Chemokines play a major role in leukocyte recruitment during the formation of tuberculous granulomas. We studied the association between genetic polymorphisms of three chemokines, monocyte chemoattractant protein-1 (MCP-1), RANTES (regulated on activation, normal T cell expressed and secreted) and macrophage inflammatory protein-1α (MIP-1α), and tuberculosis (TB). The distribution of five functionally significant single-nucleotide polymorphisms (SNPs), MCP-1 −2518A/G, RANTES −403G/A, −28C/G and In1.1T/C as well as MIP-1α +459C/T was not found to be different between patients with TB and healthy control subjects of the Hong Kong Chinese population. However, differences in linkage disequilibrium (LD) of the SNPs of RANTES and in distribution of the haplotypes of RANTES between patients with TB and healthy controls (P<0.0001) were found. Two risk haplotypes of RANTES, A-C-T and G-C-C, at positions −403, −28 and In1.1, respectively, were identified. Furthermore, combining the genotypes of RANTES −403 and In1.1, two diplotypes GA/TT (P<0.001) and GG/TC (P<0.0001) showed strong association with TB. Our findings support the association between RANTES functional polymorphisms and TB.
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB). It has been estimated that one-third of the human populations are infected by MTB. Hong Kong is a place with an intermediate burden of TB and a low prevalence of HIV infection.1 Among those infected by MTB, only 5–10% will ever develop clinical disease.2 Studies of the concordance rate of TB among monozygotic and dizygotic twins supported the importance of host genetic factors in determining the development of disease.3
Our group has previously identified the association between interleukin-12B (IL12B)4 and interferon-gamma (IFN-γ)5 genetic polymorphisms and TB. We reason that functional polymorphisms of those key cytokine genes which regulate the human immune response against MTB will influence the individual's susceptibility to developing clinical disease after infection. Recently, family studies showed the linkage of human 17q11.2 chromosomal region to susceptibility to TB.6, 7 The region consists of genes encoding β chemokines which are known to contribute to host immunity against TB. The infection of macrophages by MTB induces the production of β chemokines, particularly macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant protein-1 (MCP-1) and RANTES (regulated on activation, normal T-cell expressed and secreted). These chemokines mediate the migration and activation of T cells8 and macrophages9 to sites of MTB infection, where they form the characteristic tuberculous granuloma,10 which serves to contain the organism and prevent its dissemination to other tissues and organs.11, 12 A recent study further suggested that β chemokines might play a role in the inhibition of intracellular growth of MTB.13
Single-nucleotide polymorphisms (SNPs) in MCP-114 and RANTES15, 16 promoters influence expression of these genes, and SNPs in RANTES16, 17, 18 and MIP-1α19 genes are associated with susceptibility to HIV infection. Recently, a functionally important polymorphism is found in the intron of RANTES, In1.1T/C, which regulates the transcription level of the gene by differentially binding to alternative forms of nuclear proteins.18 We investigated five SNPs on these genes, MCP-1 −2518G/A (dbSNP: rs1024611), RANTES, −403G/A (dbSNP: rs2107538), −28C/G (dbSNP: rs2280788) and In1.1T/C (dbSNP: rs2280789), and MIP-1α +459C/T (dbSNP: rs1719134), to determine whether they are associated with susceptibility to TB in Hong Kong Chinese.
Characteristics of patients with tuberculosis and healthy control subjects
Five hundred and forty-seven Hong Kong Chinese patients with clinical TB (mean age±s.d., 47.9±18.1 years; male to female ratio 1.96:1) were prospectively recruited from 18 territory wide chest clinics under the Department of Health of Hong Kong Special Administrative Region (HKSAR) during the period 2002–2003. Active cases of TB were defined as cases proven by the presence of M. tuberculosis in sputum or other types of specimen (n=351), or observation of caseation or granuloma in biopsy specimen or detection of a positive Ziehl–Neelsen stain (n=39), or both bacteriological and histological evidences (n=22). Among these active TB cases, 86% of cases were newly diagnosed. These 412 confirmed TB cases formed the subjects of this study. Healthy control subjects (n=465; mean age±s.d., 30.9±8.8 years; male to female ratio 0.97:1) were blood donors of Hong Kong Red Cross.
