Genética Molecular Das Epidermólises Bolhosas

Recebido em 30.01.2002. / Received in January, 30th of 2002.Aprovado pelo Conselho Consultivo e aceito para publicao em 30.07.2002. / Approved by the Consultive Council and accepted for publication in July, 30th of 2002.* Trabalho realizado com bolsa de ps-doutorado da Capes e da Fundao Alexander von Humboldt, no Laboratrio de Diagnstico Gentico, Universidade de Colnia,Alemanha (Servio do Prof. Thomas Krieg) / Work done with a post-doctorate grant from Capes and the Alexander von Humbold Foundation, in the Genetic DiagnosisLaboratory, University of Cologne, Germany (Service of Prof. Thomas Krieg)

1 Professor Adjunto de Dermatologia, Universidade Federal de Pelotas. / Adjunct Professor of Dermatology, Federal University of Pelotas.

2002 by Anais Brasileiros de Dermatologia

Gentica Molecular das Epidermlises Bolhosas *Molecular Genetics of Epidermolysis Bullosa *

Hiram Larangeira de Almeida Jr.

An bras Dermatol, Rio de Janeiro, 77(5):519-532, set./out. 2002.

Almeida Jr. 519

Educao Mdica Continuada / Continuing Medical Education

Resumo: O estudo das alteraes moleculares das epidermlises bolhosas tem contribudo para que se com-preenda melhor essas enfermidades. Na epidermlise bolhosa simples a maioria dos casos est associada comalterao nas citoqueratinas basais 5 (gen KRT5) e 14 (gen KRT14), o que modifica o citoesqueleto na camadabasal da epiderme, levando degenerao dessa camada, formando bolha intra-epidrmica. Mutaes na plecti-na (gen PLEC1), componente da placa interna do hemidesmossoma, levam tambm clivagem intra-epidrmi-ca. Na epidermlise bolhosa juncional vrios gens esto envolvidos, em decorrncia da complexidade da zonada membrana basal, todos levando ao descolamento dos queratincitos basais na lmina lcida, pela disfunoda aderncia entre esses e a lmina densa. Alteraes na laminina 5 (gens LAMA3, LAMB3 e LAMC2), integrinaa6b4 (gens ITGA6 e ITGB4) e colgeno XVII (gen COL17A1) foram descritas. Por fim, na epidermlise bolhosadistrfica apenas um gen est mutado, alterando o colgeno VII (gen COL7A1), principal componente das fibri-las ancorantes, produzindo clivagem abaixo da lmina densa, variando fenotipicamente de acordo com a con-seqncia da mutao. Outra aplicao importante dessas informaes refere-se ao diagnstico pr-natal, coma perspectiva no futuro da terapia gnica.Palavras-chave: Diagnstico pr-natal; epidermlise bolhosa; gentica bioqumica; mutao; reao em cadeiada polimerase.

Summary: New data regarding the molecular aspects of the heterogeneous group of epidermolysis bullosahas brought some important information about its pathogenesis. In epidermolysis bullosa simplex themajority of mutations are localized in the genes of the basal cytokeratin 5 (gene KRT5) and 14 (gene KRT14),cytolysis at this layer with intraepidermal blister is seen under light microscopy. Mutations of plectin (genePLEC1), a protein found in the inner hemidesmosomal plaque, leads also to intraepidermal blisters. In junc-tional epidermolysis bullosa many proteins from the basal membrane zone are involved, such as laminin5 (genes LAMA3, LAMB3 and LAMC2), integrin a6b4 (genes ITGA6 and ITGB4) and collagen XVII (gene COL17A1),the dysfunction which leads to a subepidermal blister, at the level of the lamina lucida. In the third group,epidermolysis bullosa dystrophica, the mutations are localized in only one gene (gene COL7A1), where theyalter the structure of collagen VII, the principal compound of anchoring fibrils, splitting the skin under thelamina densa. This information can also be used in the prenatal diagnosis of epidermolysis bullosa, withfuture perspectives of gene therapy.Key words: prenatal diagnosis; epidermolysis bullosa; genetics, biochemical; mutation; polymerase chainreaction.

INTRODUO Antes da descoberta e padronizao da reao em

cadeia da polimerase (PCR) o seqenciamento gnico eratarefa lenta, levando-se muito tempo para analisar peque-nos segmentos. A PCR permite a amplificao rpida desegmentos de DNA, os quais so posteriormente seqencia-

INTRODUCTION Before the discovery and standardization of polyme-

rase chain reaction (PCR), gene sequencing was anarduous task, requiring a long time to analyze small seg-ments. PCR allows the fast amplification of DNA segments,which are then sequenced, thereby contributing to enor-

Figure 1: Example of gene sequencing

Figura 1: Exemplo de

seqenciamento gnico

520 Almeida Jr.


dos, tendo trazido enorme evoluo nessa rea. Por ocasiodessa reviso, ao se utilizar PCR como palavra-chave noMedline, estavam disponveis mais de 150.000 publicaescom essa tcnica, num perodo de pouco mais de 12 anos,ilustrando a importncia da mesma na pesquisa mdica.

O princpio da PCR bastante simples: o primeiropasso o isolamento de DNA, por exemplo a partir de san-gue, fazendo uso de sua insolubilidade e precipitao emalguns solventes e de sua hidrossolubilidade, havendo co-mercialmente vrios kits para essa funo.

Posteriormente uma parte do DNA obtido incubadocom uma polimerase termorresistente (j que o mesmo aquecido para que a cadeia dupla se desfaa) juntamente comseqncias conhecidas de DNA, os chamados primers (inicia-dores). Havendo no DNA em questo seqncia igual doprimer, a polimerase amplificar esse segmento de DNA. Osnucleotdeos (adenina, timidina, citosina e guanina) fazemparte da reao, para que obviamente a enzima tenha a mat-ria-prima necessria polimerizao. Ciclos de aquecimentoe resfriamento so repetidos inmeras vezes, aumentandocada vez mais o produto da PCR. Posteriormente feita ele-troforese para identificar a presena de uma banda de DNA,mostrando a positividade ou no da reao.

Numa etapa posterior o produto obtido pela PCR seqenciado, o que feito com uma variante da PCR. Oseqenciamento atualmente automatizado, sendo feitauma leitura a laser, obtendo-se grficos policromticos,representando a cor azul, citosina; a cor vermelha, timidina;o preto, guanina; e o verde, adenina (Figura 1). A compara-o do resultado obtido no paciente investigado e em seusgenitores com a seqncia normal do gen pode demonstrarmutao e o padro da herana.

Cada conjunto de trs bases do DNA codifica umaminocido para a sntese protica no ribossoma; havendouma mutao, ou seja, a troca de uma base, haver durantea sntese protica a insero de outro aminocido, alterandoa estrutura da protena, com as conseqncias que isso podeacarretar.

Inmeros gens j foram seqenciados, estando suacomposio disponvel em bancos de dados digitais. O maisutilizado o Genbank, doCentro Nacional de Infor-mao em Biotecnologia dosEstados Unidos, disponvelno endereo eletrnico:www.ncbi.nlm.nih.gov.

As mutaes so des-critas citando-se os aminoci-dos ou as bases envolvidas.Primeiro citado o aminoci-

mous progress in this field. To date, inputting PCR as a keyword in the Medline database, lists over 150,000 publica-tions using this technique, in a period of little over 12 years,thus illustrating the importance of PCR to medical research.

The principle of PCR is quite simple: the first step isto isolate the DNA, for instance taking blood and makinguse of its insolubility and precipitation in certain solventsand its water solubility, several commercial kits are availa-ble for this function.

Then, part of the DNA obtained is incubated with athermoresistant polymerase (since the DNA is heated inorder to separate the double chain) together with knownsequences of DNA, the so-called primers. If the DNA inquestion has the same sequence as that of the primer, thepolymerase will amplify this segment of DNA. The nucleoti-des (adenine, thymidine, cytosine and guanine) are part ofthe reaction, such that obviously the enzyme has the neces-sary raw material for the polymerization. Heating and coo-ling cycles are repeated innumerous times, thereby increa-sing the product of PCR more and more. After which, elec-trophoresis is performed to identify the presence of a bandof DNA, demonstrating the positivity or otherwise of thereaction.

