Host genetic factors play a significant role in determining susceptibility or resistance to Mtb infection. In our study, we focused on two scavenger receptors, SR-B1 and CD36, which are part of the host's innate immune system. We investigated the association between SNPs in these genes, namely rs4238001 in SR-B1 and rs1761667 and rs3211938 in CD36, and TB in the Pakistani population. Our findings revealed a significant association between rs3211938 and rs1761667 in CD36 and rs4238001 in SR-B1 with active TB and LTBI. These results contribute to our understanding of the genetic factors involved in TB susceptibility and may have implications for future research.
We found that the mutant genotype GG in rs3211938 was associated with resistance against active TB (p = 0.02, OR = 0.91 95%CI = 0.34–2.20), as the frequency of mutant genotype was higher in healthy controls (35.29%) compared to TB patients (19.19%). The SNP rs3211938 (T > G), located on Exon 10, introduces an amino acid change from Tyrosine to termination codon at position 325 in CD36 protein which affects the expression and function of the protein. The 'G' allele of rs3211938 is associated with the decrease in expression levels of protein and provides protection against atherogenic profile (16). We hypothesize that a decrease in expression level may result in reduced mycobacterial growth as CD36 is involved in the uptake of surfactant lipids by macrophages which promotes the growth of Mtb within macrophages (17) This mechanism potentially contributes to protection against TB. Our results are consistent with a previous study conducted on the association of CD36 and MARCO with PTB in the Chinese Han population, indicating the significant association of SNPs in the CD36 gene with resistance to PTB (18). Our research findings are further supported by an in vivo study conducted on mice, where mice lacking the CD36 gene showed resistance against mycobacteria. The absence of CD36 led to a reduced ability of mycobacteria to survive within the cells. The study also indicated that CD36 plays a role in cellular processes associated with the formation of granulomas, which aid in the initial growth and spread of bacteria (19). Therefore, we can conclude that the protective role of the GG mutant genotype in rs3211938, which is linked to a decrease in CD36 expression, subsequently contributes to the inhibition of Mtb growth and spread.
Conversely, the genotype GG of rs3211938 showed an association with susceptibility to LTBI in comparison of healthy controls vs. TB contacts (p < 0.00, OR = 0.91 95% CI = 0.34–2.20). The frequency of genotype GG was higher in TB contacts (59.09%) compared to healthy controls (35.29%). According to Il'in and Shkurupy, CD36 becomes more abundant in multi-nuclear phagocytes (MP) during periods of Mtb persistence in BCG-infected mice (20), this might be a reason for TB contacts being susceptible to latent infection. These results suggest that a single gene might play different roles in determining the susceptibility or resistance to LTBI and active TB. Our findings also align with the study investigating the association of the SP110 gene with active TB and LTBI in Taiwan, which revealed the differential role of the gene for active TB and LTBI (21).
In rs1761667, we found a higher frequency of heterozygous genotype GA in TB contacts (86.57%) than healthy controls (66.89%) suggesting a significant association of rs1761667 GA genotype with risk to LTBI (p = 0.00, OR = 3.39, 95% CI = 1.73–6.58). The SNP rs1761667 is located at Exon 1A, a nucleotide change from G > A results in decreased protein expression (16). These results align with the study conducted in the Chinese Han population to investigate the association of CD36 with carotid atherosclerosis. The results suggested the risk of disease in female patients carrying the GA genotype at rs1761667 (22). Another study suggested a strong association of the GA genotype with an increased risk of coronary heart disease in the Chongqing Han population of China (23). CD36 gene is responsible for mediating the effects of Mannose-capped lipoarabinomannan (ManLAM), leading to the release of TNF-α in peritoneal murine macrophages. Ligands of SRs have similar effects on TNF-α, and NO production as observed with ManLAM (24). CD36 also facilitates lysosomal enzyme transportation and internalizes mycobacteria (25). In accordance with these studies, a decrease in CD36 expression due to mutation might impair the cytokine production and immune response against mycobacteria resulting in susceptibility to disease. We found a weak p value for the comparison of rs1761667 genotypes between healthy controls and active TB patients (p = 0.03, OR = 0.73, 95% CI = 0.43–1.24). The difference in genotype frequency distribution between studied groups may not remain significant by increasing sample size. We conclude that rs1761667 at the CD36 gene may be important in determining the risk of LTBI but not active TB.
Interestingly, we found that SR-B1 SNP at rs4238001 was significantly associated with active TB (p = 0 00, OR = 2.18, 95% CI = 1.14–4.33). The frequency of mutant genotype AA was higher in active TB patients (21.25%) compared to healthy controls (3.63%). The SNP rs4238001 (G > A), located at Exon 1, introduces an amino acid change from Glycine to Serine at position 2 in the SR-B1 protein. This amino acid change is associated with a decrease in SR-B1 expression (26). SR-B1 engulfs Mtb in mesenchymal stem cells (MSCs), which exhibit innate control of mycobacterial replication through autophagy. MSCs are found in both human and mouse Mtb granulomas and play an important role in TB pathogenesis (27). This SNP at rs4238001 may affect the phagocytosis of Mtb in MSCs and result in an impaired control of mycobacterium. This SNP has not been studied in TB, LTBI, or any other lung disease, however, two independent research groups reported significant association of rs4238001 with coronary heart disease and low progesterone level (28, 29).
In the comparison of SR-B1 SNP at rs4238001 between healthy controls vs. TB contacts, we found that heterozygous GA genotype was significantly associated with protection against LTBI (p = 0 00, OR = 0.37, 95% CI = 0.20–0.66). SR-B1 on microfold cells (M cells) interact with Mtb EsxA enabling it to cross airway mucosa and initiate infection. Disruption in SR-B1 genes decreases Mtb binding and translocation across M cells (30). Overexpression of SR-B1 in macrophages increases Mtb and BCG binding (31). We hypothesize that changes in SR-B1 gene expression due to mutation may affect Mtb binding, resulting in protection against LTBI. Our results are consistent with the study conducted by Acton et al., in which the SNP rs4238001 was associated with protection towards atherogenic lipid profile in white men (32).
Our study possesses several noteworthy strengths. The sample size utilized in our study was calculated using scientific methods and was adequate to establish a correlation between genetic mutations and TB within the specific population. We examined two variations in each of the targeted genes that were known to impact gene expression. It is worth noting that our study is the first of its kind to explore the association between CD36 and SR-B1 gene polymorphisms with TB and LTBI. However, there were a few limitations to our study that should be acknowledged. We did not validate latent TB infection in TB contacts using Interferon Gamma Release Assay (IGRA) or QuantiFERON (QFT). Additionally, ARMS-PCR does not serve as a definitive technique for genotyping as it may fail to detect mutations at low levels and is prone to producing false positive and false negative results. The findings of this research can be confirmed by categorizing the study participants with similar genetic variations and subsequently measuring protein levels to establish the cumulative impact of these genetic variations. In conclusion, conducting follow-up studies with a larger sample size and validating the genotypes through sequencing will contribute to the verification of the current study's results.