Background: Tuberculosis is one of the leading causes of mortality and morbidity worldwide. Polymorphisms in the TLR-2 gene have a diverse influence on the innate immune response and may result in the susceptibility of active pulmonary tuberculosis diseases among particular families, ethnicities, and races. However, the genetic variation in TLR2 in the susceptibility to PTB in our population has not been previously investigated. Thus, this study assesses the association of TLR-2 gene polymorphisms with susceptibility to PTB in Northwest Ethiopian population. Material and method: A case-control study was conducted from March 5, 2022, to March 30, 2023. A total of 101 study participants were recruited in TB clinic using a convenient sampling technique.

Socio-demographic and clinical data were collected using structured questionnaires via face-toface interviews and reviewing medical records, respectively. Three milliliter of venous blood sample was collected from each participant. The DNA was extracted using an automated Maxwell® 16 kit and the gene variants in TLR-2 (G2258A and T597C) genes were genotyped using a tetra allele refractory mutation system-based PCR. The data were coded, entered into Epi-Data Manager 4.6, and exported into SPSS 25 statistical software for analysis. A binary and multivariable logistic regression analysis was done to determine the association between TLR-2 gene polymorphisms and PTB. A p-value less than 0.05 were considered significant. Results: Our findings showed that the CC (mutated) genotype in TLR2 T597C was overrepresented and significantly associated with PTB as compared to healthy control groups (CC; AOR = 4.54; p = 0.028).

The C allele of T597C polymorphism was also associated significantly with PTB as compared to LTBI (C; AOR = 2.77, p = 0.017) and healthy controls (C; AOR = 3.4, p = 0.001). While the gene variant G2258A of the TLR2 gene was not detected in our study participants. Conclusion: Our study indicated that TLR2 T597C gene variants are significantly associated with PTB susceptibility in our study groups. To confirm our findings and comprehend the mechanism of these gene variants of TLR polymorphism in PTB, future investigations in a larger sample are required. Keywords : Pulmonary tuberculosis, Toll like receptor 2, Gene polymorphism

Background Tuberculosis (TB) is one of the most communicable diseases caused by Mycobacterium tuberculosis (MTB). It is an intracellular pathogen transmitted by inhalation of aerosolized droplets containing bacteria (1). It is a global public health problem that has killed more people than any other bacterial pathogen in human history (2). According to a report from the World Health Organization (WHO), 10.6 million people were affected by TB worldwide, which increased by 4.5% from 10.1 million as compared to 2020 reports. A total of 1.6 million people died from TB in 2021. Globally, TB is the 13th leading cause of death and the second leading infectious cause of death after coronavirus disease-19 ( above HIV) (3).

The earliest encounter between host and pathogen in TB occurs at the interface between innate immune cells and MTB (4). The innate immunity to MTB is mediated by a variety of germ line-encoded pattern recognition receptors (PRRs) (5). Toll-like receptors (TLRs) are one subset of PRRs that are a cornerstone for the innate immune response (6). The TLRs are a trans-membrane protein family with leucine-rich repeats (LRR) and toll-interleukin 1 receptor (TIR) domains (7). The TLRs that play a critical role in MTB recognition are TLR2 (8). Toll-like receptor 2 can recognize a wide range of mycobacterial PAMPs, including lipoarabinomannan (LAM), lipomannan (LM), and phosphatidylinositol dimannoside (PIMs) (9). Toll Like Receptor 2 can form heterodimers with both TLR1 and TLR6 (10), which recognizes triacylated and diacylated lipopeptides, respectively (11).

