key: cord-0879282-wfljfiut authors: Zheng, Ruijuan; Zhou, Yan; Qin, Lianhua; Jin, Ruiliang; Wang, Jie; Lu, Junmei; Wang, WeiBing; Tang, Shenjie; Hu, Zhongyi title: Relationship between polymorphism of DC-SIGN (CD209) gene and the susceptibility to pulmonary tuberculosis in an eastern Chinese population date: 2010-11-14 journal: Hum Immunol DOI: 10.1016/j.humimm.2010.11.004 sha: 59061e34ef001be15318b4e286702b705b9b9547 doc_id: 879282 cord_uid: wfljfiut Dendritic cell–specific intracellular adhesion molecule–3–grabbing nonintegrin (DC-SIGN) is an important receptor for Mycobacterium tuberculosis on human dendritic cells. Previous studies have shown that the variation, especially the −871A/G and −336A/G in DC-SIGN promoter influenced the susceptibility to tuberculosis. We therefore investigated whether polymorphisms in the DC-SIGN gene were associated with susceptibility to tuberculosis in an eastern Chinese population. A total of 237 culture-positive pulmonary tuberculosis case patients and 244 controls were genotyped for −871A/G and −336A/G by pyrosequencing. Our results suggested that the 2 promoter variants of DC-SIGN gene were not associated with susceptibility to tuberculosis in Chinese. Further analysis showed that the allele -336G was associated with a protective effect against fever in pulmonary tuberculosis patients, but not against cavity formation. In addition, we compared the allelic frequencies of −871A/G and −336A/G in African, Caucasian, and Asian groups. The results showed that the tw forms of allelic frequencies detected Chinese individuals in our study were similar to the reported frequencies in other Asian populations but differed significantly from those in the African and Caucasian groups studied. The innate immune receptors, such as Toll-like receptors, Ctype lectins receptors, and Nod-like receptors, probably play a crucial role in effective immune responses to Mycobacterium tuberculosis (M tuberculosis) [1] . When the bacillus encounters the host, the specific pattern-recognition receptors on human macrophage and dendritic cell (DC) recognize the M tuberculosis and trigger phagocytic cell and then induce specific signaling pathways that lead to the induction of immune responses against the pathogens. Dendritic cellspecific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), encoded by CD209, is a C-type lectin that is primarily expressed on DC and alveolar macrophage [2, 3] . As a major recognition receptor on human DCs, DC-SIGN may influence the pathogenesis of tuberculosis. M tuberculosis interacts with DC-SIGN to activate an intracellular signaling pathway with a central role for the serine/threonine kinase Raf-1. The Raf-1-acetylationdependent signaling pathway, which induces specific gene transcription profiles, is involved in the regulation of adaptive immune response to tuberculosis [4 -6] . Genetic studies on tuberculosis have revealed that the genetic polymorphisms, potentially involved in the immune response to tuberculosis, could lead to susceptibility or resistance to tuberculosis. DC-SIGN might be a crucial part in host immunity to tuberculosis and one of the candidate genes for susceptibility to tuberculosis. In addition, it has been reported that the variants of DC-SIGN have been linked to susceptibility to several other infectious diseases, such as HIV-1 [7] , Dengue [8] , and severe acute respiratory syndrome (SARS) [9] . The research on DC-SIGN genetic variability and its association with tuberculosis mainly focused on two regions: promoter and neck. Particularly, the Ϫ336A/G loci in promoter in African populations have been studied by several groups. Barrerio et al. first reported that -336A and -871G conferred protection against tuberculosis in South African cohort [10] . Vannberg et al. further observed that -336G allele had protective effect against tuberculosis in populations from four different sub-Saharan African countries [11] . In contrast, Olesen et al., in the north African city of Tunis and Ben-Alim in the west African city of Guinea-Bissau found no association of -336 SNP polymorphism with tuberculosis [12, 13] . In non-African individuals from northwestern Colombia, GÔmez et al. reported that -336 SNP did not play a protective role against tuberculosis [14] . Some of these variants may affect the function of DC-SIGN. The variant Ϫ336A/G in the DC-SIGN gene promoter region was found to regulate promoter activity by affecting a Sp1-like binding site [8] . To date, little has been known about the association of the DC-SIGN gene variants and tuberculosis in Asian