In a subsequent stage the product obtained by PCR issequenced using a variant of PCR. The sequencing is nowautomated with laser readings providing polychromaticgraphs, with blue representing cytosine, red thymidine,black guanine and green adenine (Figure 1). Comparisonof the result obtained in the investigated patient and theirprogenitors with the normal gene sequence can demonstra-te mutation and an inherited pattern.

Each set of three bases of DNA codifies an aminoacid for the protein synthesis in the ribosome; leading to amutation, or in other words, the change of a base, duringthe protein synthesis there will be an insertion of anotheramino acid, thus altering the structure of the protein andresultant consequences.

Countless genes have already been sequenced andtheir composition is available in digital databases. Thesecan be accessed at the Genbank of the National Center of

Biotechnology Informationof the United States:www.ncbi.nlm.nih.gov.

The mutations aredescribed by citing theamino acids or bases invol-ved. Firstly the aminoa-cid/base is given that shouldconstitute the protein/gene,followed by a number, which

Almeida Jr. 521


corresponds to the location of the same in the protein/geneunder investigation and finally the aminoacid/base insertedin the place of the first, such as, for instance, Glu20Arg, inother words, the twentieth amino acid should be a glutami-ne, but the mutation leads to an insertion in the protein ofan arginine, which alters its structure. Some sequences ofbases codify the end of the protein synthesis; a mutation canlead to this interruption, the so-called premature termina-tion codon, which is described in the following manner:Lys472Stop, in other words, instead of the insertion of a lysi-ne, at the 472 position, the protein synthesis was interrup-ted. Abbreviated descriptions with only a single letter canbe found, for example such an interruption of this synthesisis denoted by an X; in the above example this would then beL472X.

The bullous dermatoses make a fascinating chapterin dermatology, they comprise acquired or congenitaldefects of the intraepidermal or dermoepidermal adhesion,leading to blisters which can be spontaneous or provokedby minimal trauma.

In epidermolysis bullosa (EB) these defects are con-genital and can be identified by gene sequencing, whichaffords a greater understanding of their molecular base,1,2

complements the clinicohistological diagnosis and maybein the medium term will partly modify the classification ofthese dermatoses.

Three subgroups of EB are recognized:2 epidermoly-sis bullosa simplex (EBS), in the which the cleaving occursinside the epidermis; junctional epidermolysis bullosa(EBJ), with subepidermal cleaving in the lamina lucida; andepidermolysis bullosa dystrophica (EBD), also subepider-mal, but below the lamina densa. EBJ and EBD cannot bedifferentiated by optical microscopy alone. There are seve-ral classifications, but in this work the second internationalconsensus has been adopted regarding the diagnosis andclassification of epidermolysis bullosa.3

Epidermolysis bullosa simplexThe group of EBS has several subtypes, according to

the intensity and location of the blisters, all of which withautosomal dominant inheritance;4 these are also called epi-dermolytic EB, since the defect is intraepidermal.3 The his-tological aspect most commonly found is degeneration ofthe basal layer, in the absence of any inflammatory infiltra-tion and without deposit of antibodies in the tissue.

The most serious form of EBS is Dowling-Mearasyndrome (EBS-DM),5 in which disseminated blisters thatalso involve the mucous membranes, are accompanied bypalmoplantar hyperkeratosis. The mildest form is Weber-Cockayne syndrome (EBS-WC) with lesions restricted to thepalmar and plantar regions.5 An intermediate form exists,again with disseminated blisters, but with a less intense pic-ture than that of EBS-DM, denominated EBS-Koebner (EBS-K), although certain authors consider this to be a mildvariant of EBS-DM.6

do/base que deveria constituir a protena/gen, seguido porum nmero, o qual corresponde localizao do mesmo naprotena/gen investigado, e por fim o aminocido/base inse-rido no lugar do primeiro, como, por exemplo, Glu20Arg,ou seja, o vigsimo aminocido deveria ser uma glutamina,mas a mutao leva insero na protena de uma arginina,o que altera sua estrutura. Algumas seqncias de basescodificam o final da sntese protica; uma mutao podelevar a essa interrupo, o chamado premature terminationcodon (cdon finalizador prematuro), a qual descrita daseguinte forma: Lys472Stop, ou seja, em vez da insero deuma lisina, na posio 472, foi interrompida a sntese pro-tica. Encontram-se tambm descries abreviadas comapenas uma letra, sendo a interrupo da sntese descritacom X; o exemplo acima ficaria L472X.

As dermatoses bolhosas so captulo fascinante dadermatologia, constitudas por defeitos adquiridos ou natosda adeso intra-epidrmica ou dermoepidrmica, levando abolhas espontneas ou provocadas por trauma mnimo,decorrentes do defeito dessa adeso.

Nas epidermlises bolhosas (EB) esses defeitos sonatos e podem ser identificados por seqenciamento gni-co, o que traz maior compreenso base molecular dasmesmas,1,2 complementa o diagnstico clnico-histolgico etalvez a mdio prazo modifique em parte a classificaodessas dermatoses.

Trs subgrupos de EB so reconhecidos:2 a epider-mlise bolhosa simples (EBS), na qual a clivagem ocorredentro da epiderme; a epidermlise bolhosa juncional(EBJ), cuja clivagem subepidrmica, na lmina lcida; e aepidermlise bolhosa distrfica (EBD), sendo tambm sube-pidrmica, mas abaixo da lmina densa. A EBJ e a EBD nopodem ser diferenciadas apenas por microscopia tica.Existem diversas classificaes, sendo adotada aqui a dosegundo consenso international sobre diagnstico e classifi-cao das epidermlises bolhosas.3

Epidermlise Bolhosa Simples O grupo da EBS tem vrios subtipos de acordo com

a intensidade e localizao das bolhas, sendo todos deherana autossmica dominante;4 so tambm chamados deEB epidermoltica, pois o defeito intra-epidrmico.3 Oaspecto histolgico mais comumente encontrado a dege-nerao da camada basal, na ausncia de infiltrado inflama-trio e sem depsito de anticorpos no tecido.

A forma mais grave da EBS a Dowling-Meara(EBS-DM),5 na qual bolhas disseminadas, envolvendo tam-bm as mucosas, so acompanhadas de hiperqueratose pal-moplantar. A forma mais leve a Weber-Cockayne (EBS-WC) com leses restritas s palmas e plantas.5 Existe umaforma intermediria com bolhas disseminadas, mas comquadro menos intenso do que o da EBS-DM, denominadoEBS - Koebner (EBS-K), que certos autores consideramvariante leve da EBS-DM.6

Algumas citoqueratinas so expressadas nas clulas

Figure 2: Schematic represen-tation of the molecule ofcytokeratin 5 and 14. Themutations of cytokeratin 5 arerepresented above the schemeand those of cytokeratin 14below, in that each line couldrepresent more than onemutation. It can be observedthat the mutations of epider-molysis bullosa simplexDowling-Meara (EBS-DM) arelocated in the extremities ofthe molecule, while most ofthose of EBS Weber-Cockayne(EBS-WC), in the non-helicalsegment between 1B and 2A.

Figura 2: Representaoesquemtica da molcula das

citoqueratinas 5 e 14. Asmutaes da citoqueratina 5

esto representadas acima doesquema e as da citoqueratina

14 abaixo, sendo que cada traopode representar mais de uma

mutao. Note-se que asmutaes da epidermlise bol-

hosa simples Dowling-Meara(EBS-DM) localizam-se nas

extremidades da molcula, e amaioria das da EBS Weber-

Cockayne (EBS-WC), no segmen-to no helicoidal entre 1B e 2A

522 Almeida Jr.


epiteliais aos pares,7 os quais formam heterodmeros, ouseja, a unio das duas molculas, configurando o citosque-leto dos epitlios, havendo especificidade de acordo com oepitlio envolvido.7 A camada basal diferencia-se de outrosepitlios e dos segmentos suprabasais da epiderme pelaexpresso das citoqueratinas 5 e 14.

