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perda de fun&#231;&#227;o do gene <span class="elsevierStyleItalic">FLG</span> associadas &#224; DA tem sido observada&#46;<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">5</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">Mais de&#160;60&#160;variantes do gene <span class="elsevierStyleItalic">FLG</span> que levam &#224; perda de fun&#231;&#227;o do gene <span class="elsevierStyleItalic">FLG</span> foram identificadas em associa&#231;&#227;o com DA&#59; as mais comuns entre os europeus s&#227;o c&#46;1537C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R501X e 2282del4&#58;S761Cfs&#42;36 e&#44; entre os africanos subsaarianos&#44; c&#46;9947C<span class="elsevierStyleHsp" style=""></span>&#62;G&#58;S3316&#42;&#46; Poucos estudos foram realizados em pacientes latino&#8208;americanos com DA&#46; Nosso objetivo foi avaliar a frequ&#234;ncia de variantes do gene <span class="elsevierStyleItalic">FLG</span> &#40;no &#233;xon&#8208;3&#41; entre pacientes com DA para comparar popula&#231;&#245;es brasileiras e internacionais e explorar suas caracter&#237;sticas cl&#237;nicas&#46;</p><p id="par0020" class="elsevierStylePara elsevierViewall">Foi realizado estudo transversal no ambulat&#243;rio de dermatologia &#40;FMABC&#59; Santo Andr&#233;&#44; S&#227;o Paulo&#41;&#46; Oitenta pacientes com DA &#40;crit&#233;rios de Hanifin e Rajka&#41; de ambos os sexos foram inclu&#237;dos e examinados por dermatologista experiente para avaliar a gravidade da doen&#231;a &#40;SCORAD&#44; EASI&#41; e coletar amostras de sangue venoso para an&#225;lise laboratorial e da mucosa oral para an&#225;lise gen&#233;tica&#46; Os participantes&#47;respons&#225;veis assinaram termo de consentimento livre e esclarecido&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">A coleta para an&#225;lise gen&#233;tica foi realizada por <span class="elsevierStyleItalic">swab</span> da mucosa da regi&#227;o bucinadora dos pacientes e colocado num tubo de ensaio est&#233;ril &#40;Oragene Collector OG&#8208;500&#174;&#44; DNA Genotek Inc&#46;&#44; Kanata&#44; Ontario&#41;&#44; que foi submetido ao sequenciamento pelo m&#233;todo de Sanger&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">A extra&#231;&#227;o de DNA foi realizada utilizando precipita&#231;&#227;o com etanol&#44; e um reagente prepIT 2P fornecido pelo kit Oragene&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">A rea&#231;&#227;o em cadeia da polimerase &#40;PCR&#41; e a an&#225;lise de seu sequenciamento foram realizadas com foco no &#233;xon&#8208;3 para identificar as variantes gen&#233;ticas mais comuns &#8211; c&#46;1537C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R501X &#40;rs61816761&#41; e c&#46;2282del4&#58;S761Cfs&#42;36 &#40;rs558269137&#41; utilizando <span class="elsevierStyleItalic">primers</span> validados da Thermo Fischer Scientific&#174; &#40;Applied Biosystems&#44; Foster City&#44; CA&#41;&#44; &#40;Hs00274028 forward&#58; 5&#8217;CTA ACA CTG GAT CCC TGG TTC CTA 3&#8217; e reverse 5&#8217; CTG AGA CAG CAG AGC CAC CAA GA 3&#8217; e Hs00395823&#44; forward&#58; 5&#8217; CAG ACC TAT CTA CCG ATT GCT CGT 3&#8217; e reverse&#58; 5&#8217; AAA TCA GGC ACTCGT&#160;CAC ACA CAG AA 3&#8217;&#41;&#46; Essa estrat&#233;gia possibilitou investigar outras variantes em &#225;reas vizinhas ao <span class="elsevierStyleItalic">loci</span> alvo&#44; mas n&#227;o cobriu toda a regi&#227;o de codifica&#231;&#227;o do &#233;xon&#8208;3 &#40;<a class="elsevierStyleCrossRef" href="#fig0005">fig&#46; 1</a>&#41;&#46; Os produtos de PCR foram purificados com esferas de DNA &#40;Agencourt &#8211; AMPure XP&#8208;Beckman Coulter&#44; Brea&#44; CA&#41;&#46; As amostras purificadas juntamente com&#160;10&#160;&#956;L desses <span class="elsevierStyleItalic">primers</span> foram utilizadas para a rea&#231;&#227;o de sequenciamento&#46; O