The Inuence of Single Nucleotide Polymorphisms of NOD2 or CD14 on Susceptibility to Tuberculosis: A Systematic Review

Background: Tuberculosis (TB) is still one of the leading causes of death worldwide. Genetic studies have pointed to the relevance of the NOD2 and CD14 polymorphic alleles in association with susceptibility or resistance to TB. Methods: A systematic review was performed on search platforms to examine the association between single nucleotide polymorphisms (SNP) and TB risk. Study quality was evaluated using the Newcastle-Ottawa Quality Scale (NOQS) Results: Thirteen studies matched the selection criteria. Of those, 9 investigated CD14 SNPs, and 6 reported a signicant association between the T allele and TT genotypes of the rs2569190 SNP and increased TB risk. In contrast, the genotype CC was found to be protective against the disease. Furthermore, in two studies, rs2569191 SNP of the CD14, G allele was described to be signicantly associated with increased TB risk. Four studies reported data uncovering the relationship between NOD2 SNPs and TB risk, with two of them reporting signicant associations of rs1861759 and rs7194886 and higher TB risk in a Chinese Han population. Paradoxically, minor allele carriers (CG or GG) of rs2066842 and rs2066844 NOD2 SNPs were associated with lower TB risk in African Americans. Conclusions: The CD14 rs2569190 and rs2569191 polymorphisms inuence TB risk depending on the allele. Furthermore, there is signicant association between NOD2 SNPs rs1861759 and rs7194886 and augmented risk of TB, especially in persons with Chinese ethnicity. The referred polymorphisms of CD14 and NOD2 genes likely play an important role in TB susceptibility and physiopathology; such effect may be affected by ethnicity.


Literature search
A systematic search was conducted between XX 2019 and June 25 2020 by two independent researchers (the authors JMC-A and DNA) in the following databases: PubMed (MEDLINE and EMBASE), Europe PubMed Central (PMC), Cochrane, Scienti c Electronic Library Online (SciELO), and Literatura Latino-Americana e do Caribe em Ciências da Saúde (Lilacs). The keywords used in the search were 'Mycobacterium tuberculosis', 'tuberculosis'; 'CD14' or 'NOD2'; and 'polymophism', 'SNPs' or 'genetic polymorphism' with various combinations. Every original research article found in the search that was in English, Spanish, or Portuguese was considered, with no restriction on the publication date. Reviews, letters to the editor, and comments were not included but were sources of additional references that did not appear in the rst search.

Selection of studies
Initially, titles and abstracts were reviewed and analyzed for eligibility (Fig. 1). Thereafter, all the eligible articles were fully read. These two steps were performed by two independent reviewers (JMC-A and DNA). The inclusion criteria were: (1) the main subject of the article must have been the genetic in uence on TB susceptibility; (2) the study must have been related to a SNP in CD14 and/or NOD2 genes. Articles that did not mention TB susceptibility, polymorphism in the genes indicated above, had non-su cient data, reviews, meta-analyses, animal model studies, letters to the editor, or which were clearly not related to the theme were excluded. Quality assessment The quality assessment of each individual study was further performed according to the Newcastle-Ottawa Quality Scale (NOQS) [20] (Table 2), which measures the quality of a study based on three aspects: selection (maximum, 4 stars), comparability (maximum, 2 stars) and exposure (maximum, 3 stars). Thus, in the processing of the article quality analysis, a maximum of 9 stars could be obtained. Publication with a total score of 0-3 was classi ed as low quality, 4-6 as moderate-quality, and ≥ 7 as high quality.  [29] c [23] Abbreviations: NA: not applicable.