As citoqueratinas 5 e 14 so reguladas pelos gensKRT5 e KRT14, localizados nos cromossomas 17 e 12, res-pectivamente. interessante observar que defeitos genti-cos distintos na EBS, um afetando a citoqueratina 5, outro,a 14,6,8,9 levam mesma alterao histolgica, pois todosesses defeitos produzem alteraes estruturais de uma ououtra citoqueratina,10 impedindo a funo estrutural dasmesmas no citoesqueleto 11 a formao dos heterodmeros,responsveis pela configurao tridimensional da clula.Essa alterao vista facilmente na histologia e culminacom a formao das bolhas, sendo esse o nico subgrupodas EB decorrente de citlise e no de defeito de adeso.

As citoqueratinas so constitudas por quatro seg-mentos helicoidais, 1A, 1B, 2A e 2B,12 tendo sido a maioriadas mutaes da EBS-DM descrita no incio do segmento 1Ae no final do segmento 2B (Figura 2)13,14 das citoqueratinasbasais. As mutaes da EBS-K tm localizao semelhan-te,15 reforando a hiptese de ser variante da EBS-DM. NaEBS-WC a maioria das mutaes localiza-se no segmentono-helicoidal entre 1B e 2A das mesmas citoqueratinas,sem que com isso se explique a localizao palmoplantardas leses.

Um quarto tipo de EBS descrito, no qual no ocor-re citlise na camada basal. a EBS com distrofia musculartardia, decorrente de alterao da plectina, presente na placainterna do hemidesmossoma (Figura 3). Como a clivagemocorre dentro da epiderme, includa nesse grupo. A plec-tina regulada pelo gen PLEC11 e est tambm envolvida nocitoesqueleto da musculatura lisa,16 da a miopatia associa-da.1,17 Outro componente da placa interna do hemidesmos-soma o antgeno do penfigide bolhoso de 230 KD de pesomolecular, no havendo at o momento descrio de muta-o no gen que o regula.1

Some cytokeratins are expressed in the epithelialcells in pairs,7 which form heterodimers, in other words, theunion of two molecules, configuring the cytoskeleton of theepithelia, with specificity according to the epithelium invol-ved.7 The basal layer differs from other epithelia and supra-basal segments of the epidermis by the expression of cyto-keratins 5 and 14.

Cytokeratins 5 and 14 are regulated by the genesKRT5 and KRT14, located in chromosomes 17 and 12, res-pectively. It is interesting to note that different geneticdefects in EBS, one affecting cytokeratin 5 and the other14,6,8,9 lead to the same histological alteration, because allthese defects produce structural alterations in one or ano-ther cytokeratin,10 impeding their structural function in thecytoskeleton11 i.e. the formation of the heterodimers, res-ponsible for the three-dimensional configuration of the cell.This alteration is easily seen in the histology and culmina-tes with the formation of blisters, making this the only sub-group of EB due to cytolysis and not to an adhesion defect.

The cytokeratins are constituted by four helical seg-ments, 1A, 1B, 2A and 2B,12 the majority of the mutations ofEBS-DM are described in the beginning of segment 1A and atthe end of segment 2B (Figure 2)13,14 of the basal cytokeratins.The mutations of EBS-K have a similar location,15 reinforcingthe hypothesis that it is a variant of EBS-DM. In EBS-WCmost of the mutations are located in the non-helical segmentbetween 1B and 2A of the same cytokeratins, though thisdoes not explain the palmoplantar location of the lesions.

A fourth type of EBS is described, in which cytolysis inthe basal layer does not occur. It is EBS with tardive musculardystrophy, due to alteration of the plectin, present in the inter-nal plaque of the hemidesmosome (Figure 3). Since the clea-ving occurs within the epidermis, it is included in this group.The plectin is regulated by the gene PLEC11 and is also invol-ved in the cytoskeleton of the smooth musculature,16 hence theassociated myopathy.1,17 Another component of the internalplaque of the hemidesmosome is the bullous pemphigoid anti-gen with 230 KD molecular weight. To date mutation in thegene that regulates this has not been described.1

Almeida Jr. 523


Junctional Epidermolysis BullosaGiven the complexity of the basal membrane zone,

alterations in several proteins involved in the dermoepider-mal adhesion can lead to the various clinical pictures ofEBJ; for these molecular alterations to be understood, it isimportant to be familiar with the substances responsible forthe adhesion between the basal keratinocytes and the colla-gen IV the lamina densa (Figure 3).

The antigen of bullous pemphigoid (180 KD) andintegrin a6b4, which are transmembranous proteins, arefound in the external plaque.18

The antigen of bullous pemphigoid (180 KD) is inreality a transmembranous collagen, denominated collagenXVII, and is regulated by the gene COL17A1.1 Each segmentof the integrin a64 is regulated by two different genes,ITGA6 and ITGB4, which are also expressed in the skin anddigestive tract.1,18

Finally, some substances present in the lamina luci-da complement this molecular net,1 of which the mostimportant is laminin 5. The laminins are heterotrimers, orthat is, they are constituted by three distinct classes of poly-peptides a, b e g,18-20 and hence regulated by three genes.Laminin 5 is composed of one a3, one b3 and one g2, regu-lated by the genes LAMA3, LAMB3 and LAMC2, respectively.

An absence or alteration of these substances produ-ces a rupture of this adhesion net, with the formation of blis-ters.2 Some mutations occur due to the so-called prematuretermination codon (PTC), which provokes an interruption ofthe protein synthesis and consequently absence of protein inthe tissue, resulting in a more serious clinical picture.

Several genophenotype correlations have alreadybeen made. Such as integrin a6b4 is expressed in the skinand intestine, mutations of which lead to forms of EBJ withatresia pilori, the clinical picture varies according to whe-ther or not it is associated to PTC.

Regarding generalized, benign and atrophic EBJ, cha-racterized by disseminated blisters with nail dystrophy, inwhich the immunohistochemistry with antibody against colla-gen XVII is negative, PTC has been demonstrated in the gene

Epidermlise Bolhosa Juncional Dada a complexidade da zona da membrana basal,

alteraes de vrias protenas envolvidas na adeso dermoe-pidrmica podem levar aos diversos quadros clnicos daEBJ; para que se compreendam essas alteraes molecula-res, importante conhecer as substncias responsveis pelaadeso entre os queratincitos basais e o colgeno IV almina densa (Figura 3).

Na placa externa do hemidesmossoma encontram-seo antgeno do penfigide bolhoso de 180 KD e a integrinaa6b4, os quais so protenas transmembranosas.18

O antgeno do penfigide bolhoso de 180 KD narealidade um colgeno transmembranoso, sendo denomina-do colgeno XVII, e regulado pelo gen COL17A1.1 Cadasegmento da integrina a64 regulado por dois gens dis-tintos, ITGA6 e ITGB4, sendo a mesma expressa na pele e notubo digestivo.1,18

Finalmente algumas substncias presentes na lminalcida complementam essa rede molecular,1 sendo a maisimportante a laminina 5. As lamininas so heterotrmeros,ou seja, so constitudas por trs classes distintas de polipep-tdeos a, b e g,18-20 e da reguladas por trs gens. A laminina5 composta por uma classe a3, uma b3 e uma g2, regula-das pelos gens LAMA3, LAMB3 e LAMC2, respectivamente.

A ausncia ou alterao dessas substncias produz aruptura dessa rede de adeso, com a formao das bolhas.2

De algumas mutaes decorre o chamado premature termi-nation codon (PTC), o que provoca a interrupo da snte-se protica e conseqentemente a ausncia da protena notecido, com quadro clnico mais grave.

Vrias correlaes genofenotpicas j foram feitas.Como a integrina a6b4 expressa na pele e no intestino,suas mutaes levam a formas de EBJ com atresia pilrica,sendo o quadro clnico varivel de acordo com sua associa-o com PTC ou no.

Na EBJ generalizada atrfica benigna, caracterizadapor bolhas disseminadas com distrofia ungueal, na qual aimuno-histoqumica com anticorpo contra o colgeno XVII negativa, foi demonstrada PTC no gen COL17A,1,21 o que

Figure 3: The adherence betweenthe basal keratinocyte and thelamina densa is made by thetransmembranous proteins basedon the external plaque of the hemi-desmosome (collagen XVII andintegrin a6b4) and by laminin 5,present in the lamina lucida. Theadherence between the laminadensa and the dermis is promotedby anchorage fibrils (collagen VII).The plectin is present in the inter-nal plaque of the hemidesmosome,and its alterations lead to theintraepidermal separation, belon-ging to the group of the epidermoly-sis bullosa simplex, as well as thealterations of the basal cytokeratins.