Analysis of association between β chemokine gene polymorphisms and tuberculosis
All five test SNPs were genotyped for 412 patients with TB and 465 healthy control subjects. Genotyping was successful in over 97% of cases and healthy controls. The distribution of the test SNPs was in Hardy–Weinberg equilibrium (HWE), except RANTES −28C/G (χ2=5.29, P=0.03). No difference in the distribution of genotype frequencies between TB cases and controls was found (Table 1).
Haplotypes of RANTES polymorphisms are associated with susceptibility to tuberculosis
Strong linkage disequilibrium (LD) was observed between each pair of SNPs, particularly between RANTES −403 and RANTES In1.1 (Table 2). Interestingly, a stronger correlation was observed in healthy control subjects compared with TB cases. The haplotype frequencies of RANTES was constructed with the three SNPs of RANTES by the expectation-maximization (EM) algorithm (Table 3). A significant difference was found in haplotype frequencies between patients with TB and control subjects (P<0.0001), which was mainly contributed by haplotypes II and V (Table 3). The genotype data of RANTES −403A/G and RANTES In1.1T/C were combined to form nine diplotypes. Actual number of patients with TB and control subjects for each diplotype was counted (Table 4). A significant difference was found in diplotype frequencies between patients with TB and control subjects (P<0.0001). Two diplotypes, F (GA/TT) and H (GG/TC), were found to be much more common in patients with TB than control subjects (Table 4).
This is the first population-based case–control study reporting the association between RANTES polymorphisms and TB. We first observed that the three RANTES SNPs were in strong LD. In particular, the LD between RANTES −403 and In1.1 in the healthy controls was 0.98. Interestingly, the LD between RANTES −403 and In1.1 in TB cases was less strong at 0.70, which inferred that a difference in distribution of haplotype of these SNPs might exist between TB cases and the healthy controls. We thus estimated the haplotype frequencies of the RANTES SNPs among the TB cases and controls (refer to Table 3) and found that there was indeed a significant difference in the distribution of RANTES haplotypes between the cases and controls (P<0.0001). Furthermore, we identified two risk RANTES haplotypes, designated II (A-C-T) and V (G-C-C), which occurred at much higher frequencies in patients than controls with no overlapping of their 95% confidence interval (Table 3). Next, we constructed diplotypes of RANTES −403 and In1.1 and confirmed significant difference in the distribution of such diplotypes between TB cases and controls. Among the nine diplotypes, F and H were much more common in TB cases than controls (Table 4). Since the most common RANTES haplotype (occurs in >60% of individuals) is haplotype VI (G-C-T), we conclude that diplotype F (GA/TT) is likely the combination of haplotypes II (A-C-T) and VI and diplotype H (GG/TC) is likely the combination of haplotypes V (G-C-C) and VI. Our findings support the association between RANTES polymorphisms and TB and identify the risk haplotypes of RANTES that confer the association.
We postulate that haplotypes II and V could be in linkage with two completely different genetic traits, which contribute to susceptibility to TB. Similar postulation was proposed in a recent study7 in which more than one functional polymorphism across the β chemokine region was found to contribute to susceptibility to TB. According to a study using luciferase assay correlating RANTES SNPs with its transcription activity,18 the two risk haplotypes, II and V, act oppositely by up- and downregulating RANTES transcription, respectively.18 RANTES concentrations in bronchoalveolar lavage fluid of pulmonary TB subjects rise acutely20 and fall during convalescence,21 indicating that upregulation of RANTES plays an important role in the immune response against MTB infection. RANTES does not function as a typical chemokine at high concentrations. At nanomolar concentrations, RANTES can signal through its specific chemokine receptors in a monomeric form, while it self-aggregates and interacts with cell-surface glycosaminoglycans (GAGs) at micromolar concentrations.22 The binding of RANTES to GAGs, which are part of the extracellular matrix, is believed to contribute to the establishment of chemotactic gradients. GAGs also mediate the oligomerization or self-aggregation of RANTES, which can result in the accidental crosslinking of cell-surface signaling molecules and in unwanted and nonspecific cell activation.22 We postulate that RANTES may function optimally at a narrow range of concentrations and production of RANTES above or below this optimal range of concentrations may be associated with impaired function of this chemokine and thus increased susceptibility to infectious disease such as TB.
