Synergetic Effect of TLR4 Gene (D299G and T399I) Polymorphisms in Susceptibility to Pulmonary Tuberculosis

Background: Toll-like receptor 4 (TLR4) is an essential component of the innate immune response to various microorganisms. Genetic polymorphisms in TLR4 are often associated with susceptibility to mycobacterial infection. Objective: The present study examines the influence of TLR4 896 A/G (Asp299Gly) and 1196 C/T (Thr399Ile) polymorphism in the susceptibility to develop pulmonary tuberculosis (PTB) in Indians. Materials & Methods: Single nucleotide polymorphisms (SNPs) of the TLR4 gene were genotyped using PCR-RFLP and sequenced in 198 patients with PTB and 60 healthy controls. Results: The frequency of mutant allele 299Gly and 399Ile of the TLR4 gene (14.4%; p < 0.048; RR=1.919; OR=2.074 & 14.6%; p < 0.020, RR=2.197; OR=2.402) was predominant in PTB patient as compared to healthy controls (7.5% & 6.7%), respectively. Data was further analysed under the dominant model [CT+TT (Thr/Ile + Ile/Ile)] at 399 position revealed preponderance of mutant allele in PTB patients (25.8%; p < 0.045; OR=2.555). On the other hand, combined evaluation of polymorphisms showed heterozygous/heterozygous (AG299+CT399) genotype was higher (16.2% vs 6.7%; p=0.063) in PTB patients than in control subjects. However homozygous/homozygous (GG299+TT399) genotype was observed only in PTB patients (3%).Conclusion: The current observation suggests the contribution of mutant allele ‘Gly’ & ‘Ile’ towards the increased risk of development of tuberculosis in North Indian population. As per our knowledge, this is first Indian study indicating the synergetic effect of two mutant (Asp299Gly and Thr399Ile) / alleles influencing the PTB infection. Further investigation on association of TLR-4 as an innate immunity biomarker with tuberculosis diseases need to be evaluated.


Introduction
Incidence of Pulmonary Tuberculosis (PTB) cases in India, in 2012 was 2.0-2.4 million. As per WHO, 2013 consensus [1] India alone, accounted for 26% of global cases. Genetic and non-genetic factors of both the bacterium and the host influence the host immune response to M.Tuberculosis. Many host susceptibility genes have been identified in the last decade. Association of numerous polymorphisms of innate immunity candidate genes implicated in host susceptibility to TB has been revealed by various studies and Toll-like receptor (TLR) 4 is one of them [2]. TLRs are trans-membrane receptors that activate cells of the innate immune systems upon recognition of pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), peptidoglycan, liporpotease etc. and they form the crucial link between the innate and adaptive immune responses. As an innate immune act, TLRs trigger signal transduction upon binding of ligands, that results in the expression of pro-inflammatory cytokines and the functional maturation of antigen presenting cells, which intern activate adaptive immunity [2,3].
The risk allele for rs4986790 is 'G' and for rs4986791 is 'T'. The SNP rs4986790 also known as 896A/G (also Asp299Gly) in the TLR4 gene is often studied along with a co-segregating SNP known as Thr399Ile, rs4986791 at C-T 1196 position [18]. Thr399Ile SNP is a non-synonymous C-T transition. Together, wide variety of both non-infectious and infectious diseases associated with these SNPs have been reported, although some cases reported conflicting or even contradictory results [12,13].
According to several studies, endotoxin-hyporesponsive phenotype is associated with the synergic effect of two variants of the TLR4 gene, Asp299Gly and Thr399Ile within the fourth exon. [11][12][13]. Moreover, the human alveolar macrophages or primary airway epithelial cells obtained from individuals with airway hypo responsiveness possess TLR4 Asp299Gly polymorphism. Extensive studies confirmed association of these TLR4 gene polymorphisms and resistance or susceptibility to many diseases and infections [2,[11][12][13]16,17]. At the molecular level, it has been shown that the TLR 4 896 A>G mutation interferes with TLR4 interaction with MyD88 and other downstream messengers [18]. These mutations also appear to affect the levels of functional TLR 4 expression, leading to a 2-fold reduction [12,19].
Several studies (in-vivo &in-vitro) have demonstrated the protective role of TLR4 in the host defence against mycobacterial infection [6,13,20] and genetic factors play a major role in the susceptibility to tuberculosis [21,22]. Only few Indian studies reported the frequency of polymorphism (Asp299Gly) of TLR 4 gene was significantly increased in PTB patients as compared to the healthy controls [2]. So, there has been great interest regarding the association of the SNPsTLR4896 A>Gand TLR41196 C>T to susceptibility for mycobacterial infection or tuberculosis disease states. The present study investigated the potential role of TLR-4 polymorphisms namely Asp299Gly and Thr399Ile as a risk factor in the development of pulmonary tuberculosis infection in North Indian population.

