1. The IFN-gamma +874A allele and RANTES -28 G allele are risk factors for SARS susceptibility. 2. The RANTES -28 G allele plays a role in the pathogenesis of SARS. 3. The polymorphisms of IL-10, TNF-alpha, IL-12, IP-10, Mig and MCP-1 are not associated with SARS susceptibility.published_or_final_versio

Lau, YL

Peiris, JS

Hong Kong Medical Journal

HKU Scholars Hub

Title Association of cytokine and chemokine gene polymorphismswith severe acute respiratory syndromeAuthor(s) Lau, YL; Peiris, JSCitation Hong Kong Medical Journal, 2009, v. 15 suppl. 2, p. 43-46Issued Date 2009URL http://hdl.handle.net/10722/170428Rights Creative Commons: Attribution 3.0 Hong Kong LicenseHong Kong Med J Vol 15 No 1 Supplement 2 February 2009      43RESEARCH FUND FOR THE CONTROL OF INFECTIOUS DISEASESDepartment of Paediatrics and Adolescent Medicine, The University of Hong KongYL LauDepartment of Microbiology, The University of Hong KongJMS PeirisRFCID project number: 03040302Principal applicant and corresponding author:Dr Yu-lung LauDepartment of Paediatrics and Adolescent Medicine, Queen Mary Hospital, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, ChinaTel: (852) 2855 4482Fax: (852) 2855 1523E-mail: lauylung@hkucc.hku.hkYL Lau 劉宇隆JSM Peiris 裴偉士 Association of cytokine and chemokine gene polymorphisms with severe acute respiratory syndromeKey Messages1. The IFN-γ +874A allele and RANTES -28 G allele are risk factors for SARS susceptibility.2. The RANTES -28 G allele plays a role in the pathogenesis of SARS.3. The polymorphisms of IL-10, TNF-α, IL-12, IP-10, Mig and MCP-1 are not associated with SARS susceptibility.Hong Kong Med J 2009;15(Suppl 2):S43-6IntroductionSevere acute respiratory syndrome (SARS) is an infectious disease caused by the SARS coronavirus,1 but the pathogenesis is still far from clear.2 We have demonstrated that genetic haplotypes associated with low-serum mannose-binding lectin (MBL) are associated with SARS,3 and our findings have been replicated recently.4 Hong Kong Chinese who are homozygotes for CLEC4M tandem repeats have recently been reported to be less susceptible to SARS. Other susceptibility genes, such as OAS-1 and MxA have also been identified. Cytokines are known to take an important role in antiviral action. Interferon (IFN)-γ from T and natural killer (NK) cells is important for driving the T helper cell type 1 (Th1) responses. It also activates monocytes and macrophages, which in turn take part in antiviral responses by producing free radicals and pro-inflammatory cytokines like tumour necrosis factor (TNF)-α. On the other hand, interleukin (IL)-10 counteracts the inflammatory response by inhibiting TNF-α production and neutrophil activation. Interleukin-12 is important for the development of the T helper type 1 (Th1) response in the initial phase of bacterial, parasitic, and viral infections. In many viral infections, IL-12 promotes viral clearance and host recovery from infection. Chemokines play an important role in cell trafficking during immune responses. Acute respiratory viruses commonly induce inflammatory chemokines in local tissue. In a previous study we confirmed that the SARS coronavirus induces upregulation of a number of inflammatory chemokines, ie Regulated upon Activation Normal T cell-Expressed and Secreted (RANTES), interferon-gamma inducible protein 10 (IP-10) and Monocyte Chemoattractant Protein-1 (MCP-1). The upregulation of these chemokines, including the monokine induced by interferon gene (Mig) may recruit inflammatory cells and leukocytes into the tissue. In this study, we