ciclo de sequenciamento foi realizado com o kit Big Dye Terminator v3&#46;1 &#40;<span class="elsevierStyleItalic">Thermo Fisher Scientific</span>&#41;&#46; Os produtos de sequenciamento foram submetidos &#224; eletroforese capilar no ABI 3500 DNA <span class="elsevierStyleItalic">Analyzer</span> &#40;Applied Biosystems&#44; Foster City&#44; CA&#41;&#46; Os dados de sequenciamento foram avaliados com o <span class="elsevierStyleItalic">software</span> Seq A&#40;14&#41; &#8211; Applied Biosystems&#44; Foster City&#44; CA&#46; O PROVEAN &#40;<span class="elsevierStyleItalic">Protein Variation Effect Analyzer</span>&#41; v1&#46;1 foi usado para prever se uma varia&#231;&#227;o de sequ&#234;ncia de prote&#237;na causada por uma substitui&#231;&#227;o <span class="elsevierStyleItalic">missense</span> afetaria a fun&#231;&#227;o da prote&#237;na &#40;dispon&#237;vel em <a href="https://provean.jcvi.org/index.php">https&#58;&#47;&#47;provean&#46;jcvi&#46;org&#47;index&#46;php</a>&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0040" class="elsevierStylePara elsevierViewall">A preval&#234;ncia de cada variante identificada foi comparada ao banco de dados p&#250;blico brasileiro de variantes gen&#244;micas &#40;ABraOm&#59; hg38 &#8211; <a href="https://abraom.ib.usp.br/">https&#58;&#47;&#47;abraom&#46;ib&#46;usp&#46;br&#47;</a>&#41; com&#160;1&#46;171&#160;amostras da popula&#231;&#227;o da mesma regi&#227;o e um banco de dados internacional &#40;genomAD&#59; v3&#46;1&#46;2 &#8211; <a href="https://gnomad.broadinstitute.org/">https&#58;&#47;&#47;gnomad&#46;broadinstitute&#46;org&#47;</a>&#41; com&#160;76&#46;156&#160;indiv&#237;duos n&#227;o aparentados de diferentes etnias&#46; A signific&#226;ncia estat&#237;stica foi definida como&#160;p &#8804; 0&#44;001&#46;<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">6</span></a></p><p id="par0045" class="elsevierStylePara elsevierViewall">Dados demogr&#225;ficos principais&#44; gravidade cl&#237;nica&#44; eosinofilia e n&#237;veis de IgE est&#227;o na <a class="elsevierStyleCrossRef" href="#tbl0005">tabela 1</a>&#46; A maioria dos pacientes apresentava DA moderada e grave&#160;&#40;79&#37;&#41;&#44; n&#237;veis elevados de IgE&#160;&#40;98&#37;&#41; e eosinofilia&#160;&#40;68&#37;&#41;&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0050" class="elsevierStylePara elsevierViewall">Os resultados da an&#225;lise gen&#233;tica da amostra de DA e a compara&#231;&#227;o com os dois controles populacionais est&#227;o na <a class="elsevierStyleCrossRef" href="#tbl0010">tabela 2</a>&#46; Vinte e seis variantes gen&#233;ticas do &#233;xon&#8208;3 do gene <span class="elsevierStyleItalic">FLG</span> foram detectadas em&#160;60&#160;pacientes &#40;75&#37;&#59; 95&#37;IC&#58;&#160;65&#37;&#8211;85&#37;&#41;&#46; Homozigose e heterozigose composta n&#227;o foram identificadas&#46;</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia><p id="par0055" class="elsevierStylePara elsevierViewall">Variantes do gene <span class="elsevierStyleItalic">FLG</span> com perda de fun&#231;&#227;o da filagrina foram observadas em oito pacientes com DA &#40;10&#37;&#59; 95&#37;&#160;IC&#160;3&#37;&#8211;17&#37;&#41;&#46; Duas variantes prevalentes no mundo &#40;c&#46;1537C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R501X e 2282delCAGT&#58;S761Cfs&#42;36&#41; foram observadas em&#160;seis&#160;pacientes &#40;7&#44;5&#37;&#41;&#46; Outra variante patog&#234;nica &#40;c&#46;2512C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R826X&#41; foi identificada em dois pacientes &#40;2&#44;5&#37;&#41;&#46; Essas tr&#234;s variantes patog&#234;nicas foram mais prevalentes na amostra de DA que nos controles brasileiros &#40;p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46; A variante 2282delCAGT&#58;S761Cfs&#42;36 &#233; comum em europeus&#44; enquanto c&#46;2512C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R826X &#233; comum entre afro&#8208;americanos&#46;</p><p id="par0060" class="elsevierStylePara elsevierViewall">Foram encontradas oito variantes sin&#244;nimas do gene <span class="elsevierStyleItalic">FLG</span>&#44; quatro delas amplamente distribu&#237;das no Brasil e no mundo&#46; Entretanto&#44; quatro delas &#40;c&#46;1665T<span class="elsevierStyleHsp" style=""></span>&#62;G&#58;rs152285733&#44; c&#46;1737A<span class="elsevierStyleHsp" style=""></span>&#62;C&#58; sin&#244;nimo&#44; c&#46;1521G<span class="elsevierStyleHsp" style=""></span>&#62;A&#58; sin&#244;nimo e c&#46;1800T<span class="elsevierStyleHsp" style=""></span>&#62;C&#58; sin&#244;nimo&#41; foram observadas como mais comuns na amostra de DA do que no mundo &#40;p &#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46; A variante c&#46;1476T<span class="elsevierStyleHsp" style=""></span>&#62;C&#58; sin&#244;nimo&#44; rara na popula&#231;&#227;o brasileira e internacional &#40;p &#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#44; &#233; comum entre afro&#8208;americanos&#46; O sin&#244;nimo c&#46;2544T<span class="elsevierStyleHsp" style=""></span>&#62;C&#44; considerado comum entre pacientes com DA e controles regionais&#44; &#233; extremamente raro no mundo &#40;p &#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall">Catorze variantes <span class="elsevierStyleItalic">missense</span> foram detectadas&#59; duas delas &#40;c&#46;1712A<span class="elsevierStyleHsp" style=""></span>&#62;G&#58;H559R&#44; c&#46;1777A<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;T581S&#41; foram mais comuns entre pacientes com DA que controles regionais e internacionais &#40;p &#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46;</p><p id="par0070" class="elsevierStylePara elsevierViewall">Nenhuma variante do gene <span class="elsevierStyleItalic">FLG</span> foi associada &#224; gravidade cl&#237;nica da DA&#44; eosinofilia ou IgE s&#233;rica elevada &#40;p<span class="elsevierStyleHsp" style=""></span>&#62; 0&#44;1&#41;&#46;</p><p id="par0075" class="elsevierStylePara elsevierViewall">Esses resultados refor&#231;am o alto polimorfismo do &#233;xon&#8208;3 do gene <span class="elsevierStyleItalic">FLG</span> e sua associa&#231;&#227;o &#233;tnica&#44; dificultando a generaliza&#231;&#227;o dos resultados gen&#244;micos em rela&#231;&#227;o aos fen&#243;tipos de DA&#44; especialmente em popula&#231;&#245;es altamente miscigenadas&#46;<a class="elsevierStyleCrossRefs" href="#bib0075"><span class="elsevierStyleSup">5&#44;7&#44;8</span></a></p><p id="par0080" class="elsevierStylePara elsevierViewall">Segundo a literatura&#44; variantes nulas eram esperadas em&#160;14&#37;&#8211;42&#37; dos pacientes com DA&#44; e dessas&#44; 20&#160;encontradas no &#233;xon&#8208;3&#46; Al&#233;m disso&#44; varia&#231;&#245;es foram relatadas do gene <span class="elsevierStyleItalic">FLG</span> entre pacientes com DA de diferentes etnias&#46;<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">8</span></a></p><p id="par0085" class="elsevierStylePara elsevierViewall">A popula&#231;&#227;o brasileira &#233; multirracial ap&#243;s&#160;500&#160;anos de miscigena&#231;&#227;o entre indiv&#237;duos da Europa Ocidental&#44; &#193;frica e Amer&#237;ndia&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">9</span></a> As variantes c&#46;2512C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R826X e 2282delCAGT&#58;S761Cfs&#42;36 nos pacientes com DA corroboram essas ancestralidades&#44; embora a variante c&#46;2544T<span class="elsevierStyleHsp" style=""></span>&#62;C&#58; sin&#244;nimo seja uma caracter&#237;stica dessa regi&#227;o&#46;</p><p id="par0090" class="elsevierStylePara elsevierViewall">Considerando associa&#231;&#227;o epidemiol&#243;gica com o desenvolvimento da doen&#231;a&#44; variantes do gene <span class="elsevierStyleItalic">FLG per</span><span