Analysis of filaggrin 2 gene polymorphisms in patients with atopic dermatitis,

Anais

Brasileiros

de

Dermatologia

www.anaisdedermatologia.org.br

INVESTIGATION

Analysis

of

filaggrin

2

gene

polymorphisms

in

patients

with

atopic

dermatitis

夽,夽夽

Amanda

Hertz

_,

_Luna

_{Azulay-Abulafia}

_,

_Adriana

_Paulino

_do

_Nascimento

_,

Cintya

Yumi

Ohara

_,

_Fabio

_Chigres

_Kuschnir

_,

_Luís

_Cristovão

_Porto

a_{PediatricDermatologyClinic,InstitutodeDermatologiaProf.RubemDavidAzulay,RiodeJaneiro,RJ,Brazil}

b_{DepartmentofDermatology,InstitutodeDermatologiaProf.RubemDavidAzulay,UniversidadedoEstadodoRiodeJaneiro,Rio} deJaneiro,RJ,Brazil

c_{HistocompatibilityandCryopreservationLaboratory,UniversidadedoEstadodoRiodeJaneiro,RiodeJaneiro,RJ,Brazil} d_{DepartmentofAllergyandImmunopathology,UniversidadedoEstadodoRiodeJaneiro,RiodeJaneiro,RJ,Brazil}

e_{DepartmentofPediatrics,FaculdadedeCiênciasMédicas,UniversidadedoEstadodoRiodeJaneiro,RiodeJaneiro,RJ,Brazil} f_{DepartmentofAllergy,UniversidadedoEstadodoRiodeJaneiro,RiodeJaneiro,RJ,Brazil}

Received8July2018;accepted26July2019 Availableonline25January2020

KEYWORDS Allergy; Dermatitis,atopic; Immunology; Polymorphism, genetic Abstract

Background: Polymorphismsofthefilaggrin2gene(rs12568784andrs16899374)areassociated with persistentatopicdermatitisinAfricanAmerican patients.Filaggrin 2isaproteinwith afunctionsimilar tofilaggrinandalsoencodedintheepidermaldifferentiationcomplexon chromosome1q21.

Objective: Toevaluatethepolymorphismsinthefilaggrin2gene(rs12568784andrs16899374) inchildrenandadultswithatopicdermatitisandtoverifytheassociationofthesewiththe severity ofthe clinicalpicture, presence ofother allergicdiseases, and socio-demographic factors.

Method: Thestudywascarriedoutwithpatientsandcontrolgroup.Questionnaireswereused toevaluateethnicity,sex,age,familyhistory,scoring,atopicdermatitis(SCORAD),amongother parameters.Genotypingofthefilaggrin2genewasperformedbyreal-timepolymerasechain reaction.

Results: Forty-eightpatientsand83controlswereevaluated.Nocorrelationwasfoundbetween thevariablesstudiedinpatientswithatopicdermatitisandpolymorphisms,nosignificant dif-ferencebetween theprevalenceofpolymorphismsinthepatientsandinthecontrolgroup p>0.05.

夽 _{How tocitethisarticle: Hertz A, Azulay-AbulafiaL, Nascimento AP,OharaCY, Kuschnir FC,Porto LC.Analysis offilaggrin 2 gene}

polymorphismsinpatientswithatopicdermatitis.AnBrasDermatol.2020;95:173---9.

夽夽_{StudyconductedattheAtopicDermatitisClinic,OliclínicaPiquetCarneiro,UniversidadedoEstadodoRiodeJaneiro,RJ,Brazil.} ∗_{Correspondingauthor.}

E-mail:[email protected](A.Hertz).

https://doi.org/10.1016/j.abd.2019.07.002

0365-0596/©2020SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.ThisisanopenaccessarticleundertheCC BYlicense(http://creativecommons.org/licenses/by/4.0/).

Studylimits:Theexclusiveuseofself-reportedethnicityinformationandthesamplesize. Results: Theresultsofthisworkcanbeanincentivefor thestudyofthepolymorphismsin atopicdermaititis,consideringthecharacteristicoftheBrazilianmultiethnicpopulation. Conclusion: ThisisanunpublishedworkinBrazilandthefirst studyintheworldtohavea controlgrouptoevaluatealterationsinthegeneoffilaggrin2.

