Tuberculosis (TB) is a chronic infection caused by Mycobacterium tuberculosis (Mtb), and it remains one of the most serious infections with high incidence and mortality, mostly in developing countries. According to the World Health Organization (WHO) report, there were an estimated number of 10 million new TB cases and 1.4 million deaths worldwide in 2019 (WHO 2020).

Early detection and appropriate treatment are the most important control strategy for TB. However, there is a low rate of TB cases detected and officially notified. In addition, smear microscopy and culture are the “gold standards” for the diagnosis of TB; however, faster and more sensitive diagnostic tests are required to improve diagnosis and patient management during infection (Acharya et al., 2020).

Emerging evidence suggests that miRNAs might be involved in the pathogenesis of a variety of human diseases, including TB (Sabir et al., 2018). MicroRNAs are an important regulator of biological processes, such as innate immune response against various infections (Wang and Cui 2012). They regulate the primary function of macrophages, dendritic cells, and natural killer cells (NKCs) and influence other regulation of proteins involved in the innate and adaptive immune pathways (Sahu et al., 2017; Tamgue et al., 2019).

Comparative miRNA expression profiles in individuals with active tuberculosis and healthy controls were studied in an Amazon population. Three different miRNA expressions, hsa-miR-4732-5p, hsa-miR-486-3p, and hsa-let-7g-5p, were identified in tuberculosis patients (Silva et al., 2021). However, different miRNA expressions can vary between individual samples during treatment and recovery (Kleinsteuber et al., 2013).

Recently, genetic variants in IL10, VDR, and microRNA genes were reported in subsequent studies in different human populations. However, there were few results of the association between these microRNA polymorphisms and tuberculosis susceptibility (Song et al., 2013; Silva et al., 2020). The aim of this study was to perform an association study between SNPs in miRNA and DROSHA and tuberculosis and evaluate whether these selected SNPs may confer susceptibility or protection to TB in the Amazon population.

There were 52 males and 126 females, with total of 178 healthy controls (mean age 70.82 ± 1.8 years; 52 males and 126 females) were enrolled. Both significant differences were found in the distribution of age and sex between cases and healthy controls. The population structure results showed significant differences in European, Amerindian, and African contributions in the cases compared to controls. As expected, the population ancestry showed a higher Amerindian and African contribution and a lower European ancestry in the opposite direction in TB patients than that in controls (Table 1).

A total of 26 SNPs were tested for the Hardy–Weinberg equilibrium with the Bonferroni correction (Table 2). In overall 26 SNPs, 10 markers were excluded for further analyses, and the remaining 16 SNPs were included for subsequent analysis by the Hardy–Weinberg equilibrium (Table 3). The results indicated that seven SNPs showed a significant association with TB risk, and those SNPs were rs10035440 in DROSHA, rs7372209 in miR26-a1, rs1834306 in miR100, rs4919510 in miR608, rs10739971 in pri-let-7a-1, rs3746444 in miR499, and rs6505162 in miR423 (Table 4).

The results of the genotype distribution showed that the SNPs rs10035440 (DROSHA) and rs1834306 (miR100) were associated with an increased risk of TB (OR = 15.58, 95% CI [5.04-48.15] and OR = 3.39, 95% CI [1.67–6.85], respectively) in the dominant model. The SNP rs7372209 (miR26-a1) was associated with an increased risk of TB in a recessive model (OR = 3.75, 95% CI [1.26–11.15]).

In addition, the SNPs rs4919510 (miR608) and rs10739971 (pri-let-7a-1) were associated with an increased risk of TB in dominant and recessive models. The SNP rs4919510 was associated with an increased risk of TB in the analysis of the dominant model (OR = 4.53, 95% CI [2.30–8.92]) and in the analysis of the recessive model (OR = 4.13, 9 5% CI [2.25–7.60]). The SNP rs10739971 was associated with an increased risk of TB in the analysis of the dominant model (OR = 6.14, 95% CI [2.88–13.08]) and in the analysis of the recessive model (OR = 7.25, 95% CI [2.13–24.69]).

However, the SNPs rs3746444 and rs6505162 were associated with a low risk of TB in the analysis of the recessive model (OR = 0.009, 95% CI [0.003–0.26]) and rs6505162, in the analysis of the dominant model (OR = 0.33, 95% CI [0.13–0,84]).

In the present study, we examined the possible association of polymorphisms in miRNA and DROSHA genes with tuberculosis susceptibility in the Brazilian Amazonian population. There were 52 males and 126 females, with total of 178 healthy controls. According to our results, we found seven SNPs in different genes that were associated with TB susceptibility. However, there are relatively few reports regarding the association of genetic variants and susceptibility to tuberculosis in an admixture of population such as the Brazilian Amazonian population.

Our previous study investigated the effect of ancestral background on TB susceptibility among the Brazilian Amazonian population. The results indicated that Amerindian (compared to African and European) ancestry populations were significantly associated as a risk factor for susceptibility to TB in the Brazilian Amazonian population (Leal et al., 2020). In the present study, we used ancestry-informative markers (AIMs) to estimate genetic ancestry and control for stratification to investigate genetic association among Brazilian admixed populations.