class="elsevierStyleItalic">se</span> n&#227;o explicam completamente a varia&#231;&#227;o na gravidade da DA&#44; eosinofilia ou n&#237;veis elevados de IgE&#44; refor&#231;ando os aspectos multifatoriais da doen&#231;a&#46;<a class="elsevierStyleCrossRefs" href="#bib0055"><span class="elsevierStyleSup">1&#44;8&#44;10</span></a></p><p id="par0095" class="elsevierStylePara elsevierViewall">Por fim&#44; identificamos variantes nulas do gene <span class="elsevierStyleItalic">FLG</span> &#40;no &#233;xon&#8208;3&#41; &#40;c&#46;1537C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;501X&#44; c&#46;2282del4&#58;S761Cfs&#42;36 e c&#46;2512C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R826X&#41; em&#160;10&#37; dos pacientes brasileiros com DA&#44; mesmo sem associa&#231;&#227;o com as principais caracter&#237;sticas cl&#237;nicas da DA&#46;</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0005">Suporte financeiro</span><p id="par0100" class="elsevierStylePara elsevierViewall">Fundo de Apoio &#224; Dermatologia do Estado de S&#227;o Paulo &#8211; Sebasti&#227;o Sampaio &#40;FUNADERSP&#41;&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Contribui&#231;&#227;o dos autores</span><p id="par0105" class="elsevierStylePara elsevierViewall">Cristina Marta Maria Laczynski&#58; Concep&#231;&#227;o e desenho&#44; aquisi&#231;&#227;o&#44; an&#225;lise e interpreta&#231;&#227;o dos dados&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">Carlos D&#8217;Apparecida Santos Machado Filho&#58; Revis&#227;o cr&#237;tica do conte&#250;do intelectual&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">H&#233;lio Amante Miot&#58; Revis&#227;o cr&#237;tica do conte&#250;do intelectual&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">Denise Maria Christofolini&#58; Revis&#227;o cr&#237;tica do conte&#250;do intelectual&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">Itatiana Ferreira Rodart&#58; Processamento do material gen&#233;tico&#44; an&#225;lise e interpreta&#231;&#227;o dos dados e revis&#227;o do conte&#250;do intelectual&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">Paulo Ricardo Coelho&#58; Revis&#227;o cr&#237;tica do conte&#250;do intelectual&#46;</p><p id="par0135" class="elsevierStylePara elsevierViewall">Todos os autores leram e aprovaram a vers&#227;o final do manuscrito&#46;</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Conflito de interesses</span><p id="par0140" class="elsevierStylePara elsevierViewall">Nenhum&#46;</p></span></span>"
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          "pt" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Gene <span class="elsevierStyleItalic">FLG</span> incluindo distribui&#231;&#227;o dos &#233;xons&#44; tamanho dos &#233;xons&#44; &#237;ntrons&#44; indica&#231;&#227;o de regi&#227;o de codifica&#231;&#227;o da profilagrina&#44; posi&#231;&#227;o dos <span class="elsevierStyleItalic">primers</span> e eletroferogramas das tr&#234;s mais frequentes variantes patog&#234;nicas encontradas em nossa amostra&#46;</p>"
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                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>I&#8211;II&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">24 &#40;30&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>III&#8211;IV&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>V&#8211;VI&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t">47&#44;84&#37;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">30&#44;25&#37;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
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Cartas ‐ Investigação
Prevalência do polimorfismo do gene filagrina (éxon‐3) em pacientes com dermatite atópica em população brasileira multirracial
Cristina Marta Maria Laczynskia,
Autor para correspondência
crislacz@hotmail.com

Autor para correspondência.
