Genetic Polymorphisms of IL17 and Chagas Disease in the ...downloads.hindawi.com/journals/jir/2017/1017621.pdf · ResearchArticle Genetic Polymorphisms of IL17 and Chagas Disease

Research ArticleGenetic Polymorphisms of IL17 and Chagas Disease inthe South and Southeast of Brazil

Pacircmela Guimaratildees Reis1 Christiane Maria Ayo2

Luiz Carlos de Mattos2 Cinara de Caacutessia Brandatildeo de Mattos2

Karina Mayumi Sakita1 Amarilis Giaretta de Moraes1

Larissa Pires Muller3 Julimary Suematsu Aquino1 Luciana Conci Macedo1

Priscila Saamara Mazini1 Ana Maria Sell13 Divina Seila de Oliveira Marques4

Reinaldo Bulgarelli Bestetti5 and Jeane Eliete Laguila Visentainer13

1Post Graduation Program of Biosciences and Physiopathology Department of Analysis Clinical and BiomedicineMaringa State University Maringa PR Brazil2Laboratory of Immunogenetics Department of Molecular Biology Medical School Sao Jose do Rio Preto SP Brazil3Laboratory of Immunogenetics Department of Basic Health Sciences Maringa State University Maringa PR Brazil4Department of Medical Clinic Londrina State University Londrina PR Brazil5Department of Cardiology and Cardiovascular Surgery Medical School Sao Jose do Rio Preto SP Brazil

Correspondence should be addressed to Jeane Eliete Laguila Visentainer jelvisentainergmailcom

Received 23 September 2016 Accepted 30 January 2017 Published 2 April 2017

Academic Editor Margarete D Bagatini

Copyright copy 2017 Pamela Guimaraes Reis et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of this studywas to investigate possible associations between genetic polymorphisms of IL17AG197A (rs2275913) and IL17FT7488C (rs763780) with Chagas Disease (CD) andor the severity of left ventricular systolic dysfunction (LVSD) in patients withchronic Chagas cardiomyopathy (CCC) The study with 260 patients and 150 controls was conducted in the South and Southeastregions of BrazilThe genotyping was performed by PCR-RFLPTheA allele andAA genotype of IL17Awere significantly increasedin patients and their subgroups (patients with CCC patients with CCC and LVSD and patients with CCC and severe LVSD) whencompared to the control group The analysis according to the gender showed that the AA genotype of IL17A was more frequentin female with LVSD and mild to moderate LVSD and also in male patients with LVSD The frequency of IL17F TC genotype washigher in male patients with CCC and severe LVSD and in female with mild to moderate LVSD The results suggest the possibleinvolvement of the polymorphisms of IL17A and IL17F in the susceptibility to chronic Chagas disease and in development andprogression of cardiomyopathy

1 Introduction

Chagas disease (CD) is a serious anthropozoonosis commonin the Americas and found mainly in endemic areas of the 21Latin American countries [1] On account of multinationalinitiatives infection prevalence is progressively decreasingand it is estimated that 6 to 8million individuals are currentlyinfected in the world with an incidence of 28000 casesa year [2] Chagas disease presents an acute phase and achronic phase After the acute phase most of the infected

patients enter in the chronic phase of the disease and about60 to 70 of infected persons are considered to have theindeterminate form (asymptomatic) of the disease [3ndash6]After several years (10 to 30) of starting the chronic phase30 to 40 of the patients develop clinical manifestationsknown as the clinical forms cardiac digestive (mainlymegaesophagus and megacolon) and cardiodigestive [5 6]The chronic Chagas cardiomyopathy (CCC) is the mostsevere form of the disease that affects 20 to 30 of theinfected individuals In endemic areas the disease is the

HindawiJournal of Immunology ResearchVolume 2017 Article ID 1017621 7 pageshttpsdoiorg10115520171017621

2 Journal of Immunology Research

Table 1 Characteristics of the chronic Chagas disease patients and controls from South and Southeast of Brazil

CD patients CCC Without CCC Control119873 = 260 119873 = 212 119873 = 48 119873 = 150

Gendera 119899 ()Male 121 (465) 97 (458) 24 (500) 74 (493)Female 139 (535) 115 (542) 24 (500) 76 (507)

Ageb

Min-max 31ndash90 31ndash90 38ndash76 28ndash100Mean plusmn SD (year) 629 plusmn 100 639 plusmn 102 586 plusmn 78 623 plusmn 174

CCC patients with chronic Chagas cardiomyopathy Min minimum age Max maximum age SD standard deviationaNo statistically significant difference was observed between the groups for genderbStatistically significant difference was observed between the groups for age CCC versus without CCC

main death cause in patients aged between 30 and 50 years[5 7]

It is known that genetic variability and immunologicresponse influence the pathogenesis of the chronic phase ofthe disease Associations were observed in several cytokinegenes [8] with the susceptibility or protection against thedevelopment or progression of the CD andor its clinicalformsThe IL-17 is a proinflammatory cytokine secreted by Tcells activated and expressed in different tissuesThis cytokinetakes part in inflammatory responses mediated by T cellsand plays an important role in the tissue homeostasis anddiseases progression [9] The IL-17F presents a high degreeof homology with the IL-17A (57 identical) [9] and seemsto have a biological action similar to IL-17A in vitro and invivo though significantly weaker [10] The genes that codifythem are mapped in the same chromosome in the position6p12 [9 11]