Study Design
We conducted this study at National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India during the period of 2012-2014. The inclusion criteria for the recruitment of PTB patients were defined as follows: patients had to present with clinical manifestations suggestive of tuberculosis, i.e., the presence of productive cough, low-grade fever, night sweats, weight loss and chest pain. Pulmonary tuberculosis was diagnosed on the basis of the presence of clinical symptoms or chest radiography or the presence of acid fast bacilli (AFB) in sputum smear or M. tuberculosis positive culture on Löwenstein-Jensen medium. A detailed clinical history, sex, and age were also collected from the requisition form that accompanied with specimens. On the other hand the healthy control population was recruited from people who come for general health examinations or non TB cases (absence of clinical symptoms of active PTB, asymptomatic with no previous history of TB or ATT) or attendants of patients or volunteers with normal chest X-ray. Healthy controls were further subjected to Mantoux test and categorized into PPD negative and PPD positive.

Current study was approved by Research and Ethics
Committee of the Institute. The study groups involved 258 subjects consisting of 198 individuals with PTB and 60 healthy controls. Peripheral blood samples were collected in EDTA tubes from all PTB patients and unrelated healthy controls after obtaining consent as per ethical guidelines.

TLR4 Genotyping
Genomic DNA was extracted from EDTA anticoagulated peripheral blood leukocytes by salting out procedure (Miller et al., 1988). Single nucleotide polymorphisms (SNPs) of TLR4gene (896 A/G; Asp299Gly and 1196 C/T; Thr399Ile) were detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) according to previously described method (2,23)]. Amplification of human DNA was performed by PCR in a total 25 μl reaction volume containing 1X PCR buffer, 0.2 mM dNTPs, 50 ng of each primer (Table 1) (TLR 4 Asp299Gly and Thr399Ile), 1U of Taq polymerase (Genei, India) and 50-100 ng of extracted DNA. The amplification protocol used was; denaturation at 95 °C for 4 min, followed by 30 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 30 sec. The amplified products were visualized on 2% agarose gels. Amplified fragments of TLR 4 Asp299Gly and TLR4 Thr399Ile polymorphism were digested overnight at 37°C with 1U of NcoI and HinfI restriction endonucleases, respectively. The restricted amplicons were visualized on 3% agarose gel.

TLR4 Amplicon Sequencing
For the confirmation of TLR 4 Asp299Gly (rs4986790) and Thr399Ile (rs4986791) polymorphisms, ten randomly selected PCR products were purified and got sequenced commercially with the reverse primers using ABI Prism Big Dye terminator Cycle sequencing system (PE Applied Biosystem, USA). The chromatograms were visualized using BioEdit [24]. Sequence alignments were done using Clustal OMEGA [25].

Statistical Analysis
To analyze the association of two polymorphisms (Asp299Gly and Thr399Ile) of TLR-4 gene, the allelic and genotypic frequencies of both polymorphisms were compared between the PTB patients and the healthy subjects and assessed using MSTAT statistical software by Chi-square (χ2) and Fischer test. Chi-square test was used to compare the difference in the distribution of mutations between PTB affected individuals and the control group. Strength of association between mutations and the disease/ control group was computed by Odds ratio (95% confidence interval). The p values < 0.05 was considered to be statistically significant.

Results
In this study, we investigated the role of TLR4 polymorphism at position A896G (Asp299Gly) and C1196T (Thr399Ile) in the study groups involved 258 subjects consisting of 198 individuals with tuberculosis (age range: 18-76 years; mean age 34.4; 178 men and 20 women) and 60 healthy controls (age range: 18-65; mean age: 44; 36 men and 24 women) from the outdoor and wards National Institute of Tuberculosis & Respiratory Diseases, Delhi, using PCR-RFLP method.