©2020SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

Introduction

Atopic dermatitis (AD) is a frequent inflammatory skin disease that affects approximately20% of children, com-promisingthequalityoflifeofthepatientandthefamily. Mutations in the gene encoding the filaginous epidermal protein have been shown to be an important factor in thedevelopment of thisdisease.1 _{Filaggrinis also}

associ-atedwiththepersistenceandcoexistenceofotherallergic diseases.2

Thefilaggrin(FILAGGRIN:FILamentAGGRegatingproteIN) isaproteinfoundincorneocytesresponsibleforaggregating keratin in the formation of stratum corneum.3 _{It is}

pro-duced froma precursor, pro-filaggrin, which is located in thegranularlayerof theepidermis inthegranulesof ker-atohyalin. Pro-filaggrin is subsequently dephosphorylated, resulting in the formation of filaggrin monomers having keratinfilamentaggregationproperties.Filaggrin degrada-tiongenerates NaturalMoisturizing Factors (NMFs), which include free amino acids, Urocanic Acid (UCA), and Car-boxylicPyrrolidonicAcid(PCA).NMFspromotewateruptake andentrapment,andmaintenanceofacidicpHoftheskin, essentialfor theprocessofhydration andmaintenanceof theskinbarrier.4

The gene encoding filaggrin is located onchromosome 1q21,inaregioncalledepidermaldifferentiationcomplex.3

Accordingtotheliterature,27.5%ofCaucasianAmericans, 48% of Europeans,31.4% of Chinese and 20% of Japanese withADpresentmutationsinthefilaggringene.5

Currently,47mutationswithlossoffunctionhavealready been identified in the gene encoding filaggrin in patients with AD. Profiles other than mutations were identified in patients from Europe and Asia.6 _{Among the mutations}

described, the most commonly found in Caucasians are R501Xand2282del4(comprising50%ofthetotal),in addi-tiontoR2447XandS3247X.7_{IntheJapanesepopulation,the}

main mutationswere 3321delAand S2554X.8 _These

muta-tionsseemtovaryaccordingtoethnicgroups,forexample, in the African-American population studies have shown a much lower prevalence than in Caucasians.9 _{Gao et al.}

foundthatR501Xwasfoundin3.2%of187African-American patients with AD and no association with 2282 of the 4 patients.10 _{Margolis et al. evaluatedthe genotype of 370}

AfricanAmericanswithADandfoundmutationsinonly,1% ofpatients (R501X, 3.2%;2282del4, 0.5%;S3247X, 3%and R2447X,1.4%).7,8

Filaggrin 2 is a protein whose function is similar to filaggrinand also encodedin the region called epidermal differentiation complex, according to Makino et al. the expression of filaggrin 2 protein is decreased in patients withAD.11 _{Thereisanassociation betweenpolymorphisms}

in the filaggrin 2 gene (rs 12568784 and rs 1689937411) andpersistentADinAfricanAmericanpatientswithatopic dermatitis.12

Theaimofthisstudywastoevaluatethepolymorphisms inthefilaggrin2gene(rs12568784andrs16899374)in chil-dren and adults with atopic dermatitis and to verify the associationofthesewiththeseverityoftheclinicalpicture, presenceof otherallergicdiseases andsocio-demographic factors.

Methods

A cross-sectional study involving patients of both sexes, agedbetween 1and 27years,diagnosed withatopic der-matitis, according to United Kingdom Working Party (UK) criteria,13_{recruitedfromoutpatientclinicsforatopic}

der-matitis. Patients with other chronic diseases of the skin (psoriasis, amongothers) or systemic diseases thataffect the skin, such as primary immunodeficiencies, systemic lupuserythematosuswereexcluded.

Accordingtotheorderofarrivalintheclinic,thepatients answeredaquestionnairewiththefollowingvariables: gen-der, age, ethnicity,onset ofAD, familyhistory of allergic diseases and presence of asthma, food allergy and rhini-tis.TheISAACquestionnaire,validatedforourlanguage14,15

wasusedtoevaluatethepresenceofasthmaandrhinitis.In addition,theseverityofADwasanalyzedbySCORAD (Scor-ing AtopicDermatitis).14,15 _{The severity levelsofAD were}

definedaslight:SCORADupto25points;moderatebetween 25---50points;andserious,above50points.16

After signing of consent terms, venous blood samples werecollectedintubescontainingEDTA.Inordertoincrease thereliabilityofthestudyandbecauseoftheethical dif-ficulty of creating a pediatric control group with blood collection,controlsamplesfromthesamepopulationgroup fromabonemarrowbankwerechosen,whichwerematched bysexandethnicity.