Our results suggested that the SNPs rs10035440 (DROSHA), rs4919510 (miR608), rs10739971 (pri-let-7a-1), rs7372209 (miR26-a1), and rs1834306 (miR100) were associated to an increased risk of TB. With respect to SNPs rs3746444 (miR499) and rs6505162 (miR423), these genetic variants were associated to a decreased risk for TB susceptibility in active-TB patients.

Previous studies have revealed that deregulation of miRNAs and miRNA-related genes occur in many diseases (de Souza et al., 2020; Silva et al., 2020). DROSHA initiates the biosynthesis of miRNAs by processing pri-miRNAs. Single nucleotide polymorphisms (SNPs) in miRNA biosynthesis genes, DROSHA, were indicated to be correlated with cancer risk as pediatric ALL (Gutierrez-Camino et al., 2014; López-López et al., 2014).

Some studies suggested that genetic variations were associated with cancer, HBV infection, and sepsis. Dai et al. (2018) reported that the rs4919510 polymorphism in miRNA-608 was associated with a decreased risk of collateral cancer (Dai et al., 2018). López-López et al. (2014) indicated significant associations between rs4919510 polymorphism in miRNA-608 and toxicity for cancer treatment (López-López et al., 2014). Al-Qahtani et al. (2017) found that miR-608 rs4919510 was associated with an increased risk of disease progression in patients with HBV (Al-Qahtani et al., 2017). Zhang et al. (2015) indicated that miR-608 rs4919510 was found to be strongly associated with a higher risk of developing sepsis and multiple organ dysfunction (Zhang et al., 2015).

miRNA let-7 is involved in numerous cellular processes, inflammation, immunity, protective functions, and viral infections (Bernstein et al., 2021). Li et al. (2016) reported the relationship of the pri-let-7a-1 rs10739971 polymorphism with the prognosis of gastric cancer patients in northern China (Li et al., 2016). M. tuberculosis suppresses the innate immune response in macrophages, when suppressing miRNA let-7f expression, and modulates the immune response to TB infection by elevating target expression, A20, an inhibitor of the NF-κB pathway (Kumar et al., 2015).

Ni et al. (2014) and Kleinsteuber et al. (2013) reported that M. tuberculosis can modulate the expression of host miRNA and limit the response of macrophages during TB infection. miRNA 26-A targets a component (P300) of the IFN-y signaling cascade and was found to be differently expressed during tuberculosis infection (Kleinsteuber et al., 2013; Ni et al., 2014). miRNA-26a regulates innate immune signaling, macrophages polarization, and trafficking of Mycobacterium tuberculosis to lysosomes during TB infection (Sahu et al., 2017). It also plays a role in intestinal inflammation (Zhang et al., 2021), preeclampsia (Eskandari et al., 2019), and cancer (Ying et al., 2016).

Studies have shown that miRNA-100 is associated with the adaptive immune system (Negi et al., 2015), anti-inflammatory function in the vascular system (Pankratz et al., 2018), and the occurrence, development, and cancer invasion (Liu et al., 2012; Huang et al., 2020). Genetic variation in miRNA-100 rs1834306 was found to be an increased risk of gastrointestinal disease (Zhu et al., 2020), increased risk of Hepatitis B virus (Motawi et al., 2019, 1834306), and a decreased risk for esophageal squamous cell carcinoma (Zhu et al., 2015) in several studies.

Li et al. (2011) reported that there was no association between miR-499 rs3746444 and pulmonary tuberculosis (PTB) risk in the Han population; however, subjects carrying the C allele showed a decreased PTB risk. Li et al. also found that there was an association between rs3746444 and PTB in the Tibetan population, and individuals carrying the C allele exhibited an increased PTB risk (Li et al., 2011). Naderi et al. (2015) indicated a lack of association between miRNA-146a rs2910164 and miRNA-499 rs3746444 gene polymorphisms and susceptibility to pulmonary tuberculosis (Naderi et al., 2015). Additionally, miR-499 rs3746444 was related to other diseases, including acute lymphoblastic leukemia (de Souza et al., 2020), asthma (Dong et al., 2020, 49), and inflammatory arthritis (Wang and Pan 2019).

Studies have evaluated the association between the SNP miRNA-423 rs6505162 and susceptibility to colorectal carcinoma (Jia et al., 2018) and coronary artery disease (Jha et al., 2019). A recent study showed that the expression of miRNA-423 was significantly increased in the serum of patients with tuberculosis, and the upregulation of miR-423-5p could suppress autophagosome–lysosome fusion in macrophages by posttranscriptional regulation of VPS33A (Tu et al., 2019).

In this study, it was possible to determine the genotypic frequencies for the SNP in selected genes as well as associations related to risk or protection for tuberculosis infection. We found seven genetic variants significantly associated in individuals with tuberculosis from the Amazon region. Our study revealed that the SNPs rs10035440 (DROSHA), rs7372209 (miR26-a1), rs1834306 (miR100), rs4919510 (miR608), and rs10739971 (pri-let-7a-1) were significantly associated with a high risk of TB. In addition, the SNPs rs3746444 (miR499) and rs6505162 (miR423) were significantly associated with a low risk of developing tuberculosis. Our study contributes to a better understanding of TB pathogenesis and may promote the development of new diagnostic tools against M. tuberculosis infection. Future studies must be carried out to evaluate the functional effects of polymorphisms of the selected genes as well as the inclusion of other SNPs and large sample study, to corroborate the findings for tuberculosis in our country.