif., USA) in a final volume of 50 µl. Following an initial denaturation at 95°C for 5 min, samples were subjected to 30 cycles of denaturation at 95°C for 30 s, annealing at 57-60°C (according to the requirements of the primer set) for 30 s and elongation at 72°C for 30 s, followed by a final elongation step at 72°C for 10 min. The reaction products of the PCRs and enzyme restrictions were analyzed on 3% agarose gel (+705A/G, +1158T/C, +1637G/A, +1664 C/T) or 8% polyacrylamide gel (+1196G/C). All patient genotypes showing unrestricted fragments in PCR-RFLP were confirmed by sequencing.

 Statistical Analysis

The associations between the various polymorphisms and TB were investigated using the two-tailed ² test. Differences with p values of <0.05 were considered significant. Odds ratios and 95% confidence intervals were calculated to assess the relative disease risk conferred by the genotypes. Haplotypes were generated using Haploview (version 2.05), and SPSS (version 11.0) was used for all statistical analyses.

 Results

 Baseline Clinical Characteristics of the TB Patients

Table 1 shows the baseline characteristics of the 115 TB patients. Median age was 36 years and 65 of the 115 TB patients were male. Thirty-six (31.3%) patients had at least one family member with a history of TB. Seventy-nine (68.7%) patients had pulmonary TB including TB pleuritis and the other 36 (31.3%) had extrapulmonary TB.

Table 1. Baseline characteristics of study subjects

 Genotype Frequencies of the 5 SNPs in TB Patients and Controls

Of the 5 missense mutations, C1664T alone was not polymorphic (table 2). The genotype distributions of the 4 other SNPs were in Hardy-Weinberg equilibrium. No difference in genotype distributions was observed between patients and controls. The most common haplotype in both groups was A-T-G (705-1158-1196), and the proportions of this haplotype in the two groups were similar (69.6% in TB patients vs. 63.9% in controls, p = 0.94) (table 3).

Table 2. Allele frequencies of the 5 SNPs in TB patients and controls

Table 3. Haplotype frequencies in TB patients and controls

 Discussion

This study denies the proposed association between polymorphisms in IL-12 receptor 1 gene and TB [18, 19] in the Korean population. We genotyped 5 SNPs that have been alleged to cause missense mutations, +705A/G (Q214R), +1158T/C (M365T), +1196G/C (G378R), +1637G/A (A525T) and +1664 C/T (P534S), in the IL-12 receptor 1 gene using PCR-RFLP in TB patients and healthy controls, and found no association between these SNPs and TB in Koreans.

IL-12 promotes cell-mediated immunity to intracellular pathogens by inducing type-1 helper T cell responses and interferon- production by binding to high-affinity -1/-2 heterodimeric IL-12 receptor complexes on T cells and natural killer cells [21]. Moreover, mutations causing IL-12 receptor 1 deficiencies have been reported to cause severe forms of disseminated infection by salmonella and mycobacteria [22,23,24]. The report that a missense mutation in IL-12 receptor 1 caused abdominal TB in a child [25] stimulated researchers to establish a relationship between SNPs in the IL-12 receptor 1 gene and TB. Although SNPs in the 3'UTR of the IL-12 receptor 1 gene were reported not to be associated with TB [26], Akahoshi et al. [18] reported that SNPs in the coding sequence of the IL-12 receptor 1 gene are associated with TB in the Japanese and Moroccan populations [19]. However, we failed to confirm this association in the Korean population.

In TB susceptibility association studies, conflicting results are not uncommon. For example, missense mutations of D543N in the NRMAP1 gene were proposed to be a genetic risk factor for TB in Gambia [6], and this was later confirmed in the Japanese [7]. However, this association was denied in New Guinea [8] and Taiwanese [10] populations. In addition, the association between the TaqI polymorphism in vitamin D binding receptor and TB proposed in the Gambian population [11] was not reproduced in Gujarati Hindus [12] or in the Cambodian people [27].