Polymorphism in genes encoding cytokines may influ-ence the level of cytokines production and consequentlycause different immunological responses to different dis-eases Previous studies show that genetic polymorphismsof IL17A G197A and IL17F T7488C affect the productionof IL-17A and F respectively [12 13] Such polymorphismshave already been associated with autoimmune and inflam-matory diseases as rheumatoid arthritis [14] periodontitis[15] and cancer both gastric [16] and breast cancer [17]To our knowledge only one study involving the SNPsof IL17A and the CD [18] was found so far and if weconsider the SNPs of IL17F there are no related articlespublished yet For this reason our study aims to investi-gate whether the genetic polymorphisms of IL17A G197A(rs2275913) and IL17F T7488C (rs763780) were related toCD andor the severity of the left ventricular systolic dys-function (LVSD) in patients with CCC from North andNortheast regions of Parana and the Northeast region of SaoPaulo (states located in the South and Southeast of Brazilresp)

2 Material and Methods

21 Patients and Controls For this study 260 patients withchronic CDwere selected from different municipalities in theNorth and Northwest regions of Parana and in the Northwestregion of Sao PauloThe patients were cared for in the Chagas

Disease Laboratory in the State University of Maringa theClinical Hospital in Londrina and the Base Hospital of theMedical School in Sao Jose do Rio Preto All patients weresubmitted to a resting electrocardiogram (ECG) exam and atwo-dimensional echocardiography Patients who presenteda normal ECG were classified as patients without CCCand patients with electrocardiographic changes common toCCC were classified as patients with CCC The severity ofthe LVSD was measured according to the left ventricularejection fraction (LVEF) and the Teichhoolz method wasapplied following the II Brazilian Guideline for Severe HeartDiseases [19] Patients with CCC were classified consideringthe (LVEF) in three different groups patients without LVSD(LVEF gt 60) patients with mild to moderate LVSD (LVEF40ndash60) and patients with severe LVSD (LVEF lt 40) Toall statistical analysis were considered the following groupsall Chagas disease patients (CD) chronic Chagas cardiomy-opathy patients (CCC) without Chagas cardiomyopathypatients (without CCC) chronic Chagas cardiomyopathypatients with LVSD (with LVSD) chronic Chagas cardiomy-opathy patients without LVSD (without LVSD) patients withmild to moderate LVSD (Mildmoderate LVSD) and patientswith severe LVSD (severe LVSD)

The control group was composed of 150 individualshealthy and nonrelated patientrsquos spouses and contacts retire-ment communitiesrsquo residents with negative serology to Tcruzi antigens The clinicopathological features of patientsand controls are presented in Table 1 No significant differ-ences were observed among groups in terms of gender butdifferences in age were observed between CCC and withoutCCC patients (639 plusmn 102 versus 586 plusmn 78 respectively119875 le 005) Due to the significant miscegenation of Brazilianpopulation we consider patients and controls as a mixedethnic group (CaucasiansMulattos and Blacks) according toParra et al (2003) [20] Mean age gender rates and residencein the same geographical areas were carefully matching toselect the groups

The laboratory diagnosis of CD in patients and con-trols was made by ELISA (Enzyme-Linked ImmunoSorbentAssay) test in serum or plasma using the immunoas-say ldquoChagasrdquo from Abbott Laboratories (Santiago Chile)In cases of weak reagent the diagnosis was confirmedby the indirect immunofluorescence test (IIFT) with theIMUNOCRUZI antigen (Biolab Rio de Janeiro Brazil) or

Journal of Immunology Research 3

ELISAcruzi (bioMerieus SA Brazil) respecting themanufac-turerrsquos instructions

The Ethics committees from each institution haveapproved this study as seen in the protocols they have reg-istered (0122010-COPEP-UEM CAAE 02960093000ndash09FAMERP - 0092011) and written informed consent wasobtained from all subjects prior to participation

22 DNA Extraction and Genotyping The extractionmethodused in this research was the salting-out adapted [21] Thegenomic DNA was extracted from 250 120583L of buffy-coatobtained from 5mL of peripheral blood collected in tubeswith EDTA (Ethylenediaminetetraacetic acid) Thematerialrsquosconcentration and purity were determined by NanoDrop2000 equipment (Thermo Scientific Wilmington USA)

The SNPs in IL-17A (rs2275913) and IL-17F (rs763780)were genotyped using PCR-RFLP (Polymerase Chain Re-action-Restriction Fragment Length Polymorphism) [15]The primers sequences to IL17A G197A were sense 51015840-AAC-AAGTAAGAATGAAAAGAGGACATGGT-31015840 and anti-sense 51015840-CCCCCAATGAGGTCATAGAAGAATC-3 whileto IL17F T7488C they were sense 51015840-ACCAAGGCTGCT-CTGTTTCT-31015840 and antisense 51015840-GGTAAGGAGTGGCAT-TTCTA-31015840 The reaction of DNA amplification was made ina total volume of 30 120583L containing 100 ng of genomic DNA10 120583M from each primer 200120583M from each dNTP 20mMof MgCl2 3 120583L of 10x PCR buffer and 15U of Taq DNApolymerase (Invitrogen Life Technologies Grand Island NYUSA)The PCR products were digested during one hour sub-mitted to 37∘Cwith the enzyme XagI (Fermentas Canada) toIL17A G197A and the enzyme NlaIII (New England Biolabs)to IL17F T7488C and subsequently separated by agarose gelelectrophoresis to 35 with SYBR Green (Invitrogen LifeTechnologies Grand Island NY USA)