populations. Therefore, we initiated a case-control study to investigate the relationship between DC-SIGN gene variants (Ϫ871A/G and Ϫ336A/G) and susceptibility to tuberculosis in China, where the incidence and mortality rates of tuberculosis have increased in the past decade. The samples were comprised of 237 patients with pulmonary tuberculosis and 244 healthy controls. All blood samples were collected in Shanghai Pulmonary Hospital located in Eastern China from December 2007 to November 2008. The diagnosis of tuberculosis case depended on clinical radiological evidences (such as X-ray or computed tomography [CT] scan) and sputum culture positivity. All patients were human immunodeficiency virus (HIV) negative. A body temperature greater than 37.5ЊC was defined as indicating fever in the pulmonary tuberculosis patients. The average age of the 237 patients was 46.6 years (SE, 17.74 years); 155 subjects (65.4%) were male and 82 subjects (34.6%) were female. Control subjects were healthy individuals and were matched to patients by gender, age, ethnicity, and socioeconomic status. The study was approved by the Ethics Committees of the Shanghai Pulmonary Hospital affiliated with Tongji University, Shanghai China. The genomic DNA was extracted from 2 ml ethylenediaminetetraacetic acid (EDTA)-treated blood by using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). The DC-SIGN Ϫ871A/G (rs735239) and Ϫ336A/G (rs4804803) SNP assays were performed by pyrosequencing using a PSQ96MA system (Pyrosequencing AB, Uppsala, Sweden). The primers sequences were designed by PSQ Assay Design Software Version 1.1 and are presented in Table 1 . The PCR reaction was performed in a total volume of 50 l of reaction mix containing 50 ng of genomic DNA, 20 pmol of F and R primers, 0.2 mM of dNTP mixture, and 1.0 U of Taq polymerase. The PCR program amplified the DC-SIGN Ϫ871A/G was as follows: 5 minutes' denaturation at 95ЊC; 50 cycles of 30 seconds at 95ЊC, 30 seconds at 54ЊC, and 30 seconds at 72ЊC, with a final extension of 10 minutes at 72ЊC. The amplified condition of the DC-SIGN Ϫ336A/G was 95ЊC for 5 minutes, followed by 50 cycles of 95ЊC for 30 seconds, 58ЊC for 30 seconds, 72ЊC for 30 seconds, and then 1 cycle at 72ЊC for 10 minutes. Single strand separation was performed using a Vacuum Prep Tool and Vacuum Prep worktable (Biotage, Uppsala, Sweden) according to the manufacturer's recommendations. Pyrosequencing was performed in an automated PSQ96 MA system using PyroGold SNP reagent kit containing enzymes and substrates ((Biotage, Uppsala, Sweden). The allele and genotype frequencies were determined by direct counting. The significant differences of allele and genotype frequencies were calculated using Pearson 2 test. Age was considered as quantitative variables and determined by using the Student t test. The associations of genotype and patients' clinical features of tuberculosis were carried out by 2 test. Further analysis was performed by using a stepwise binary logistic regression correction to adjust for the confounding factors of age and gender when the unadjusted p value was significant. The analyses were performed by using the SPSS (SPSS, Inc., Chicago, IL). The Hardy-Weinberg equilibrium of each polymorphism was determined by the program HWE. Pairwise linkage disequibilibrium (LD) between SNPs was calculated using HaploView statistical software. The power analysis was performed by Gpower3.0 software. All statistical analyses were two-tailed, and p Ͻ 0.05 was considered statistically significant. The allele and genotype frequencies of the DC-SIGN allele at -871and -336 loci in 237 tuberculosis patients and 244 controls are presented in Table 2 . For the two polymorphisms tested in this study within the DC-SIGN gene, genotype distributions were in Hardy-Weinberg equilibrium in both patients and controls (all p Ͼ 0.05). For the -336 variant, genotypes GG and GA were less frequently observed in tuberculosis patients and controls, only one variant of genotype GG being observed in controls and none in patients. For the -871 variant, genotype AA was more frequent than genotypes AG and GG in patients and controls. However, statistical analyses showed that there was no significant difference in genotype and allele frequencies between tuberculosis patients and controls at the investigated loci. With regard to the power analysis of the whole study, the study had power of 99.2% to yield a statistically significant result using post hoc analysis when ␣ had been set at 0.05, effect size set at 0.2, and the total sample size was 481 for