Polymorphisms and Susceptibility to Tuberculosis in Sudanese Patients


 Background: Genetic factors are important contributors to the development of a wide range of complex disease. Polymorphisms in genes encoding for toll like receptors (TLRs) usually influence the efficiency of the immune response to infection and are associated with disease susceptibility and progression. Therefore, we aims to describe the first association between TLR1, TLR2, TLR4 TLR6 , TLR8 , TLR9 ,and TLR10 genes polymorphisms and susceptibility to tuberculosis in Sudanese patients.Methodology: Here we performed a case study which included 160 tuberculosis patients and 220 healthy matched controls from Sudan. In the study population, we evaluated the possible association between 86 markers in TLR1, TLR2, TLR4 TLR6 , TLR8 , TLR9 ,and TLR10 genes polymorphisms and susceptibility to tuberculosis disease in Sudanese population using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) . Result: From our results It appeared that in the tuberculosis population the TLR1 rs5743557, TLR1 rs4833095, TLR1 rs5743596, TLR2 rs5743704, TLR2 rs5743708, TLR2 rs3804099 , TLR4 rs4986790 ,TLR4 rs4986791, TLR6 rs5743810 , TLR8 rs3764879 , TLR8 rs3764880, TLR9 rs352165, TLR9 rs352167, TLR9 rs187084 and TLR10 rs4129009 were significantly more often encountered (p<0.0001) than in the control population and were associated with tuberculosis in the Sudanese population. For the other polymorphisms tested, no association with tuberculosis was found in the population tested. Conclusion: This indicates that the genotypes obtained for TLR1 rs5743557, TLR1 rs4833095, TLR1 rs5743596, TLR2 rs5743704, TLR2 rs5743708, TLR2 rs3804099 , TLR4 rs4986790 ,TLR4 rs4986791, TLR6 rs5743810 , TLR8 rs3764879 , TLR8 rs3764880, TLR9 rs352165, TLR9 rs352167, TLR9 rs187084 and TLR10 rs4129009 allele have a significant role in the genetic susceptibility to development tuberculosis in Sudanese population.

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][23][24][25][26]. Genetic variations of TLR1, TLR2, TLR4, TLR6,  TLR8, TLR9 and TLR10 have been  Hence, the identi cation of mutations in these receptors could be used as a marker to screen the individuals who are at risk.
Therefore the aim of the present study is to determine the possible association between 86 single-nucleotide polymorphisms (SNPs) of TLR1, TLR2, TLR4 TLR6, TLR8, TLR9 ,and TLR10 with pulmonary tuberculosis in a sub-Saharan Sudanese 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).
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 (

Ethics statement
The present study was approved by the Ethics Committee of University of Khartoum, Khartoum, Sudan (5/2018). Written informed consents were obtained from all participants in the study.

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.

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.
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, TLR1 rs4833095, TLR1 rs5743596, TLR2 rs5743704, TLR2 rs5743708, TLR2 rs3804099 , TLR4   rs4986790 ,TLR4 rs4986791, TLR6 rs5743810 , TLR8 rs3764879 , TLR8 rs3764880, TLR9 rs352165, TLR9 rs352167, TLR9  rs187084 and TLR10 rs4129009 were signi cantly 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. 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 in uence their expression, function and alters the recognition or signaling mechanism, which leads to the disease susceptibility [47,48]. 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 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, 49] and, East Asian Population for TLR1 rs5743557 [32], but not in populations originating from in North China.
[32] and African American subpopulation [38]. 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 di cult. 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  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 [39, 52, 53] but not European subgroup [51]. Zhang et al. [32] showed a signi cant 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. [36,54] TLR2 rs3804099 was associated with susceptibility to TB meningitis rather than with susceptibility to pulmonary TB in a casecontrol study of a Chinese cohort.
[55] 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 [52].
Several studies have demonstrated critical role of TLR4 in M. tuberculosis recognition and veri ed necessity of these TLR for development of a protective response against M. tuberculosis infection [40]. Variants in TLR4, rs4986790 and rs4986791, were investigated for their association with susceptibility or resistance to pulmonary tuberculosis [27,28,30,34,45]. It is known that for TLR6 rs5743810 SNP have a protective effect against TB development. The T allele was found by Shey et al.
[60] to reduce NF-kB signalling which led to an altered level of IL-6 production, while Randhawa et al. [61] 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 al. [32] where the T allele and TT genotype was also associated with resistance to TB disease.
The TLR8 polymorphism plays a signi cant part in the immune response .in regulating the induction of interferon (IFN) and in ammatory cytokines [46,51]. 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  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 [52, 62]. TLR9 is an intracellular pathogen recognition receptor (PRR) that recognizes non-methylated cytosine-phosphate-guanine (CpG) motifs in bacterial DNA [52,62]. Based on NCBI SNP database, twelve SNPs have been identi ed for TLR9 gene. Studies have been 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 [67, 68]. Bharti et al found that rs187084 locus may be associated with susceptibility to TB in Indian population [68] The SNP rs187084 and rs5743836 SNPs located in the promoter are the most important and have been associated with various in ammatory diseases is located in the promoter of TLR9 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 In conclusion our result indicates that the genotypes obtained for TLR1 rs5743557, TLR1 rs4833095, TLR1 rs5743596, TLR2   rs5743704, TLR2 rs5743708, TLR2 rs3804099, TLR4 rs4986790 ,TLR4 rs4986791, TLR6 rs5743810, TLR8 rs3764879, TLR8  rs3764880, TLR9 rs352165, TLR9 rs352167, TLR9 rs187084 and TLR10 rs4129009

Declarations
Acknowledgements We would like to thank all health centers' staf for their kind collaboration and assistance. And also great thanks to all participants contributed to this work.
Authors' contributions: NAM was provided conceptual framework for the project, participated in data collection and analysis , participated in the molecular performance and writing the manuscript Funding: Not applicable.
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.