Association of Toll-like Receptors 1, 2, 4, 6, 8, 9 and 10 Genes Polymorphisms and Susceptibility to Pulmonary Tuberculosis in Sudanese Patients

Introduction

Tuberculosis (TB) has been declared a major public health problem worldwide, due to the emergence of almost untreatable strains of Mycobacterium tuberculosis (M. tuberculosis). In addition to that, the only existing vaccine against TB, the BCG vaccine, fails to protect against pulmonary TB, the most important form of disease manifestation.¹ It kills approximately 10 million people and nine million new TB cases are reported worldwide annually.^1,2 Moreover, in the last two decades, three major coronavirus epidemics have been reported worldwide.³ The burden of COVID-19, HIV, and TB is one of the major and persistent global health challenges of the twenty-first century.^4–10

Figure 1 PCR-RFLP assay for analyzing the TLR4-rs4986790 A/G polymorphisms in Sudanese pulmonary Tuberculosis patients. Notes: Lane M shows DNA marker; lanes 1 and 6 show AA genotype; lanes 2, 4, and 7 show GA genotype; lanes 3, 5, and 8 show GG genotype.

Figure 2 PCR-RFLP assay for analyzing theTLR8-rs3764879 G/C polymorphisms in Sudanese pulmonary  Tuberculosis patients. Notes: Lane M shows DNA marker; lanes 2 and 4 show GG genotype; lanes 3 and 6 show CC genotype; lanes 1, 5, and 7 show CG genotype.

Genetic factors are important contributors to the development of a wide range of complex disease.^11–21 A person who is susceptible to a particular infectious disease, such as TB, the risk of developing the disease is higher than one who has not inherited the genetic risk factor.^11–21 These evidences on the influence of host genetic factors in TB susceptibility led to the development of strategies to identify candidate genes or susceptibility loci in the human genome. Identification of polymorphisms in genes has enabled linkage and association studies to be used in explaining individual variation in susceptibility to and severity of TB in humans.

In this context, toll-like receptors (TLRs) are a family of proteins that are expressed either on the extra cellular cell surface (TLR1, 2, 4, 5, 6) or in the cytosol or on endosomal membranes (TLR3, 7, 8, 9) of macrophages and dendritic cells. TLRs are essential for recognition of a broad repertoire of pathogen-associated molecular patterns (PAMPs) on macrophages and dendritic cells and play an important role in the innate responses against M. tuberculosis.^22–26 Genetic variations of TLR1, TLR2, TLR4, TLR6, TLR8, TLR9 and TLR10 have been associated with the susceptibility to TB in different ethnic groups.^27–33 In contrast, other studies have failed to demonstrate significant associations of TLRs polymorphisms with TB.^34–37

However, to date, no TLR mutations or single-nucleotide polymorphisms (SNPs) have been established as accepted risk factors for PTB among different ethnic populations. Besides, only one study have been performed on Sudanese patients to understand the role of TLR2 polymorphism in PTB.³¹ Therefore, to confirm the role of TLR variants in the increased risk for PTB we conducted the candidate gene association study by investigating 86 SNPs present in TLR1, TLR2, TLR4 TLR6, TLR8, TLR9 and TLR10 genes in a sub-Saharan Sudanese pulmonary tuberculosis patients.

Materials and Methods

Study Population

A prospective, cross sectional, case-control study was carried out during the period between 2019 and 2020 at Abu-Angah Hospital, Khartoum, Sudan. 160 patients with active pulmonary TB and 220 healthy controls were included. EDTA blood samples were taken from all patients and healthy controls. All tuberculosis patients had microbiological (by culture and/or smear) or radiological evidence of M. tuberculosis disease (Table 1).

Table 1 Characteristics of the Study Population

The healthy controls had no evidence of tuberculosis disease by clinical examination, and were matched on age, gender and BCG status (Table 1).

The collected blood samples were tested for other infectious diseases and that included hepatitis B (HBsAg, InTec products, INC, China), hepatitis C (Rapid Anti-HCV Test, InTec products, INC, China), syphilis (RAPIDAN TESTER, product code: RTTP01, Turkey), and HIV (HIV1, 2 Cassette test, Clinotech Diagnostics & Pharmaceuticals, Canada). Blood samples were stored at −20 ° C until use. Nasopharyngeal swab for was used to define Corona Virus Disease-2019 (COVID-19).

Ethics Statement

The present study was approved by the Ethics Committee of University of Khartoum, Khartoum, Sudan (5/2018). This study adheres to the Declaration of Helsinki (1964). Written informed consents were obtained from all participants in the study or legally responsible guardians for participants less than 18 years old.