Figura 3: A aderncia entre oqueratincito basal e a lmina

densa feita pelas protenas trans-membranosas a partir da placa

externa do hemidesmossoma(colgeno XVII e integrina a6b4) epela laminina 5, presente na lmi-

na lcida. A aderncia entre almina densa e a derme pro-

movida pelas fibrilas ancorantes(colgeno VII). A plectina est pre-

sente na placa interna dohemidesmossoma, e suas alter-

aes levam clivagem intra-epidrmica, pertencendo ao

grupo da epidermlise bolhosasimples, assim como as alteraes

das citoqueratinas basais

Figure 4: Diagram of thesynthesis of collagen VIIand anchoring fibrils. After the loss of segmentNC2 the formation ofantiparallel dimers occurs,which group together andform anchorage fibrils.

Figura 4: Esquema da sntesedo colgeno VII e das fibrilasancorantes. Aps a perda do

segmento NC2 ocorre a formao dos dmeros

antiparalelos, os quais seagrupam e formam

as fibrilas ancorantes.

524 Almeida Jr.


correlaciona com a ausncia tecidual do colgeno XVII.Alguns autores denominam essa forma, por ter curso bran-do e expectativa de vida normal, de EBJ no-Herlitz. 21,22

Semelhante s mutaes dos componentes dohemidesmossoma descritas acima, as mutaes da lamini-na tambm provocam o descolamento da epiderme. Amaior parte das mutaes dos gens da laminina 5 leva PTC, provocando ausncia da protena e quadro clnicointenso,23,24 caracterizado por leses disseminadas afetandotambm as mucosas,23 com sobrevida curta em funo dascomplicaes bacterianas, denominadas EBJ tipo Herlitzou EBJ letal.

As alteraes j foram demonstradas no trs gensque codificam a laminina5, 25 no havendo diferena fenot-pica de acordo com o segmento envolvido,26 sugerindo quetodos so importantes para a funo adesiva da mesma.26,27

Oitenta por cento das mutaes residem no gen LAMB3,22,24,28-30 havendo duas delas recorrentes (R635X e R42X),26,28 asquais perfazem metade das mutaes no LAMB3.1 Nessegen j foram relatadas 35 mutaes diferentes.22

Existem relatos de mutao da laminina 5 em pacien-tes nos quais a imuno-histoqumica consegue demonstrardiminuio da laminina 5, e no ausncia, como na EBJ-Herlitz, e, em decorrncia disso, o quadro clnico no tograve.31,32 Essas formas tm sido tambm denominadas EBJno-Herlitz,33 as quais clinicamente so semelhantes s for-mas decorrentes da mutao do colgeno XVII.33

Todas as formas de EBJ so autossmicas recessivas.23

Epidermlise Bolhosa Distrfica A caracterstica clnica principal das EBD so as

cicatrizes decorrentes da perda tecidual, pois a clivagemocorre abaixo da lmina densa.34 Como nos outros grupos,h variantes de acordo com o quadro clnico; apesar dessasvariantes, o defeito gentico foi localizado em um nicogen, o COL7A1.35 Esse gen responsvel pela codificaodo colgeno VII, principal constituinte das fibrilas ancoran-tes,35 as quais participam da aderncia da lmina densa coma derme (Figura 3), da tambm a denominao EB dermo-ltica. Nesse grupo existem formas com herana autossmi-ca dominante e recessiva.

Para que se entenda acorrelao entre gentipo efentipo na EBD necessrioque se entenda tambm opapel do colgeno VII na ade-

COL17A,1,21 which correlates with the tissular absence of colla-gen XVII. Some authors denominate this form non-Herlitz EBJ,as it presents a mild course and normal life expectancy.21,22

Similar to the mutations in components of the hemides-mosome described above, mutations in the laminin also pro-voke dislocation of the epidermis. Most of the mutations of thegenes of laminin 5 lead to PTC, provoking absence of the pro-tein and intense clinical picture,23,24 characterized by dissemi-nated lesions also affecting the mucous membranes,23 with lowsurvival in function of bacterial complications, denominatedHerlitz syndrome or epidermolysis bullosa lethalis.

The alterations have already been demonstrated inthe three genes that codify laminin 5,25 without a phenotypedifference according to the segment involved,26 suggestingthat all are important for its adhesion function.26,27 Eightypercent of the mutations reside in the gene LAMB3,22,24,28-30

two of these are recurrent (R635X and R42X),26,28 whichamount to half of the mutations in LAMB3.1 In this genealone 35 different mutations have already been reported.22

There are reports of mutation in laminin 5 amongpatients in which the immunohistochemistry demonstrateda reduction in laminin 5, but not a complete absence, as inHerlitz Syndrome, and consequently the clinical picture wasnot so serious.31,32 These forms have also been denominatednon-Herlitz EBJ,33 which are clinically similar to the formsarising from mutation of collagen XVII.33

All forms of EBJ are inherited as an autosomalrecessive trait.23

Epidermolysis bullosa dystrophica The main clinical characteristic of EBD is scarring

after tissue loss, since the separation occurs below thelamina densa.34 As in the other groups, there are variantsreflecting the clinical picture; in spite of these variants, thegenetic defect is located in a single gene, COL7A1.35 Thisgene is responsible for codifying collagen VII, the mainrepresentative of the anchoring fibrils,35 which participatein the adherence of the lamina densa to the dermis (Figure3), hence it is also denominated dermolytic EB. In thisgroup there are forms inherited as autosomal dominant and

recessive traits. In order to unders-

tand the correlation betweengenotype and phenotype inEBD it is necessary to also

Figura 5: Representaoesquemtica da molcula docolgeno VII. A maioria das

mutaes da epidermlise bol-hosa distrfica recessiva

Halloupeau-Siemens (EBD-RHS) premature termination codon

(PTC) levando ausncia damolcula no tecido. Na forma

recessiva Mitis (EBD-RM) osdefeitos localizam-se no final dosegmento colgeno e no NC-2.

Na forma dominante (EBD-D) assubstituies de glicina repre-

sentadas embaixo ocorremgeralmente no segundo segmen-

to colgeno. Cada trao pode rep-resentar mais de uma mutao.

Figure 5: Schematic representa-tion of the molecule of collagenVII. The majority of mutationsin epidermolysis bullosa dys-trophica recessive Halloupeau-Siemens (EBD-RHS) are prema-ture termination codon (PTC)leading to the absence of themolecule in the tissue. In therecessive mitis form (EBD-RM)the defects are located at theend of the collagen segment andin NC-2. In the dominant form(EBD-D) the substitutions ofglycine shown below usuallyoccur in the second collagensegment. Each line can repre-sent more than one mutation.

Almeida Jr. 525


understand the role of collagen VII in the dermoepidermaladhesion.

This is produced by the keratinocytes and has a tri-ple helix configuration of collagen, preceded and followedby non-collagen segments (NC-1 and NC-2, respectively).36, 37

In the center of the triple helix there is small non-collagensegment, which probably provides flexibility to the protein.Later, at the extracellular level, a fusion occurs betweentwo of these molecules with loss of the NC-2 segment, for-ming antiparallel dimers. The union of several dimersforms the anchoring fibrils34,36,37 (Figure 4).

Three subtypes of EBD are well characterized: reces-sive EBD Halloupeau-Siemens (EBD-RHS), with an intenseclinical picture, producing acral retractions with synechiaeof the digits and involvement of the digestive tract;37 recessi-ve EBD mitis (EBD-RM), in which the clinical picture is muchless intense in comparison with that of EBD-RHS, with loca-lized lesions in the areas of greatest trauma, such as theknees and extremities; and the dominant form (EBD-D), witha similar picture to that of EBD-RM,37 associated to naildystrophy and, in some cases, with white papular lesions.