23 Statistical Analysis The allele and genotype frequenciesof IL17A G197A and IL17F T7488C were estimated andthe genotype distribution was evaluated to Hardy-Weinbergbalance [22] The association tests were realized to thecodominant dominant recessive overdominant and log-additive genetic inheritance models The 119875 le 005 valueswere considered statistically significant to Chi-square testwith Yates correction and logistic regression The statisticalcomparisons between these groups were realized and theestimated risk to develop CD andor CCC in individualswho hold genetic polymorphisms was calculated by deter-mination of OD (Odds Ratio) with 95 of confidenceinterval adjusted by gender and age All statistical analysiswas performed using the software SNPStats (httpbioinfoiconcologianetindexphp)[23] andtheOpenEpiprogramver-sion 303a (httpwwwopenepicomMenuOE_Menuhtm)

3 Results

The ratio distributions of genotype frequency for all ana-lyzed genes were in Hardy-Weinberg equilibrium (119875 gt005) In order to evaluate the possible association of IL17AG197A and IL17F T7488C SNPs and Chagas disease theallele and genotype frequencies between patients (CD) and

their subgroups (CCC without CCC with LVSD withoutLVSDMildmoderate LVSD severe LVSD) and controls werecompared (Table 2) Statistically significant differences wereobserved for A allele and AA genotype of IL17A but nosignificant difference was found to IL17F

The A allele frequency of IL17A was significantly higherin the CD patients when compared to controls (119875 = 0032OR = 146 95 CI = 105ndash205) The same was found whenCCC patients (119875 = 0021 OR = 152 95 CI = 108ndash215)patients with CCC and LVSD (119875 = 0009 OR = 173 95 CI= 115ndash259) and patients with CCC and severe LVSD (119875 =0009 OR = 197 CI = 120ndash321) were compared to controls

The AA genotype was more frequent in the CD patientsthan in the control group and statistically significant dif-ferences were observed in more than one model of geneticinheritance (Codominant 119875 = 0019 OR = 453 95 CI= 131ndash1573 Recessive 119875 = 00089 OR = 412 95 CI =120ndash1413 Log-additive 119875 = 002 OR = 150 95 CI =106ndash213) Same results can be seen when the subsets arecompared CCC versus controls (Codominant 119875 = 001 OR= 516 95 CI = 147ndash1814 Recessive 119875 = 00048 OR = 46795 CI = 135ndash1618 Log-additive 119875 = 0013 OR = 157 95CI = 109ndash224) patients with CCC and LVSD versus controls(Codominant 119875 = 0006 OR = 681 95 CI = 177ndash2629Dominant 119875 = 0034 OR = 173 95 CI = 104ndash287Recessive 119875 = 00045 OR = 573 95 CI = 152ndash2164 Log-additive 119875 = 00046 OR = 185 95 CI = 120ndash285) patientswith CCC and severe LVSD versus controls (Codominant119875 = 00047 OR = 964 95 CI = 228ndash4085 Recessive119875 = 0002 OR = 818 95 CI = 200ndash3351 Log-additive 119875 =0057 OR = 211 95 CI = 124ndash360) For all comparisonsthe recessive inherence model was the best according Akaikeinformation criteria (AIC) It means that two copies of A arenecessary to change the risk so GA or GG have the sameeffect No difference was observed when allele and genotypefrequencies of IL17A were compared between patients withCCC and patients without CCC Likewise no associationwas observed when the progression of cardiac forms wasconsidered the different forms (without LVSD with LVSDmildmoderate LVSD and severe LVSD) were comparedwith each other and no statistically significant difference wasnoticed

After stratifying according to gender significant differ-ences were observed for IL17A and IL17F genotype frequen-cies when the progression of cardiac form was evaluatedTheIL17AAA genotype was more frequent in female with LVSD(OR = 663 95 CI = 121ndash3640) and with mildmoderateLVSD (OR = 757 95 CI = 107ndash5340) than in the controlgroup although not significant males with LVSD also hadhigher frequency of AA genotype compared to controls (135versus 784 resp) (Table 3) In relation to IL17F the TCgenotype was more frequent in male patients with severeLVSD when compared to other groups without LVSD (OR= 482 95 CI = 155ndash1498) with mildmoderate LVSD (OR= 600 95 CI = 118ndash3063) without CCC patients (OR =670 95 CI = 119ndash3753) and controls (OR = 340 95 CI =124ndash931) In female statistical difference was not observedalthough TC was higher in mildmoderate LVSD (17)when compared to others patients and control (Table 4)


Table 2 Genotypes and allele frequencies distribution of IL17A rs2275913 and IL17F rs763780 in Chagas disease patients and controls in apopulation from South and Southeast of Brazil

Allelegenotype 119899()

CD patients CCC WithoutLVSD

WithLVSD

MildmoderateLVSD

SevereLVSD

WithoutCCC Control

119873 = 260 119873 = 212 119873 = 109 119873 = 103 119873 = 52 119873 = 51 119873 = 48 119873 = 150