DNA Isolation

Genomic DNA was isolated from blood samples with the large volume kit for the MagNA Pure system (Roche, Almere, The Netherlands) according to the manufacturer’s descriptions. The isolated DNA was stored at −20°C.

Genotyping

Genomic variants of TLR1 (rs5743604, rs5743611, rs5743618, rs76600635, rs5743551, rs5743557, rs5743565, rs5743566, rs5743580, rs5743594 rs4833095, rs5743595, rs5743596); TLR2 (rs1816702, rs5743704 rs5743708, rs7656411, rs11938228, rs893629, rs1898830, rs121917864, rs4696480, rs3804099, rs5743699 rs3804100); TLR4 (rs7869402, rs1927907, rs1927911, rs1927914, rs6478317, rs55912718, rs5030719, rs10759931, rs10759933, rs2770150, rs1554973, rs11536878, rs11536879, rs7873784, rs11536889, rs4986790, rs4986791, rs11536897, rs11536898); TLR6 (rs3796508, rs5743810, rs5743831, rs1039559, rs6531670, rs5743788, rs5743794); TLR8 (rs4830805, rs4830808, rs3747414, rs3761624, rs1548731, rs2109134, rs3788935, rs1013150, rs5744068, rs3764879, rs5744080, rs3764880, rs17256081, rs5741883, rs2407992, rs178998); TLR9 (rs5743836, rs164637, rs352139, rs352140, rs352143, rs352162, rs352165, rs352167, rs187084); and TLR10 (rs4129009, rs7694115, rs10856839, rs11466645, rs4274855, rs11096955, rs11096956, rs11096957, rs7698870, and rs10776483) genes were detected by PCR followed by restriction enzyme fragment analysis (PCR-RFLP) (Figures 1 and 2). All PCR primers are stated in Table 2. Each of the PCRs consisted of a pre-denaturation step of 4 minutes at 94°C and 40 cycles each of 30 seconds denaturation at 94°C, 30 seconds annealing at 55°C and 30 seconds elongation at 72°C. This was followed by a post-elongation step of 7 minutes at 72°C. Restriction endonucleases were selected using the NEBcutter software (http://nc2.neb.com/NEBcutter2/). Restriction endonucleases were obtained from Fermentas (st. Leon-rot, Germany), and Roche (Penzberg, Germany) and were used as described by the manufacturer. Restriction fragments were visualized by electrophoresis on 2% agarose gels (Hispanagar, Sphaero Q, Leiden, The Netherlands).

Table 2 PCR Primers and Restriction Enzymes for genotyping the Different Single Nucleotide Polymorphisms

The criteria for the selection of SNPs for this study were based on their previously reported to change the level or function of corresponding gene products and influence susceptibility/resistance to infections. Also, the selection was based on the publically available information on the polymorphisms of TLR genes available in the 1000 Genomes project for the South Asian population (http://www.internationalgenome.org).³⁸

Statistical Analysis

The mean age of the patient population and the control population were compared by the unpaired t-test. Gender, occupation and BCG-vaccination status between the patient and control population were compared with the Fisher exact test. Verification of Hardy-Weinberg equilibrium (HWE) was performed with Pearson’s χ² test. The effect of the TLR1; TLR2; TLR4; TLR6; TLR8; TLR9; and TLR10 polymorphisms on susceptibility to tuberculosis were assessed with the Fisher exact test. P-value of <0.05 was deemed statistically significant. All statistical analyses were performed using SPSS for Windows v11.0 statistical analysis software. This study was in accordance with the principles of the Helsinki Declaration (1964). This study was in accordance with the principles of the Helsinki Declaration (1964) This study was in accordance with the principles of the Helsinki Declaration (1964)

Results

Characteristics of Tuberculosis Patients and Healthy Control Subjects

One hundred and sixty Sudanese tuberculosis patients were included into the study. The diagnosis of tuberculosis was based on the presence of MTB in a positive Ziehl-Nielson (ZN) smear of a sputum specimen and/or by positive culture with tuberculosis and radiological evidence (chest X-ray) (Table 1). The control population comprised 220 healthy unrelated people from the same endemic area in Sudan, they were matched on gender and BCG-status (Table 1) and showed no signs of any lung disease. Unfortunately, the occupation of the control population differed from that of the patient population.