Electron microscopy and immunohistochemical cha-racterization with antibodies against collagen VII showalteration in the anchoring fibrils to the extent of theirabsence in EBD-RHS and reduction in the milder forms,EBD-RM and EBD-D.38 In some cases the immunohistoche-mistry is positive but without anchoring fibrils revealed byelectron microscopy, which demonstrates the presence ofpart of the molecule, but with structural alteration.38

The identification of the mutations responsible for EBDhas brought a greater understanding of this spectrum (Figure 5).

In EBD-RHS the genetic alteration is a PTC, with con-sequent interruption in the synthesis of collagen VII,39 whichcorrelates with the intensity of the clinical picture and withthe findings of electron microscopy and immunohistoche-mistry, in which the anchoring fibrils are not detected.34,37

In EBD-RM the greater part of the mutations occur atthe end of the collagen segment and in NC-2, interfering inthe formation of the antiparallel dimers and altering the

so dermoepidrmica.O mesmo produzido pelos queratincitos e possui

uma tripla hlice de colgeno, precedida e seguida por seg-mentos no colgenos (NC-1 e NC-2, respectivamente).36,37

No centro da tripla hlice h pequeno segmento no colge-no, o qual provavelmente d flexibilidade protena.Posteriormente, no nvel extracelular, ocorrer com duasdessas molculas uma fuso com perda do segmento NC-2,formando dmeros antiparalelos. A unio de vrios dmerosforma as fibrilas ancorantes 34,36,37 (Figura 4).

Trs subtipos da EBD esto bem caracterizados: aEBD recessiva Halloupeau-Siemens (EBD-RHS), comquadro clnico intenso, produzindo retraes acrais comsinquia dos dgitos e acometimento do tubo digestivo;37 aEBD recessiva Mitis (EBD-RM), na qual a intensidade doquadro clnico bem menor em comparao ao da EBD-RHS, com leses localizadas nas reas de maior trauma,como joelhos e extremidades; e a forma dominante (EBD-D), com quadro semelhante ao da EBD-RM,37 associadacom distrofia ungueal e, em alguns casos, com lesesalbo-papulides.

A microscopia eletrnica e a caracterizao imuno-histoqumica com anticorpos contra colgeno VII mostramalterao nas fibrilas ancorantes, indo da ausncia das mes-mas, na EBD-RHS, diminuio nas formas mais leves,EBD-RM e EBD-D.38 Em alguns casos a imuno-histoqumica positiva, sem que se observe as fibrilas ancorantes namicroscopia eletrnica, o que demonstra a presena de parteda molcula, mas com alterao de sua estrutura.38

A identificao das mutaes responsveis pela EBDtrouxe maior compreenso a esse espectro (Figura 5).

Na EBD-RHS a alterao gentica uma PTC, comconseqente interrupo na sntese do colgeno VII,39 o quecorrelaciona com a intensidade do quadro clnico e com osachados de microscopia eletrnica e imuno-histoqumica,com os quais no se detectam as fibrilas ancorantes. 34,37

Na EBD-RM a maior parte das mutaes ocorre nofinal do segmento colgeno e no NC-2, interferindo na for-mao dos dmeros antiparalelos, alterando a conformao

526 Almeida Jr.


da protena, estando, porm, presente, decorrendo um qua-dro clnico mais leve e a presena de fibrilas ancorantes namicroscopia eletrnica.37,40

Na EBD-D a alterao caracterstica a substituiode uma glicina no segmento colgeno,41,42 alterando suaestabilidade e talvez propiciando sua degradao. 36,37,43

Como na EBD-RM, as fibrilas ancorantes esto presentes,mas com sua funo comprometida. A maior parte dasmutaes localiza-se logo depois do segmento no colge-no do centro da tripla hlice;42 a mutao G2043R a maiscomumente descrita.36,41 Tambm j foi demonstrado que aalterao funcional da fibrila ancorante depende da locali-zao da substituio da glicina, 34,44 o que, por sua vez, con-tribui para a variabilidade clnica. No existe explicaoconvincente a respeito de por que a substituio de glicina herdada de forma dominante.

A maioria dos casos da forma pr-tibial da EBD autossmica dominante, tendo sido descrita tambm a subs-tituio de glicina.45 Casos recessivos foram igualmentepublicados,45 podendo ser considerados variantes das for-mas leves de EBD, no se sabendo o porqu da ocorrncialocalizada das leses.

Cerca de 100 mutaes diferentes j foram descritasna EBD,34 sendo encontradas em 80% dos casos examina-dos.37 Assim como nas outras formas de EB, algumas muta-es no se enquadram no esquema acima exposto, pois,por exemplo, algumas substituies de glicina foram encon-tradas na EBD-RM;37,40,41 razo de, nesses casos, os genitoresque apresentam essa substituio de glicina serem normais,ou seja, a mutao no ser dominante, s se expressando deforma recessiva, com a herana de dois alelos mutados, eat o momento no pde ser esclarecida.41

Alguns quadros clnicos intermedirios, de difcil clas-sificao clnica, j foram tambm descritos com essas muta-es incomuns, como, por exemplo, EBD recessiva com umaPTC em um alelo e uma substituio de glicina em outro.46

Discusso Os novos aspectos moleculares, tanto gnico quanto

protico, mostram o quo variado o espectro da EB(Tabela 1). Na EBS os defeitos genticos das citoqueratinasbasais produzem alterao histolgica em funo da modi-ficao do citoesqueleto na camada basal da epiderme,sendo que a alterao da plectina, componente da placainterna do hemidesmossoma, tambm leva clivagem intra-epidrmica. Na EBJ vrios gens esto envolvidos, devido complexidade da zona da membrana basal, mas todoslevam ao descolamento dos queratincitos basais da lminadensa, ou seja, a clivagem ocorre na lmina lcida. Por fim,na EBD apenas um gen est mutado, alterando o colgenoVII, sendo a clivagem abaixo da lmina densa, mas, mesmo,assim variando fenotipicamente, de acordo com a conse-qncia da mutao.

Apesar de contribuir com importantes avanos nacompreenso dessas enfermidades, o seqenciamento gni-

compliance of the protein, though these continue present,giving rise to a milder clinical picture and the presence ofanchoring fibrils in the electron microscopy.37,40

In EBD-D, the characteristic alteration is the substi-tution of a glycine in the collagen segment,41,42 altering itsstability and maybe propitiating its degradation.36,37,43 As inEBD-RM, the anchorage fibrils are present, but their func-tion is impaired. Most of the mutations are located imme-diately after the non-collagen segment of the center of thetriple helix;42 the G2043R mutation is the most commonlydescribed.36,41 Likewise it has already been demonstratedthat the functional alteration of the anchoring fibrilsdepends on the location in which the glycine is substitu-ted,34,44 which in turn contributes to the clinical variability.As yet, there is no convincing explanation as to why theglycine substitution is an inherited dominant trait.

The majority of cases involving the pretibial form ofEBD are autosomal dominant and the substitution of glyci-ne has also been described.45 Recessive cases have beenpublished,45 these could equally be considered variants ofthe mild forms of EBD, the reason behind the localizedoccurrence of the lesions is not known.

About 100 different mutations have already beendescribed in EBD,34 and are found in 80% of the cases exa-mined.37 As in other forms of EB, some mutations are notdefined within the above described outline, because, for ins-tance, some substitutions of glycine have been found inEBD-RM;37,40,41 to date, it has yet to be clarified why in thesecases the progenitors that present such glycine substitutionmay be normal, or in other words, the mutation is not domi-nant and is only expressed in a recessive manner, with theinheritance of two changed alleles.41

Various intermediate clinical pictures, presenting diffi-cult clinical classification, have already been described withsuch uncommon mutations, for instance, recessive EBD withPTC in one allele and a glycine substitution in the other.46

Discussion The new molecular aspects, involving both genes

and proteins, demonstrate just how varied the spectrum ofEB can be (Table 1). In EBS the genetic defects of the basalcytokeratins produce a histological alteration due to themodification of the cytoskeleton in the basal layer of theepidermis, in that alteration of the plectin, a component ofthe internal plaque of the hemidesmosome, also leads to theintraepidermal separation. In EBJ several genes are invol-ved, due to the complexity of the basal membrane zone, butall lead to the dislocation of the basal keratinocytes of thelamina densa, in other words, the cleaving occurs in thelamina lucida. Finally, in EBD only one gene is modified,altering the collagen VII, cleaving below the lamina densa,but even so with phenotype variation, according to the con-sequence of the mutation.