IL17A G197AG 369 (712) 297 (704) 159 (729) 138 (676) 72 (706) 66 (647) 72 (750) 235 (783)A 149 (288)a 125 (296)b 59 (271) 66 (324)c 30 (294) 36 (353)d 24 (250) 65 (217)GG 130 (502) 104 (493) 58 (532) 46 (451) 24 (470) 22 (431) 26 (542) 88 (587)GA 109 (421) 89 (422) 43 (395) 46 (451) 24 (470) 22 (431) 20 (417) 59 (393)AA 20 (77)e 18 (85)f 8 (73) 10 (98)g 3 (60) 7 (138)h 2 (41) 3 (20)IL17F T7488CT 484 (931) 394 (929) 207 (949) 187 (908) 97 (933) 90 (882) 90 (937) 282 (940)C 36 (69) 30 (71) 11 (51) 19 (92) 7 (67) 12 (118) 6 (63) 18 (60)TT 224 (862) 182 (858) 98 (899) 84 (816) 45 (865) 39 (765) 42 (875) 132 (880)TC 36 (138) 30 (142) 11 (101) 19 (184) 7 (135) 12 (235) 6 (125) 18 (120)CCC patients with chronic Chagas cardiomyopathy LVSD left ventricular systolic dysfunction Recessive model AA versus GA + GG OR odds ratio CIconfidence interval Adjustment of the genotypic differences for the effect of age and gender was applieda119875 = 0032 OR = 146 and 95 CI = 105ndash205 CD patients versus controls

b119875 = 0021 OR = 152 and 95 CI = 108ndash215 CCC versus controls

c119875 = 0009 OR = 173 and 95 CI = 115ndash259With LVSD versus controls

d119875 = 0009 OR = 197 and 95 CI = 120ndash321 Severe LVSD versus controls

eRecessive model 119875 = 0009 OR = 412 95 CI = 120ndash1413 CD patients versus controlsfRecessive model 119875 = 0005 OR = 467 95 CI = 135ndash1618 CCC versus controlsgRecessive model 119875 = 0005 OR = 573 95 CI = 152ndash2164 With LVSD versus controlshRecessive model 119875 = 0002 OR = 818 95 CI = 200ndash3351 Severe LVSD versus controls

Table 3 Genotype frequencies of IL17A rs2275913 in Brazilian patients with LVSD in chronic Chagas cardiomyopathy stratified accordingto gender

Gender IL17A G197AWithLVSD119899 ()

MildmoderateLVSD119899 ()

Control119899 ()

MaleGG 23 (451) 13 (591) 43 (5811)GA 24 (4706) 9 (409) 30 (4054)AA 4 (784) 0 1 (135)

FemaleGG 23 (451) 11 (3793) 45 (5921)GA 22 (4314) 15 (5172) 29 (3816)AA 6 (1176)a 3 (1035)b 2 (263)

LVSD chronic Chagas cardiomyopathy patients with left ventricular systolic dysfunction OR odds ratio CI confidence intervalData adjusted by ageOnly significant results are showedaOR = 663 and 95 CI = 121ndash3640 with LVSD versus controlbOR = 757 and 95 CI = 107ndash5340 mildmoderate LVSD versus control

Considering the variable age no significant difference wasobserved between IL17 SNPs and CD andor the severity ofthe left ventricular systolic dysfunction (LVSD)

4 Discussion

The identification of genes that are candidates for susceptibil-ity or protection against CD has major implications not onlyto better understand the pathogenesis of the disease but alsoto control and develop therapeutic strategies In this study apossible association between the genetic polymorphisms ofIL17A G197A and IL17F T7488C with CD and the severity

of CCC was investigated in a population from South andSouthwest regions in Brazil

In this study the IL17A A allele and the AA genotypewere more frequent in CD and CCC patients female withLVSD or mildmoderate LVSD and male with LVSD whencompared to control The risk to severe LVSD was observedin male carrying the IL17F TC genotype The IL17 polymor-phism could be correlated to the risk of disease indicatingsusceptibility to chronic Chagas disease and increasing riskof severe cardiomyopathy when gender was considered inmultivariate analyses The mutant allele A of IL17A wasassociated with a higher production of IL-17 [12] and the


Table 4 Genotype frequencies of IL17F rs753780 in Brazilian Chagas disease patients with chronic cardiomyopathy stratified according togender

Gender IL17F T7488CWithoutLVSD119899 ()


SevereLVSD119899 ()

Without CCC119899 ()

Controls119899 ()

Male TT 40 (8696) 21 (913) 19 (655) 22 (458) 64 (8649)TC 6 (1304)a 2 (87)b 10 (345) 2 (42)c 10 (1351)d

Female TT 58 (9206) 24 (828) 20 (909) 20 (417) 68 (8947)TC 5 (794) 5 (172) 2 (91) 4 (83) 8 (1053)

CCC chronic Chagas cardiomyopathy LVSD left ventricular systolic dysfunction OR odds ratio CI confidence intervalData adjusted by ageOnly significant results are showedaOR = 482 and 95 CI = 155ndash1498 severe LVSD versus without LVSDbOR = 602 and 95 CI = 118ndash3078 severe LVSD versus mildmoderate LVSDcOR = 670 and 95 CI = 119ndash3753 severe LVSD versus without CCC patientsdOR = 340 and 95 CI = 124ndash931 severe LVSD versus controls

IL-17F activity is similar to IL-17A although significantlyweaker [10 12 13] Based on these findings it is possible toinfer that the higher production of IL-17 a proinflammatorycytokine could contribute to tissue damage and might berelated to the development and progression of CCC in thispopulation