Distribution of TLR1, TLR2, TLR4, TLR6, TLR8, TLR9 and TLR10 Genes Polymorphisms

To detect the possible deficiencies in TLR1; TLR2; TLR4; TLR6; TLR8; TLR9; and TLR10 production among tuberculosis patients, genotype (Table 3) and allele frequencies (Table 4) in the promoters of the genes encoding for TLR1, TLR2, TLR4, and TLR6 were determined. To determine if the SNPs reached Hardy-Weinberg equilibrium (HWE), the Pearson’s χ² test was performed. It appeared that in the patients and control population, all genotype distributions were in Hardy-Weinberg disequilibrium.

Table 3 Genotype Distributions and Hardy Weinberg Equilibrium in Sudanese Tuberculosis Patients and Healthy Controls

Table 4 Allele Frequencies of Tuberculosis Patients in Comparison to a Matching Healthy Control Population

To determine if there was an association between any of the studied SNPs and tuberculosis, the allele frequencies between the control population and the patient population were compared with the Fisher Exact test. It appeared that in the tuberculosis population the TLR1 (rs5743557, rs4833095, rs5743596), TLR2 (rs5743704, rs5743708, rs3804099), TLR4 (rs4986790, rs4986791), TLR6 (rs5743810), TLR8 (rs3764879, rs3764880), TLR9 (rs352165, rs352167, rs187084) and TLR10 (rs4129009) were significantly more often encountered (p<0.0001) (Table 4) than in the control population and were associated with tuberculosis in the Sudanese population (Table 4). For the other polymorphisms tested, no association with tuberculosis was found in the population tested (Table 4).

Discussion

Association Between TLR1, TLR2, TLR4, TLR6, TLR8, TLR9 and TLR10 Genes Polymorphisms and Tuberculosis

Tuberculosis is a complex disease in which environmental, immunological and genetic factors are contributed. It has been estimated that 10% of the infected population with M. tuberculosis may develop TB disease sometime in their life, suggesting that the majority of those infected are endowed with a protective immune response. Previous association studies have indicated a potential involvement of genetic variation within innate immune response genes as risk factors for TB. In particular, TLR1,³⁹ TLR2,^40–45 TLR4,⁴⁶ with TLR8⁴⁷ and TLR9³⁰ genetic variants have been associated with TB, suggesting that TLR-mediated responses may be important for protection to M. tuberculosis infection in humans.

TLRs are a family of PRRs consisting of 12 members in human and other mammals. TLRs play a crucial role in the recognition of M. tuberculosis, this immune activation occurs only in the presence of functional TLRs. Variants of TLRs may influence their expression, function and alters the recognition or signaling mechanism, which leads to the disease susceptibility.^48,49 The polymorphisms of TLRs have been hypothesized to affect the tuberculosis susceptibility. However, the direct evidence remains controversial.

Therefore, in the present study, we genotyped 86 SNPs including TLR1, TLR2, TLR4, TLR6, TLR8, TLR9 and TLR10 in the Sudanese tuberculosis patients to determine whether they are associated with susceptibility to TB in Sudanese tuberculosis patients.

Our results describes novel mutations in the TLR1, TLR2, TLR4, TLR6, TLR8, TLR9 and TLR10 genes and describes their association with pulmonary tuberculosis infection. Studies of the genetic factors involved in complex diseases have not yet provided clear explanations for the onset of such diseases, though they may help to identify the risk factor to get the infection.

Our results revealed that TLR1 rs5743557, TLR1 rs4833095, and TLR1 rs5743596 allele were more frequently found in the patients population compared to the healthy controls population. The association of the TLR1 rs5743557, TLR1 rs4833095 and TLR1 rs5743596 allele with tuberculosis were also found in other populations originating from Caucasian, Indian population and South Asian for TLR1 rs4833095^32,50 and, East Asian Population for TLR1 rs5743557,³² but not in populations originating from in North China³² and African American subpopulation.³⁹ Further, in vitro experiments showed that the TLR1 rs4833095 is in strong linkage disequilibrium (LD) with TLR1 rs5743618 and determining how these two SNPs contribute to TB susceptibility has proven difficult. TLR1 rs5743618 -GT genotype was related to reduction in surface expression of TLR1 in monocytes and granulocytes. In addition, after stimulated by inactivated H37Rv, samples from children with the rs5743618-GT genotype showed a decreased production of TNF-α and CXCL10, invariable production of IL-6 and IL-8 and increased production of IL-10.⁵¹ In Spanish population, rs5743618 G allele and GG genotype influenced the susceptibility to PTB;³³ in Han Chinese population, Ma et al reported that rs5743618 was not associated with adult TB.³⁹ Zhang et al³² conducted a meta-analysis on TLR1 rs5743618 and also found no association with TB susceptibility, which corresponds with our results.³² Salie et al performed an association study of 23 polymorphisms in five TLR genes (TLR1, TLR2, TLR4, TLR8, and TLR9) in TB cases and healthy controls in a South African population. The study found that TLR1 rs5743618, TLR8 rs3764879, and TLR8 rs3764880 polymorphisms were associated with TB susceptibility in both sexes.⁵²