Distribution of Genotypic and Allelic Frequency of TLR 4 (Asp299Gly and Thr399Ile) Polymorphism in Healthy Controls vs PTB Patients
Population distribution of the TLR 4 alleles revealed that two mutations occurred with a frequency of nearly 7% in the healthy Indian population (Asp 299 Gly, 7.5% and Thr 399 Ile, 6.7%) (Tables 2 & 3) 3.1.1. Asp299Gly Genotyping The restriction digestion for mutant allele at position 896 A/G (Asp299Gly) yielded two fragments of size 223 bp and 26 bp (Fig. 1). The allelic and genotypic frequencies of TLR4 mutation were analyzed in patients with PTB and healthy controls ( Fig. 2; Table 2). The allelic frequency of Asp299Gly was found to be significantly increased (14.3%) in PTB patients compared with healthy control (7.5%) group [(χ2 value = 3.924; Odds ratio = 2.074, p value = 0.048, 95% CI = 0.954-4.65; RR = 1.919 (0.960-4.084)]. The genotypic frequency of heterozygous (AG) and homozygous mutant (GG) was also found to be increased in PTB patients as compared to the healthy controls (21.5% vs 11.7%; p = 0.084) and (3.5% vs 1.7%; p = 0.464), respectively but not significant.

Thr399Ile Genotyping
The restriction of mutant allele at position 1196 C/T (Thr399Ile) yielded two fragments of sizes 377 bp and 29 bp (Fig. 3). The distribution of Thr399Ile polymorphism alleles and genotypes in PTB patients and healthy controls was analysed ( Fig. 4; Table 3). The frequency of the mutant T (399Ile) allele was 14.5% in the PTB patients as compared with 6.7% in controls and were significantly different [χ2 value =5.257; Odds ratio = 2.402; 95% CI= 1.066-5.623; p-value =0.020; RR=2.197 (1.057-4.897)]. An upsurge was also observed in the genotypic heterozygous frequency (CT) in the PTB patients as compared to healthy controls of CT (22% vs 13.3%, p=0.133) and TT genotypes (3.5% vs 0%, p=0.140) but it was found statistically non significant.  As the frequency of variant genotypes of both of the polymorphisms was quite low, we adopted a dominant model and the association of tuberculosis was calculated.

Combined Effect of Asp299Gly and Thr399Ile
Further analysis of the combined effect of Asp299Gly and Thr399Ile in our study indicated that overall in 8 PTB patients, either risk allele 'T' for Thr399Ile or risk allele 'G' for Asp299Gly of TLR4 gene were present which may be associated with susceptibility to PTB in North Indian population. Interestingly, 6 PTB patients were carrying both homozygous mutant GG of 299 A/G TLR4 (Asp299Gly) & TT of 399 C/T TLR4 (Thr399Ile), simultaneously. And the frequency of homozygous mutant (GG+TT) in PTB patients (N = 6/198; 3%; p = 0.172) was increased as compared to healthy controls (N = 0/60; 0%). Similarly, frequency of heterozygous mutant (AG + CT) polymorphism were also increased in PTB patient (N = 32/198; 16.2%; p = 0.063) as compared with controls (N = 4/60; 6.7%), respectively. Statistically frequency for both homozygous & heterozygous mutant was found not significant.

DNA Sequencing for TLR4 Asp299Gly and Thr399Ile Polymorphism
The PCR products harbouring the TLR4variants from RFLP analysis were confirmed through DNA sequencing. These included four samples from PTB patients (one heterozygous and 3 homozygous mutants) for both Asp299Gly & Thr399Ile polymorphisms harbouring the variant. The sequencing results demonstrated that all sequenced samples had Asp (codon GAT) at amino acid 299 in wild type and Gly (codon GGT) in heterozygous and in homozygous mutants. The sequence from the heterozygous mutant revealed the "T" nucleotide was replaced with T/C (Fig. 5a) and with "C" in homozygous mutant (Fig. 5b). The sequencing results confirmed that all sequenced samples had Thr at amino acid 399 (codon ACC) in wild type and Ile (codon ATC) in heterozygous and homozygous mutant. The wild type G nucleotide in heterozygous sample was replaced with G/A nucleotide (Fig. 6a) and with "A" nucleotide in homozygous mutant (Fig. 6b) in samples by DNA sequencing. There was 100% concordance between the results of RFLP typing and DNA sequencing.