To determine the genetic variation of filaggrin 2, the genomic DNA of the individuals participating in the study waspurifiedusingtheQIAamp DNABloodMiniKit (Qiagen, Valencia,CA)accordingtothemanufacturer’sinstructions. Theprincipleofthiskitisbasedontheextractionofgenomic DNAfromnucleatedbloodcellsandsubsequentbindingof the DNA to a glass fiber matrix contained in a MicroSpin column.Briefly, after digestionof the cellmembranes by proteinaseK,thenucleicmaterialwastransferredtoa sil-ica membrane where it wassubjected to several washes withalcoholicsolutions.Theelutionwasdonebythe addi-tionofultrapurewater.ThegenomicDNAwasquantifiedby measuringtheabsorbanceattheopticaldensityof260nm

(OD260)usingtheNanoDropLitespectrophotometer (Ther-moScientific).Thefinalpreparationwasstoredat−20◦C.

Genotyping of the filaggrin 2 gene (rs16833974 and rs12568784) was performed by real-time PCR using com-merciallyavailableTaqMan®_{(ABIassay;AppliedBiosystems)}

probelabeledwiththeVICandFAMfluorophores foreach allele, C3425690910 for rs16833974 and C11261511 to rs12568784.Briefly, the reactionwasperformed in afinal volumeof 10␮Lcontaining40ngof genomicDNA,Taqman SNPGenotypingAssays 40XandTaqmanGenotypingMaster Mix according to themanufacturer’s instructions. Initially the sampleswere subjected toapre-PCR reading stepat 60◦Cfor30s,followedbythedenaturationstepat95◦Cfor 10min.Thentheamplificationstageconsistedof40cycles of 95◦Cfor 15s and 60◦C for 1min, for annealing of the primerandextensionofthefragments.Afteramplification oftheDNAfragments,thesamplesweresubmittedtothe finalpost-PCRreadingstepat60◦Cfor30s.ThePCRreaction wasperformed withthe aid of the ABI StepOnePlus Real-TimePCRSystem(AppliedBiosystems,FosterCity,CA).The resultsoftheallelicdiscriminationanalysiswereplottedon ascatter plotcontrastingthefluorescenceofeach probe. Inordertoincrease thereliabilityof thestudyinrelation tomutationanalysis,controlsamplesfromabonemarrow bankwereanalyzed.

Statisticalanalysis

A sample of 48 patients with a larger control group (1.7 times)of83patientswasusedforabetterstatistical anal-ysis. Thefrequency andrelativefrequency statisticswere usedtopresentthevariablesindescriptivetables,inorder tounderstandtheprofileofthesampleandalsoofthe con-trols.Inordertoverifypossibleassociations,theChi-square test, orFisher’sexact test,wasusedtoidentifyevidence thatshows thesignificanceofcertainvariablesinrelation tothegeneticmutationstudiedandSCORAD(Scoringatopic dermatitis)level.

Thesignificancelevelof0.05,p-value<0.05wasusedto evaluatetheassociationsbetweenthedependentvariable (mutationsinthefilaggrin2gene)andtheother variables ofinterestinthestudy.TheprogramsMicrosoftExcel2010 andSoftwareR,version3.3.1 (RCoreTeam2015,Vienna, Austria)wereemployedfororganizing,creatingtablesand statisticalanalysisofdata.Thestudyfollowedtheprinciples oftheHelsinkiDeclarationandwasapprovedbytheethics committeeunderopinionn◦36934514.9.0000.5259.

Results

FromNovember2015toFebruary2016datawerecollected from48patients,54%male.Thecontrolgroupconsistedof 83samples.Thetotalpolymorphismprevalencers16833974 inthegroupofpatientswas16.7%(14.6%heterozygoteand 2.1%homozygous)and15.7%inthecontrolgroup(15.7% het-erozygous)andthetotalprevalenceofrspolymorphismrs 12568784were:43.7%ofthepatients(35.4%heterozygous and8.3%homozygous)andcontrol:46.9%(37.3% heterozy-gousand9.6%homozygous).Themaincharacteristicsofthe sampleandthecontrolgrouparedescribedintable1.