hypothesised that polymorphisms of the cytokine genes, IFN-γ, IL-10, TNF-α, IL-12, and the chemokine genes, RANTES, IP-10, Mig and MCP-1, might be associated with SARS. These genes were chosen for their key roles in antiviral action and inflammation regulation and their polymorphisms based on their potential regulation of gene expression.MethodsThis study was conducted from September 2005 to August 2006.Patient populationsThe study was approved by the Clinical Research Ethics Committee of the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. This study included 495 Hong Kong Chinese patients with SARS (211 males and 284 females, mean±SD age was 40.74±15.73 years) and 578 ethnically matched healthy controls from the Red Cross (343 males and 235 females, mean±SD age was 30.05±9.49 years). At least 95% of the patients were documented as having SARS-CoV antibody seroconversion and/or detectable SARS-CoV RNA in respiratory secretions by RT-PCR. The death group consisted of 57 patients who died from SARS and their mean±SD age was Lau and Peiris44      Hong Kong Med J Vol 15 No 1 Supplement 2 February 200956.2±15.3 years, with 33 males and 24 females.GenotypingIFN-γ +874A/T, IL-10 -1082G/A, IL-10 -592A/C, IL-12B promoter 318/322, IL-12B intron 2 218/221, IL-12B intron 4 268/272, IL-12B 3’UTR A/C and TNF-α -308 G/A were genotyped using the TaqMan system (Applied Biosystems, CA, USA). RANTES -28C/G and MCP-1 -2518A/G were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RANTES -403A/G, RANTES In1.1T/C, IP-10 nt1811A/G, IP-10 nt2867C/A and Mig nt367A/G were genotyped using the MassARRAY system (Sequenom, CA, US).Statistical analysisA two-step analysis was used to determine the association of polymorphisms with SARS. The genotype frequencies and allele frequencies of all the genes were compared between SARS patients and controls using a 3x2 Chi squared test and a 2x2 Chi squared test respectively. Logistic regression was then used to calculate the odds ratios (OR) [95% confidence interval (CI)] and corresponding P values of different genotype frequencies among SARS patients and controls by adjusting for age and sex as co-variables. Association with SARS infection outcomes (death vs survival) was then tested by comparing the genotype frequencies and allele frequencies of all the genes between the death group and the survival group of SARS patients using a 3x2 Chi squared test and a 2x2 Chi squared test respectively. The genotype frequencies of all the single nucleotide polymorphisms (SNPs) were tested for Hardy-Weinberg equilibrium (HWE) separately in SARS patients and controls using the Chi squared test. The significant P value for multiple testing was adjusted with Bonferroni’s correction and all statistical analyses were performed using SAS, version 8.02 and SAS/Genetics (SAS Institute, NC, US).Genotype SNP No. (%) OR (95% CI)* P value*SARS (n=476) Control (n=449)IFN-γ +874 A/A 332 (69.8) 203 (45.2) 5.19 (2.78 - 9.68) <0.001A/T 127 (26.7) 189 (42.1) 2.57 (1.35 - 4.88)T/T 17 (3.6) 57 (12.7) ReferenceIL-10 -1082 A/A 439 (92.2) 411 (91.5) NS†A/G 35 (7.4) 38 (8.5)G/G 2 (0.4) 0 (0)IL-10 -592 A/A 244 (51.3) 209 (46.6) NSA/C 188 (39.5) 214 (47.7)C/C 44 (9.2) 26 (5.8)TNF-a -308 GG 403 (84.7) 377 (83.9) NSGA 70 (14.7) 70 (15.6)AA 3 (0.6) 2 (0.5)SARS (n=470) Control (n=558)IL-12B promoter 318/322 318/318 122 (26.0) 145 (26.4) NS318/322 252 (53.6) 238 (43.4)322/322 96 (20.4) 166 (30.2)IL-12B intron 2 218/221 218/218 153 (32.6) 160 (29.3) NS218/221 234 (49.8) 251 (45.9)221/221 83 (17.7) 135 (24.7)IL-12B intron 4 268/272 268/268 70 (14.9) 133 (23.8) NS268/272 222 (47.2) 237 (42.5)272/272 147 (31.3) 150 (26.9)Others 31 (6.6) 38 (6.81)IL-12B 3’UTR A/A 157 (33.4) 155 (27.9) NSA/C 235 (50.0) 294 (52.9)C/C 78 (16.6) 107 (19.2)AlleleIFN-γ +874 A 791 (83.1) 595 (66.3) 2.23 (1.75 - 2.83) <0.001T 161 (16.9) 303 (33.7)IL-10 -1082 A 913 (95.9) 860 (95.8) NSG 39 (4.1) 38 (4.2)IL-10 -592 A 676 (71.0) 632 (70.4) NSC 276 (29.0) 266 (29.6)TNF-a -308 G 876 (92.0) 824 (91.8) NSA 76 (8.0) 74 (8.2)IL-12B promoter 318/322 318 496 (52.8) 528 (48.1) NS322 444 (47.2) 570 (51.9)IL-12B intron 2 218/221 218 540 (57.5) 571 (52.3) NS221 400 (42.6) 521 (47.7)IL-12B intron 4 268/272 268 376 (40.0) 517 (46.4) NS272 533 (56.7) 560 (50.3)Others 31 (3.3) 37 (3.30)IL-12B 3’UTR A 549 (58.4) 604 (54.3) NSC 391 (41.6) 508 (45.7)Table 1. Genotype frequencies and allele frequencies of cytokines in SARS patients and controls* P value and OR (95% CI) were calculated with the use of logistic regression models, adjusted for sex and age. After correction by Bonferroni method, the significant P value should be less than 0.003† NS denotes not significantAssociation of cytokine and chemokine gene polymorphisms with SARSHong Kong Med J Vol 15 No 1 Supplement 2 February 2009      45ResultsOur case-control study for cytokines genotyped the SNPs IFN-γ +874A/T, IL-10 -1082G/A, IL-10 -592A/C, TNF-α -308G/A, IL-12B promoter 318/322, IL-12B intron 2 218/221, IL-12B intron 4 268/272 and IL-12B 3’UTR A/C in Chinese patients with SARS and in healthy controls. All SNPs were in HWE (P>0.05) in SARS patients and controls using the Chi squared test and the genotype distributions and allele frequencies of these SNPs are shown in Table 1. The IFN-γ +874A allele was overrepresented in the SARS patients (83.1%) when compared with the controls (66.3%) [P<0.001]. It was also significantly associated with susceptibility to SARS in a dose-dependent manner (P<0.001), ie individuals with IFN-γ +874 AA and AT genotype had OR of 5.19 (95% CI, 2.78-9.68) and 2.57 (95% CI, 1.35-4.88) of developing SARS respectively. The SNPs of IL-10, TNF-α, and IL-12 SNPs were chosen for their potential regulation of protein expression levels. Nonetheless we found no significant association between these SNPs and SARS (Table 1). We also compared the genotype and allele frequencies of all the polymorphisms of the SARS patient death and survival groups but no significant associations were established. For the study on chemokines, RANTES -28C/G, RANTES -403A/G, RANTES In1.1T/C, IP-10 nt1811A/G, IP-10 nt2867C/A, Mig nt367A/G, and MCP-1 -2518 A/G were genotyped. Their genotype and allele frequencies are shown in Table 2. The RANTES -28 CG and GG genotypes were significantly associated with SARS susceptibility with OR of 3.28 (95% CI, 2.32-4.64) and 3.06 (95% CI, 1.47-6.39) respectively (P<0.0001) [Table 2]. The RANTES -28 G allele was also significantly increased in SARS patients (P<0.0001, OR=2.80, 95% CI, 2.11-3.71) [Table 2]. Our data did not show any significant association with the SNPs of IP-10, Mig and MCP-1 (Table 2). All genotype frequencies of the chemokine polymorphisms in SARS patients and controls were in HWE except for the RANTES -28C/G frequency in the controls. To confirm that there was no genotyping error that may have contributed to the HWE observation, direct DNA sequencing was performed on 20 to 30 samples for each SNP. No ambiguous results were obtained. We further compared the genotype and allele frequencies of the RANTES -28C/G between the SARS death and survival groups. The RANTES -28 G allele was associated with death from SARS in a gene-dosage