Selection of articles
Our primary search identi ed a total of 326 articles (Fig. 1). Through the study selection process, 13 articles met the inclusion criteria and were included in the systematic review [3,9,[21][22][23][24][25][26][27][28][29][30][31]. The majority of the studies evaluated CD14 gene polymorphisms (n = 9), whereas four studies analyzed NOD2 polymorphisms. All of the selected studies adopted the case-control design, in which the case was de ned as patients with tuberculosis, whether pulmonary, extrapulmonary, or both and controls were de ned as individuals not infected with Mtb. The majority of articles investigated the relationship between presence of polymorphisms and the risk of developing only pulmonary TB (n = 7), whereas ve studies tested association of SNPs with pulmonary and other forms of TB, and one assessed the relationship with susceptibility to spinal TB (Table 1).
In this systematic review, data on 4,054 TB patients were examined, whereas 3,993 individuals were identi ed as controls. The median sample size (IQR) per study was 267 (123-401) and 187 (127-413) for TB patients and healthy controls, respectively. The detailed characteristics of each study are shown in Table 1.
It is also possible to visualize in the Fig. 2 that the CD14 polymorphisms were studied in diverse populations from various ethnicities, including Mexican, Colombian, Polish, Turkish, Iranian, South Korean and Chinese. In contrast, the NOD2 polymorphisms were studied in 3 restricted populations: North Americans, Chinese and Ugandan.

Quality Assessment
The quality scores of the studies, assessing the risk of bias, are displayed in Table 2. Five studies were of good quality and eight were of moderate quality (Table 2).

CD14 polymorphisms and risk of tuberculosis
In the present review, CD14 polymorphisms were the most frequently studied in the context of TB susceptibility. In total, 9 studies reported potential associations, presenting data for different populations. The sample sizes varied between 3987-1969 TB cases and 2018 controls. The studies reported data on 7 CD14 SNPs: rs2569190 [3, 9, 21-27], rs2569191 [22,23], rs3138078 [22], rs2915863 [22], rs3138076 [22], rs5744455 [22] and rs5744454 [22]. Of the 9 publications which investigated this gene locus, six studies reported a signi cant association between the T allele of SNP rs2569190 and higher odds of TB [3,[21][22][23][24]26]. The T allele was signi cantly more frequent in TB patients compared with healthy controls in subpopulations from China [21][22][23], Mexico [3], Iran [24] and South Korea [26]. Also, analyzes of odds ratio (OR) revealed an increased risk of TB when this allele was present, in case of both pulmonary and extrapulmonary disease. Of note, three of such studies [21][22][23] reported this aforementioned association in Chinese participants, reinforcing the idea that this CD14 allele may indeed increase the risk of developing TB in this particular population.
With regards to genotypes, the CD14 TT genotype was signi cantly more common in TB patients than in healthy controls and increased the risk of TB in ve [3,21,23,24,26] of the six studies which analyzed the rs2569190 SNP. Only in one Chinese study, Zhao et al [23], the occurrence of the T allele in homozygosis was reported not to be related with a signi cant risk of TB in the OR analyzes (OR = 1). Nevertheless, in the same study, the presence of T allele alone was shown to be not only more frequent in TB cases, but also was described as a risk factor for TB development in the same population (OR = 1.4; 95% CI = 1.148-1.708; p = 0.001). In addition, Zheng et al [23] described that the association of TT with the CT genotype (TT + CT) was found signi cantly more frequent in spinal TB patients than in controls (85.00% vs 44.17%; P < 0.05), with an OR of 2.10 (CI 95%= 1.09-3.85). Therefore, it is possible to conclude that the CD14 T allele may directly impact the risk of TB development. When such allele is present in the genetic loci, either in heterozygosis or homozygosis, the odds to progress with active TB is shown to be high.
In contrast, Zhao et al [23] reported that frequencies of the CD14 genotypes CT and CC in the rs2569190 polymorphism were lower in TB cases than in controls. Logistic regression analysis of such results demonstrated that presence of CT and CC genotypes are likely protective against TB (OR = 0.46 and 0.63, respectively; 95% CI = 0.34-0.63 and 0.42-0.93). In agreement with these latter ndings, Alavi-Naini et al. [24] described that the C allele in homozygosis was a protection factor in a sub-analysis of Iranian subjects, with an OR of 0.44 (95% CI 0.23-0.83; p = 0.006), whereas the CT genotype was not signi cantly associated with a lower risk for TB (OR = 1.03, 95% CI = 0.6-1.7; p = 0.5). Thus, the CC genotype might be a protective factor in the TB pathophysiology.