Thegeneticpolymorphismdistributionofrs16833974and rs12568784accordingtothesociodemographicvariablesof thesampleandthecontrolgrouparerespectivelyshownin

table2.

Themainvariablesstudiedwere:gender,ethnicity, SCO-RAD,familyhistoryofatopyandassociatedallergicdiseases. Asforpolymorphismrs16833974,statisticallysignificant dif-ferenceswere foundregardingthe ethnicityvariable,and thedarkertheskincolorthegreaterthechanceofhaving thepolymorphism,p-value=0.031(Table3).

Asforpolymorphismrs12568784,thefollowingvariables were alsostudied: sex, ethnicity, SCORAD,family history of atopy and associated allergic diseases (food allergy, asthmaandrhinitis)andnostatisticalsignificancewasfound amongthevariablesexceptfortheparentvariable.Thetest

hadp-value=0.032,whichwaslowerthanthesignificance

level of 0.05 (5%).Given this,it is possible toaffirm the existence of statistical evidences that prove some causal relationbetweenpolymorphismandAtopy(Father). Accord-ing to the data in the table we can see a reduction in polymorphism rs12568784 in the presence of Atopia (Pai) (Table4).

A total of 46 crosses of interest were performed in order to verify statistical significance between polymor-phism and family history, sex, ethnicity, disease severity, atopy (asthma, rhinitis, familial allergy) and comparison withcontrolofmutationfrequency,ethnicityandsex. We soughttoperformevaluationseparatelyfromheterozygous and homozygous polymorphisms, but no association was found.

Discussion

Therelationshipbetween polymorphismsofthefilaggrin2 gene and AD has recently been reported.11 _{According to}

theliterature,filaggrin2 polymorphismmayinfluencethe severityofthedisease.11_{Inthisstudy,wesoughttoanalyze}

somepointsofthisassociationinabroadway,throughthe exploratoryanalysis andcorrelation of clinical and demo-graphicdata.

Itisimportanttoconsiderthatthecontrolgroupusedin thestudywasof convenience,withsamples ofbone mar-rowbank,duetothedifficultyoforganizingacontrolgroup ofchildren.Agenderandethnicitypairingwasperformed between thegroups. This is the first work that uses con-trolstoevaluate polymorphismsof filaggrin2 and DA.No statisticaldifferenceswerefoundbetweentheprevalence ofpolymorphismsin thegroup ofpatients withAD andin thecontrolgroup.In addition,therewasnodifferencein severityinpatientswithpolymorphism.

Likewise, when we analyzed the correlations between ageat onsetofADandpolymorphisms,therewereno sta-tistical differences. As described by Margolis et al., no associationof polymorphism withother atopic-valued dis-eases(asthma,rhinitis,foodallergy)wasalsofound.12

Familyhistoryisanimportantriskfactorforthe develop-mentofAD.Approximately70%ofpatientswithADpresent afamilyhistoryofatopicdiseases.Theprobabilityof devel-opingAD is about two to threetimes greater in children withoneatopicparent,andincreasestothreetofivetimes ifbothparents areatopic.17 _{Ingeneral,the maternal}

his-Table1 Distributionofsocio-demographicdataofpatientsandcontrols

Sociodemographicsvariables Patients Control

n % n % Sex F 22 46% 33 40% 0.58 M 26 54% 50 60% Ethnicity White 17 35% 29 35% Brown 12 25% 26 31% 0.51 Africanamerican 18 38% 28 34% Unidentified 1 2% 0 0% Age

Infant(0---2years) 4 8% 21---30years 55 66%

Children(3---9years) 28 58% 31---45years 21 25%

Adolescent(10---19years) 13 27% >45years 6 7%

Adult(20yearsormore) 3 6% Unidentified 1 1%

Table2 Distributionofthers16833974 and rs12568784polymorphismaccording tothe sociodemographicvariables ofthe

patientsandcontrolgroup

Sociodemographicsvariables Polymorphismrs16833974

Patients Control

Present Absent p-Value Present Absent p-Value

n % n % n % n % Sex F 4 8% 18 38% 1 5 6% 28 34% 0.14 M 4 8% 22 46% 8 10% 42 51% Ethnicity White 1 2% 16 33% 0.08 1 1% 28 34% 0.03 Brown 1 2% 11 23% 4 5% 22 27% AfricanAmerican 6 13% 12 25% 8 10% 20 24% Unidentified 0 0% 1 2% 0 0% 0 0%