Considering the IL-17 biological function in Chagasdisease Guedes et al [24] showed that the neutralization ofIL-17 in mice BALBc infected with T cruzi has resulted in ahigher recruitment of inflammatory cells to the cardiac tissuein the acute phase of the infection leading to an increase inmyocarditis and consequently premature death despite thereduction of the local parasitism Miyazaki et al [25] havereported the importance of the IL-17 in the T cruzi infectionand the cardiac inflammation control in CD They observedthat in the experimental acute infectionwithT cruzi disabledmice in IL-17 presented a higher mortality rate and para-sitemia when compared to the group control (C57BL6 wildtype) as well as a lower expression of cytokines as IFN-120574 IL-6 andTNF-120572 suggesting a protective role of IL-17 in the acutephase of the disease The neutralization of IL-17 also resultedin a higher production of IL-12 IFN-120574 TNF-120572 chemokinesand its receptors indicating that the IL-17 may perform arole in the control of cardiac inflammation through themodulation of Th1 response On the other hand Magalhaeset al [26] showed that in Chagas patients with cardiac formthe total lymphocytes and the Th17 cells presented a lowexpression of IL-17A in comparison to the patients with theindeterminate form and control group and the analysis ofcorrelation between IL-17A and the cardiac function showedthat the high expression of this cytokine was associated with abetter clinical outcome in the human CD according to valuesof the ejection fraction and left ventricular diastolic diameterindicating a protective role against the severity of CCC

Five SNPs of IL17A were analyzed in patients with CD ina population of an endemic region of Colombia gtThe SNPrs8193036 was associated with the protection against T cruziinfection and the development of CCC Meanwhile for theSNP rs2275913 the same SNP evaluated in this study the fre-quency of allele A was higher in patients than in controls and

significant differencewas observed although significancewaslost after the correction [18] We observed that IL17A A alleleand AA genotype were higher in Chagas disease as well inCCC with or without LVSD but no difference was observedbetween CD or CCC patients However after stratifyingaccording to gender female with IL17AAA genotype had riskof developingmildmoderate LVSD (approximately seven) asmale to develop LVSD (although not significant) and malewith IL17F TC genotype had higher risk to develop severeLVSD compared to other cardiac form and controls

A study conducted by Peng et al [27] in Chinese patientswith dilated cardiomyopathy did not find association withIL17A G197A and IL17F T7488C polymorphism Howeverafter stratification by gender the IL17F was associated withdilated cardiomyopathy in male patients that present theTC-CC genotypes suggesting that the presence of therare allele (C) might be associated with the disease inthese patients In this study we found that the IL17F TCgenotype was associated with developing severe LVSD inmale patients when the sample stratification by gender wasdone

The risk of development severe cardiac form in malewith CD was showed in two Brazilian studies Rassi et al[28] showed that gender (male) and left ventricular systolicdysfunction on echocardiography are potential risk factorsfor death in subjects with CD They evaluated a cohortof 424 Brazilian outpatients followed for about eight yearsand confirmed the results in 153 patients of other Braziliancommunity hospital Fae et al [29] observed a higher riskof developing severe forms of cardiomyopathy in men (OR= 875) corroborating the results of this study

The present study has potential limitations The majorlimitation was the number of patients limiting the signifi-cance of results and consequently no strong association couldbe found principally when independent multiple compar-isons were carried out However the risk of population strat-ification bias due to differences in ethnic background wasminimized by matching patients with controls individualsof the same ethnic background Mean age gender ratesand residence in the same geographical areas were carefully


matching to select the groups Another limitation was thatIL17 gene expression or serum levels were not evaluated

5 Conclusions

In these South and Southeast Brazilian patients the IL17Apolymorphisms AA genotype and A allele were associatedwith susceptibility to chronic CD and the severity of theleft ventricular systolic dysfunction (LVSD) In additionthe IL17A AA genotype was associated with mildmoderateLVSD in female patients whereas the IL17F TC genotypewasassociated with severe LVSD in male patients These resultssuggest the possible involvement of the polymorphisms ofIL17A and IL17F in the susceptibility to chronic CD and indevelopment and progression of CCC Additional studies areneeded to confirm these results and for understanding thefunctional role polymorphism in CD

Disclosure

Partial data from this study were presented in the 20thCongress of the Brazilian Society of Bone Marrow Trans-plantation Fortaleza Ceara Brazil in August 24ndash27 2016available in httpamborgbr_ebook2016RAMBRamb-su-plementar-agosto-2016indexhtml1z and identified as P-114 The opinions assumptions and conclusions or recom-mendations expressed in this material are the responsibilityof the authors and do not necessarily reflect the views of theFAPESPThe present address of Reinaldo Bulgarelli Bestetti isDepartamento de Medicina Universidade de Ribeirao PretoAv Costabile Romano No 2201 14096-900 Ribeirao PretoSP Brazil

Competing Interests

The authors declare that there is no conflict of interestsinvolved

Acknowledgments

The authors appreciate the participation and cooperation ofall volunteers (patients and controls) and of the RegionalBlood Bank in Maringa the Clinical Hospital in Lond-rina the Chagas Disease Laboratory in State University ofMaringa (UEM) the Base Hospital of Medical School inSao Jose do Rio Preto and the technicians of the Immuno-genetics Laboratory (LIG-UEM) for the assistance with thesample collection This study was financially supported byFundacao Araucaria (State of Parana Research Founda-tion) CNPq CAPES and FAPESP (Sao Paulo ResearchFoundation) Grants nos 201108075-4 201306580-9 andLIG-UEM