After recognizing PAMPs in a particular cell type, the cytosolic TIR domain associated with myeloid differentiation protein 88 (MyD88) and Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF) adaptor molecules are activated. Then, MyD88 recruits interleukin 1 receptor-associated kinase 4 (IRAK- 4) that phosphorylates IRAK1, which in turn activates TNF receptor-associated factor 6 (TRAF6). Both proteins leave the receptor complex and interact with TGF-β-activated kinase 1 (TAK1). Then, TAK1 becomes phosphorylated and activates the I-κB kinase (IKK) complex comprising IKKα, IKKβ, and IKKγ, and mitogen-activated protein kinase (MAPK) leading to the activation of NF-κB and c-Jun N-terminal kinases (JNK) transcription factor signaling pathways, respectively (12). The activation of these transcription factor signaling cascades promotes the production of pro-inflammatory cytokines (IL- 6, IL-12, TNF-α), type I interferon, and chemokine expression; and plays an essential role in the modulation of the host’s innate and adaptive immunity (13).

Toll like receptor 2 is a membrane protein that is expressed on the surface of cells, encoded by the TLR2 gene, which has four exons and is located on chromosome 4q31-33 in humans. It encodes a protein with 784 amino acid residues (14). Theoretically, any genetic variation in the LRR and TIR domains could influence how TLR molecules recognize the PAMPs and translate their signals (15). The most studied single nucleotide polymorphisms (SNPs) found on the conserved TIR domain in the C-terminal end of the TLR2 gene is G2258A (rs5743708) (16). It is a non-synonymous SNP that causes amino acid substitution from arginine (Arg) to glutamine (Gln) at the position of 753 (Arg753 Glu) (16).

The Arg753 Glu polymorphism could resulting in impaired tyrosine phosphorylation of TLR2, TLR2-TLR6 dimerization, suppression of transcription factors such as NF-κB activation, and decreasing secretion of important inflammatory mediators (17). It leads to a reduced response of macrophages to bacterial peptides and results in an attenuation of the host immune response, thus increasing susceptibility to TB (18, 19, 20). Another SNP found in the extracellular domain of the LRR of TLR2 is T597C, located at the position of 199 (Asn199 Asn). It is a synonymous SNP that did not result asparagine (Asn) amino acid alteration (21). Synonymous SNPs can modulate mRNA structure (22), influence protein folding, and ultimately alter protein function (23).

This type of SNP may also lower TLR expression, attenuate the host’s innate immune response, and result in decreased macrophage function in response to infection with MTB (21). According to estimates, approximately 90% of TB infected individuals will stay asymptomatic with latent infection (24), whereas only 10% of persons infected with MTB will develop active TB disease at some point in their lives, implying that the majority of those infected have a protective immune response (24, 25). Different studies concluded that TB is a genetically primed and determined infectious disease, and the host genetic polymorphism is the mechanism that leads to the progression from infection to TB disease (26, 27, 28).

Host genetic variations involved in innate immunity are thought to be the most probable factor predisposing individuals to the development of TB (29) and may play a significant role in the modulation of immune responses to MTB infection and the clinical progression of TB (30). Mutations in TLR producing genes could cause structural and functional changes in these receptors, altering immunological responses and influencing disease development (31). The ability of certain individuals to respond properly to TLR ligands may be impaired by SNPs within TLR genes that affect immune regulation, resulting in a shift in balance between pro- and anti-inflammatory cytokines and altered susceptibility to the infectious disease that might contribute to the pathogenesis of complex diseases including TB (32, 33).

This allows the bacterium to evade elimination by the immune system, and the disease subsequently progresses (34). Geneticvariations,especiallySNPs,canaffectthedevelopment of disease in individuals and responses to pathogens, drugs, and vaccines (35). In Ethiopia, even though it has a significant health impact, there is no genetic evidence published in the country related to the TLR gene polymorphisms of TB infected people. Thus, this study aimed to determine potential genetic biomarkers of TLR2 gene with the susceptibility of PTB patients in Northwest Ethiopia. The difference in polymorphism within genes involved in the host immune response has been proposed as a plausible reason to explain why some people resist infection more successfully than others and play a major role in determining differential susceptibility to major infectious diseases (36, 37).