Lohmueller et al. [28 ] argued that a sizable fraction of reported associations are supported by strong evidence of replication and that observed inconsistencies are usually due to false-negative results produced by underpowered studies. However, the present study fulfills the suggested strategies for genetic association studies [29] and involves 115 TB patients and 151 controls, which is comparable to the 98 patients and 197 controls in the study of Akahoshi et al. [18]. Furthermore, two large-scale association studies involving more than 300 TB patients [11, 27] suggest a possible ethnicity-specific association among different racial groups as well. Thus, a failure to replicate associations between polymorphisms of IL-12 receptor 1 and TB in Korean population might be understood in this context. A meta-analysis of the various results available should elucidate the association between the IL-12 receptor 1 polymorphism and TB.

In conclusion, we found no association between the polymorphisms of the IL-12 receptor 1 gene and TB in Koreans. Given that an association was previously reported in the Japanese and Moroccan populations, these results may reflect racial differences with respect to TB susceptibility. A large-scale association study is needed to more precisely elucidate the true association between polymorphisms of IL-12 receptor 1 and TB in both populations.

 Acknowledgements

This work was supported by the Seoul National University Hospital Research Fund (grant No. 09-2003-005-0).

  References

1.

Corbett EL, Watt CJ, Walker N, Maher D, Williams BG, Raviglione MC, Dye C: The growing burden of tuberculosis: Global trends and interactions with the HIV epidemic. Arch Intern Med 2003;163:1009-1021.

2.

Murray JF: Cursed duet: HIV infection and tuberculosis. Respiration 1990;57:210-220.

3.

Murray JF: Tuberculosis and HIV infection: A global perspective. Respiration 1998;65:335-342.

4.

Murray CJ, Styblo K, Rouillon A: Tuberculosis in developing countries: Burden, intervention and cost. Bull Int Union Tuberc Lung Dis 1990;65:6-24.

5.

Comstock GW: Tuberculosis in twins: A re-analysis of the Prophit survey. Am Rev Respir Dis 1978;117:621-624.

6.

Bellamy R, Ruwende C, Corrah T, McAdam KP, Whittle HC, Hill AV: Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N Engl J Med 1998;338:640-644.

7.

Gao PS, Fujishima S, Mao XQ, Remus N, Kanda M, Enomoto T, Dake Y, Bottini N, Tabuchi M, Hasegawa N, et al: Genetic variants of NRAMP1 and active tuberculosis in Japanese populations. International Tuberculosis Genetics Team. Clin Genet 2000;58:74-76.

8.

Cervino AC, Lakiss S, Sow O, Hill AV: Allelic association between the NRAMP1 gene and susceptibility to tuberculosis in Guinea-Conakry. Ann Hum Genet 2000;64:507-512.

9.

Ryu S, Park YK, Bai GH, Kim SJ, Park SN, Kang S: 3'UTR polymorphisms in the NRAMP1 gene are associated with susceptibility to tuberculosis in Koreans. Int J Tuberc Lung Dis 2000;4:577-580.

10.

Liaw YS, Tsai-Wu JJ, Wu CH, Hung CC, Lee CN, Yang PC, Luh KT, Kuo SH: Variations in the NRAMP1 gene and susceptibility of tuberculosis in Taiwanese. Int J Tuberc Lung Dis 2002;6:454-460.

11.

Bellamy R, Ruwende C, Corrah T, McAdam KP, Thursz M, Whittle HC, Hill AV: Tuberculosis and chronic hepatitis B virus infection in Africans and variation in the vitamin D receptor gene. J Infect Dis 1999;179:721-724.

12.

Wilkinson RJ, Llewelyn M, Toossi Z, Patel P, Pasvol G, Lalvani A, Wright D, Latif M, Davidson RN: Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: A case-control study. Lancet 2000;355:618-621.

13.

Wilkinson RJ, Patel P, Llewelyn M, Hirsch CS, Pasvol G, Snounou G, Davidson RN, Toossi Z: Influence of polymorphism in the genes for the interleukin (IL)-1 receptor antagonist and IL-1beta on tuberculosis. J Exp Med 1999;189:1863-1874.

14.

Bellamy R, Ruwende C, Corrah T, McAdam KP, Whittle HC, Hill AV: Assessment of the interleukin 1 gene cluster and other candidate gene polymorphisms in host susceptibility to tuberculosis. Tuber Lung Dis 1998;79:83-89.

15.

Selvaraj P, Sriram U, Mathan Kurian S, Reetha AM, Narayanan PR: Tumour necrosis factor alpha (-238 and -308) and beta gene polymorphisms in pulmonary tuberculosis: Haplotype analysis with HLA-A, B and DR genes. Tuberculosis (Edinb) 2001;81:335-341.