Noteworthy, three articles did not nd any evidence for a signi cant association between SNP rs2569190 and TB development -Ayaslioglu et al [25], Druszczyñska et al [27] and Pacheco et al [9]. There was no signi cant difference in terms of genotype frequency and allele distribution between TB patients and controls, or between distinct TB clinical forms. Moreover, no association was found between the CD14-159C/T polymorphism and TB clinical severity in studies that evaluated Turkish, Caucasian Polish or White and Mestizo Colombian patients. In the Turkish study, one hypothesis described for the lack of association was the small sample size (88 TB cases and 116 controls) [25]. Moreover, Druszczyñska et al [27] have not speci ed whether TB patients and controls enrolled were from the same region, which could possibly have in uenced the ndings. Discrepancies related to ethnicity or other epidemiologic or environmental characteristics among the diverse populations investigated may have contributed to these con icting results.
Additional investigations evaluating the CD14 SNP rs2569191 revealed signi cant associations with odds of TB in 2 distinct publications from China [22,23]. Both studies reported that the G allele of A-1145G was more prevalent in TB cases than in controls, indicating an increased TB risk. In one of these studies [22], individuals with the GG genotype of A-1145G were more likely to present with TB (OR = 2.78, 95% CI = 1.79-4.32; p = 0.016). Paradoxically, the second investigation [23] suggested that the frequencies of genotypes AG and AA were lower in TB cases than in controls, arguing for a protective role against TB (OR = 0.60 and 0.44, respectively; 95% CI = 0.44-0.83 and 0.29-0.65). Therefore, CD14 SNP rs2569191 may play an important role in TB vulnerability in persons from China through either being associated with increased or decreased susceptibility, depending on the genotype.
The others CD14 SNPs (rs3138078, rs2915863, rs2569192, rs3138076, rs5744455 and rs5744454) were evaluated by only one study [22]. In such investigation, the alleles signi cantly associated with TB were only the G allele of rs2915863 and the G allele of rs3138078. For the SNPs rs2569192; rs3138076; rs5744455 and rs5744454 there were no statistically relevant associations. Since no other study has analyzed these SNPs, it is not possible to conclude whether they have a potential in uence on susceptibility to TB.

NOD2 polymorphisms and TB
In regard to the NOD2 gene, the results from the studies were very diverse, with the SNPs being associated with either increased or decreased susceptibility to TB in each one of the study populations based on ethnicity, age group or biological sex. In such studies, a total of 2085 TB cases and 2347 controls were investigated. These studies were performed in different countries including China, Uganda and North America, with the latter being focused on African Americans.
Two publications reported data on NOD2 SNPs in China [29,30], with a total of 2651 individuals. Zhao et al [29], demonstrated that the frequency of the TG genotype in rs1861759 SNP was substantially associated with TB (OR = 2.16; 95% CI = 1.31-3.58; p = 0.0023) in the Han population. Interestingly, the same study did not identify this relationship between the TG genotype and TB in Chinese participants from Kazak or Uygur ancestry. A different study investigating Han Chinese individuals [30] identi ed the NOD2 SNP rs7194886 as a risk factor for TB. Patients with the SNP rs7194886 who presented the CT or TT genotypes were more likely to present with the disease when compared to individuals with the CC genotype, with an OR of 1.35 (95% CI = 1.05-1.72). In addition, this same study found that the frequency of the rs7194886 T allele was associated with TB risk (OR = 1.25, 95% CI = 1.00-1.57). Furthermore, the study evaluated that the effects of the rs7194886 polymorphism seems to be greater in either smokers or men [30], with an OR of 1.53 (95% CI = 1.07-2.18) and 1.44 (95% CI = 1.08-1.92) respectively. In addition, the Haplotype rs9302752 C-rs7194886 T was linked with a higher risk of presenting with sputum culture-positive TB (p = 0.039).