, Carlos D’Apparecida Santos Machado Filhoa, Hélio Amante Miotb, Denise Maria Christofolinic, Itatiana Ferreira Rodartc, Paulo Ricardo Criadoa
a Disciplina de Dermatologia, Centro Universitário FMABC, Santo André, SP, Brasil
b Departamento de Dermatologia, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP, Brasil
c Departamento de Genética, Centro Universitário FMABC, Santo André, SP, Brasil
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&#233; altamente polim&#243;rfico&#44; na regi&#227;o do complexo de diferencia&#231;&#227;o epid&#233;rmica &#40;1q21&#46;3&#41;&#44; codificando as prote&#237;nas mais importantes envolvidas na homeostase da barreira cut&#226;nea&#46; <span class="elsevierStyleItalic">FLG</span> &#233; o principal fator gen&#233;tico associado &#224; DA&#44; e seu &#233;xon&#8208;3 transcreve a maior parte da prote&#237;na profilagrina&#46; Altera&#231;&#245;es da barreira cut&#226;nea est&#227;o presentes em pacientes com DA sem altera&#231;&#245;es do gene <span class="elsevierStyleItalic">FLG</span>&#59; entretanto&#44; a presen&#231;a de variantes que levam &#224; perda de fun&#231;&#227;o foram associadas a fen&#243;tipos cl&#237;nicos como doen&#231;a persistente de in&#237;cio precoce&#44; asma e sensibiliza&#231;&#227;o al&#233;rgica&#46;<a class="elsevierStyleCrossRefs" href="#bib0055"><span class="elsevierStyleSup">1&#44;4</span></a> Intensa disparidade &#233;tnica na frequ&#234;ncia de variantes que levam &#224; perda de fun&#231;&#227;o do gene <span class="elsevierStyleItalic">FLG</span> associadas &#224; DA tem sido observada&#46;<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">5</span></a></p><p id="par0015" class="elsevierStylePara elsevierViewall">Mais de&#160;60&#160;variantes do gene <span class="elsevierStyleItalic">FLG</span> que levam &#224; perda de fun&#231;&#227;o do gene <span class="elsevierStyleItalic">FLG</span> foram identificadas em associa&#231;&#227;o com DA&#59; as mais comuns entre os europeus s&#227;o c&#46;1537C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R501X e 2282del4&#58;S761Cfs&#42;36 e&#44; entre os africanos subsaarianos&#44; c&#46;9947C<span class="elsevierStyleHsp" style=""></span>&#62;G&#58;S3316&#42;&#46; Poucos estudos foram realizados em pacientes latino&#8208;americanos com DA&#46; Nosso objetivo foi avaliar a frequ&#234;ncia de variantes do gene <span class="elsevierStyleItalic">FLG</span> &#40;no &#233;xon&#8208;3&#41; entre pacientes com DA para comparar popula&#231;&#245;es brasileiras e internacionais e explorar suas caracter&#237;sticas cl&#237;nicas&#46;</p><p id="par0020" class="elsevierStylePara elsevierViewall">Foi realizado estudo transversal no ambulat&#243;rio de dermatologia &#40;FMABC&#59; Santo Andr&#233;&#44; S&#227;o Paulo&#41;&#46; Oitenta pacientes com DA &#40;crit&#233;rios de Hanifin e Rajka&#41; de ambos os sexos foram inclu&#237;dos e examinados por dermatologista experiente para avaliar a gravidade da doen&#231;a &#40;SCORAD&#44; EASI&#41; e coletar amostras de sangue venoso para an&#225;lise laboratorial e da mucosa oral para an&#225;lise gen&#233;tica&#46; Os participantes&#47;respons&#225;veis assinaram termo de consentimento livre e esclarecido&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">A coleta para an&#225;lise gen&#233;tica foi realizada por <span class="elsevierStyleItalic">swab</span> da mucosa da regi&#227;o bucinadora dos pacientes e colocado num tubo de ensaio est&#233;ril &#40;Oragene Collector OG&#8208;500&#174;&#44; DNA Genotek Inc&#46;&#44; Kanata&#44; Ontario&#41;&#44; que foi submetido ao sequenciamento pelo m&#233;todo de Sanger&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">A extra&#231;&#227;o de DNA foi realizada utilizando precipita&#231;&#227;o com etanol&#44; e um reagente prepIT 2P fornecido pelo kit Oragene&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">A rea&#231;&#227;o em cadeia da polimerase &#40;PCR&#41; e a an&#225;lise de seu sequenciamento foram realizadas com foco no &#233;xon&#8208;3 para identificar as variantes gen&#233;ticas mais comuns &#8211; c&#46;1537C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R501X &#40;rs61816761&#41; e c&#46;2282del4&#58;S761Cfs&#42;36 &#40;rs558269137&#41; utilizando <span class="elsevierStyleItalic">primers</span> validados da Thermo Fischer Scientific&#174; &#40;Applied Biosystems&#44; Foster City&#44; CA&#41;&#44; &#40;Hs00274028 forward&#58; 5&#8217;CTA ACA CTG GAT CCC TGG TTC CTA 3&#8217; e reverse 5&#8217; CTG AGA CAG CAG AGC CAC CAA GA 3&#8217; e Hs00395823&#44; forward&#58; 5&#8217; CAG ACC TAT CTA CCG ATT GCT CGT 3&#8217; e reverse&#58; 5&#8217; AAA TCA