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CCR5 chemokine receptor gene variants in chronic Chagas' disease

2014; Elsevier BV; Volume: 176; Issue: 2 Linguagem: Inglês

10.1016/j.ijcard.2014.07.043

1874-1754

Amanda Priscila de Oliveira, Cássia Rúbia Bernardo, Ana Vitória Silveira Camargo, Daniel Fernando Villafanha, Carlos Eugênio Cavasini, Cinara Cássia Brandão de Mattos, Moacir Fernandes de Godoy, Reinaldo B. Bestetti, Luíz Carlos de Mattos,

Research on Leishmaniasis Studies

Chronic Chagas heart disease affects approximately 30% of patients infected by the protozoan Trypanosoma cruzi around 20 years after infection. The clinical manifestations of this disease are malignant arrhythmias [[1]Bestetti R.B. Santos C.R. Machado-Júnior O.B. et al.Clinical profile of patients with Chagas' disease before and during sustained ventricular tachycardia.Int J Cardiol. 1990; 29: 39-46Abstract Full Text PDF PubMed Scopus (34) Google Scholar], chronic systolic heart failure [[2]Bestetti R.B. Otaviano A.P. Cardinalli-Neto A. da Rocha B.F. Theodoropoulos T.A. Cordeiro J.A. Effects of B-blockers on outcome of patients with Chagas' cardiomyopathy with chronic heart failure.Int J Cardiol. 2011; 151: 205-208Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar], sudden cardiac death [[3]Bestetti R.B. Cardinalli-Neto A. Sudden cardiac death in Chagas' heart disease in the contemporary era.Int J Cardiol. 2008; 131: 9-17Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar], and thromboembolism [[4]Biolo A. Ribeiro A.L. Clausell N. Chagas cardiomyopathy—where do we stand after a hundred years?.Prog Cardiovasc Dis. 2010; 52: 300-316Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar]. Tissue damage resulting from inflammatory infiltrates and persistence of T. cruzi in myocardial tissue is involved in the pathogenesis of cardiomyopathy. However, the precise pathogenic mechanism of Chagas' heart disease is not completely elucidated [4Biolo A. Ribeiro A.L. Clausell N. Chagas cardiomyopathy—where do we stand after a hundred years?.Prog Cardiovasc Dis. 2010; 52: 300-316Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 5Cunha-Neto E. Nogueira L.G. Teixeira P.C. et al.Immunological and non-immunological effects of cytokines and chemokines in the pathogenesis of chronic Chagas disease cardiomyopathy.Mem Inst Oswaldo Cruz. 2009; 104: 252-258Crossref PubMed Scopus (78) Google Scholar]. The CC chemokine receptor 5 (CCR5) expressed by monocytes, macrophages and TH1 cells is a receptor for the CCL3, CCL4 and CCL5 chemokines [6Sallusto F. Mackay C.R. Lanzavecchia A. The role of chemokine receptors in primary, effector, and memory immune responses.Annu Rev Immunol. 2000; 18: 593-620Crossref PubMed Scopus (927) Google Scholar, 7Szpakowska M. Fievez V. Arumugan K. van Nuland N. Schmit J.C. Chevigné A. Function, diversity and therapeutic potential of the N-terminal domain of human chemokine receptors.Biochem Pharmacol. 2012; 84: 1366-1380Crossref PubMed Scopus (65) Google Scholar]. Genetic variants of the CCR5 gene may be involved in the differential susceptibility for Chagas cardiomyopathy [8Calzada J.E. Nieto A. Beraún Y. Martín J. Chemokine receptor CCR5 polymorphisms and Chagas' disease cardiomyopathy.Tissue Antigens. 2001; 58: 154-158Crossref PubMed Scopus (69) Google Scholar, 9Fernández-Mestre M.T. Montagnani S. Layrisse Z. Is the CCR5-59029-G/G genotype a protective factor for cardiomyopathy in Chagas disease?.Hum Immunol. 2004; 65: 725-728Crossref PubMed Scopus (31) Google Scholar, 10Flórez O. Martín J. González C.I. Genetic variants in the chemokines and chemokine receptors in Chagas disease.Hum Immunol. 