Despite its important contribution to progress in theunderstanding of these illnesses, gene sequencing should be

Almeida Jr. 527


used together with clinical, histological, electron microscopyand immunohistochemical findings in the diagnosis of EB.47

Another important application for molecular gene-tics is in prenatal diagnosis (PND),2,48 examining fetal DNAobtained from the chorion rather than the fetal skin. PNDperformed on the basis of the lesions requires the collectionof a skin specimen, which should be representative of the ill-ness, in order to avoid a false-negative result, one shouldwait until the eighteenth or twentieth week.43 Sequencinghas the advantage that it can be performed around the tenthweek, which means that a more precocious decision to ter-minate the gestation can be made in those countries inwhich this procedure is permitted. Furthermore, complica-tions arising from fetoscopy with biopsy occur in betweenfour to 7% of cases compared to 1% in chorionic biopsy.43

Genetic sequencing has already been used in PNDfor all forms of EB,23,24,43,49-51 and has already been performedbefore implantation, based on a cell obtained from an embr-yo with a number of cells varying from five to eight.52

Genetic counseling is another important applicationof this new information, since it helps to explain the inheri-tance pattern, especially when dealing with frequent andwell-known mutations. Also in the case of de novo muta-tions, when the DNA exam of the progenitors is normal andthe mutation is only found in the patient, it can be affirmedthat the risk factor for the next gestation is very low.

co deve ser utilizado em conjunto com a clnica, histologia,microscopia eletrnica e a imuno-histoqumica no diagns-tico da EB.47

Outra aplicao importante da gentica molecularocorre no diagnstico pr-natal (DPN),2,48 examinando-se oDNA fetal obtido do crion e no a pele fetal. O DPN feitoa partir das leses necessita da obteno de fragmento dapele, o qual deve ser representativo da enfermidade, paraevitar resultado falso-negativo, devendo-se esperar at adcima oitava ou vigsima semana.43 O seqenciamentotem a vantagem de poder ser feito em torno da dcimasemana, o que permite, nos pases em que a gestao podeser interrompida nessas situaes, deciso mais precoce.Alm disso as complicaes da fetoscopia com bipsiaocorrem entre quatro e 7% dos casos, e na bipsia corini-ca em 1%.43

O seqenciamento gentico j foi utilizado no DPNde todas as formas de EB,23,24,43,49-51 j tendo sido tambm rea-lizado antes da implantao, a partir de clula obtida deembrio com nmero de clulas varivel de cinco a oito.52

Aconselhamento gentico outra aplicao importante des-sas novas informaes, pois ajuda a esclarecer o padro deherana, principalmente tratando-se de mutaes freqentese bem conhecidas. Tambm no caso de mutaes de novo,quando o exame do DNA dos genitores normal e a muta-

EBS

EBJ

EBD

Dowling-Meara Dowling-Meara

Weber-Cockaine Weber-Cockaine

EBS - distrofia muscular EBS - muscular dystrophy

Herlitz Herlitz

No-Herlitz Non-Herlitz

EBJ - atresia pilrica EBJ - atresia pilori

Halloupeau-Siemens Halloupeau-Siemens

EBD-Recessiva Mitis EBD-Recessive Mitis

EBD-Dominante EBD-Dominant form

Citoqueratina 5 ou 14Cytokeratin 5 or 14

Citoqueratina 5 ou 14Cytokeratin 5 or 14

PlectinaPlectin

Laminina 5Laminin 5

Colgeno XVIICollagen XVII

Integrina alpha6 beta4Integrin alpha6 beta4

Colgeno VIICollagen VII



KRT5 ou KRT14KRT5 or KRT14

KRT5 ou KRT14KRT5 or KRT14

PLEC1PLEC1

LAMA3, LAMB3 ou LAMC2LAMA3, LAMB3 or LAMC2

COL17A1COL17A1

ITGA6 ou ITGB4ITGA6 or ITGB4

COL7A1COL7A1

COL7A1COL7A1

COL7A1COL7A1

17 ou 1217 or 12

17 ou 1217 or 12

8

18, 1 ou 118. 1 or 1

10

2 ou 172 or 17

3

3

3

Protena alterada Altered protein

Gen envolvido Gene involved

Cromosoma Chromosome

SubtipoSubtype

Tabela 1: Principais tipos de EB, com as protenas, gens e cromossomas envolvidos.Tabel 1:- Main types of EB, with the involved proteins, genes and chromosomes.

528 Almeida Jr.


o s encontrada no paciente, pode-se afirmar que o riscopara uma prxima gestao muito baixo. Com relao prole do paciente, depender do tipo da mutao encontra-da, dominante ou recessiva,42 ou seja, presente em um salelo ou em dois.

Em funo dessas recentes informaes sobre a expres-so gnica,53 novas perspectivas teraputicas existem para asEB, embora ainda em fase experimental. J h relatos da mani-pulao ex vivo de queratincitos de portadores de EBJ, inca-pazes de produzir a cadeia 3 da laminina 5, os quais, apstransferncia gnica, se mostraram capazes ainda que transi-toriamente de sintetiz-la, abrindo novas perspectivas tera-puticas para esse grupo de genodermatoses.54 Modelo animalcom ratos transgnicos, simulando a doena humana, temacrescentado informaes relevantes na pesquisa das EB. 10,12

Alguns autores consideram que as correlaes entregentipo e fentipo estejam apenas comeando 34 e que aexpanso dos bancos de dados sobre as alteraes gnicas sejade extrema importncia, pois permitir, cada vez mais, que semelhore essa correlao e talvez at se reclassifique, com baseem aspectos moleculares, parte das genodermatoses.38 q

Regarding the patient's offspring, this will depend on whe-ther the type of mutation found, is dominant or recessive,42

present in only one allele or both.In function of this recent information regarding gene

expression,53 there are new therapeutic perspectives for EB,although these are still in an experimental phase. Therehave already been reports of ex vivo manipulation of kera-tinocytes from patients with EBJ, unable to produce the 3chain of laminin 5, which, after gene transfer, were demons-trated to be capable albeit transitorily of synthesizing it,thereby opening new therapeutic perspectives for this groupof genodermatoses.54 Animal models using transgenic miceto simulate human disease, has been contributing informa-tion relevant to the research of EB.10,12

Some authors consider that research into the corre-lation between genotype and phenotype is just at the begin-ning34 and that the expansion of the databases on gene alte-rations is of extreme importance, since it will enable anever increasingly improved correlation and perhaps even areclassification of some genodermatoses based on molecu-lar aspects.38 q

REFERNCIAS / REFERENCES1. Pulkkinen L, Uitto J. Mutation analysis and molecular geneticsof epidermolysis bullosa. Matrix Biol 1999;18:29-42.2. Uitto J, Pulkkinen L. Molecular genetics of heritable blisteringdisorders. Arch Dermatol 2001; 137: 1458-61.3. Fine JD, Eady RAJ, Bauer EA, et al. Revised classificationsystem for inherited epidermolysis bullosa: report of the secondinternational consensus meeting on diagnosis ans classification ofepidermolysis bullosa. J Am Acad 2000;42:1051-66.4. Mller FB, Kster W, Tuderman LB, Korge BP. Novel K5 andK14 mutations in German patients with the Weber-Cockaynevariant of epidermolysis bullosa simplex. J Invest Dermatol 1998;111:900-2.5. Horn HM, Tidman MJ. The clinical spectrum of epidermolysisbullosa simplex. Br J Dermatol 2000; 142: 468-72.6. Shemanko CS, Mellerio JE, Tidman MJ, Lane EB, Eady RAJ.Severe palmo-plantar hyperkeratosis in Dowling-Meara epider-molysis bullosa simplex caused by a mutation in the keratin 14gene (KRT14). J Invest Dermatol 1998; 111:893-5.7. Irvine AD, Mclean WHI. Human keratin diseases: the increa-sing spectrum of disease and sublety of the phenotype-genotypecorrelation. Br J Dermatol 1999; 140: 815-28. 8. Sasaki Y, Shimizu H, Akiyama M, et al. A recurrent keratin 14mutation in Dowling-Meara epidermolysis bullosa simplex . Br JDermatol 1999; 141: 747-8.9. Livingston RJ, Sybert VP, Smith LT, Dale BA, Presland RB,Stephens K. Expression of a truncated keratin 5 may contribute tosevere palmo-plantar hyperkeratosis in epidermolysis bullosasimplex patients. J Invest Dermatol 2001; 116:970-4.10. Peters B, Kirfel J, Bssow H, Vidal M, Magin TM. Completecytolysis and neonatal lethality in keratin 5 knockout mice revealits fundamental role in skin integrity and in epidermolysis bullosasimplex. Mol Biol Cell 2001; 12: 1775-89.