GGC ACTCGT&#160;CAC ACA CAG AA 3&#8217;&#41;&#46; Essa estrat&#233;gia possibilitou investigar outras variantes em &#225;reas vizinhas ao <span class="elsevierStyleItalic">loci</span> alvo&#44; mas n&#227;o cobriu toda a regi&#227;o de codifica&#231;&#227;o do &#233;xon&#8208;3 &#40;<a class="elsevierStyleCrossRef" href="#fig0005">fig&#46; 1</a>&#41;&#46; Os produtos de PCR foram purificados com esferas de DNA &#40;Agencourt &#8211; AMPure XP&#8208;Beckman Coulter&#44; Brea&#44; CA&#41;&#46; As amostras purificadas juntamente com&#160;10&#160;&#956;L desses <span class="elsevierStyleItalic">primers</span> foram utilizadas para a rea&#231;&#227;o de sequenciamento&#46; O ciclo de sequenciamento foi realizado com o kit Big Dye Terminator v3&#46;1 &#40;<span class="elsevierStyleItalic">Thermo Fisher Scientific</span>&#41;&#46; Os produtos de sequenciamento foram submetidos &#224; eletroforese capilar no ABI 3500 DNA <span class="elsevierStyleItalic">Analyzer</span> &#40;Applied Biosystems&#44; Foster City&#44; CA&#41;&#46; Os dados de sequenciamento foram avaliados com o <span class="elsevierStyleItalic">software</span> Seq A&#40;14&#41; &#8211; Applied Biosystems&#44; Foster City&#44; CA&#46; O PROVEAN &#40;<span class="elsevierStyleItalic">Protein Variation Effect Analyzer</span>&#41; v1&#46;1 foi usado para prever se uma varia&#231;&#227;o de sequ&#234;ncia de prote&#237;na causada por uma substitui&#231;&#227;o <span class="elsevierStyleItalic">missense</span> afetaria a fun&#231;&#227;o da prote&#237;na &#40;dispon&#237;vel em <a href="https://provean.jcvi.org/index.php">https&#58;&#47;&#47;provean&#46;jcvi&#46;org&#47;index&#46;php</a>&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0040" class="elsevierStylePara elsevierViewall">A preval&#234;ncia de cada variante identificada foi comparada ao banco de dados p&#250;blico brasileiro de variantes gen&#244;micas &#40;ABraOm&#59; hg38 &#8211; <a href="https://abraom.ib.usp.br/">https&#58;&#47;&#47;abraom&#46;ib&#46;usp&#46;br&#47;</a>&#41; com&#160;1&#46;171&#160;amostras da popula&#231;&#227;o da mesma regi&#227;o e um banco de dados internacional &#40;genomAD&#59; v3&#46;1&#46;2 &#8211; <a href="https://gnomad.broadinstitute.org/">https&#58;&#47;&#47;gnomad&#46;broadinstitute&#46;org&#47;</a>&#41; com&#160;76&#46;156&#160;indiv&#237;duos n&#227;o aparentados de diferentes etnias&#46; A signific&#226;ncia estat&#237;stica foi definida como&#160;p &#8804; 0&#44;001&#46;<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">6</span></a></p><p id="par0045" class="elsevierStylePara elsevierViewall">Dados demogr&#225;ficos principais&#44; gravidade cl&#237;nica&#44; eosinofilia e n&#237;veis de IgE est&#227;o na <a class="elsevierStyleCrossRef" href="#tbl0005">tabela 1</a>&#46; A maioria dos pacientes apresentava DA moderada e grave&#160;&#40;79&#37;&#41;&#44; n&#237;veis elevados de IgE&#160;&#40;98&#37;&#41; e eosinofilia&#160;&#40;68&#37;&#41;&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0050" class="elsevierStylePara elsevierViewall">Os resultados da an&#225;lise gen&#233;tica da amostra de DA e a compara&#231;&#227;o com os dois controles populacionais est&#227;o na <a class="elsevierStyleCrossRef" href="#tbl0010">tabela 2</a>&#46; Vinte e seis variantes gen&#233;ticas do &#233;xon&#8208;3 do gene <span class="elsevierStyleItalic">FLG</span> foram detectadas em&#160;60&#160;pacientes &#40;75&#37;&#59; 95&#37;IC&#58;&#160;65&#37;&#8211;85&#37;&#41;&#46; Homozigose e heterozigose composta n&#227;o foram identificadas&#46;</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia><p id="par0055" class="elsevierStylePara elsevierViewall">Variantes do gene <span class="elsevierStyleItalic">FLG</span> com perda de fun&#231;&#227;o da filagrina foram observadas em oito pacientes com DA &#40;10&#37;&#59; 95&#37;&#160;IC&#160;3&#37;&#8211;17&#37;&#41;&#46; Duas variantes prevalentes no mundo &#40;c&#46;1537C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R501X e 2282delCAGT&#58;S761Cfs&#42;36&#41; foram observadas em&#160;seis&#160;pacientes &#40;7&#44;5&#37;&#41;&#46; Outra variante patog&#234;nica &#40;c&#46;2512C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R826X&#41; foi identificada em dois pacientes &#40;2&#44;5&#37;&#41;&#46; Essas tr&#234;s variantes patog&#234;nicas foram mais prevalentes na amostra de DA que nos controles brasileiros &#40;p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46; A variante 2282delCAGT&#58;S761Cfs&#42;36 &#233; comum em europeus&#44; enquanto c&#46;2512C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R826X &#233; comum entre afro&#8208;americanos&#46;</p><p