Sociodemographicsvariables Polymorphismrs12568784

Patients Control

Present Absent p-Value Present Absent p-Value

n % n % n % n % Sex F 11 23% 11 23% 0.56 19 23% 14 17% 0.17 M 10 21% 16 33% 20 24% 30 36% Ethnicity White 7 15% 10 21% 0.38 10 12% 19 23% 0.51 Brown 3 6% 9 19% 17 20% 9 11% AfricanAmerican 10 21% 8 17% 12 14% 16 19% Unidentified 1 2% 0 0% 0 0% 0 0%

toryofADismorepredictiveforthedevelopmentofADin relationtopaternalatopy.18_{Inourstudy,85%ofthepatients}

presented positive family history for atopy, corroborating theliterature.

In general, no statistical differences were observed betweenfamilyhistoryofatopyandfilaggrinpolymorphisms 2, except for the frequency of the polymorphism muta-tion rs 12568784 in the presence of paternal atopy. This data can only refer to a statistical finding, due to

Table3 Variablesstudiedinthepatient’sgroupandpolymorphismrs16833974

Variables Polymorphismrs16833974 Total p-Value

Present Absent n % n % n % Sex F 4 50% 18 45% 22 46% 1 M 4 50% 22 55% 26 54% Total 8 100% 40 100% 48 100% Scorad Mild 6 75% 13 33% 19 40% 0.026 Moderate 2 25% 15 38% 17 35% Severe 0 0% 10 25% 10 21% Unidentified 0 0% 2 5% 2 4% Total 8 100% 40 100% 48 100% HFAtopia Yes 8 100% 33 83% 41 85% 0.56 No 0 0% 5 13% 5 10% Unidentified 0 0% 2 5% 2 4% Total 8 100% 40 100% 48 100% Mother Yes 5 63% 18 45% 23 48% 0.7 No 3 38% 17 43% 20 42% Unidentified 0 0% 5 13% 5 10% Total 8 100% 40 100% 48 100% Father Yes 6 75% 16 40% 22 46% 0.24 No 2 25% 19 48% 21 44% Unidentified 0 0% 5 13% 5 10% Total 8 100% 40 100% 48 100% Ethnicity White 1 13% 16 40% 17 35% 0.031 Brown 1 13% 11 28% 12 25% AfricanAmerican 6 75% 12 30% 18 38% Unidentified 0 0% 1 3% 1 2% Total 8 100% 40 100% 48 100% Asthma Yes 4 50% 18 45% 22 46% 0.66 No 2 25% 19 48% 21 44% Unidentified 2 25% 3 8% 5 10% Total 8 100% 40 100% 48 100% Foodallergy Yes 1 13% 15 38% 16 33% 0.37 No 5 63% 22 55% 27 56%

the small sample number and possibly without biologi-calplausibility.More studies are neededto elucidate this finding.

AccordingtoMargolisetal.(2014),filaggringene poly-morphisms2,rs12568784andrs16833974,areassociated with the persistence of AD in African Americans and are absentorarerarelyfoundinindividualsofEuropeanorigin.12

In the study population, there wasstatistical significance among the patients with autodeclared black AD and the polymorphismsrs16833974.

TheBrazilianpopulationhasahighdegreeof miscegena-tionand followsdifferentethnicpatternswhen compared topopulation groups from other countriesand continents suchasChina,theUnitedStates,JapanandEurope,where therearealreadystudieswiththeprevalenceoffilaggrin.5

Therefore,itisdifficulttoestablishaclassificationby eth-nicitiesinourcountry.Theexclusiveuseofinformationof theself-declared ethnicgroup isnot thebestmethod for ethnicclassification.19

Table4 Variablesstudiedinthepatient’sgroupandpolymorphismrs1256878

Variables Polymorphismrs12568784 Total p-Value

Present Absent n % n % n % Sex F 11 52% 11 41% 22 46% 0.56 M 10 48% 16 59% 26 54% Total 21 100% 27 100% 48 100% Scorad Mild 9 43% 10 37% 19 40% 0.91 Moderate 5 24% 12 44% 17 35% Severe 5 24% 5 19% 10 21% Unidentified 2 10% 0 0% 2 4% Total 21 100% 27 100% 48 100%

