A case-control study on correlation between the single nucleotide polymorphism of CLEC4E and the susceptibility to tuberculosis among Han people in Western China

Tuberculosis (TB) is the leading cause of death among infectious diseases worldwide. China, which is the third highest-burdened country, accounted for 8.4% of the total global cases in 2019 [1]. The pathogen of tuberculosis is the Mycobacterium tuberculosis (MTB). Although approximately one-third of the people worldwide are infected with MTB, it is worth noting that only 3–10% of them eventually develop active clinical TB during their lifetime [2]. The occurrence or development of tuberculosis is determined by the complex interaction between three factors, the MTB strain itself, environmental, and host genetic factors [3‐5]. Host genetics has been revealed to be important in determining disease progression and outcomes after MTB infection by many animal models studies, twin and family studies, as well as numerous case-control studies [6].

When MTB invades the host, it initially faces the innate immunity, which is modulated by the innate immunity genes. Pattern recognition receptors (PRRs) are key signaling molecules of the innate immune system that affect the initial identification of MTB [7]. Numerous studies have shown that genetic variation of the of PPRs, such as C-type lectin receptors (CLRs), Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs) or their adapter protein-coding gene, is involved in modulating MTB-mediated immune responses and participate in determining the outcomes of MTB infection [8‐10].

Macrophage-inducible C-type lectin (Mincle) is a newly described macrophage-inducible CLR, encoded by the C-type Lectin Receptor 4E (CLEC4E). Trehalose-6,6′-dimycolate (TDM), also known as cord factor, is the most abundant cell wall glycolipid of MTB that is important for the initial identification of MTB. It has been reported that Mincle could be considered as the mammalian receptor for TDM from MTB [11]. Lu et al found impaired production of interleukin-6 together with tissue necrosis factor in TDM-stimulated macrophages from Mincle^−/− mice exposed to Malassezia spp [12]. Moreover, in response to a TB vaccine containing trehalose-6,6′-dibehenate (TDB), a synthetic analog of TDM, Mincle has been shown to have a pivotal role in the generation of Th1/Th17 cell immune responses and granuloma formation [13]. These studies all suggested that Mincle has a significant role in recognition of mycobacteria.

So far, only two studies have investigated the relationship between the CLEC4E gene and TB susceptibility in humans [14, 15]. Yet, these two studies both had a small sample size, and their results were controversial. In order to evaluate the possible function of CLEC4E gene variants in TB, more studies need to be conducted. Moreover, such research has never been carried out among the western China population. Consequently, this relatively large-scale study was designed to investigate whether the single nucleotide polymorphisms (SNPs) in the CLEC4E gene were associated with susceptibility to TB in a Han population from Western China.

The role of host’s genetic factor in tuberculosis susceptibility has gained increasing attention in TB research over recent years. Mincle is an indispensable receptor for TDM-induced innate immune responses (such as granuloma genesis) and in vitro macrophage activation during mycobacterial infection [25]. In the present study of the Western Han Chinese population, rs10841856 minor G allele of CLEC4E, which was the coding gene of Mincle, significantly increased the susceptibility to tuberculosis, especially among male subjects. Interestingly, Deo et al [14] suggested that for rs10841847, the minor G allele was a risk factor of pulmonary tuberculosis infection in a northern Chinese population. According to the 1000 Genomes Project, rs10841847, which is also an intronic variation of CLEC4E, is in strong linkage disequilibrium (LD) with rs10841856 (D′ = 0.95). Our findings on the association with TB risk of rs10841856 in male individuals supported the suggestion of the involvement of CLEC4E genetic polymorphism in TB. Nevertheless, Bowker et al [15] genotyped four tagSNPs of CLEC4E, reporting no differences in these SNPs between South Africa TB patients and controls. Such different observations might reflect the existence of many confounding factors, including ethnic background and sample size.

TB has a higher incidence in males than in females. In 2018, males accounted for 68%, while females accounted for only 31% of TB patients in China [1]. In our study, TB was also more common in males than in female individuals. Recently, Haiko et al [26] conducted a Genome-Wide association study that emphasizes the importance of sex-stratification analysis, because strong sex-specific effects are found on both autosomes and X chromosomes, and these effects should be considered when studying the association with SNPs and TB. When the whole data were stratified according to sex, in rs10841856, the G allele was a risk genotype for TB, especially in males. A significant difference was also found only in males when the association was calculated under the dominant model. This study showed the impact of sex on TB for CLEC4E rs10841856. Sex-specific effects of gene SNPs have been previously described in some diseases, including TB [27‐29] As far as we know, this is the first report that described sex-specific interactions for variants in CLEC4E, which could be used as a basis for replication studies in independent populations.

The rs10841856 polymorphism is located in the intronic region. Although genetic polymorphisms in intron regions are not generally thought to cause changes in the encoded amino acids, they may affect splicing, transcription, and expression of genes [30‐32]. According to data from the GTEx project, rs10841856 might be an eQTL of CLEC4E and RP11-561P12.5. Rs10841856 polymorphism decreased the expression of CLEC4E and increased the expression of RP11-561P12.5 in whole blood. The decreased expression of CLEC4E was associated with bacterial infection and has been observed in several studies [33, 34]. For MTB infection, Pahari et al [35]. observed that CLEC4E agonist could improve host immunity and reduced bacterial load in the lungs of the infected mice. They elucidated the novel role of CLEC4E in inducing autophagy during defending MTB infection. Rs10841856 might be associated with CLEC4E expression decrease, which may weaken the defense ability against MTB. RP11-561P12.5 is a lncRNA located at chromosome 12: 8700957-8720209, adjoining CLEC4E. Although there are scarce reports on the biological functions of RP11-561P12.5, according to the lncRNASNP2 database, RP11-561P12.5 may bind to miR-197-3p. Van Rensburg et al [36] demonstrated that the neutrophil-associated miR-197-3p showed significantly lower transcript levels in TB cases; meanwhile, miR-197-3p acted as a binding site on the 3’UTR region of IL-22 receptor IL22RA1, thereby affecting the production of IL-22 [37]. IL-22 can inhibit MTB growth within macrophages [38] and promotes the innate immune responses, thereby limiting damage during pathogen infections [39]. The rs10841856 polymorphism influences the expression of RP11-561P12.5. We speculated that by binding to miR-197-3p, lncRNA RP11-561P12.5 might have a similar mechanistic effect on the production of IL-22 that are also involved in the occurrence of TB.

No association of the other 3 SNPs (rs4480590, rs10770847, and rs10770855) was found with tuberculosis in this study after Bonferroni correction. To date, there was no TB-related research on rs4480590, rs10770847 and rs10770855. These three SNPs may not be related to TB risk in the Western China Han population. However, multicenter studies with large samples are needed to further verify these findings.

The present study has some limitations. Firstly, SNPs were mainly detected in the intrinsic region. Thus, variants in exons and regulatory genetic sequences should be taken into consideration, which means that more comprehensive and systematic variants of association studies are needed in the future. Secondly, the individuals involved in our study were all from the Western China Han population, which suggests that as same as for any novel genetic association, our findings should be replicated in other population and functional tests, and pathway analyses are required to validate our findings further.

In conclusion, the strong association was observed between the G allele and the dominate model of rs10841856 and the susceptibility of TB among males in a western Chinese Han population. Rs10841856 and its strong LD SNPs are associated with a decrease in CLEC4E and an increase in RP11-561P12.5. Accordingly, rs10841856 in CLEC4E might be a novel mutation that has a significant role in increasing the risk of TB among the male Han population from Western China.