The role of Interferon-γ Receptor-1 Gene (-56 T < C ) polymorphism in development of susceptibility to pulmonary Tuberculosis in Central Sudan

Background: It is estimated that every year 8 million new cases of tuberculosis occur globally and that 2 to 3 million people die annually from the disease. Human genetic variation is an important determinant of the outcome of infection with Mycobacterium tuberculosis. The aim of this study is to determine genetic susceptibility to tuberculosis among patients and their control by identifying the frequency of some IFNγR1 polymorphisms in development of susceptibility to pulmonary tuberculosis in Gezira state of Central Sudan. Methods: Polymorphism (-56 T< C) was analyzed in both 126 confirmed positive TB cases and 200 matched controls using PCR-RFLP method. Results: The results showed that the distribution of IFNγR1-56 T < C genotype differs significantly between tuberculosis patients and controls and those carrying the mutant C allele were associated with two times reduced risk for susceptibility to Tuberculosis (P-value = 0.017, odds ratio = 1.6, 95% CI = 1.025 – 1.441), that mean the mutation IFNγR1 -56 T < C gives protection two times more than the wild type allele. However, more studies in IFNγR1 gene, and polymorphisms in IFNγ gene, needs to be performed with larger sample size


INTRODUCTION
During the last few years it has become clear that; the genotype of the host plays an important role in the evolution and outcome of infectious diseases.Considering tuberculosis, there still no satisfactory explanation for the fact that small proportion of individuals infected with Mycobacterium tuberculosis develop a severe clinical disease while others developed immunity against mycobacterial infections.Differences between Mycobacterium isolates from different patients with different clinical phenotypes of tuberculosis have not been fully demonstrated.Many host genes are involved in the control of the internal environment which the Mycobacterium faces and should favor the survival internally within the host genes that confer some protection against the infections.Considerably, more evidences are available to indicate the importance of host factors in determining the clinical presentations of mycobacterial infection.The full knowledge of these factors is important not only to understand how the disease evolves within an individual, but also of great importance to understand, from an epidemiological point of view, how it behaves in a given population.
Many of the cytokine-binding receptors function in the immune and hematopoietic systems (Casanova et al., 2001).
The level of secretion of both monokines (IFNg IL-12) and lymphokines (IFNg) by peripheral blood mononuclear cells has been reported to be low in IFNgR1-deficient children.
Typically, these patients do not develop well differentiated and well circumscribed mycobacterial granulomas.This provides further evidence that impaired IFNg mediated immunity affects both phagocytes and lymphocytes (Casanova et al., 2001).
The polymorphisms in IFNgR1 inhibit signaling that introduced by IFNg to phagocytes that produced more activity against M. tuberculosis in many studies that confirm this fact.(Newport, 2004, Newport et al., 1996).
Complete IFN-g receptor ligandbinding chain (IFNgR1) deficiency is a life threatening autosomal recessive immune disorder.Affected children invariably die of mycobacterial infection, unless bone marrow transplantation is undertaken.Pathogenic IFNgR1 mutations identified to date include nonsense, splice mutations, frame shift deletions and insertions.All result in a premature stop codon upstream from the segment encoding the transmembrane domain, precluding cell surface expression of the receptors.

MATERIALS AND METHODS
The (5`-GGGCGTGGGCGGGGTCAA-`3) IFNgR -56 T→ C R: (5`-CCTCCCTCCCTCTCGTCC-`3) PCR condition as initial temperature 95 C º for 5 minute, followed by 35 cycles of 94 C º as melting temperature for one minute, 66 C º as annealing temperature for one minute and 72 C º as extension temperature, then final prolongation step at 72 C º for 5 minutes.

Digestion with Restriction Enzyme (Eco 47Щ)
In transition replacement of cyctine to thiamin in position -56 create a new restriction site for Eco 47Щ.In a total volume of 15 µl, 5 µl PCR products was digested overnight with 3µl Eco 47Щ enzyme and mixed with1.5 µl Buffer3 10x , 0.2 µl BSA 50µg and 7.8 µl deionized water.
The Digested DNA samples were mixed with 5 µl loading dye before being loaded on a 10 % Non-denaturaning polyacrylamide gel and electrophoresed at 100 V for two hours.Then the gel was stained in 0.1 µg/ml ethidium bromide solution for 10-15 minutes and visualized under UV light in Gel Documentation System (GDS).
The genotype was assigned according to the length of obtained fragments.Complete cleavage of 285 bp into 193 and 92 bp fragments is characteristic profile of the homozygote mutant allele (CC), incomplete cleavage into 285 bp, 193 bp and 92 bp is of the heterozygote (TC).While absolute absence of digestion is characteristic of the homozygote wild (TT).

Statistical Analysis
Concordance of genotype frequencies with Hardy-Weinberg equilibrium was tested by a χ2 goodness-of-fit test.The baseline value of these groups was compared using the unpaired t test.The statistical tests were performed using statistical package of social sciences (SPSS)
The distribution of IFNgR1-56 T<C genotype show no significant difference between TB patients and controls (P = 0.071).The distribution of IFNgR1 -56 C >T genotype differ significantly between TB patients and controls (P = 0.017) (Fig. 2).

WT hetro hetro hetro hetro hetro WT hetro mutant
That indicate the T allele is associate with two times increased risk to pulmonary tuberculosis than C allele (P-value = 0.017, odds ratio = 1.6, 95% CI = 1.025 -1.441).

DISCUSSION
Tuberculosis is a social disease that is extremely sensitive to changes in the standard of living.The disease is an infection that is primarily spread and transmitted directly from human being to human being.The degree of crowdness and the congested houses are therefore important factors in the spreading of the disease (Puranen, 2003) that is the same factors that is found in rural community beside the direct contact between the populations.In this study the distribution of study subject according to locality showed that 89 (71%) were from rural area and 37 (29%) were from the urban areas.
Different genotypes seem to respond differently to environmental risk factors.Host genetic factors have an important role in the development of clinical disease following infection with tuberculosis, but inheritance of TB susceptibility in the general population is non-Mendelian.The correlation between the molecular pathology, mycobacterial virulence, and clinical phenotype in inherited IFNgR1 deficiency suggests that more subtle variation in IFNgR1 could contribute to M .tuberculosisdisease susceptibility in an outbred population (Awomoyi et al. 2004).
The distribution of IFNgR1 -56 C >T genotype differs significantly between TB patients and controls and those carrying the mutant C allele were associated with two times reduced risk for susceptibility to TB (P-value = 0.017, odds ratio = 1.6, 95% CI = 1.025 -1.441).
That means the mutation IFNgR1-56 CC give protection two times according to this result, that consistent with result of the study uses population collections from the Gambia, Guinea Bissau, and the Republic of Conakry.All cases were confirmed by either two consecutive smear-positive samples or a positive Mycobacterium tuberculosis (MTB) culture, and that found there is evidence from an in vitro model of cell expression that constructs bearing the IFNgR1-56 C allele produce less transcriptional activity in a standard

Fig. 2 :
Fig. 2: The percentages of Allele frequency of IFNγR1 -56 in study subjects and controls study was conducted in Wad Medani Chest Hospital during the period between June 2005 and Augustus 2006.Ethical approval was obtained from both the Federal Ministry of Health in Khartoum and the Research Committees of the University of Gezira.Consent was taken from the study subjects.