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Synergetic Effect of TLR4 Gene (D299G and T399I) Polymorphisms in Susceptibility to Pulmonary Tuberculosis

Discussion
The susceptibility to tuberculosis in connection with TLR4 is focussed on two SNPs. The first is 896 th base adenine substitution with guanine of 299 th codon, which results in replacement of aspartic acid by glycine (Asp299Gly), and the other is at 1,196 bp substitution of cytosine with thymine, which results in a threonine to isoleucine replacement at 399 th codon (Thr399Ile) [26,27]. Evidence suggests that polymorphisms within TLRs may reduce its affinity to M. tuberculosis derived ligands causing an impairment of the immune response against TB by altering the signal transduction of anti-MTB immune responses [22].
In this regard, the present study provides the evidence that polymorphisms in TLR4 gene contribute to PTB susceptibility in North Indians. The combined effect of two common polymorphisms (Asp299Gly and Thr399Ile) of TLR4 gene is being reported first time in this study, which reports the increase in the likelihood to acquire PTB. Our results indicated that in distribution of population of the TLR 4 alleles revealed that two mutations occurred with a frequency of nearly 7% in the healthy Indian population (Asp299Gly, 7.5% and Thr399Ile, 6.7%) (Tables 2 & 3; Fig.  2 & 4). Our results are in concordance with the published study by Najmi et al., (2010) [2] in North Indian population.
On the other hand, the distribution of allelic frequency varies from the range of various frequency of allele has been reported in different population (1 -14.3% for TLR4 299 and 3 -13.8% for TLR4 399) [12,16]. Further, the comparative analysis revealed that the allelic frequencies of mutant 299Gly and 399Ile of TLR4 gene were significantly raised [ [6,11,28]. Notably in contrast, some studies reported that no association was found between these polymorphisms and susceptibility to PTB [11,16,17,29]. In our study we observed an upsurge in the genotypic frequencies of mutant allele at both positions (Asp299Gly & Thr399Ile) of TLR 4 gene in PTB patients as compared to healthy controls but were not statistically significant. The second statistical approach which we adopted in current study is the dominant model and the association with PTB.  (Table-2). An earlier study [22] also reported the significant association with tuberculosis for TLR9 gene after adopting the dominant model and additive model. In contrast, Verma et al., (2011)[30] adopted a recessive model where the frequency of variant genotypes of both the polymorphism of TLR 4 gene was quite low and found the significantly increased risk of Neurocysticercosis.
Furthermore, the combined evaluation of both mutant genotype (299Gly; GG and 399Ile; TT) showed that the frequency of heterozygous/heterozygous (AG+ CT) genotype [16.2% vs 6.7% (p = 0.063)] and homozygous/homozygous (GG+TT) genotype was higher (3% vs 0%; p = 0.172) in PTB patients than in control subjects but this difference failed to reach the level of statistical significance between the PTB patients & healthy control (Table 4). Both TLR4 gene polymorphisms act in a dependent manner by affecting each other to impart susceptibility to tuberculosis. It has also been reported previously that TLR4 Asp299Gly polymorphism occurred in combination with the polymorphism at Thr399Ile amino acid position [12,16,31]. Another study also reported the synergetic effect of genotypes TLR4 C1196T and TLR9 T1486C polymorphisms & found increased risk of PTB susceptibility [11].
Sequencing results demonstrated 100% concordance with genotyping results of both of Asp299Gly and Thr399Ile polymorphisms. GAT (Asp) → GGT (Gly) and ACC (Thr) → ATC (Ile) substitution in heterozygous and homozygous mutant was observed at nucleotide position 896 and 1196, respectively. The same polymorphisms were reported to have a role to play not only in tuberculosis, but also in other diseases too [12,27,32].
It may be feasible that, in cases of tuberculosis, both of these polymorphisms are involved in altering the immune function, which includes control of inflammatory cascades, elaboration of effect or molecules, and interaction with adaptive immune responses. The current observation highlight the contribution of mutant allele 'Thr' towards the increased risk of development of tuberculosis in North Indian population.

Conclusions
In conclusion, the present study suggests that Asp299Gly and Thr399Ile polymorphisms of TLR4 gene are involved as risk factors for PTB in our population. The presence of Gly and Ile increased the risk for the development of tuberculosis. Therefore, the results prove to be an effort in the direction of providing relevant information that the carriers of these mutations or genetic variations in TLR4 could contribute to the likelihood of developing PTB. This incidence of both homozygous as well as heterozygous polymorphisms to an extent could be of diagnostic value in the evaluation of genetic disease patterns. As per our knowledge this is the first Indian study reporting synergetic effect of two mutant allele of TLR4 in PTB infection. Further studies are needed to interpret the better role of TLRs in conjunction with other biomarkers.