• 1.

    Grange JM, Lethaby JI, 2004. Leprosy of the past and today. Seminar Respir Crit Care Med 25: 271281.

  • 2.

    Shields E, Russell DA, Vance MAP, 1987. Genetic epidemiology of the susceptibility to leprosy. J Clin Invest 79: 11391143.

  • 3.

    Scollard DM, Adams LB, Gillis TP, Krahenbuhl JL, Truman RW, Williams DL, 2006. The continuing challenges of leprosy. Clin Microbiol Rev 19: 338381.

  • 4.

    Alcais A, Sanchez FO, Thuc NV, Lap VD, Oberti J, Lagrange PH, Schurr E, Abel L, 2000. Granulomatous reaction to intradermal injection of lepromin is linked to the human NRAMP1 gene in Vietnamese leprosy sibships. J Infect Dis 181: 302308.

    • Search Google Scholar
    • Export Citation
  • 5.

    Atkinson SE, Khanolkar Young S, Marlowe S, Jain S, Reddy RG, Suneetha S, Lockwood DN, 2004. Detection of IL-13, IL-10 and IL-6 in the leprosy skin lesions of patients during prednisolone treatment for type 1 reactions. Int J Lepr Other Mycobact Dis 72: 2734.

    • Search Google Scholar
    • Export Citation
  • 6.

    Rastogi N, Goh KS, Berchel M, 1999. Species-specific identification of Mycobacterium leprae by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol 37: 20162019.

    • Search Google Scholar
    • Export Citation
  • 7.

    World Health Organization, 1997. Expert committee on Leprosy. Chemotherapy of Leprosy. WHO Technical Report Service No. 847. Geneva: WHO, 1113.

    • Search Google Scholar
    • Export Citation
  • 8.

    Merza M, Farnia P, Anoosheh S, Varahram M, Kazampour M, Pajand O, Saeif S, Mirsaeidi M, Masjedi MR, Velayati AA, Hoffner S, 2009. The NRAMPI, VDR and TNF-alpha gene polymorphisms in Iranian tuberculosis patients: the study on host susceptibility. Braz J Infect Dis 13: 252256.

    • Search Google Scholar
    • Export Citation
  • 9.

    Kardum LB, Etokebe GE, Knezevic J, 2005. Interferon-γ receptor -1 gene promoter polymorphisms (G 611A; T-56C) and susceptibility to tuberculosis. Scand J Immunol 63: 142150.

    • Search Google Scholar
    • Export Citation
  • 10.

    Velazquez CG, Roa RL, Rizo VD, 2010. Abnormalities in intracellular processing and expression of interferon-gamma receptor in adherent cells from lepromatous leprosy patients. J Interferon Cytokine Res 3: 99105.

    • Search Google Scholar
    • Export Citation
  • 11.

    Newport MJ, Huxley CM, Huston S, Catherine M, Hawrylowic Z, Oostra BA, Williamson R, Levin M, 1996. A mutation in the interferon-γ receptor gene and susceptibility to mycobacterial infection. N Engl J Med 335: 19411948.

    • Search Google Scholar
    • Export Citation
  • 12.

    Lee BS, Kim BC, Jin SH, Park YG, Kim SK, Kang TJ, Chae GT, 2003. Missense mutation 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 55: 177181.

    • Search Google Scholar
    • Export Citation
  • 13.

    Santos AR, Suffys PN, Vanderborght PR, Moraes MO, Vieira LM, Cabello PH, Bakker AM, Matos HJ, Huizinga TW, Ottenhoff TH, Sampaio EP, Sarnoa EN, 2002. Role of tumor 8 necrosis factor-α and interleukin-10 promoter gene polymorphisms in Leprosy. J Infect Dis 186: 16871691.

    • Search Google Scholar
    • Export Citation
  • 14.