16.

Dorman SE, Holland SM: Interferon-gamma and interleukin-12 pathway defects and human disease. Cytokine Growth Factor Rev 2000;11:321-333.

17.

Doffinger R, Altare F, Casanova JL: Genetic heterogeneity of Mendelian susceptibility to mycobacterial infection. Microbes Infect 2000;2:1553-1557.

18.

Akahoshi M, Nakashima H, Miyake K, Inoue Y, Shimizu S, Tanaka Y, Okada K, Otsuka T, Harada M: Influence of interleukin-12 receptor beta1 polymorphisms on tuberculosis. Hum Genet 2003;112:237-243.

19.

Remus N, El Baghdadi J, Fieschi C, Feinberg J, Quintin T, Chentoufi M, Schurr E, Benslimane A, Casanova JL, Abel L: Association of IL12RB1 polymorphisms with pulmonary tuberculosis in adults in Morocco. J Infect Dis 2004;190:580-587.

20.

Lee SB, Kim BC, Jin SH, Park YG, Kim SK, Kang TJ, Chae GT: Missense mutations of the interleukin-12 receptor beta 1(IL12RB1) and interferon-gamma receptor 1 (IFNGR1) genes are not associated with susceptibility to lepromatous leprosy in Korea. Immunogenetics 2003;55:177-181.

21.

Trinchieri G: Proinflammatory and immunoregulatory functions of interleukin-12. Int Rev Immunol 1998;16:365-396.

22.

de Jong R, Altare F, Haagen IA, Elferink DG, Boer T, van Breda Vriesman PJ, Kabel PJ, Draaisma JM, van Dissel JT, Kroon FP, et al: Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients. Science 1998;280:1435-1438.

23.

Altare F, Durandy A, Lammas D, Emile JF, Lamhamedi S, Le Deist F, Drysdale P, Jouanguy E, Doffinger R, Bernaudin F, et al: Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency. Science 1998;280:1432-1435.

24.

Caragol I, Raspall M, Fieschi C, Feinberg J, Larrosa MN, Hernandez M, Figueras C, Bertran JM, Casanova JL, Espanol T: Clinical tuberculosis in 2 of 3 siblings with interleukin-12 receptor beta1 deficiency. Clin Infect Dis 2003;37:302-306.

25.

Altare F, Ensser A, Breiman A, Reichenbach J, Baghdadi JE, Fischer A, Emile JF, Gaillard JL, Meinl E, Casanova JL: Interleukin-12 receptor beta1 deficiency in a patient with abdominal tuberculosis. J Infect Dis 2001;184:231-236.

26.

Ma X, Reich RA, Gonzalez O, Pan X, Fothergill AK, Starke JR, Teeter LD, Musser JM, Graviss EA: No evidence for association between the polymorphism in the 3' untranslated region of interleukin-12B and human susceptibility to tuberculosis. J Infect Dis 2003;188:1116-1118.

27.

Delgado JC, Baena A, Thim S, Goldfeld AE: Ethnic-specific genetic associations with pulmonary tuberculosis. J Infect Dis 2002;186:1463-1468.

28.

Lohmueller KE, Pearce CL, Pike M, Lander ES, Hirschhorn JN: Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease. Nat Genet 2003;33:177-182.

29.

Silverman EK, Palmer LJ: Case-control association studies for the genetics of complex respiratory diseases. Am J Respir Cell Mol Biol 2000;22:645-648.

  Author Contacts

Jae-Joon Yim, MD
Department of Internal Medicine, Seoul National University College of Medicine
28 Yongon-Dong, Chongno-Gu
Seoul, 110-744 (Republic of Korea)
Tel. +82 2 760 2059, Fax +82 2 762 9662, E-Mail yimjj@snu.ac.kr

  Article Information

Received: July 6, 2004
Accepted after revision: November 22, 2004
Number of Print Pages : 4
Number of Figures : 0, Number of Tables : 3, Number of References : 29

  Publication Details

Respiration (International Journal of Thoracic Medicine)

Vol. 72, No. 4, Year 2005 (Cover Date: July-August 2005)

Journal Editor: Bolliger, C.T. (Cape Town)
ISSN: 0025-7931 (print), 1423-0356 (Online)

For additional information: http://www.karger.com/res

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