Zhou et al. BMC Infectious Diseases (2021) 21:788 https://doi.org/10.1186/s12879-021-06448-2 RESEARCH ARTICLE Open Access A case-control study on correlation between the single nucleotide polymorphism of CLEC4E and the susceptibility to tuberculosis among Han people in Western China Wenjing Zhou, Lijuan Wu, Jiajia Song, Lin Jiao, Yi Zhou, Juan Zhou, Nian Wang, Tangyuheng Liu, Zhenzhen Zhao, Hao Bai, Tao Wu and Binwu Ying* Abstract Background: Tuberculosis (TB) is one of the leading causes of morbidity and mortality in Western China. Preclinical studies have suggested the protective effect of the C-type lectin receptor of family 4 member E (CLEC4E) from TB. Herein, we investigated the association between CLEC4E gene variants and TB susceptibility in a western Chinese Han population. Methods: We genotyped four single nucleotide polymorphisms (SNPs) rs10841856, rs10770847, rs10770855 and rs4480590 in the CLEC4E gene using the improved multiplex ligation detection reaction (iMLDR) assay in 900 TB cases and 1534 healthy controls. Results: After stratifying the whole data by sex, it was found that males exhibited mutant allele G of rs10841856 was more strongly associated with increased TB risk after Bonferroni correction (OR = 1.334, 95% CI: 1.142–1.560; P < 0.001 after adjusting for age; p = 0.001 after Bonferroni correction). The genetic model analysis found that rs10841856 was associated with the increased risk of TB among males under the dominant model (OR = 1.557, 95% CI = 1.228–1.984, P < 0.001 after adjusting for age, P < 0.001 after Bonferroni correction). Bioinformatics analysis suggested that rs10841856 might fall in putative functional regions and might be the expression quantitative trait loci (eQTL) for CLEC4E and long noncoding RNA RP11-561P12.5. Conclusions: Our study revealed that rs10841856 in the CLEC4E gene might be related to increased TB risk, especially the dominant genetic model among male Han individuals from Western China Keywords: Tuberculosis, CLEC4E, Single nucleotide polymorphisms, Disease susceptibility * Correspondence: [email protected] Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhou et al. BMC Infectious Diseases (2021) 21:788 Page 2 of 8 Introduction among the western China population. Consequently, this Tuberculosis (TB) is the leading cause of death among relatively large-scale study was designed to investigate infectious diseases worldwide. China, which is the third whether the single nucleotide polymorphisms (SNPs) in highest-burdened country, accounted for 8.4% of the the CLEC4E gene were associated with susceptibility to total global cases in 2019 [1]. The pathogen of tubercu- TB in a Han population from Western China. losis is the Mycobacterium tuberculosis (MTB). Although approximately one-third of the people worldwide are in- Methods fected with MTB, it is worth noting that only 3–10% of Subjects them eventually develop active clinical TB during their Chinese Western Han individuals with TB recruited lifetime [2]. The occurrence or development of tubercu- from West China Hospital of Sichuan University be- losis is determined by the complex interaction between tween January 2014 and February 2016 were enrolled. three factors, the MTB strain itself, environmental, and TB was diagnosed according to TB guidelines [16] based host genetic factors [3–5]. Host genetics has been re- on their laboratory test results, clinical symptoms, and vealed to be important in determining disease progres- radiological examination. The inclusion criteria are typ- sion and outcomes after MTB infection by many animal ical symptoms and signs of tuberculosis and meet at models studies, twin and family studies, as well as nu- least one of the following conditions: (1) smear positive merous case-control studies [6]. for at least two separate clinical specimens and/or cul- When MTB invades the host, it initially faces the in- ture positive for MTB and/or examination positive for nate immunity, which is modulated by the innate im- MTB nucleic acid (TB-DNA) (2) CT and other imaging munity genes. Pattern recognition receptors (PRRs) are examinations showed typical manifestations of active tu- key signaling molecules of the innate immune system berculosis. (3) pathological diagnosis supports tubercu- that affect the initial identification of MTB [7]. Numer- losis lesions. Patients suffer from immunodeficiency, ous studies have shown that genetic variation of the of autoimmune