The identification of SNPs in host genes associated with susceptibility and resistance to TB plays an important role in investigating potential mechanisms underlying host immune responses to TB disease and will provide a rational basis for developing new approaches to treatment or novel therapies to treat the diseases (34, 36, 38). To date, several studies have already investigated the potential association between genetic polymorphisms in TLRs and susceptibility to TB in several human populations. However, results are in many instances inconsistent and inconclusive, especially when they were conducted in different populations, geography, and environments (39, 40), emphasizing the need for more studies from different human populations (40). It was believed the study has ultimately hope that through genetic testing, individuals infected for TB will be identified before the onset of the disease – at a time when primary prevention strategies would be safely administered.

It is most likely that such predictive genetic testing would be offered to individuals with LTBI before it was made available to the general population. It will add value to our current perspectives of treating, promising vaccine design, or preventing TB diseases. As far as we know, there has not been a study on the TLR2 gene polymorphism-related genetic risk factors for PTB in Ethiopia. Hence, this study aimed to assess the association between TLR2 gene polymorphisms and the susceptibility of PTB patients in Northwest, Ethiopia. In this study, the genotype distribution of T597C and G2258A was determined, and their association with PTB disease susceptibility was analyzed. METHOD AND MATERIAL Study area The study was conducted in the TB clinic at the University of Gondar comprehensive specialized hospital.

The hospital is located in the Central Gondar zone, Gondar Town Administration.ThetownislocatedinAmharaNationalRegional State, 727 kilometers (km) from Addis Ababa, the capital city of Ethiopia. The town is situated at a latitude and longitude of 12° 36′ N and 37° 28′ E, respectively, with an elevation of 2133 meters above sea level. Currently, the population of Gondar town in 2023 is estimated to be 413,000 (41). The University of Gondar comprehensive specialized hospital is one of the oldest health institutions in Ethiopia, produces many professionals, and has also given huge service to the community since 1954 (42). It is a multidisciplinary, specialized teaching hospital with more than 1,000 beds and approximately 2,000 members of staff.

Currently, it provides health care services for more than seven million inhabitants in its catchment area (43). The TB clinic is one of the services delivered by the hospital for the diagnosis of patients with suspected TB, and it provides anti-TB treatment. Study design and study period A case control study was conducted at UoGCSH from March 5, 2022, to March 30, 2023. Source population All adult TB patients visiting the TB clinic at the UoGCSH were used as the source population. Study population All adult patients confirmed with PTB in the TB clinic were taken as case groups during the study period. In addition, people who have no history of TB or active TB diseases collected from Gondar blood bank were taken as a control for the comparison groups.

Sample size and sampling techniques All eligible study participants who fulfilled the inclusion criteria during the study period were included. Thus, a convenient sampling technique was used, and a total of 101 study participants (35 PTB patients, 20 LTBI, and 46 HCs) were enrolled in the study. Eligibility criteria Inclusion criteria Adult patients (age ≥ 18) confirmed with PTB during the study period were included as case groups in the study. Furthermore, adult individuals (age ≥ 18) who were free from clinical symptoms of active PTB and individuals with no medical history of TB were included as a control group in the study. Exclusion criteria In case groups, a patient with extra-PTB was excluded.

Moreover, individuals with known chronic diseases such as diabetes mellitus and rheumatoid arthritis and viral infections like HIV were excluded from the study. Individuals with a history of TB, a history of anti-TB treatment, and clinical manifestations of active TB were excluded from control study participant recruitment. In addition, individuals with known chronic diseases such as diabetes mellitus and rheumatoid arthritis and viral infections like HIV were also excluded from the control group. Data collection tool and laboratory procedure Socio-demographic and clinical data The data was collected by nurse professionals working in TB clinics. The socio-demographic and behavioral factor related data of PTB patients was collected through a face-to-face interview using structured questionnaires.