References

[1] WHO ldquoChagas disease (American trypanosomiasis)mdashFactsSheetrdquo in The Weekly Epidemiological Records vol 87 pp 519ndash522 World Health Organization Geneva Switzerland 2012

[2] PAHO Chagas Disease (American Trypanosomiasis) RegionalOffice for the Americas of the World Healthg OrganizationWashington DC USA 2014

[3] A Moncayo ldquoChagas disease current epidemiological trendsafter the interruption of vectorial and transfusional transmis-sion in the southern cone countriesrdquo Memorias do InstitutoOswaldo Cruz vol 98 no 5 pp 577ndash591 2003

[4] A M Macedo C R Machado R P Oliveira and S D JPena ldquoTrypanosoma cruzi genetic structure of populations andrelevance of genetic variability to the pathogenesis of chagasdiseaserdquoMemorias do Instituto Oswaldo Cruz vol 99 no 1 pp1ndash12 2004

[5] A Rassi Jr A Rassi and J A Marin-Neto ldquoChagas diseaserdquoThe Lancet vol 375 no 9723 pp 1388ndash1402 2010

[6] A Rassi A Rassi and J Marcondes de Rezende ldquoAmericanTrypanosomiasis (ChagasDisease)rdquo InfectiousDisease Clinics ofNorth America vol 26 no 2 pp 275ndash291 2012

[7] A Rassi Jr A Rassi and J A Marin-Neto ldquoChagas heartdisease pathophysiologic mechanisms prognostic factors andrisk stratificationrdquo Memorias do Instituto Oswaldo Cruz vol104 no 1 pp 152ndash158 2009

[8] C M Ayo M M D O Dalalio J E L Visentainer et alldquoGenetic susceptibility to chagas disease an overview about theinfection and about the association between disease and theimmune response genesrdquo BioMed Research International vol2013 Article ID 284729 13 pages 2013

[9] T A Moseley D R Haudenschild L Rose and A HReddi ldquoInterleukin-17 family and IL-17 receptorsrdquoCytokine andGrowth Factor Reviews vol 14 no 2 pp 155ndash174 2003

[10] N Hizawa M Kawaguchi S-K Huang and M NishimuraldquoRole of interleukin-17F in chronic inflammatory and allergiclung diseaserdquo Clinical and Experimental Allergy vol 36 no 9pp 1109ndash1114 2006

[11] T Korn E Bettelli M Oukka and V K Kuchroo ldquoIL-17 andTh17 cellsrdquo Annual Review of Immunology vol 27 pp 485ndash5172009

[12] J L Espinoza A Takami K Nakata et al ldquoA genetic variant inthe IL-17 promoter is functionally associated with acute graft-versus-host disease after unrelated bone marrow transplanta-tionrdquo PLoS ONE vol 6 no 10 Article ID e26229 2011

[13] M Kawaguchi D Takahashi N Hizawa et al ldquoIL-17F sequencevariant (His161Arg) is associatedwith protection against asthmaand antagonizes wild-type IL-17F activityrdquo Journal of Allergyand Clinical Immunology vol 117 no 4 pp 795ndash801 2006

[14] G B N Nordang M K Viken J E Hollis-moffatt et alldquoAssociation analysis of the interleukin 17A gene in Caucasianrheumatoid arthritis patients from Norway and New ZealandrdquoRheumatology vol 48 no 4 pp 367ndash370 2009

[15] J M V Zacarias E A Sippert P Y Tsuneto J E L VisentainerC D O E Silva and A M Sell ldquoThe influence of interleukin17A and IL17F polymorphisms on chronic periodontitis diseasein Brazilian patientsrdquo Mediators of Inflammation vol 2015Article ID 147056 8 pages 2015

[16] X Wu Z Zeng B Chen et al ldquoAssociation between polymor-phisms in interleukin-17A and interleukin-17F genes and risksof gastric cancerrdquo International Journal of Cancer vol 127 no 1pp 86ndash92 2010

[17] LWang Y Jiang Y Zhang et al ldquoAssociation analysis of IL-17Aand IL-17F polymorphisms in Chinese han women with breastcancerrdquo PLoS ONE vol 7 no 3 Article ID e34400 2012


[18] D A L Rodriguez L E Echeverrıa C I Gonzalez and JMartin ldquoInvestigation of the role of IL17A gene variants inChagas diseaserdquo Genes and Immunity vol 16 no 8 pp 536ndash540 2015

[19] O P Dutra H W Besser H Tridapalli et al ldquoSociedadeBrasileira de Cardiologia II Brazilian guideline for severe heartdiseaserdquo Arquivos Brasileiros de Cardiologia vol 87 no 2 pp223ndash232 2006

[20] F C Parra R C Amado J R Lambertucci J Rocha C MAntunes and S D J Pena ldquoColor and genomic ancestry inBraziliansrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 100 no 1 pp 177ndash182 2003

[21] S W M John G Weitzner R Rozen and C R Scriver ldquoArapid procedure for extracting genomic DNA from leukocytesrdquoNucleic Acids Research vol 19 no 2 article 408 1991

[22] Sun Wei Guo and E A Thompson ldquoPerforming the exact testof Hardy-Weinberg proportion for multiple allelesrdquo Biometricsvol 48 no 2 pp 361ndash372 1992