The study involving African American patients [31] reported an association between rs2066842, rs2066844, and rs5743278 NOD2 SNPs and odds of TB. The study participants who were carriers of the CC genotype in rs2066842 and rs2066844 SNPs were less likely to have TB, with an OR of 0.55 (95% CI = 0.32-0.94) and 0.27 (95% CI = 0.08-0.88), respectively. In converse, individuals who were heterozygous (CG) in rs5743278 exhibited an increased chance of having TB (OR = 2.16, 95% CI = 1.01-4.72).
Furthermore, in the adult African population from Uganda [28], presence of the SNP rs17313265 seemed to increase the susceptibility to TB (OR = 1.98, 95% CI = 1. 23, 3.19). Notably, increased frequency of the rs6500328 and rs2111234 SNPs was found in persons who did not TB, suggesting that such SNPs may be linked to resistance against Mtb infection, with OR of 2.44 (95% CI = 1.01. 5.88) and 1.56 (95% CI = 1.07, 2.28), respectively.

Discussion
The molecular basis of infectious diseases is an indispensable approach to understand how the gene regulation ultimately determines clinical outcomes. In recent decades, a great deal of research has been done on the associations between genetic polymorphisms and susceptibility to TB [32,33]. Most of the target loci are PRRs contributing to mycobacterial diseases, especially TB [34]. CD14 and NOD2 are considered to be key PRRs in the innate immune system [35]. In this systematic review, a variety of studies were identi ed evaluating seven CD14 SNPs and four evaluating nine NOD2 SNPs, and those genetic variants were associated with Mtb infection.
We identi ed a signi cant association between the T allele of CD14 SNP rs2569190 and TB, in which this allele increased the risk of having pulmonary and extrapulmonary TB in different ethnic groups. This polymorphism is located in the promoter region of the CD14 gene [35] and the T allele was attributed to a negative regulator of in vitro T-cell proliferation and decreased production of cytokines, including interferon-y (IFN-γ) [26]. IFN-γ is an essential cytokine for the control of mycobacterial infection [36], and for that reason it is possible to hypothesize that rs2569190 may result in an environment that favors development of TB. Interestingly, other studies also related this SNP with occurrence of ischemic stroke [37], cardiovascular disease [38] and asthma [39], indicating that these polymorphisms could actually lead to a more profound alteration in immune responses that may affect a large number of clinical conditions.
Another important association of CD14 polymorphism with TB was described in this review, involving the SNP rs2569191. In this setting, the G allele of A-1145G was more prevalent in TB cases than in controls and thus related to increased TB risk in two Chinese studies. An interesting link between such SNP and circulating concentrations of IgE has been proposed. In a study performed in patients with asthma, the authors identi ed a heightened IgE concentrations in those who had the G allele of A-1145G [40]. Moreover, other studies identi ed the IgE as a marker of the Mtb infection, in which pre-treatment levels of serum total IgE concentrations in TB patients were signi cantly higher than in healthy individuals; such levels decreased after successful antitubercular treatment [41,42]. It is possible that the polymorphism rs2569191 plays an important role in TB susceptibility which may be related to IgE concentrations. Future studies in other ethnic groups are warranted to directly test this hypothesis.
NOD2 is one of the most well-studied genes in the context of the innate immune response against microbial pathogens [43]. This gene accounts for a cytoplasmic receptor belonging to the NOD-like receptor family [44] and it is known to participate in the induction of in ammation during Mtb infection [45]. In experimental conditions, Mtb recognition is NOD2-dependent [46]. Mice genetically de cient in NOD2 are shown to be more susceptible to TB [47]. Here, we found two studies [29,30] in the Chinese population that reported three different polymorphisms (rs1861759, rs7194886 and rs9302752) associated with increased TB risk. The rs1861759 (synonymous variant) TG genotype has been described to be related to higher risk of TB [29]. Individuals with the rs7194886 CT or TT genotype are more likely to develop TB [30]. The rs9302752 C allele has been linked to a higher risk of being sputum culture-positive TB. To our knowledge, these three polymorphisms do not appear in other TB studies. Curiously, such SNPs have been also related to increased risk of leprosy [48].