diseases, or other infectious diseases were PPRs, such as C-type lectin receptors (CLRs), Toll-like excluded. Healthy controls were enrolled from the same receptors (TLRs), RIG-I-like receptors (RLRs), and population in the same period from the Physical Exam- NOD-like receptors (NLRs) or their adapter protein- ination Center in West China Hospital of Sichuan Uni- coding gene, is involved in modulating MTB-mediated versity. They were all healthy according to normal immune responses and participate in determining the laboratory test, physical examination and imaging exam- outcomes of MTB infection [8–10]. ination. Individuals with TB history or non-Han popula- Macrophage-inducible C-type lectin (Mincle) is a tion were excluded. Finally, 900 TB cases and 1534 newly described macrophage-inducible CLR, encoded by healthy controls were enrolled. the C-type Lectin Receptor 4E (CLEC4E). Trehalose- The study protocol has been reviewed and approved 6,6′-dimycolate (TDM), also known as cord factor, is the by the Ethics Committee of West China Hospital of most abundant cell wall glycolipid of MTB that is im- Sichuan University. Written informed consent was portant for the initial identification of MTB. It has been obtained from all participants before performing any reported that Mincle could be considered as the mam- study-related procedure. malian receptor for TDM from MTB [11]. Lu et al found impaired production of interleukin-6 together with tis- SNP selection and genotyping sue necrosis factor in TDM-stimulated macrophages Genetic variation information of the CLEC4E and inter- − − from Mincle / mice exposed to Malassezia spp [12]. genic regions of its upstream and downstream were ob- Moreover, in response to a TB vaccine containing tained from the dbSNP database https://www.ncbi.nlm. trehalose-6,6′-dibehenate (TDB), a synthetic analog of nih.gov/SNP/. Haploview V4.2 was then employed to TDM, Mincle has been shown to have a pivotal role in run the TagSNPs with a threshold of r2 greater than or the generation of Th1/Th17 cell immune responses and equal to 0.8 from rescored SNPs. TagSNP with a minor granuloma formation [13]. These studies all suggested allele frequency of (MAF) > 0.20 according to 1000 Ge- that Mincle has a significant role in recognition of nomes Project in East Asian population was selected. mycobacteria. Peripheral blood samples were collected from 2434 in- So far, only two studies have investigated the relation- dividuals and transferred to the biological specimen ship between the CLEC4E gene and TB susceptibility in bank of resources of “Tuberculosis Researches” in the humans [14, 15]. Yet, these two studies both had a small Department of Laboratory Medicine, West China Hos- sample size, and their results were controversial. In pital, Sichuan University for preservation, and the demo- order to evaluate the possible function of CLEC4E gene graphic information of these individuals was gathered. variants in TB, more studies need to be conducted. Genomic DNA was extracted by the QIAamp® DNA Moreover, such research has never been carried out Blood Mini Kit (Qiagen, Hilden, Germany). Improved Zhou et al. BMC Infectious Diseases (2021) 21:788 Page 3 of 8 multiplex ligation detection reaction (iMLDR) method Table 1 Basic characteristics of the participants enrolled in the (Genesky Biotechnologies Inc., Shanghai, China) [17] study was used to genotype SNPs. 10% of samples were Characteristic TB, n (%) Control, n (%) P-value randomly selected for re-genotyping to check for Sex concordance and the reproducibility of the genotyping Male 542 (60.22) 821 (53.52) <0.001 was 100%. Female 358 (39.78) 713 (46.48) Statistical analysis Male/Female ratio 1.514 1.151 The Chi-square test, independent t-test, and Mann- Age (years) Whitney U test were applied for general variables. All Median 41 (27,57) 36 (29,45) <0.001 these calculations were performed by SPSS 20.0 (IBM, Male, Median 44.5 (26,58) 38 (30,47) <0.001 Chicago, USA). The Hardy-Weinberg equilibrium Female, Median 37 (27,54) 34 (29,43) <0.001 (HWE), differences of genotype distribution, and allele Age group frequency of candidate SNPs between the TB group and control group or between age and sex subgroup were < 40 417 (46.33) 922 (60.10) <0.001 analyzed using PLINK 1.07 software [18]. Unconditional ≥ 40 483 (53.67) 612 (39.90) logistic regression models were used to test for the dom- Age group-sex 0.036 inant model and recessive model.