[23] X Sole E Guino J Valls R Iniesta and V Moreno ldquoSNPStatsaweb tool for the analysis of association studiesrdquoBioinformaticsvol 22 no 15 pp 1928ndash1929 2006

[24] P M da Matta Guedes F R S Gutierrez F L Maia et al ldquoIL-17 produced during Trypanosoma cruzi infection plays a centralrole in regulating parasite-inducedmyocarditisrdquoPLoSNeglectedTropical Diseases vol 4 no 2 article e604 2010

[25] Y Miyazaki S Hamano S Wang Y Shimanoe Y Iwakura andH Yoshida ldquoIL-17 is necessary for host protection against acute-phase Trypanosoma cruzi infectionrdquo The Journal of Immunol-ogy vol 185 no 2 pp 1150ndash1157 2010

[26] L M D Magalhaes F N A Villani M D C P Nunes K JGollob M O C Rocha and W O Dutra ldquoHigh interleukin 17expression is correlated with better cardiac function in humanChagas diseaserdquoThe Journal of Infectious Diseases vol 207 no4 pp 661ndash665 2013

[27] Y Peng B Zhou Y-Y Wang et al ldquoAnalysis of IL-17 gene poly-morphisms in Chinese patients with dilated cardiomyopathyrdquoHuman Immunology vol 74 no 5 pp 635ndash639 2013

[28] A Rassi Jr A Rassi W C Little et al ldquoDevelopment andvalidation of a risk score for predicting death in Chagasrsquo heartdiseaserdquo New England Journal of Medicine vol 355 no 8 pp799ndash808 2006

[29] K C Fae S A Drigo E Cunha-Neto et al ldquoHLA and120573-myosinheavy chain do not influence susceptibility to Chagasrsquo diseasecardiomyopathyrdquo Microbes and Infection vol 2 no 7 pp 745ndash751 2000

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Characteristics of the chronic Chagas disease patients and controls from South and Southeast of Brazil

CD patients CCC Without CCC Control119873 = 260 119873 = 212 119873 = 48 119873 = 150

Gendera 119899 ()Male 121 (465) 97 (458) 24 (500) 74 (493)Female 139 (535) 115 (542) 24 (500) 76 (507)

Ageb

Min-max 31ndash90 31ndash90 38ndash76 28ndash100Mean plusmn SD (year) 629 plusmn 100 639 plusmn 102 586 plusmn 78 623 plusmn 174

CCC patients with chronic Chagas cardiomyopathy Min minimum age Max maximum age SD standard deviationaNo statistically significant difference was observed between the groups for genderbStatistically significant difference was observed between the groups for age CCC versus without CCC

main death cause in patients aged between 30 and 50 years[5 7]

It is known that genetic variability and immunologicresponse influence the pathogenesis of the chronic phase ofthe disease Associations were observed in several cytokinegenes [8] with the susceptibility or protection against thedevelopment or progression of the CD andor its clinicalformsThe IL-17 is a proinflammatory cytokine secreted by Tcells activated and expressed in different tissuesThis cytokinetakes part in inflammatory responses mediated by T cellsand plays an important role in the tissue homeostasis anddiseases progression [9] The IL-17F presents a high degreeof homology with the IL-17A (57 identical) [9] and seemsto have a biological action similar to IL-17A in vitro and invivo though significantly weaker [10] The genes that codifythem are mapped in the same chromosome in the position6p12 [9 11]

Polymorphism in genes encoding cytokines may influ-ence the level of cytokines production and consequentlycause different immunological responses to different dis-eases Previous studies show that genetic polymorphismsof IL17A G197A and IL17F T7488C affect the productionof IL-17A and F respectively [12 13] Such polymorphismshave already been associated with autoimmune and inflam-matory diseases as rheumatoid arthritis [14] periodontitis[15] and cancer both gastric [16] and breast cancer [17]To our knowledge only one study involving the SNPsof IL17A and the CD [18] was found so far and if weconsider the SNPs of IL17F there are no related articlespublished yet For this reason our study aims to investi-gate whether the genetic polymorphisms of IL17A G197A(rs2275913) and IL17F T7488C (rs763780) were related toCD andor the severity of the left ventricular systolic dys-function (LVSD) in patients with CCC from North andNortheast regions of Parana and the Northeast region of SaoPaulo (states located in the South and Southeast of Brazilresp)

2 Material and Methods

21 Patients and Controls For this study 260 patients withchronic CDwere selected from different municipalities in theNorth and Northwest regions of Parana and in the Northwestregion of Sao PauloThe patients were cared for in the Chagas

Disease Laboratory in the State University of Maringa theClinical Hospital in Londrina and the Base Hospital of theMedical School in Sao Jose do Rio Preto All patients weresubmitted to a resting electrocardiogram (ECG) exam and atwo-dimensional echocardiography Patients who presenteda normal ECG were classified as patients without CCCand patients with electrocardiographic changes common toCCC were classified as patients with CCC The severity ofthe LVSD was measured according to the left ventricularejection fraction (LVEF) and the Teichhoolz method wasapplied following the II Brazilian Guideline for Severe HeartDiseases [19] Patients with CCC were classified consideringthe (LVEF) in three different groups patients without LVSD(LVEF gt 60) patients with mild to moderate LVSD (LVEF40ndash60) and patients with severe LVSD (LVEF lt 40) Toall statistical analysis were considered the following groupsall Chagas disease patients (CD) chronic Chagas cardiomy-opathy patients (CCC) without Chagas cardiomyopathypatients (without CCC) chronic Chagas cardiomyopathypatients with LVSD (with LVSD) chronic Chagas cardiomy-opathy patients without LVSD (without LVSD) patients withmild to moderate LVSD (Mildmoderate LVSD) and patientswith severe LVSD (severe LVSD)