    Roy S, Frodsham A, Saha B, Hazra SK, Taylor M, Hill AVS, 1999. Association of vitamin D receptor genotype with leprosy type. J Infect Dis 179: 187191.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

Interferon-Gamma Receptor-1 Gene Promoter Polymorphisms and Susceptibility to Leprosy in Children of a Single Family

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  • Pediatric Respiratory Diseases Research Centre, Tehran, Iran; Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD),UNION and WHO Collaborating Centre, Shahid Beheshti University of Medical Sciences (Medical Campus), Tehran, Iran

The autosomal recessive disorder, because of a single mutation in interferon-γ receptor-1(IFNGR1) at position −56, was found to be associated with susceptibility to leprosy in children of the same family. The existence of such heterozygous carriers might explain the crucial role of IFNGR1 in the host defense against intracellular pathogens such as Mycobacterium leprae. The single nucleotide polymorphisms (SNPs) in major candidate genes, i.e., natural resistance-associated macrophage protein 1 (NRAMP1), vitamin D receptor (VDR), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), interleukin-12-receptor 1 (IL-12R1), were not found to be associated with this disease.

Introduction

Leprosy is an infectious disease caused by the Mycobacterium leprae.1 Clinically, leprosy is manifested as a disease spectrum with two polar forms, lepromatous-LL and tuberculoid-TT.2 The spectrum is further divided into infectious multibacillary, paucibacillary, and transitional borderline forms.2 The precise mechanism of transmission of Mycobacterium leprae is unknown. Recent molecular studies have begun to explain the importance of immune response to M. leprae at two fundamental levels. In the first level the overall susceptibility and resistance to this organism have been described, and at the second level a range of cellular immunity and delayed hypersensitivity have been shown among individuals who are infected.3,4 Generally, they revealed a predominance of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) transcripts in tuberculoid lesions, and IL-4, IL-5, and IL-10 in lepromatous ones.2,3 Alternatively, in cytokine genes several single nucleotide polymorphisms (SNPs) have been confirmed to play a critical role in triggering host-pathogen interactions.5 In this study, the influence of major candidate genes, i.e., SNPs in the natural resistance-associated macrophage protein 1 (NRAMP1) gene, vitamin D receptor (VDR), TNF-α, IL-10, interleukin-12-receptor 1 (IL-12R1), and IFNGR1 were investigated within infected children of a single family.

In 2010, an Iranian boy, 12 years of age, was admitted to a Department of Pediatric in National Research Institute of TB and Lung Diseases, Tehran, Iran. He presented with massive lesions with localized drainage on the left and right foot and complaining about edema (Figure 1). The problem started 8 years ago with the appearance of a small blister on his toes. A presumptive diagnosis of fungal infection was made at local consultation that did not improve despite topical and systemic antifungal therapy. On admission to our hospital, nerve trunks of the upper and lower limbs were normal. The routine laboratory tests showed, erythrocyte sedimentation rate (ERS) of 110 mm/h with 9.5g/dL of hemoglobin. Antinuclear antibodies (ANAs) and rheumatoid factor (RF) were negative. Radiological investigation was normal (thorax and skeleton). Histopathological examination of skin biopsy stated the chronic lymphohistiocytic process associated with vasculitius. The result of smear examination for acid-fast bacillus (AFB) was reported to be scanty positive. Bacterial DNA was prepared by a microbead method and 7 μL of the supernatant was used for polymerase chain reaction (PCR) using primers Tb11 (5′-ACCAACGAT GGTGTCCAT) and Tb12 (5′-CTTGTCGAACCGC-ATACCCT).6 The PCR amplified a 439-bp fragment of hsp65. Digestion of PCR product upon restriction enzymes (BstEII and HaeIII) produced fragments of 315, 135 bp and 265,130 bp, respectively. Therefore, the PCR-restriction fragment length polymorphism (PCR-RFLP) using hsp65 is simple, fast, and useful for the positive identification of M. leprae, which still remains the only non-cultivable Mycobacterium.6

Figure 1.
Figure 1.

Infection with Mycobacterium leprae.