Our result of MCP-1 −2518 allele contrasts with that of Flores-Villanueva et al.,23 who found association of the MCP-1 −2518 allele G with susceptibility to TB in Mexicans and Koreans. The positive association seen in Koreans has to be interpreted with caution due to small sample size (129 TB cases and 162 healthy controls) of this study. However, there appear genuine difference between Chinese and Mexicans in the role of MCP-1 promoter allele polymorphism in conferring risk to TB.
In conclusion, we observed difference in the strength of LD of RANTES SNPS between patients with TB and healthy controls. We subsequently found the association between specific haplotypes of RANTES −403, −28 and In1.1 and TB in Hong Kong Chinese. Future studies using a higher density of SNPs in the genomic region incorporating these RANTES SNPs may yield additional information on genetic susceptibility to TB.
Patients and methods
The present study was approved by the Ethics Committees of the Faculty of Medicine, the University of Hong Kong and the Department of Health, HKSAR. Patients were recruited, after obtaining informed consent, from territory-wide chest clinics under the Department of Health of HKSAR during the period 2002–2003 and the healthy controls were blood donors of Hong Kong Red Cross during the period 2002–2004. All active TB cases had microbiological or histological evidence of M. tuberculosis infection. No subjects had HIV infection.
Genomic DNA was isolated from frozen whole blood-ethylene diamine tetraacetic acid of patients and buffy-coat lymphocytes of controls using QIAamp DNA Blood Mini Kit (Qiagen Inc., CA, USA), according to the manufacturer's instructions.
Polymerase chain reaction-restriction fragment length polymorphism (RFLP) methods, as described previously, were used to genotype patients and controls for MCP-1 −2518G/A,14 RANTES −403G/A, −28C/G,24 and In1.1 T/C,17 as well as, MIP-1α +459C/T.19 Genotypes were confirmed by direct sequencing with the same primers for RFLP of MCP-1 and MIP-1α genotyping, and with the following primers to amplify the promoter sequence of RANTES: forward, 5′-IndexTermTGA CCA GGA TGA AAG CAA GA-3′ and reverse, 5′-IndexTermCTT CAT GGT ACC TGT GGG AGA-3′, and the first intron of RANTES: forward, 5′-IndexTermCCT GGT CTT GAC CAC CAC A-3′, and reverse, 5′-IndexTermGCT GAC AGG CAT GAG TCA GA-3′. All restriction enzymes were purchased from New England Biolabs Inc., Beverly, MA, USA.
All statistical analyses were performed using SAS version 8.02 and SAS/Genetics software (SAS Institute, Cary, NC, USA). The genotype frequencies of all SNPs were tested for HWE and were compared between patients with TB and control subjects by χ2 tests. The level of statistical significance was set at <0.05. LD, as measured by correlation coefficient (r2), between SNPs was calculated. Haplotype reconstruction was performed using the EM algorithm and difference in haplotype distribution between cases and controls was detected with permutation of 1000 times. The test statistic (T) was defined as T=−2(ln Lpc−ln Lp−ln Lc), where ln Lpc is the natural logarithm of the maximum-likelihood estimates for patients with TB plus control subjects, ln Lp is the natural logarithm of the maximum-likelihood estimates for patients with TB and ln Lc is the natural logarithm of the maximum-likelihood estimates for control subjects. Diplotypes were also constructed to compare the difference between cases and controls.
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We thank Ms Ivy Ng for sequencing primer designs and technical support. The present study was approved by the Ethics Committees of the Faculty of Medicine, The University of Hong Kong and the Department of Health, Hong Kong Special Administrative Region (HKSAR), China. All authors do not have any commercial or other association that might pose a conflict of the interest in this research. This study was supported by research grants to AKSC (HKU CRCG#10205036 and 10205662). SFC was supported by Research Postgraduate Studentship of The University of Hong Kong. Part of the information in this paper was presented at the 3rd Hong Kong Medical Genetics Conference in April 2005 in Hong Kong Special Administrate Region, China and at the European Human Genetics Conference 2005 in May 2005 in Prague, Czech Republic. Information in this paper formed part of the MPhil thesis of SFC at The University of Hong Kong.
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Chu, S., Tam, C., Wong, H. et al. Association between RANTES functional polymorphisms and tuberculosis in Hong Kong Chinese. Genes Immun 8, 475–479 (2007). https://doi.org/10.1038/sj.gene.6364412
- genetic susceptibility