2012; 73: 852-858Crossref PubMed Scopus (31) Google Scholar]. In this study, we investigated the relationship of the CCR5Δ32 (rs333) and CCR5 59029 A/G (rs1799987) polymorphisms of the CCR5 gene in patients with chronic Chagas' disease, with and without left ventricular systolic dysfunction (LVSD); left ventricular ejection fraction (LVEF) is currently the most important predictor of all-cause mortality of patients with this condition [[11]Theodoropoulos T.A.D. Bestetti R.B. Otaviano A.P. Cordeiro J.A. Rodrigues V.C. Silva A.C. Predictors of all-cause mortality in chronic Chagas' heart disease in the current era of heart failure therapy.Int J Cardiol. 2008; 128: 22-29Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. This study was approved by the Research Ethics Committee of the Medicine School in São José do Rio Preto (# 009/2011) and informed consent was obtained from all patients. A total of 168 consecutive male and female patients were enrolled from the Cardiomyopathy Outpatient Service of Hospital de Base of the Fundação Faculdade de Medicina de São José do Rio Preto. Anti-T. cruzi antibodies were detected by immunosorbent assay (ELISA) according to the manufacturer's instructions (ELISAcruzi; bioMerieux S.A. Brazil). Patients were divided into three groups according to the LVEF; patients with LVEF >60%, between 60% and 40% and those with LVEF 50% indicated normal left ventricular systolic function, LVEF between 30% and 50% indicated mild to moderate LVSD, and a LVEF <30% was consistent with severe LVSD. Genomic DNA was attained using a commercial kit for silica column extraction (PureLink™ Genomic DNA Mini Kit, Invitrogen, Carlsbad, California/USA) and following the manufacturer's instructions. The CCR5Δ32 polymorphism of the CCR5 gene was identified by polymerase chain reaction (PCR), according to the protocol of Huang et al. [[13]Huang Y. Paxton W.A. Wolinsky S.M. et al.The role of a mutant CCR5 allele in HIV-1 transmission and disease progression.Nat Med. 1996; 2: 1240-1243Crossref PubMed Scopus (1206) Google Scholar]. The polymorphism of the promoter region of the CCR5 gene (CCR5-59029 A/G) was identified by PCR-restriction fragment length polymorphism (PCR-RFLP) as described by McDermott et al. [[14]McDermott D.H. Zimmerman P.A. Guignard F. Kleeberger C.A. Leitman S.F. Murphy P.M. CCR5 promoter polymorphism and HIV-1 disease progression.Lancet. 1998; 352: 866-870Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar]. Comparisons of proportions between groups were made by the chi-square test using the software GraphPad Instat (version 3.06). Differences were considered statistically significant for a p-value < 0.05. The CCR5Δ32 polymorphism was investigated in 168 patients: 152 (90.5%) were wild type homozygotes, and 16 (9.5%) heterozygotes. Homozygotes for this deletion were not found. Table 1 shows data on allele and genotype frequencies. The CCR5 59029 A/G polymorphism was analyzed in 155 patients. The genotype frequencies were: AA (39.4%), AG (36.1%) and GG (24.5%). The data on allele and genotype frequencies are shown in Table 2.Table 1Genotype and allele frequencies of the CCR5∆32 polymorphism in Chagas' disease patients with normal left ventricular systolic function, and mild to moderate, and severe left ventricular systolic dysfunction.GenotypesNormalMild/moderate LVSDSevere LVSDχ2DFpaCalculated by χ2; LVSD: Left ventricular systolic dysfunction; DF: Degree of freedom.CCR5∆32No.%No.%No.