dependent manner (P=0.014), with -28 CG and GG individuals having a 2.12-fold (95% CI, 1.11-4.06) and 4.01-Genotype SNP No. (%) OR (95% CI)* P value*SARS (n=495) Control (n=578)RANTES -403 AA 54 (10.9) 56 (9.7) NS†AG 223 (45.0) 262 (45.3)GG 218 (44.0) 260 (45.0)RANTES -28 CC 316 (63.8) 491 (84.9) Reference <0.0001CG 154 (31.1) 73 (12.6) 3.28 (2.32 - 4.64)GG 25 (5.0) 14 (2.4) 3.06 (1.47 - 6.39)RANTES ln1.1 CC 54 (10.9) 54 (9.3) NSCT 217 (43.8) 257 (44.5)TT 224 (45.3) 267 (46.2)IP-10 nt1811 AA 1 (0.2) 0 (0) NSAG 38 (7.7) 29 (5.0)GG 456 (92.1) 549 (95.0)IP-10 nt2867 AA 1 (0.2) 0 (0) NSAC 38 (7.7) 31 (5.4)CC 456 (92.1) 547 (94.6)Mig nt367 AA 1 (0.2) 0 (0%) NSAG 38 (7.7) 34 (5.9)GG 456 (92.1) 544 (94.1)SARS (n=478) Control (n=421)MCP-1 -2518 AA 115 (24.1) 113 (26.8) NSAG 225 (47.1) 213 (50.6)GG 138 (28.9) 95 (22.6)AlleleRANTES -403 A 331 (33.4%) 374 (32.4%) NSG 659 (66.6%) 782 (67.7%)RANTES -28 C 786 (79.4%) 1055 (91.3%) 2.80 (2.11 - 3.71) <0.0001G 204 (20.6%) 101 (8.7%)RANTES ln1.1 C 325 (32.9%) 365 (31.6%) NST 665 (67.2%) 791 (68.4%)IP-10 nt1811 A 40 (4.0%) 29 (2.5%) NSG 950 (96.0%) 1127 (97.5%)IP-10 nt2867 A 40 (4.0%) 31 (2.7%) NSC 950 (96.0%) 1125 (97.3%)Mig nt367 A 40 (4.1%) 34 (2.9%) NSG 938 (95.9%) 1122 (97.1%)MCP-1 -2518 A 455 (47.6) 439 (52.1) NSG 501 (52.4) 403 (47.9)Table 2. Genotype frequencies and allele frequencies of chemokines in SARS patients and controls* P value and OR (95% CI) were calculated with the use of logistic regression models, adjusted for sex and age. After correction by Bonferroni method, the significant P value should be less than 0.003† NS denotes not significantLau and Peiris46      Hong Kong Med J Vol 15 No 1 Supplement 2 February 2009RANTES -28C/G No. (%) OR (95% CI)* P value*Death (n=57) Survival (n=438)Genotype 0.014CC 26 (45.6) 290 (66.2) ReferenceCG 25 (43.9) 129 (29.5) 2.12 (1.11 - 4.06)GG 6 (10.5) 19 (4.3) 4.01 (1.30 - 12.4)Allele 0.002C 77 (67.5) 709 (80.9)G 37 (32.5) 167 (19.1) 2.10 (1.30 - 3.39)Table 3. Genotype and allele frequencies of RANTES -28C/G among death and survival groups in SARS patients* P value and OR (95% CI) were calculated with the use of logistic regression models, adjusted for sex and agefold (95% CI, 1.30-12.4) increased risk of death from SARS respectively (Table 3). No association with death from SARS was detected for the other chemokine genes studied.DiscussionIt has been reported previously that the IFN-γ +874A allele is associated with infectious diseases, revealing its potential role in host defences against microbial infections. The mechanism by which the IFN-γ +874A/T allele influences susceptibility to SARS may depend on its role in the regulation of IFN-γ production. The T allele of IFN-γ +874A/T provides a binding site for the transcription factor nuclear factor-kB (NF-kB), which is able to regulate IFN-γ expression. It is possible that low IFN-γ production may impair the anti-viral response to SARS-CoV, rendering these individuals more susceptible to infection with this virus. Our observation that the IFN-γ +874A allele was significantly associated with SARS-CoV infection suggests a genetic risk factor for SARS. The role of IFN-γ in the antiviral response to SARS-CoV has also been supported by recent studies showing that IFN-γ can inhibit the replication of SARS-CoV in combination with IFN-b in vitro. RANTES is responsible for the recruitment of eosinophils, lymphocytes, monocytes and basophils at the site of inflammation and is involved in many viral infections. We found that the -28 G allele of RANTES was associated with susceptibility to and death from