Familyhistoryatopia

Yes 20 95% 21 78% 41 85% 0.059 No 0 0% 5 19% 5 10% Unidentified 1 5% 1 4% 2 4% Total 21 100% 27 100% 48 100% Mother Yes 11 52% 12 44% 23 48% 1 No 9 43% 11 41% 20 42% Unidentified 1 5% 4 15% 5 10% Total 21 100% 27 100% 48 100% Father Yes 14 67% 8 30% 22 46% 0.032 No 6 29% 15 56% 21 44% Unidentified 1 5% 4 15% 5 10% Total 21 100% 27 100% 48 100% Ethnicity White 7 33% 10 37% 17 35% 0.38 Brown 3 14% 9 33% 12 25% AfricanAmerican 10 48% 8 30% 18 38% Unidentified 1 5% 0 0% 1 2% Total 21 100% 27 100% 48 100% Asthma Yes 8 38% 14 52% 22 46% 0.54 No 10 48% 11 41% 21 44% Unidentified 3 14% 2 7% 5 10% Total 21 100% 27 100% 48 100% Foodallergy Yes 6 29% 10 37% 16 33% 0.75 No 12 57% 15 56% 27 56% Unidentified 3 14% 2 7% 5 10% Total 21 100% 27 100% 48 100%

The genomes of most Brazilians are miscegenated and genetic markers are capable of providing valuable new insights into the current structure of the Brazilian population.19

Futureprospectsofancestral analysisofbloodsamples from our study may allow a better understanding of the associationbetweenfilaggrin2andDA,sincetheprevalence

offilaggringenemutationsshowethnicdifferencesinboth thegeneralpopulationandindividualswithDA.20

Thesmallsamplesizemayhavelimitedthesignificance ofourresultsandperhapsalargersampleand/orancestral studymaycontributetotheclarificationbetween individu-alswithADandfilaggrinpolymorphisms2.Theperformance oftheproteicalstudyoffilaggrin2,sincestructural

differ-ences may be due topost-transcriptional controls, would alsocontributetoabetterevaluationoffilaggrin2.

Todate,therearenostudiesoffilaggrin2andDAgene polymorphismsinLatin America,reinforcingtherelevance ofour studyandthe noveltyof thisinitiative inBrazil. In addition,this is the firstworldwide study using a control grouptoevaluatechangesinthefilaggringene2.

Conclusion

Very little is known about the profile of filagrins 2 poly-morphismsassociatedwithADinthehighlymixedBrazilian population.Theresultsof thiswork canbeaconsiderable incentivetoresearchonpolymorphismsandADinthe coun-try,collaboratingtoabetterunderstanding,notonlyofways oftreatingthedisease,butalsoofthegeneticprofileofthe population

Asforpolymorphismrs16833974,statisticallysignificant differenceswerefoundregardingtheethnicityvariable,and thedarkertheskin colorthegreaterthechance ofhaving thepolymorphism,p-value=0.031.

Financial

support

ThisworkwascarriedoutwiththefinancialsupportofFundo deApoioàDermatologia(FUNADERM)oftheBrazilian Soci-etyofDermatology.

Authors’

contributions

Amanda Hertz: Conception and planning of the study; obtaining,analysis,andinterpretationofthedata;critical reviewoftheliterature.

LunaAzulay-Abulafia:Effectiveparticipationinresearch orientation.

AdrianaPaulinodoNascimento:Conceptionandplanning ofthestudy;obtaining,analysis,andinterpretationofthe data.

CintyaYumiOhara:Conceptionandplanningofthestudy. FabioChigresKuschnir:Criticalreviewoftheliterature; criticalreviewofthemanuscript.

Luís Cristovão Porto: Conception and planning of the study;obtaining,analysis,andinterpretationofthedata.

Conflicts

of

interest

Nonedeclared.

References

1.Baurecht H, Irvine AD, Novak N, Illig T, Bühler B, Ring J, et al. Toward a majorrisk factor for atopic eczema: meta-analysisoffilaggrinpolymorphismdata.JAllergyClinImmunol. 2007;120:1406---12.

2.IrvineAD,McLeanWH,LeungDY.Filaggrinmutationsassociated withskinandallergicdiseases.NEnglJMed.2011;365:1315.

3.SandilandsA,SutherlandC,IrvineAD,McLeanWH.Filaggrinin thefrontline:roleinskinbarrierfunction anddisease.JCell Sci.2009;122:1285---94.