%CCR5/CCR578(91.8)40(93.0)34(85.0)1.88020.391CCR5/CCR5∆327(8.2)3(7.0)6(15.0)Total85(100.0)43(100.0)40(100.0)AllelesCCR5163(95.9)83(96.5)74(92.5)1.78620.409CCR5∆327(4.1)3(3.5)6(7.5)a Calculated by χ2; LVSD: Left ventricular systolic dysfunction; DF: Degree of freedom. Open table in a new tab Table 2Genotype and allele frequencies of the CCR5 59029 A/G polymorphism in Chagas' disease patients with normal left ventricular systolic function, and mild to moderate, and severe left ventricular systolic dysfunction.GenotypesNormalMild/moderate LVSDSevere LVSDχ2DFpaCalculated by χ2; LVSD: Left ventricular systolic dysfunction; DF: Degree of freedom.CCR5 59029A/GNo.%No.%No.%AA30(37.5)16(40.0)15(42.9)0.30220.860AG29(36.3)14(35.0)13(37.1)0.03820.981GG21(26.2)10(25.0)7(20.0)0.52120.771Total80(100.0)40(100.0)35(100.0)AllelesA89(55.6)46(57.5)43(61.4)0.67120.715G71(44.4)34(42.5)27(38.6)a Calculated by χ2; LVSD: Left ventricular systolic dysfunction; DF: Degree of freedom. Open table in a new tab The CCR5Δ32 deletion results in a non-functional receptor not detected on the cell surface, whereas heterozygous individuals for this polymorphism have reduced levels of CCR5 expression [15Liu R. Paxton W.A. Choe S. et al.Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.Cell. 1996; 86: 367-377Abstract Full Text Full Text PDF PubMed Scopus (2567) Google Scholar, 16Wu L. Paxton W.A. Kassam N. et al.CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro.J Exp Med. 1997; 185: 1681-1691Crossref PubMed Scopus (639) Google Scholar]. The results of this study show no association of genotypes and alleles of the CCR5Δ32 polymorphism between groups of patients with chronic Chagas' disease. A study suggested that the CCR5/CCR5Δ32 genotype can protect against inflammatory cardiomyopathy, a consequence of the chronic phase of Chagas' disease [[9]Fernández-Mestre M.T. Montagnani S. Layrisse Z. Is the CCR5-59029-G/G genotype a protective factor for cardiomyopathy in Chagas disease?.Hum Immunol. 2004; 65: 725-728Crossref PubMed Scopus (31) Google Scholar] but this study did not confirm this due to the low frequency of the CCR5/CCR5Δ32 genotype. The CCR5 59029 A/G polymorphism of the promoter region alters the expression of CCR5 on the surface of leukocytes. It has been demonstrated that the A allele has higher promoter activity in vitro than the G allele and that the increased expression of CCR5 results from the AA genotype [14McDermott D.H. Zimmerman P.A. Guignard F. Kleeberger C.A. Leitman S.F. Murphy P.M. CCR5 promoter polymorphism and HIV-1 disease progression.Lancet. 1998; 352: 866-870Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 17Shieh B. Liau Y.E. Hsieh P.S. Yan Y.P. Wang S.T. Li C. Influence of nucleotide polymorphisms in the CCR2 gene and the CCR5 promoter on the expression of cell surface CCR5 and CXCR4.Int Immunol. 2000; 12: 1311-1318Crossref PubMed Scopus (79) Google Scholar]. The genotypes and alleles of the CCR5 59029 A/G polymorphism did not differ between the groups of Chagas' disease patients included in this study. Our results contrast with the results reported by other studies. Calzada et al. [[8]Calzada J.E. Nieto A. Beraún Y. Martín J. Chemokine receptor CCR5 polymorphisms and Chagas' disease cardiomyopathy.Tissue Antigens. 2001; 58: 154-158Crossref PubMed Scopus (69) Google Scholar] found a higher frequency of the AG genotype and the presence of the G allele in asymptomatic patients compared to patients with cardiomyopathy. These authors suggested that the G allele could protect against the development of Chagas cardiomyopathy. Another study found a low frequency of the GG genotype in patients with arrhythmias compared to asymptomatic patients [[9]Fernández-Mestre M.T. Montagnani S. Layrisse Z. Is the CCR5-59029-G/G genotype a protective factor for cardiomyopathy in Chagas disease?.Hum Immunol. 