the two groups. In addition, we performed the analyses at the haplotype level. A 2 test first revealed that the global distribution of haplotype frequencies was not significantly different between PTB patients and controls (p ϭ 0.36). The analysis of haplotypes containing the two SNPs Ϫ336A/G and Ϫ871A/G showed no significant difference between the patients and controls (Table 3) . The DC-SIGN Ϫ871A/G and Ϫ336A/G SNP were further examined in the patients for association with clinical features of tuberculosis; results are presented in Table 4 . A 2 test first revealed a Adjusting for the potential confounding factor of age and gender, analysis of logistic regression demonstrated there was significant difference between DC-SIGN -336G and fever (p ϭ 0.037). The fever rate was 17% and 49% in -336G allele carriers and A allele carriers, respectively. The fever rate of G allele carriers decreased 32% compared with A allele carriers. In post hoc analysis, the study had power of 95% to yield a significant effect if ␣ had been set at 0.05, effect size set at 0.3, and the total sample size was 149 for patients with and without fevers. However, the significant association between DC-SIGN-336GG genotype and cavity formation described in previously study had not been observed in our research. There was no significant difference between the polymorphism of DC-SIGN Ϫ871A/G and clinical features (data not shown). To find whether allelic distribution was different among various ethnic origins, we compared the allelic frequencies between African populations, Caucasian populations reported by Barreiro et al., and the Chinese populations in the control group in our study, and found a significant difference in the distribution of DC-SIGN allelic frequencies among these ethnic groups. The allele -336G had the lowest frequencies in Asians compared with Africans and Caucasians ( Table 5 ). The frequencies of -871G in Asians were higher than in Africans and lower than in Caucasians. The allelic frequencies of two forms detected in our study were similar to the frequencies of Asian populations previously reported by Barreiro et al. and Sakuntabhai et al. [8, 10] . DC-SIGN is a type II transmembrane lectin receptor and has gained great attention in recent years because of its involvement in multiple aspects of immune-related function. DC-SIGN is involved not only in various DC functions, such as differentiation, migration, antigen capture and T-cell priming, but also in induction of intracellular signaling pathways via several motifs [15, 16] that can cause extensive modulation of immune responses [6] . In addition, DC-SIGN recognizes several pathogens, such as viruses (HIV-1, dengue, West Nile virus, and measles virus) [17, 18, 19, 20] , bacteria (Helicobacter pylori, Streptococcus pneumoniae) [15, 21] , fungi (Candida albicans and Aspergillus fumigatus) [22] and parasites (Leishmania and Schistosoma mansoni) [23] contributing to generation of pathogen-tailored immune responses [24] , induces immunosuppressive response by disarming the MAPK pathway in DCs [25] , and enhances TH1 differentiation. In this study, we detected the polymorphisms at -871 and -336 loci situated in the promoter region of the DC-SIGN gene in total 481 individuals. The two variants and haplotype of Ϫ871A/G and Ϫ336A/G in the DC-SIGN gene were not associated with the susceptibility to pulmonary tuberculosis in Chinese population, although the frequency of A allele in patients for both SNPs increased somewhat. The association between DC-SIGN polymorphisms and susceptibility to tuberculosis has been described in many studies, which showed positive association in South African and sub-Saharan African populations [10, 11] , whereas no association was observed in the Tunisian and Colombian populations studied [12, 14] . The host genetic background might be an explanation for this discrepancy [26, 27] . Our results showed that the allelic distribution of DC-SIGN gene in Asian populations differed from that of African and Caucasian populations and the difference between African and Asian groups was more prominent. The highest -336G allelic frequency was found in African populations and lowest was observed in Asians. Of our 481 samples, the genotype -336 GG was detected only in 1 sample. In the control group, the frequency of the -336G allele was 7.3%, whereas a high frequency (35-45%) was observed in African populations. Similarly, the allele -871G was absent in sub-Saharan African populations, whereas it had a frequency of 23.4% in our study [8, 10, 28, 29] . Although the two SNPs