The control group was composed of 150 individualshealthy and nonrelated patientrsquos spouses and contacts retire-ment communitiesrsquo residents with negative serology to Tcruzi antigens The clinicopathological features of patientsand controls are presented in Table 1 No significant differ-ences were observed among groups in terms of gender butdifferences in age were observed between CCC and withoutCCC patients (639 plusmn 102 versus 586 plusmn 78 respectively119875 le 005) Due to the significant miscegenation of Brazilianpopulation we consider patients and controls as a mixedethnic group (CaucasiansMulattos and Blacks) according toParra et al (2003) [20] Mean age gender rates and residencein the same geographical areas were carefully matching toselect the groups

The laboratory diagnosis of CD in patients and con-trols was made by ELISA (Enzyme-Linked ImmunoSorbentAssay) test in serum or plasma using the immunoas-say ldquoChagasrdquo from Abbott Laboratories (Santiago Chile)In cases of weak reagent the diagnosis was confirmedby the indirect immunofluorescence test (IIFT) with theIMUNOCRUZI antigen (Biolab Rio de Janeiro Brazil) or







3 Results










WithLVSD

MildmoderateLVSD

SevereLVSD

WithoutCCC Control

119873 = 260 119873 = 212 119873 = 109 119873 = 103 119873 = 52 119873 = 51 119873 = 48 119873 = 150









Control119899 ()

MaleGG 23 (451) 13 (591) 43 (5811)GA 24 (4706) 9 (409) 30 (4054)AA 4 (784) 0 1 (135)

FemaleGG 23 (451) 11 (3793) 45 (5921)GA 22 (4314) 15 (5172) 29 (3816)AA 6 (1176)a 3 (1035)b 2 (263)



4 Discussion








SevereLVSD119899 ()


Controls119899 ()

Male TT 40 (8696) 21 (913) 19 (655) 22 (458) 64 (8649)TC 6 (1304)a 2 (87)b 10 (345) 2 (42)c 10 (1351)d

Female TT 58 (9206) 24 (828) 20 (909) 20 (417) 68 (8947)TC 5 (794) 5 (172) 2 (91) 4 (83) 8 (1053)











5 Conclusions


Disclosure


Competing Interests


Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results










WithLVSD

MildmoderateLVSD

SevereLVSD

WithoutCCC Control

119873 = 260 119873 = 212 119873 = 109 119873 = 103 119873 = 52 119873 = 51 119873 = 48 119873 = 150









Control119899 ()

MaleGG 23 (451) 13 (591) 43 (5811)GA 24 (4706) 9 (409) 30 (4054)AA 4 (784) 0 1 (135)

FemaleGG 23 (451) 11 (3793) 45 (5921)GA 22 (4314) 15 (5172) 29 (3816)AA 6 (1176)a 3 (1035)b 2 (263)



4 Discussion








SevereLVSD119899 ()


Controls119899 ()

Male TT 40 (8696) 21 (913) 19 (655) 22 (458) 64 (8649)TC 6 (1304)a 2 (87)b 10 (345) 2 (42)c 10 (1351)d

Female TT 58 (9206) 24 (828) 20 (909) 20 (417) 68 (8947)TC 5 (794) 5 (172) 2 (91) 4 (83) 8 (1053)











5 Conclusions


Disclosure


Competing Interests


Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















WithLVSD

MildmoderateLVSD

SevereLVSD

WithoutCCC Control

119873 = 260 119873 = 212 119873 = 109 119873 = 103 119873 = 52 119873 = 51 119873 = 48 119873 = 150









Control119899 ()

MaleGG 23 (451) 13 (591) 43 (5811)GA 24 (4706) 9 (409) 30 (4054)AA 4 (784) 0 1 (135)

FemaleGG 23 (451) 11 (3793) 45 (5921)GA 22 (4314) 15 (5172) 29 (3816)AA 6 (1176)a 3 (1035)b 2 (263)



4 Discussion








SevereLVSD119899 ()


Controls119899 ()

Male TT 40 (8696) 21 (913) 19 (655) 22 (458) 64 (8649)TC 6 (1304)a 2 (87)b 10 (345) 2 (42)c 10 (1351)d

Female TT 58 (9206) 24 (828) 20 (909) 20 (417) 68 (8947)TC 5 (794) 5 (172) 2 (91) 4 (83) 8 (1053)











5 Conclusions


Disclosure


Competing Interests


Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















SevereLVSD119899 ()


Controls119899 ()

Male TT 40 (8696) 21 (913) 19 (655) 22 (458) 64 (8649)TC 6 (1304)a 2 (87)b 10 (345) 2 (42)c 10 (1351)d

Female TT 58 (9206) 24 (828) 20 (909) 20 (417) 68 (8947)TC 5 (794) 5 (172) 2 (91) 4 (83) 8 (1053)











5 Conclusions


Disclosure


Competing Interests


Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions


Disclosure


Competing Interests


Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Genetic Polymorphisms of IL17 and Chagas Disease in the ...downloads.hindawi.com/journals/jir/2017/1017621.pdf · ResearchArticle Genetic Polymorphisms of IL17 and Chagas Disease