11. Ma L, Yamada S, Wirtz D, Coulombe PA. A hot-spot mutationalters the mechanical properties of keratin filament networks. NatCell Biol 2001; 3: 503-6.12. Cao T, Longley MA, Wang XJ, Roop DR. An inducible mousemodel for epidermolysis bullosa simplex: implications for genetherapy. J Cell Biol 2001; 152: 651-6.13. Batta K, Rugg EL, Wilson NJ, et al. A keratin 14 knockoutmutation in recessive epidermolysis bullosa simplex resulting inless severe disease. Br J Dermatol 2000; 143: 621-7.14. Mller FB, Almeida Jr. HL, Schumann H, et al. An update onkeratin mutations in epidermolysis bullosa simplex Dowling-Meara (in press) .15. Liovic M, Stojan J, Bowden PE, et al. A novel keratin 5 muta-tion (K5V186L) in a family with EBS-K: a conservative substitu-tion can lead to development of different disease phenotypes. JInvest Dermatol 2001; 116: 964-9.16. Bauer JW, Rouan F, Kofler B, et al. A compound heterozy-gous one amino-acid insertion/nonsense mutation in the plectingene causes epidermolysis bullosa simplex with plectin defi-ciency. Am J Pathol 2001; 158: 617-25.17. Kurose K, Mori O, Hashisuka H, Shimizu H, Owaribe K,Hashimoto T. Cultured keratinocytes from plectin/HD1-deficientepidermolysis bullosa simplex showed altered ability of adhesionto the matrix. J Dermatol Sci 2000; 24: 184-9.18. Nievers MG, Schaapveld RQJ, Sonnenberg A. Biology andfunction of hemidesmossomes. Matrix Biol 1999; 18:5-17.19. Aumailley M, Krieg T. Laminins: a family of diverse multi-functional molecules of basement membranes. J Invest Dermatol1996;106:209-14.20. Aumailley M, Rousselle P. Laminins of the dermo-epidermaljunction. Matrix Biol 1999; 18:19-28.21. Ruzzi L, Pas H, Posteraro P, et al. A homozygous nonsense

Almeida Jr. 529


composed of more exons than any previously characterized gene.Genomics 1994; 21: 169-79. 36. Mellerio JE, Alanis JCS, Talamantes ML, et al. A recurrentglycine substitution mutation, G2043R, in the type VII collagengene (COL7A1) in dominant dystrophic epidermolysis bullosa. BrJ Dermatol 1998; 139: 730-7.37. Jrvikallio A, Pulkkinen L, Uitto J. Molecular basis of dystro-phic epidermolysis bullosa: mutations in the type VII collagengene (COL7A1). Human Mut 1997; 10: 338-47.38. Kon A, Pulkkinen L, Yamamoto AI, Hashimoto I, Uitto J.Novel COL7A1 mutations in dystrophic forms of epidermolysisbullosa. J Invest Dermatol 1998; 111:534-7.39. Christiano AM, Anhalt G, Gibbons S, Bauer EA, Uitto J.Premature termination codons in the type VII collagen gene(COL7A1) underlie severe, mutilating recessive dystrophic epi-dermolysis bullosa. Genomics 1994; 21: 160-8.40. Ryoo YW, Kim BC, Lee KS. Characterization of mutations ofthe type VII collagen gene (COL7A1) in recessive dystrophic epi-dermolysis bullosa mitis (M-RDEB) from three Korean patients. JDermatol Sci 2001; 26: 125-32.41. Rouan F, Pulkkinen L, Jonkman MF, et al. Novel and de novoglycine substitution mutations in the type VII collagen gene(COL7A1) in dystrophic epidermolysis bullosa: implications forgenetic counseling. J Invest Dermatol 1998; 111:1210-13.42. Lee YY, Li C, Chao SC, Pulkkinen L, Uitto J. A de novo glyci-ne substitution mutation in the collagenous domain of COL7A1 indominant dystrophic epidermolysis bullosa. Arch Dermatol Res2000; 292: 159-63.43. Klingberg S, Mortimore R, Parkes J, et al. Prenatal diagnosisof dominant dystrophic epidermolysis bullosa, by COL7A1 mole-cular analysis. Prenat Diagn 2000; 20: 618-22.44. Murata T, Masunaga T, Shimizu H, et al. Glycine substitutionmutations by different amino acids in the same codon of COL7A1lead to heteregeneous clinical phenotypes of dominant dystrophicepidermolysis bullosa. Arch Dermatol Res 2000; 292: 477-81.45. Betts CM, Posteraro P, Costa AM, et al. Pretibial dystrophicepidermolysis bullosa: a recessively inherited COL7A1 splice sitemutation affecting procollagen VII processing. Br J Dermatol1999; 141: 833-39.46. Nordal EJ, Mecklenbeck S, Hausser I, Skranes J, TudermanLB, Dahl TG . Generalized dystrophic epidermolysis bullosa:identification of a novel, homozygous glycine substitution,G2031S, in exon 73 of COL7A1 in monozygous triplets.Br JDermatol 2001; 144: 151-7.47. Mcgrath JA, Ashton GHS, Mellerio JE, et al. Moderation ofphenotypic severity in dystrophic and junctional forms of epider-molysis bullosa through in-frame skipping of exons containingnon-sense or frameshift mutations. J Invest Dermatol 1999;113:314-21.48. Klausegger A, Pulkkinen L, Gubo GP, et al. Is screening of thecandidate gene necessary in unrelated partners of members offamilies with Herlitz junctional epidermolysis bullosa? J InvestDermatol 2001; 116:474-5.49. Christiano AM, Pulkkinen L, Mcgrath JA, Uitto J. Mutation-based prenatal diagnosis of Herlitz junctional epidermolysis bullo-sa. Prenat Diagn 1997; 17: 343-54.50. Hovnonian A, Hilal L, Bardon CB, et al. DNA-based prenataldiagnosis of generalized recessive dystrophic epidermolysis bullo-sa in six pregnancies at risk for recurrence. J Invest Dermatol1995; 104:456-61.