Blood specimen collection Under aseptic techniques, three milliliters and seven milliliters of venous blood were collected from PTB patients and asymptomatic healthy individuals, respectively. Four milliliters of blood from asymptomatic healthy individuals were distributed to four QFT test tubes (one ml for each tube) for LTBI screening, and the remaining three ml from asymptomatic healthy individuals and PTB patients’ blood were transferred to EDTA test tubes by following the appropriate blood collection procedure. The blood in the EDTA tube was properly mixed with anticoagulants to avoid blood clots before laboratory investigations were made. Each tube was labeled with a unique patient code for easy identification. The blood samples in the EDTA tube were kept under -800C until DNA isolation was done.

QuantiFERON-TB Gold plus Assay The QuantiFERON-TB Gold Plus assay (Qiagen, Germany) was used to measure IFN-γ production from T cells following in vitro stimulation with MTB specific antigens, including ESAT-6 and CFP-10). Four milliliters (4 ml) of blood were transferred to four separate tubes labeled as (Nil, MTB antigen-1, MTB antigen-2, and Mitogen) for antigen stimulation, which were then incubated for 20 hours. The Mitogen tube was used as a positive control, while the nil tube was used to adjust the background value for non-specific IFN-γ detection in blood samples by subtracting from the TB antigen tubes and Mitogen tubes. After a period of incubation, the tubes were centrifuged at 2500 relative centrifugal force (RCF) for 15 minutes, and the plasma samples were separated and transferred into a Nunc tube, and stored at -800 C until transported to Armaur Hansen Research Institute (AHRI), Addis Ababa, with the appropriate cold chain system for IFN-γ analysis.

Finally, the presence of LTBI was determined by measuring IFN-γ from plasma using an enzyme-linked immunosorbent assay (ELISA), and the results were interpreted using the TB QFT version 2.71.2 software (44). DNA extraction The DNA extraction was done on the whole blood samples collected from study participants using the automated Maxwell® 16 kit (Promega Corporation, USA), following the manufacturer’s instructions. The Maxwell® 16 instrument purifies samples using a novel paramagnetic particle, that provides a mobile solid phase that optimizes sample capture, washing, and purification of gDNA (45). Three hundred microliters (µl) of whole blood were added to the tube, and then 300 µl of lysis buffer and 30 µl of proteinase K were added and vortexed for 10 seconds, followed by a quick spine for 15 seconds.

Then, it was incubated at 56 0C for 20 minutes at 400 rpm (45). In the meantime, the Maxwell® 16 cartridge was prepared as follows: The cartridges were placed in the Maxwell 16 cartridge rack and then pressed down on the cartridge to snap it into position. Empty elution tubes were placed for each cartridge, and then 60 µl of elution buffer was added to each elution tube. The seal was carefully peeled back only to the upper first well, and all the incubated material of each sample was transferred to well #1 of each cartridge, and one plunger was placed into the well of each cartridge. Finally, the rack was placed in the Maxwell extractor, and the extraction run started.

Following the extraction, the extracted product was transferred to the tube and stored at -80 0C until the PCR amplification (45). The extracted DNA concentrations were measured using a Quibit fluorometer (Invitrogen, USA) (46). Tag SNP and its primer sequence selection We searched the SNP locations of TLR2 gene in the SNP database of the National center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/SNP). Moreover, based on previously published data on the association of TLR-2 gene with the susceptibility of TB from different studies, two SNPs from TLR-2 genes (G2258A and T597C), were selected. The SNP selection was based on their location, possible functional effect, and associations with PTB diseases. We used four primers (two external primers and two inner primers) for each SNP.

Primer sequences were taken from previous literature, and the primer sequence details for all variants are provided in Table 1. Primers were purchased as lyophilized oligonucleotides (Sigma Aldrich, Germany), and then reconstituted with sterile deionized water to make 100µM concentrations of stock solution. Then, dilutions were made from this stock to reach 10µM and 20 µM working solution and stored at -20 0C. Table 1: SNPs with their primer sequences and PCR products size used for genotyping TLR2 (G2258A and T597C) gene. SNP ID SNP position (nucleotide/ AA residue) Chromosome position Gene location Primer sequence 5’- 3’ PCR product size

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