Citation: The American Society of Tropical Medicine and Hygiene 84, 4; 10.4269/ajtmh.2011.10-0515

The treatment was initiated according to World Health Organization (WHO) recommendations, with 100 mg/d of dapson, 50 mg/d of clofazimine, and rifampacin 600 mg/month.7 After only 1 month of treatment the skin lesions became smaller and flatter.7 Further classical investigation showed two more cases of leprosy in family members (his maternal uncle and his sister).They were registered in different hospitals. They had already developed deformities in their feet and hands. In one of them, the foot was spontaneously and in another one was surgically amputated. Genetic investigation was performed on all three cases and other family members that were in close contact, but did not develop leprosy (mother, father, brothers, sisters, uncle, nephew, and nieces). The SNPs at INT4, D543, 3′UTR of NRAMP1 gene, SNPs in restriction sites of BsmI and FokI of VDR gene, and SNPs of TNF-α at −238, −308, −244, −857, −863 positions, SNPs of IL-10 at position −1082, −819, −592, SNPs of IL-12 RB1 at +705, +1158, +1196, +1637, +1664, and IFNGR1at position −179 and −56 were investigated using PCR-RFLP.8 The frequency of the genotypes were estimated by direct gene counting and the data analyzed using Pearson's χ2 test. The results were compared with previously published data8. Allele frequencies of NRAMP1 gene polymorphisms for INT4, D543, and 3′UTR were the same in the patient and his close family members with or without leprosy (G/G; G/G; TGTG+/+). Similarly, no difference was observed between allele frequencies of VDR for FokI and BsmI gene polymorphisms. The observed allele frequencies of IL-10 (−1082 G to A; −819 C to T; −592 C to A), IL-12 (−705A to G; −1158 T to C; −1196 C to T; −1637 G to A; −1665 C to T), and TNF-α (−238 G to A; −308 G to A) were also not statistically significantly (Table 1). Although, all three cases that developed leprosy had an SNP at IFNGR1 at −56 positions. This type of mutation was absent in other family members and in healthy control population of Iran (Table 1).

Table 1

Gene polymorphisms in leprosy patients and their close family members. The results were compared with healthy populations

SNPsGenotypePatients (N = 3)Close family member (N = 12)Healthy populations* (N = 60)P valueχ2
TNF−238G/G3 G/G (100%)8 G/G (66%)57 G/G (95%)0.007χ2 = 9.8
G/A or A/A04 G/A (33%)3 G/A (5%)
TNF−308
G/G2 G/G (66%)1 G/G (8.3%)56 G/G (93%)0.001χ2 = 43.32
TNF−244G/A or A/A1 G/A (33%)11 G/A (91%)4 G/A (6%)
G/G3 G/G (100%)12 G/G (100%)60 G/G (100%)
TNF−857
C/C3 C/C (100%)10 C/C (83%)42 C/C (70%)0.1χ2 = 4.57
TNF−863C/T or T/T02 C/T (16.6%)18 C/T (30%)χ2 = 1.36
C/C3 C/C (100%)5 C/C (41%)39 C/C (65%)0.5
C/A or A/A1 G/A (33%)7 C/A (58%)21 C/A (35%)
IFN−179G/G3 G/G (100%)12 G/G (100%)60 G/G (100%)
G/T or T/T000
IFN−56T/T012 T/T (100%)60 T/T (100%)
T/C or C/C3 C/C (100%)00
IL-10−1082G/G008 G/G (13.3%)0.32χ2 = 2.24
G/A or A/A2 G/A (66%), 1 A/A (33%)11G/A (91%), 1 A/A (8.3%)45 G/A (75%), 7 A/A (11%)
IL-10−819C/C1 C/C (33%)1 C/C (8%)5 C/C (8%)
C/T or T/T2 C/T (66%)7 C/T (58%), 4 T/T (33%)43 C/T (71%), 12/T (20%)0.34χ2 = 2.14
IL-10−592C/C1 C/C (33%)1 C/C (8.3%)5 C/C (8%)0.34χ2 = 2.14
C/A or A/A2 C/A (66%)7 C/A (58%), 4 A/A (33%)40 C/A (66%), 20 A/A (33%)
IL-12−705A/A3 A/A (100%)12 A/A (100%)60 A/A 100%0.34χ2 = 2.14
A/G or G/G0000.34χ2 = 2.14
IL-12−1158T/T1 T/T (33%)1 T/T (8.3%)5 T/T (8%)
T/C or C/C2 C/A (66%)7 C/A (58%) 4 A/A (33%)45 C/A (75%), 10 A/A (16%)
IL-12−1196C/C1 C/C (33%)1 C/C (8%)5 C/C (8%)0.34χ2 = 2.14
C/T or T/T2 C/A (66%)7 C/A (58%) 4 A/A (33%)45 C/A (75%), 10 A/A (16%)
IL-12−1637G/G3 G/G (100%)12 G/G (100%)60 G/G (100%)
G/A or A/A000
IL-12−1665C/C3 C/C (100%)12 C/C (100%)60 C/C (100%)
C/T or T/T000
NRAMP INT4G/G3 G/G (100%)12 G/G (100%)60 G/G (100%)
G/C or C/C000
NRAMP D543G/G3 G/G (100%)12 G/G (100%)44 G/G (73%)0.08χ2 = 5.0
G/A or A/A0016 G/A (27%)
NRAMP 3′UTRTGTG+/+3 +/+ (100%)12+/+ (100%)57+/+ (95%)0.68χ2 = 0.78
+/del or del/del003+/del (5%)