In an African-American study [31], it was observed that the three NOD2 polymorphisms exhibit impact on TB risk. The polymorphism rs2066844 represents missense mutations which variants are located in the C-terminal region and cause defective production of proin ammatory mediators [49]. The rs2066842 is a missense variant but, when presented alone, is not related with altered gene function [50]. Importantly, the two polymorphisms in our systematic review related to protection against TB in the context of the presence of allele C [31]. Nevertheless, the presence of the T allele in such mutations was contrastingly associated with increased TB risk. The association between the presence of the T allele and augmented pathology has been described for other diseases such as Crohn's disease [51] and gastric cancer [52]. The genotyping heterozygous (CG) of rs5743278 has been linked to higher risk of TB [51]. It is possible that the rs5743278 SNP causes an amino acid change from a low hydrophobic arginine to a highly hydrophobic tryptophan, modifying the stability of the NOD2 structure or its ability to properly interact with Mtb ligands [51]. Finally in our review one study in a population from Uganda described the NOD2 rs17313265 polymorphism as being related with increased susceptibility to TB in adults whereas the SNPs rs6500328 and rs2111234 exhibited an association with resistance to Mtb infection [28]. Furthermore, these three polymorphisms have not been reported in other studies with TB patients. The results presented so far indicate that NOD2 polymorphisms may be dramatically affected by ethnicity and/or ancestry. This scenario reinforces the need of additional investigations performed in a variety of ethnic populations and especially with study design using family-based control subjects to eliminate the bias in strati cation of the populations.
To our knowledge, the present study is the rst systematic review to explore the relation of all CD14 and NOD2 SNP polymorphisms with the susceptibility to pulmonary and/or extrapulmonary TB in different ethnicities. A strength of our study was the comprehensive search strategy, which used detailed inclusion and exclusion criteria. Moreover, methodological quality was assessed in duplicate using the Newcastle-Ottawa scale [20], which reduced the subjectivity of the selection of studies and allowed for precise evaluation of the risk of bias in several domains. There was no report categorized as low quality, resulting in a review with reduced risk of bias. Another important strength is that the inclusion of the SNPs was not limited to one speci c locus or ethnicity, allowing the study to observe different loci associated with TB risk or not in various populations.
Our study has some limitations. It was not possible to perform a meta-analysis mainly because a considerable part of the SNP polymorphisms appeared in only one study at a time and only a few were reported in more than one publication. Additionally, some studies lacked the Odds Ratio analyses, and for this reason we were able to perform only a detailed systematic investigation. However, the need for a study to compile and critically revise these results in different ethnicities reinforces the importance of our work, regardless of quantitative analyzes.
This review revealed that there is strong evidence that the T allele of rs2569190 CD14 polymorphism increases the risk of pulmonary and/or extrapulmonary TB in perons from different ethnicities. In addition, the CD14 SNP rs2569191 and the NOD2 SNPs rs1861759 and rs7194886 are shown here to be associated with a high TB risk in the Chinese population. In contrast, Minor allele carriers (CG or GG) of rs2066842 and rs2066844 were at low risk of TB in African Americans. Since such genes account for key molecules of the immune system, the referred polymorphisms of CD14 and NOD2 genes likely play an important role in TB physiopathology. These results add knowledge to the eld by reinforcing the genetic in uence on susceptibility to active TB. Such knowledge, if validated by larger studies, may help development of tools for assessment of TB risk and hopefully predict clinical outcomes in precision medicine approaches.