Our results also revealed that TLR2 rs5743708, TLR2 rs3804099 were more frequently found in the patient population compared to the healthy control population. The association of the TLR2 rs5743708, with tuberculosis were also found in other populations originating from Asian and Hispanic populations^40,53,54 but not European subgroup.⁵² Zhang et al³² showed a significant association with TB susceptibility for the rs5743708 A allele and AA genotype across different ethnic groups in Asians and Europeans, but decreased risk in the Hispanic population.³²

In contrast, the TLR2 rs5743708 and TLR2 rs5743704 were not associated with TB meningitis in an Indian population.^36,55 TLR2 rs3804099 was associated with susceptibility to TB meningitis rather than with susceptibility to pulmonary TB in a case-control study of a Chinese cohort.⁵⁶ Another study investigated possible associations of 16 polymorphisms of six TLR genes and TIRAP with TB susceptibility in a Chinese population. It found that TLR2 rs5743708, polymorphism was associated with pulmonary TB.⁵³

Several studies have demonstrated critical role of TLR4 in M. tuberculosis recognition and verified necessity of these TLR for development of a protective response against M. tuberculosis infection.⁴¹ Variants in TLR4, rs4986790 and rs4986791, were investigated for their association with susceptibility or resistance to pulmonary tuberculosis.^{27,28,30,34,46}

The results of our study are consistent with the reports of Najmi et al in India²⁸ Ferwerda et al in Tanzania⁴⁶ and Pulido et al in Spain⁵⁷ but not similar to that reported previously in other Sudanese³¹ and South India⁵⁸ Asian populations³⁴ European Caucasians,³⁴ North and South American^34,39, African populations^34,39, Gambian TB population⁵⁹, a south-eastern Chinese population,⁶⁰ Mexico,³⁵ and USA.³⁰

It is known that for TLR6 rs5743810 SNP have a protective effect against TB development. The T allele was found by Shey et al⁶¹ to reduce NF-kB signalling which led to an altered level of IL–6 production, while Randhawa et al⁶² showed that it leads to increased IFN-γ production and thus protection against M. tuberculosis. These functional studies correlate with the results found in this meta-analysis as well as that of Zhang et al³² where the T allele and TT genotype was also associated with resistance to TB disease.

The TLR8 polymorphism plays a significant part in the immune response in regulating the induction of interferon (IFN) and inflammatory cytokines.^47,52 Our results revealed that TLR8 rs3764879 and rs3764880 allele were more frequently found in the patients population compared to the healthy controls population. The association of the TLR8 rs3764879 and rs3764880 alleles with tuberculosis were also found in other populations originating from Russian and Indonesian populations.⁴⁷ Furthermore, associations have also been found in Turkish male children,⁶³ Pakistan population,⁶⁴ and South African population.⁵² However, neither Kobayashi et al²⁷ nor Chimusa et al⁶⁵ showed any association between rs3764880 and TB susceptibility.

Ethnic differences in TLR polymorphisms may in part reflect the ethnic diversity of host TB susceptibility. Davila et al found that around 30% of the Indonesian male subjects carried the A allele (rs3764880) associated with risk for TB, whereas this same allele was present in 78% of Russian patients.⁴⁷ 34.3% of the Turkish male children with pulmonary TB had the A allele associated with risk for TB, which is similar to Indonesian population.⁶³ The rs3764880 polymorphism of TLR8 was observed more than 16% among the healthy Chinese adult population by Cheng et al.⁶⁶

The gene of TLR9 is located on chromosome 3p21.3. The total length of TLR9 gene is approximate 5 kb. Its coding gene has two exons, and the major coding region is in the second exon.^53,67 TLR9 is an intracellular pathogen recognition receptor (PRR) that recognizes non-methylated cytosine-phosphate-guanine (CpG) motifs in bacterial DNA.^53,67 Based on NCBI SNP database, twelve SNPs have been identified for TLR9 gene. Studies have indicated certain race population with special genotype of TLR9 polymorphism might have higher risk for TB. Our results revealed that TLR9 rs352165, rs352167 and rs187084 allele were more frequently found in the patients population compared to the healthy controls population. Sanchez et al found that TLR9 rs352165, rs352167 were not associated with TB risk in a Colombian population.³⁷