id="par0060" class="elsevierStylePara elsevierViewall">Foram encontradas oito variantes sin&#244;nimas do gene <span class="elsevierStyleItalic">FLG</span>&#44; quatro delas amplamente distribu&#237;das no Brasil e no mundo&#46; Entretanto&#44; quatro delas &#40;c&#46;1665T<span class="elsevierStyleHsp" style=""></span>&#62;G&#58;rs152285733&#44; c&#46;1737A<span class="elsevierStyleHsp" style=""></span>&#62;C&#58; sin&#244;nimo&#44; c&#46;1521G<span class="elsevierStyleHsp" style=""></span>&#62;A&#58; sin&#244;nimo e c&#46;1800T<span class="elsevierStyleHsp" style=""></span>&#62;C&#58; sin&#244;nimo&#41; foram observadas como mais comuns na amostra de DA do que no mundo &#40;p &#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46; A variante c&#46;1476T<span class="elsevierStyleHsp" style=""></span>&#62;C&#58; sin&#244;nimo&#44; rara na popula&#231;&#227;o brasileira e internacional &#40;p &#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#44; &#233; comum entre afro&#8208;americanos&#46; O sin&#244;nimo c&#46;2544T<span class="elsevierStyleHsp" style=""></span>&#62;C&#44; considerado comum entre pacientes com DA e controles regionais&#44; &#233; extremamente raro no mundo &#40;p &#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46;</p><p id="par0065" class="elsevierStylePara elsevierViewall">Catorze variantes <span class="elsevierStyleItalic">missense</span> foram detectadas&#59; duas delas &#40;c&#46;1712A<span class="elsevierStyleHsp" style=""></span>&#62;G&#58;H559R&#44; c&#46;1777A<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;T581S&#41; foram mais comuns entre pacientes com DA que controles regionais e internacionais &#40;p &#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46;</p><p id="par0070" class="elsevierStylePara elsevierViewall">Nenhuma variante do gene <span class="elsevierStyleItalic">FLG</span> foi associada &#224; gravidade cl&#237;nica da DA&#44; eosinofilia ou IgE s&#233;rica elevada &#40;p<span class="elsevierStyleHsp" style=""></span>&#62; 0&#44;1&#41;&#46;</p><p id="par0075" class="elsevierStylePara elsevierViewall">Esses resultados refor&#231;am o alto polimorfismo do &#233;xon&#8208;3 do gene <span class="elsevierStyleItalic">FLG</span> e sua associa&#231;&#227;o &#233;tnica&#44; dificultando a generaliza&#231;&#227;o dos resultados gen&#244;micos em rela&#231;&#227;o aos fen&#243;tipos de DA&#44; especialmente em popula&#231;&#245;es altamente miscigenadas&#46;<a class="elsevierStyleCrossRefs" href="#bib0075"><span class="elsevierStyleSup">5&#44;7&#44;8</span></a></p><p id="par0080" class="elsevierStylePara elsevierViewall">Segundo a literatura&#44; variantes nulas eram esperadas em&#160;14&#37;&#8211;42&#37; dos pacientes com DA&#44; e dessas&#44; 20&#160;encontradas no &#233;xon&#8208;3&#46; Al&#233;m disso&#44; varia&#231;&#245;es foram relatadas do gene <span class="elsevierStyleItalic">FLG</span> entre pacientes com DA de diferentes etnias&#46;<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">8</span></a></p><p id="par0085" class="elsevierStylePara elsevierViewall">A popula&#231;&#227;o brasileira &#233; multirracial ap&#243;s&#160;500&#160;anos de miscigena&#231;&#227;o entre indiv&#237;duos da Europa Ocidental&#44; &#193;frica e Amer&#237;ndia&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">9</span></a> As variantes c&#46;2512C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R826X e 2282delCAGT&#58;S761Cfs&#42;36 nos pacientes com DA corroboram essas ancestralidades&#44; embora a variante c&#46;2544T<span class="elsevierStyleHsp" style=""></span>&#62;C&#58; sin&#244;nimo seja uma caracter&#237;stica dessa regi&#227;o&#46;</p><p id="par0090" class="elsevierStylePara elsevierViewall">Considerando associa&#231;&#227;o epidemiol&#243;gica com o desenvolvimento da doen&#231;a&#44; variantes do gene <span class="elsevierStyleItalic">FLG per</span><span class="elsevierStyleItalic">se</span> n&#227;o explicam completamente a varia&#231;&#227;o na gravidade da DA&#44; eosinofilia ou n&#237;veis elevados de IgE&#44; refor&#231;ando os aspectos multifatoriais da doen&#231;a&#46;<a class="elsevierStyleCrossRefs" href="#bib0055"><span class="elsevierStyleSup">1&#44;8&#44;10</span></a></p><p id="par0095" class="elsevierStylePara elsevierViewall">Por fim&#44; identificamos variantes nulas do gene <span class="elsevierStyleItalic">FLG</span> &#40;no &#233;xon&#8208;3&#41; &#40;c&#46;1537C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;501X&#44; c&#46;2282del4&#58;S761Cfs&#42;36 e c&#46;2512C<span class="elsevierStyleHsp" style=""></span>&#62;T&#58;R826X&#41; em&#160;10&#37; dos pacientes brasileiros com DA&#44; mesmo sem