SNPs = single nucleotide polymorphisms.

Healthy population: here referred to those cases that had no mycobacterial-infection, using purified protein derivative (tuberculin) (PPD), smear, and x-ray chest examination.

These heterozygous carriers (T to C at IFN−56 position) might explain differences in susceptibility to M. leprae.

The IFN-γ induces cellular activation by binding to a receptor complex consisting of at least two subunits: the IFN-γ- binding subunit (IFNGR1) and a chromosome 21-encoded transmembrane accessory factor (IFNR2).9,10 Recently, Newport and others,11 showed mutations in the gene for IFN-γ are able to limit the growth of mycobacteria in vitro, and there has been a synergistic effect of IFN-γ and TNF-α. It was interesting to note that the investigated patients in this study had no other infections with pathogenic bacteria or fungi and have coped with viral infections normally. The specificity of the defect in these children suggests that there must be a redundancy of immunologic mechanisms against most of these pathogens but not against M. leprae. Therefore, present cases not only provide important information on the role of the IFNGR1 Pathway in human immunity, but also identify a gene with a potentially crucial role in determining susceptibility to M. leprae. Few investigators showed an association between mutation of IFNGR1 and lepromatous leprosy patient, but others did not support the idea.10,12 Generally, the diverse genetic results of the disease acquisition might be explained by distinct genetic susceptibly to the disease among different populations.2 Clearly, the complete absence of IFNGR1 on cell surface induces so profound an immune defect that such mutations are unlikely to explain M. leprae susceptibility in the general population. However, the existence of heterozygous carriers of the defect or more subtle variations in this gene might explain differences in M. leprae susceptibility within the population.

In our study IFNGR1 at position −56 T/C polymorphism was positively associated with an increased susceptibility to leprosy. Although polymorphisms of NRAMP, VDR, and TNF-α have been associated with increased susceptibility to leprosy in some studies, we did not find a mutation in these genes.2,13,14 Additionally, we could not find a link between IL10 and IL12R1 and the disease in our study. Overall, there are many literatures about clinical manifestations and patient's immune response of adult leprosy. However, the molecular basis of the genetic vulnerability of leprosy in children is still limited. The association of IFNGR1 polymorphism with susceptibility to leprosy should be studied with a larger sample size to elucidate the exact role of IFNGR1 polymorphisms in this disease.

ACKNOWLEDGMENTS:

We thank the patient and his family members who cooperated with our medical team during sample collection.

  • 1.

    Grange JM, Lethaby JI, 2004. Leprosy of the past and today. Seminar Respir Crit Care Med 25: 271281.

  • 2.

    Shields E, Russell DA, Vance MAP, 1987. Genetic epidemiology of the susceptibility to leprosy. J Clin Invest 79: 11391143.

  • 3.

    Scollard DM, Adams LB, Gillis TP, Krahenbuhl JL, Truman RW, Williams DL, 2006. The continuing challenges of leprosy. Clin Microbiol Rev 19: 338381.

  • 4.

    Alcais A, Sanchez FO, Thuc NV, Lap VD, Oberti J, Lagrange PH, Schurr E, Abel L, 2000. Granulomatous reaction to intradermal injection of lepromin is linked to the human NRAMP1 gene in Vietnamese leprosy sibships. J Infect Dis 181: 302308.