SARS. RANTES -28C/G is located at the NF-κB binding site, suggesting that this SNP may be involved in the regulation of RANTES expression. Further in vitro studies have shown that the RANTES -28 G allele enhances NF-κB binding that leads to elevation of promoter activity and increases RANTES expression in CD8+ T cells, CD4+ T cells and monocytes/ macrophages. Together with our observation that the -28 G allele is associated with SARS, we conclude a high level of RANTES may predispose individuals to developing SARS. Too high a level of RANTES may intensify lung inflammation and lead to lymphopaenia, increasing the chance of secondary infection and hence the death rate among SARS patients. Therefore, we speculate that the RANTES -28 G allele that associates with higher levels of RANTES may enhance inflammation and lead to severe clinical outcomes in SARS. Indeed, the RANTES -28 G allele did show a strong association with death in Hong Kong Chinese patients with SARS (Table 3). We did not find a significant association between the SNPs of IL-10, TNF-α, IL-12, IP-10, Mig, and MCP-1 and SARS. Nevertheless, we cannot entirely exclude the roles of these cytokines and chemokines in susceptibility to SARS, because other SNPs in these genes may also be involved in gene expression regulation. Further studies on other SNPs able to alter gene expression levels are required to ascertain the relationship of these SNPs with SARS.ConclusionsWe demonstrated that the IFN-γ +874A allele is significantly associated with SARS susceptibility in a dose-dependent manner.5 Due to its role in regulating IFN-γ expression, this allele may be involved in the pathogenesis of SARS by altering IFN-γ production. In addition, we demonstrated that the RANTES -28 G allele, which correlates with high RANTES production, was associated with SARS susceptibility.6 It was also associated with adverse SARS outcomes, suggesting that a high RANTES level may play a role in the pathogenesis of SARS.6AcknowledgementsThis study was supported by the Research Fund for the Control of Infectious Diseases (RFCID: 03040302), Food and Health Bureau, Hong Kong SAR Government. In addition, this project was partially supported by the Outstanding Researcher Awards (YLL and JSMP), Postgraduate Studentships from The University of Hong Kong, and Edward Sai Kim Hotung Paediatric Education and Research Fund. Part of the results of this report was published in references 5 and 6.References1. Peiris JS, Lai ST, Poon LL, et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003;361:1319-25.2. Lau YL, Peiris JS. Pathogenesis of severe acute respiratory syndrome. Curr Opin Immunol 2005;17:404-10. 3. Ip WK, Chan KH, Law HK, et al. Mannose-binding lectin in severe acute respiratory syndrome coronavirus infection. J Infect Dis 2005;191:1697-704.4. Zhang H, Zhou G, Zhi L, et al. Association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection. J Infect Dis 2005;192:1355-61.5. Chong WP, Ip WK, Tso GH, et al. The interferon gamma gene polymorphism +874 A/T is associated with severe acute respiratory syndrome. BMC Infect Dis 2006;6:82.6. Ng MW, Zhou G, Chong WP, et al. The association of RANTES polymorphism with severe acute respiratory syndrome in Hong Kong and Beijing Chinese. BMC Infect Dis 2007;7:50.

Association of cytokine and chemokine gene polymorphisms with severe acute respiratory syndrome

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Association of cytokine and chemokine gene polymorphisms with severe acute respiratory syndrome

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