associa&#231;&#227;o com as principais caracter&#237;sticas cl&#237;nicas da DA&#46;</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0005">Suporte financeiro</span><p id="par0100" class="elsevierStylePara elsevierViewall">Fundo de Apoio &#224; Dermatologia do Estado de S&#227;o Paulo &#8211; Sebasti&#227;o Sampaio &#40;FUNADERSP&#41;&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Contribui&#231;&#227;o dos autores</span><p id="par0105" class="elsevierStylePara elsevierViewall">Cristina Marta Maria Laczynski&#58; Concep&#231;&#227;o e desenho&#44; aquisi&#231;&#227;o&#44; an&#225;lise e interpreta&#231;&#227;o dos dados&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">Carlos D&#8217;Apparecida Santos Machado Filho&#58; Revis&#227;o cr&#237;tica do conte&#250;do intelectual&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">H&#233;lio Amante Miot&#58; Revis&#227;o cr&#237;tica do conte&#250;do intelectual&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">Denise Maria Christofolini&#58; Revis&#227;o cr&#237;tica do conte&#250;do intelectual&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">Itatiana Ferreira Rodart&#58; Processamento do material gen&#233;tico&#44; an&#225;lise e interpreta&#231;&#227;o dos dados e revis&#227;o do conte&#250;do intelectual&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">Paulo Ricardo Coelho&#58; Revis&#227;o cr&#237;tica do conte&#250;do intelectual&#46;</p><p id="par0135" class="elsevierStylePara elsevierViewall">Todos os autores leram e aprovaram a vers&#227;o final do manuscrito&#46;</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Conflito de interesses</span><p id="par0140" class="elsevierStylePara elsevierViewall">Nenhum&#46;</p></span></span>"
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                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>Masculino&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">40 &#40;50&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleItalic">Idade &#40;anos&#41;&#44; m&#233;dia &#40;DP&#41;</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">16 &#40;12&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " colspan="2" align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleVsp" style="height:0.5px"></span></td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " colspan="2" align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleItalic">Fototipo&#44; n &#40;&#37;&#41;</span></td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>I&#8211;II&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">24 &#40;30&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>III&#8211;IV&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>V&#8211;VI&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">5 &#40;6&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleVsp" style="height:0.5px"></span></td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " colspan="2" align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleItalic">Etnia&#44; n &#40;&#37;&#41;</span></td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>Caucasiana&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">54 &#40;68&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">1 &#40;1&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">25 &#40;31&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">17 &#40;21&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>Moderada&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">45 &#40;56&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t"><span class="elsevierStyleHsp" style=""></span>Grave&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">18 &#40;23&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t"><span class="elsevierStyleItalic">EASI&#44; m&#233;dia &#40;DP&#41;</span>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t"><span class="elsevierStyleItalic">N&#237;veis elevados de IgE</span><a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a><span class="elsevierStyleItalic">&#44; n &#40;&#37;&#41;</span>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">79 &#40;98&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t"><span class="elsevierStyleItalic">Eosin&#243;filos</span><span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">&#62;5&#37;&#44; n &#40;&#37;&#41;</span>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">HT&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">rs2011331&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">T454A&nbsp;\t\t\t\t\t\t\n
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ISSN: 26662752
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