    • Search Google Scholar
    • Export Citation
  • 5.

    Atkinson SE, Khanolkar Young S, Marlowe S, Jain S, Reddy RG, Suneetha S, Lockwood DN, 2004. Detection of IL-13, IL-10 and IL-6 in the leprosy skin lesions of patients during prednisolone treatment for type 1 reactions. Int J Lepr Other Mycobact Dis 72: 2734.

    • Search Google Scholar
    • Export Citation
  • 6.

    Rastogi N, Goh KS, Berchel M, 1999. Species-specific identification of Mycobacterium leprae by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol 37: 20162019.

    • Search Google Scholar
    • Export Citation
  • 7.

    World Health Organization, 1997. Expert committee on Leprosy. Chemotherapy of Leprosy. WHO Technical Report Service No. 847. Geneva: WHO, 1113.

    • Search Google Scholar
    • Export Citation
  • 8.

    Merza M, Farnia P, Anoosheh S, Varahram M, Kazampour M, Pajand O, Saeif S, Mirsaeidi M, Masjedi MR, Velayati AA, Hoffner S, 2009. The NRAMPI, VDR and TNF-alpha gene polymorphisms in Iranian tuberculosis patients: the study on host susceptibility. Braz J Infect Dis 13: 252256.

    • Search Google Scholar
    • Export Citation
  • 9.

    Kardum LB, Etokebe GE, Knezevic J, 2005. Interferon-γ receptor -1 gene promoter polymorphisms (G 611A; T-56C) and susceptibility to tuberculosis. Scand J Immunol 63: 142150.

    • Search Google Scholar
    • Export Citation
  • 10.

    Velazquez CG, Roa RL, Rizo VD, 2010. Abnormalities in intracellular processing and expression of interferon-gamma receptor in adherent cells from lepromatous leprosy patients. J Interferon Cytokine Res 3: 99105.

    • Search Google Scholar
    • Export Citation
  • 11.

    Newport MJ, Huxley CM, Huston S, Catherine M, Hawrylowic Z, Oostra BA, Williamson R, Levin M, 1996. A mutation in the interferon-γ receptor gene and susceptibility to mycobacterial infection. N Engl J Med 335: 19411948.

    • Search Google Scholar
    • Export Citation
  • 12.

    Lee BS, Kim BC, Jin SH, Park YG, Kim SK, Kang TJ, Chae GT, 2003. Missense mutation 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 55: 177181.

    • Search Google Scholar
    • Export Citation
  • 13.

    Santos AR, Suffys PN, Vanderborght PR, Moraes MO, Vieira LM, Cabello PH, Bakker AM, Matos HJ, Huizinga TW, Ottenhoff TH, Sampaio EP, Sarnoa EN, 2002. Role of tumor 8 necrosis factor-α and interleukin-10 promoter gene polymorphisms in Leprosy. J Infect Dis 186: 16871691.

    • Search Google Scholar
    • Export Citation
  • 14.

    Roy S, Frodsham A, Saha B, Hazra SK, Taylor M, Hill AVS, 1999. Association of vitamin D receptor genotype with leprosy type. J Infect Dis 179: 187191.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to Parissa Farnia, Mycobacteriology Research Centre, NRITLD/UNION and WHO Collaborative Centre for TB and Lung Diseases, Shahid Beheshti University (Medical Campus), Tehran, 19556, P.O:19575/154, Iran. E-mail: pfarnia@hotmail.com

Financial support: This study was sponsored by a grant from MRC/NRITLD (010-18-007).

Authors' addresses: Ali A. Velayati, Soheila Khalilzadeh, and Maryam Hasanzadh, Pediatric Respiratory Diseases Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences (Medical Campus), Tehran, Iran, E-mails: mycopf@hotmail.com, SKhalil@hotmail.com, and Hasanz2@yahoo.com. Parissa Farnia, Amir M. Farahbod, and Maryam F. Sheikolslam, Mycobacteriology Research Centre, NRITLD/UNION and WHO Collaborative Centre for TB and Lung Diseases, Shahid Beheshti University (Medical Campus), Tehran, Iran, E-mails: pfarnia@hotmail.com, amirmfar@yahoo.com, and m.sheikholslami@gmail.com.

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