4.MócsaiG,GáspárK,NagyG,IrinyiB,KapitányA,BíróT,etal. Severeskininflammationandfilaggrinmutationsimilarlyalter theskinbarrierinpatientswithatopicdermatitis.BrJ Derma-tol.2014;170:617---24.

5.PolcariI,BeckerL,SteinSL,SmithMS,PallerAS.Filaggringene mutationsinAfricanAmericanswithbothichthyosisvulgarisand atopicdermatitis.PediatrDermatol.2014;31:489---92.

6.Flohr C, England K, Radulovic S, McLean WH, Campbel LE, Barker J, et al. Filaggrin loss-of-function mutations are associated with early-onset eczema, eczema severity and transepidermalwaterlossat3monthsofage.BrJDermatol. 2010;163:1333---6.

7.MargolisDJ, ApterAJ,GuptaJ,HoffstadO, PapadopoulosM, CampbellLE,etal.Thepersistenceofatopic dermatitisand filaggrin(FLG)mutationsinaUSlongitudinalcohort.JAllergy ClinImmunol.2012;130:912---7.

8.VercelliDMD.Advancesinasthmaandallergygeneticsin2007. JAllergyClinImmunol.2008;122:267---71.

9.BieberT.Atopicdermatitis.NEnglJMed.2008;358:1483---94.

10.GaoPS,RafaelsNM,HandT,MurrayT,BoguniewiczM,HataT, etal. Filaggrinmutationsthat conferriskofatopic dermati-tisconfer greaterriskfor eczemaherpeticum.JAllergy Clin Immunol.2009;124:507---13.

11.MakinoT,MizawaM,YamakoshiT,TakaishiM,ShimizuT. Expres-sion of filaggrin-2 protein in the epidermis of human skin diseases:acomparativeanalysiswithfilaggrin.BiochemBiophys ResCommun.2014;449:100---6.

12.Margolis DJ, Gupta J, Apter AJ, Ganguly T, Hoffstad O, PapadopoulosM,etal.Filaggrin-2variationisassociatedwith morepersistentatopicdermatitisinAfricanAmericansubjects. JAllergyClinImmunol.2014;133:784---9.

13.Williams HC, Burney PG, Hay RJ, Archer CB, Shipley MJ, Hunter JJ,et al. The U.K. WorkingParty’s Diagnostic Crite-ria for Atopic Dermatitis. I.Derivation ofa minimum setof discriminatorsforatopicdermatitis.BrJDermatol.1994;131: 383---96.

14.SoléD,VannaAT,YamadaE,RizzoMC,NaspitzCK.International Study of Asthma and Allergies in Childhood (ISAAC) written questionnaire: validation of the asthma component among Brazilian children. J Investig Allergol ClinImmunol. 1998;8: 376---82.

15.VannaAT,YamadaE,ArrudaLK,Naspitz CK,SoléD. Interna-tionalstudy ofasthmaand allergies inchildhood: validation oftherhinitissymptomquestionnaireandprevalenceof rhini-tisinschoolchildreninSãoPauloBrazil.PediatrAllergyImmunol. 2001;12:95---101.

16.OranjeAP,GlazenburgEJ,WolkerstorferA,deWaardvander Spek FB. Practical issueson interpretation ofscoring atopic dermatitis:theSCORADindex,objectiveSCORADandthe three-itemseverityscore.BrJDermatol.2007;157:645---8.

17.WadondaKabondoN,SterneJA,GoldingJ,KennedyCT,Archer CB, Dunnill MG,ALSPAC StudyTeam.Association ofparental eczema, hay fever, and asthma with atopic dermatitis in infancy:birthcohortstudy.ArchDisChild.2004;89:917---21.

18.RuizRG, KemenyDM,PriceJF.Higherriskofinfantile atopic dermatitisfrommaternalatopythanfrompaternalatopy.Clin ExpAllergy.1992;22:762---6.

19.Cardena MMSG,SantosAR, SantosS, MansurAJ, Pereira AC, FridmanC.Relac¸ãoentreaetniaauto-declarada,haplogrupo mitocondrialeancestralidadegenômicaemindivíduosdo sud-estebrasileiro.SaúdÉticaJustic¸a.2013;18:62---6.

20.WingeMCG,BradleyM.Prevalenceoffilaggringenemutations: anevolutionaryperspective.In:ThyssenJP,MaibachHI,editors. Filaggrin,vol.1.2014.p.119---28.