In previous study, a meta-analysis was performed to assess the association between seven extensively studied TLR9 polymorphisms (rs187084, rs352165, rs5743836, rs5743842, rs352139, rs352140 and rs352167) and TB risk. The analysis revealed an association between certain TLR9 polymorphism and TB risk. The studies included Indians, Iranian and West African, Indonesians, Vietnamese, Chinese and Mexicans. The results showed that rs187084 and rs5743836 polymorphisms were not associated with TB risk, while the association between rs352139 polymorphism and TB risk may vary by race.^68,69

Bharti et al found that rs187084 locus may be associated with susceptibility to TB in Indian population.⁶⁹ The SNP rs187084 and rs5743836 SNPs located in the promoter are the most important and have been associated with various inflammatory diseases located in the promoter of TLR9 gene.^70–72 Previous functional analyses have shown that both rs187084 and rs5743836 SNPs influence the transcription of TLR9 by regulation of promoter activity.^70,72,73 Some studies found that the rs187084 in TLR9 showed no association with TB in Vietnam and Iran.^74,75

Our result revealed that rs5743836, rs164637, rs352139, rs352140, rs352143, and rs352162 allele may not be risk factors for susceptibility to pulmonary tuberculosis in Sudanese populations. On the other hands rs352139 has been strongly associated with susceptibility to TB in Indonesian⁷² and Vietnamese populations,^27,73 African-Americans and in Mexican Amerindians but not with Caucasians and African patients from Guinea-Bissau.³⁰ Sanders et al found that TLR9 rs5743836 and TLR9 rs352140 alleles have protective effect against meningococcal meningitis in Dutch children.⁷⁶

The rs5743836 in TLR9 showed a strong association with tuberculosis in African-Americans and Caucasians,³¹ while the association was not found in Vietnam⁷⁵ or Mexico population⁷⁴ and Chinese population.⁵³ Different variants of TLR9 rs352140 polymorphisms are described in the genome database³⁸ and in in the Asian population, in Japan.⁷⁷

TLR9 rs352142 polymorphism was positively associated with meningeal TB, while variant TWF2 rs352143 was associated with pulmonary TB in a Vietnamese cohort.⁷⁵

Recently, a number of studies indicated that TLR10 serves as a modulatory pattern-recognition receptor with mainly inhibitory properties on TLR2-derived immune responses, which are involved in the progression of TB.^39,40 Our result revealed that TLR10 rs4129009 that was associated with TB susceptibility in Sudanese tuberculosis patients. Various studies found that single-nucleotide polymorphisms in TLR10 were associated with susceptibility to tuberculosis in different ethnic groups. Ma et al demonstrated that polymorphisms of TLR10 were significantly associated with TB in African and European Americans.³⁹

Bulat-Kardum et al found that the rs11096957 AA genotype was associated with a predisposition to TB in the Caucasian population.⁷⁸ However, in the present study, we did not observe association between rs11096957 and risk of TB, no association was also found in Han Chinese population.⁷⁹ This inconsistent result is likely due to the ethnic difference.

There are some limitations in our study which need to be mentioned. The sample size was relatively limited and the methods were limited to PCR-RFLP. Moreover, some genotypes and alleles of SNPs were found to have low frequency in the studied population which may limit the statistical power. Moreover, we did not correlate the TLR SNPs with demographic characteristics or with others clinical parameters. Moreover, functional exploration inferring these candidate SNPs should be conducted further to confirm our findings. With these limitations, the study provides evidence for the association of SNPs present in TLR1, TLR2, TLR4, TLR6, TLR8, TLR9 and TLR10 genes with pulmonary tuberculosis. The presence of associated SNPs with pulmonary tuberculosis may give us the clue that it may provide susceptibility to pulmonary tuberculosis.

In conclusion, since TLRs are the key modulator of inflammatory processes, the associated specific TLRs with PTB may be a potential target for attenuation of specific TLR pathways to reduce the severity of the disease and testing for TLR SNPs may be helpful for early prediction of the course of the disease and early identification of patients who at risk. This the first published results that studies 86 SNPs and their association to pulmonary tuberculosis. Future studies are warranted in a larger sample to replicate our findings and understand the mechanism of association of TLR polymorphism in PTB.