might be associated with resistance to TB in Africans, we failed to duplicate this association in the Chinese population. We speculated that for the two SNPs to take effect, other genes or combination of genes need to be involved. Further analysis about the relationship of the 2 variants in DC-SIGN gene promoter and the clinical features of the cases suggested a significant association between G allele in -336 loci and protection against fever in PTB patients in a Chinese population. Fever is a typical symptom of active tuberculosis. The potential role of DC-SIGN on fever tuberculosis disease is novel and warrants further investigation. M tuberculosis infection induces fever, and this symptom is attributed to an increased production of predominant endogenous pyrogen, such as interleukin (IL)-6, IL-1, and interferon (INF)-␥. Moreover, M tuberculosis Manose-dependent binding to CD209 leads to Raf-1-dependent upregulation of IL-6 [15] . However, the DC-SIGN -336G variant affects a Sp1 binding site and decreases the expression of DC-SIGN resulting in less efficient cytokine and chemokine secretion [8, 30] . It is hypothesized that individuals homozygous for the -336G allele may, in part, be protected from expressing low levels of DC-SIGN and therefore less prone to the IL-6 secretion induced by Manose-dependent binding to DC-SIGN. In conclusion, no significant association with tuberculosis was observed for the two variants of Ϫ871A/G and Ϫ336A/G in DC-SIGN gene in a Chinese population. However, the -336G allele was weakly associated with PTB patients' fever. In addition, our results offered the SNP frequencies in an eastern Chinese population, which might be useful for the study in ethnic groups. Mycobacterial interaction with innate receptors: TLRs, C-type lectins, and NLRs DC-SIGN, a related gene, DC-SIGNR and CD23 form a cluster on 19p13 Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses Innate signaling by the C-type lectin DC-SIGN dictates immune responses DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB Association of DC-SIGN promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type 1 infection A variant in the CD209 promoter is associated with severity of dengue disease Polymorphisms in the C-type lectin genes cluster in chromosome 19 and predisposition to severe acute respiratory syndrome coronavirus (SARS-CoV) infection Promoter variation in the DC-SIGN encoding gene CD209 is associated with tuberculosis CD209 genetic polymorphism and tuberculosis disease Promoter and neck region length variation of DC-SIGN is not associated with susceptibility to tuberculosis in Tunisian patients DC-SIGN (CD209), PENTRAXIN 3 and vitamin D receptor gene variants associate with pulmonary tuberculosis risk in West Africans Analysis of DC-SIGN (CD209) functional variants in patients with tuberculosis Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis, HIV-1 and Helicobacter pylori Salp15 binding to DC-SIGN inhibits cytokine expression by impairing both nucleosome remodeling and mRNA stabilization DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells Reconstruction of dengue virus in complex with the carbohydrate recognition domain of DC-SIGN The location of asparagine-linked glycans on West Nile virions controls their interactions with CD209 (dendritic cell-specific ICAM-3 grabbing nonintegrin) Measles virus targets DC-SIGN to enhance dendritic cell infection DC-SIGN specifically recognizes Streptococcus pneumoniae serotypes 3 and 14 The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells Cutting edge: Carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells Pathogen recognition by DC-SIGN shapes adaptive immunity Sakazakii targets DC-SIGN to induce immunosuppressive responses in dendritic cells by modulating MAPKs Genetics of susceptibility to human infectious disease The human model: A genetic dissection of immunity to infection in natural conditions DC-SIGN and DC-SIGNR genetic diversity among different ethnic populations: Potential implications for pathogen recognition and disease susceptibility The heritage of pathogen pressures and ancient demography in the human innateimmunity CD209/CD209L region CD209 DC SIGN -336AϾG promoter polymorphism and severe acute respiratory syndrome in Hong Kong Chinese We thank all patients and participants of this study. We also thank Shenjie Tang for sample assistance and WeiBing Wang for statistical analysis. This work was supported by the Grants from Science and Technology Commission of Shanghai Municipality (nos. 08DZ2271300, 08DZ2291500, and 08JC1420000).