2004; 65: 725-728Crossref PubMed Scopus (31) Google Scholar]. These authors suggested that this genotype may protect against the development of cardiomyopathy symptoms in individuals infected with T. cruzi [[9]Fernández-Mestre M.T. Montagnani S. Layrisse Z. Is the CCR5-59029-G/G genotype a protective factor for cardiomyopathy in Chagas disease?.Hum Immunol. 2004; 65: 725-728Crossref PubMed Scopus (31) Google Scholar]. The results of the work of Flórez et al. [[10]Flórez O. Martín J. González C.I. Genetic variants in the chemokines and chemokine receptors in Chagas disease.Hum Immunol. 2012; 73: 852-858Crossref PubMed Scopus (31) Google Scholar] did not show any significant association between the CCR5 59029 A/G polymorphism and protection against the development of Chagas' disease cardiomyopathy, however, the development of Chagas disease cardiomyopathy was associated with the HHA haplotype in which the G allele was present. The authors suggest that in this type of study it may be more appropriate to estimate haplotypes, as several polymorphisms in the promoter region may influence the CCR5 receptor expression levels and the cell type in which it is expressed [10Flórez O. Martín J. González C.I. Genetic variants in the chemokines and chemokine receptors in Chagas disease.Hum Immunol. 2012; 73: 852-858Crossref PubMed Scopus (31) Google Scholar, 18Mummidi S. Bamshad M. Ahuja S.S. et al.Evolution of human and non-human primate CC chemokine receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and simian immunodeficiency virus.J Biol Chem. 2000; 275: 18946-18961Crossref PubMed Scopus (145) Google Scholar]. In this study, the heart disease was evaluated according to the degree of LVSD. In other studies, the aim was not to investigate any possible relationship between CCR5Δ32 and CCR5 59029 A/G polymorphisms and left ventricular impairment, and so the criteria to classify patients were different [8Calzada J.E. Nieto A. Beraún Y. Martín J. Chemokine receptor CCR5 polymorphisms and Chagas' disease cardiomyopathy.Tissue Antigens. 2001; 58: 154-158Crossref PubMed Scopus (69) Google Scholar, 9Fernández-Mestre M.T. Montagnani S. Layrisse Z. Is the CCR5-59029-G/G genotype a protective factor for cardiomyopathy in Chagas disease?.Hum Immunol. 2004; 65: 725-728Crossref PubMed Scopus (31) Google Scholar, 10Flórez O. Martín J. González C.I. Genetic variants in the chemokines and chemokine receptors in Chagas disease.Hum Immunol. 2012; 73: 852-858Crossref PubMed Scopus (31) Google Scholar]. These distinct strategies may have contributed to the discrepant results reported in this study compared to other research. In conclusion, the CCR5Δ32 (rs333) and CCR5 59029 A/G (rs1799987) polymorphisms had no relationship with LVSD in patients with chronic Chagas' heart disease in this study population. Thus, these polymorphisms do not seem to influence left ventricular impairment. The authors declare no conflicts of interest. This work was supported by FAPESP (São Paulo Research Foundation) Grants #2011/08075-4, #2011/19439-7, #2012/20735-2, and #2012/05580-2 and Brazilian Ministry of Education—CAPES PhD Scholarship (Coordination of Improvement of Higher Education Personnel, Brazil). Many thanks to Igor Isnardi Barreto for technical support (FAPESP #2012/03679-1) and to David Hewitt to proofread the English. Many thanks to Professor Stephen Henry, Auckland University of Technology, New Zealand, by the comments.