mutation in type XVII collagen gene (COL17A1) uncovers analternatively spliced mRNA accounting for an unusually mildform of non-Herlitz junctional epidermolysis bullosa. J InvestDermatol 2001; 116:182-7.22. Nakano A, Pfendner E, Pulkkinen L, Hashimoto I, Uitto J.Herlitz junctional epidermolysis bullosa: novel and recurrentmutations in the LAMB3 gene and the population carrier fre-quency. J Invest Dermatol 2000; 115:493-8.23. Vailly J, Pulkkinen L, Miguel C, et al. Identification of ahomozygous one-basepair deletion in exon 14 of the LAMB3gene in a patient with Herlitz junctional epidermolysis bullosaand prenatal diagnosis in a family at risk for recurrence. J InvestDermatol 1995; 104:462-6.24. Takizawa Y, Shimizu H, Pulkkinen L, et al. Novel mutationsin the LAMB3 gene shared by two japanese unrelated familieswith Herlitz junctional epidermolysis bullosa, and their applica-tion for prenatal testing. J Invest Dermatol 1998; 110:174-8.25. Takizawa Y, Shimizu H, Pulkkinen L, et al. Novel prematuretermination codon mutations in the laminin _2-chain gene(LAMC2) in Herlitz junctional epidermolysis bullosa. J InvestDermatol 1998; 111:1233-4.26. Pulkkinen L, Meneguzzi G, Mcgrath JA, et al. Predominanceot the recurrent mutation R635X in the LAMB3 gene in europeanpatients with Herlitz junctional epidermolysis bullosa has impli-cations for mutation detection strategy. J Invest Dermatol 1997;109:232-7.27. Mcgrath JA, Kivirikko S, Ciatti S, Moss C, Christiano AM,Uitto J. A recurrent homozygous nonsense mutation within theLAMA3 gene as a cause of Herlitz junctional epidermolysis bul-losa in patients of pakistani ancestry: evidence for a foundereffect. J Invest Dermatol 1996; 106:781-4.28. Cserhalmi PB, Horvath A, Boros V, et al. Identification ot theLAMB3 hotspot mutation R635X in a hungarian case of Herlitzjunctional epidermolysis bullosa. Exp Dermatol 1997;6:70-4.29. Takizawa Y, Shimizu H, Pulkkinen L, et al. Combination of anovel frameshift mutation (1929delCA) and a recurrent nonsensemutation (W610X) of the LAMB3 gene in a japanese patient withHerlitz junctional epidermolysis bullosa, and their application forprenatal testing. J Invest Dermatol 1998; 111:1239-40.30. Hauschild R, Wollina U, Tuderman LB. Junctional epider-molysis bullosa gravis (Herlitz): diagnostic and genetic aspects. JEu Acad Dermatol Venereol 2001; 15: 73-6.31. Christiano AM, Pulkkinen L, Eady RAJ, Uitto J. Compoundheterozygosity for nonsense and missense mutations in theLAMB3 gene in nonlethal junctional epidermolysis bullosa. JInvest Dermatol 1996; 106:775-7.32. Mcgrath JA, Pulkkinen L, Christiano AM, Leigh IM, EadyRAJ, Uitto J. Altered laminin 5 expression due to mutations in thegene encoding the 3 chain (LAMB3) in generalized atrophicbenign epidermolysis bullosa. J Invest Dermatol 1995; 104:467-74.33. Inoue M, Tamai K, Shimizu H. A homozygous missensemutation in the cytoplasmatic tail of 4 integrin, G931D, that dis-rupts hemidesmossome assembly and underlies non- Herlitz junc-tional epidermolysis bullosa without pyoric atresia? J InvestDermatol 2000; 114:1061-3.34. Tuderman LB, Hpfner B, Hauasli NH. Biology of anchoringfibrils: lessons from dystrophic epidermolysis bullosa. MatrixBiol 1999; 18: 43-54.35. Christiano AM, Hoffman GG, Honet LCC, et al . Structuralorganization of the human type VII collagen gene (COL7A1),

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51. Rugg EL, Baty D, Shemanko CS, et al. DNA based prenataltesting for the skin blistering disorder epidermolysis bullosa sim-plex. Prenat Diagn 2000;20:371-7.52. Friedman PBC, Tang Y, Adler A, Krey L, Grifo JA, ChristianoAM. Preimplantation genetic diagnosis in two families at risk forrecurrence of Herlitz junctional epidermolysis bullosa. ExpDermatol 2000; 9:290-7.

53. Khavari PA. Gene therapy for genetic skin disease. J InvestDermatol 1998; 110:462-6.54. Vailly J, Palacios LG, DellAmbra E, et al. Corrective genetransfer of keratinocytes from patients with junctional epidermoly-sis bullosa restores assembly of hemidesmossomes in reconstruc-ted epithelia. Gene Ther 1998; 5: 1322-32.

ENDEREO PARA CORRESPONDNCIA: / MAILING ADDRESS:Prof. Dr. Hiram Larangeira de Almeida Jr.Departamento de Medicina EspecializadaFaculdade de Medicina da UFPELAv. Duque de Caxias 250Pelotas RS 96030 002 [email protected]

Almeida Jr. 531


1. Qual a herana gentica no grupo da EBS? a) autossmica dominanteb) autossmica recessivac) ligada a X dominanted) ligada a X recessivae) polignica

2. Com respeito EBS qual dos achados abaixo NO encontrado?

a) degenerao da camada basal b) clivagem sub-epidrmicac) ausncia de infiltrado inflamatrio d) ausncia de anticorpos no tecidoe) citlise

3. Quais das protenas abaixo est alterada no grupo daEBS?

a) citoqueratina 14b) citoqueratina 10c) laminina 5 d) colgeno XVIIe) colgeno VII

4. Qual manifestao extra-cutnea est descrita na EBS?a) atresia de pilorob) porfria cutnea tardac) paralisia espsticad) distrofia muscular tardiae) estenose de esfago

5. Em que parte do gen das citoqueratinas basais localiza-sea maioria das mutaes da EBS- Weber-Cockayne?

a) no incio do segmento 1A b) no final do segmento 2B c) no segmento no-helicoidal entre 1B e 2Ad) no incio do segmento 2Be) no final do segmento 1A

6. Qual a herana gentica no grupo da EBJ? a) autossmica dominanteb) autossmica recessivac) ligada a X dominanted) ligada a X recessivae) polignica

7. Em qual das protenas abaixo NO foi ainda descritamutao?

a) plectinab) antgeno do penfigide bolhoso de 180 KDc) antgeno do penfigide bolhoso de 230 KDd) laminina 5e) integrina a6b4

8. Qual manifestao extra-cutnea est descrita na EBJ?a) atresia de pilorob) megaclonc) estenose de esfagod) distrofia muscular tardiae) acloridria

9. Assinale a alternativa falsa.a) As mutaes tipo premature termination codon (PTC),levam interrupo da sntese proteica com ausncia da protena no tecido e quadro clnico mais graveb) As mutaes j foram demonstradas nos trs gens quecodificam a laminina 5, no havendo diferena fenotpicade acordo com o segmento envolvidoc) O antgeno do penfigide bolhoso de 180 KD um colgeno transmembranosod) No grupo da EBJ no ocorrem mutaes recurrentese) A integrina a6b4 uma protena transmembranosa

10. Qual protena est envolvida na EBJ tipo Herlitz ou EBJLetal?

a) integrina a6b4b) colgeno XVII c) laminina 5d) plectinae) colgeno VII

11. Em qual das formas abaixo da EB apenas um gen estmutado?

a) Epidermlise Bolhosa Simplesb) Epidermlise Bolhosa Juncionalc) Epidermlise Bolhosa Epidermolticad) Epidermlise Bolhosa Distrficae) Epidermlise Bolhosa Adquirida

12. Qual alterao foi demostrada na EBJ generalizada atr-fica benigna?

a) PTC no gen COL17A1b) mutao no gen LAMB 3c) mutao nos gens ITGA6 e ITGB4d) PTC no gen LAMC2e) PTC no gen COL7A1

13. A laminina 5 um:a) heterodmerob) dmero antiparaleloc) colgeno transmembranosod) heterotrmeroe) componente da placa interna do hemidesmossoma.

Questes e Resultado das Questes / Questions and Answers to Questions

532 Almeida Jr.


d) Na EBD-Dominante a alterao encontrada uma substituio de glicina no segmento colgenoe) A localizao da substituio de glicina no contribui para a variabilidade clnica na EBD-Dominante

18. Qual das formas de EB no decorrente do defeito deadeso e sim por citlise?

a) Epidermlise Bolhosa Simples b) Epidermlise Bolhosa Juncionalc) Epidermlise Bolhosa Distrficad) Epedermlise Bolhosa Dermolticae) Epidermlise Bolhosa Adquirida

19. Com relao utilizao do sequenciamento gnico apartir do corion no diagnstico pr-natal, assinale a alterna-tiva falsa.

a) No necessita de bipsia da peleb) As complicaes ocorrem em 1 % dos casosc) Pode ser realizado ainda antes da implantaod) Pode ser utilizado em todas as formas de EBe) Deve ser feito na 20a semana

20. Assinale a alternativa correta.a) Nas mutaes de novo o risco para a prole do paciente muito baixo

b) A manipulao ex vivo de clulas de portadores de EBno possvelc) Modelo animal com ratos transgnicos tem ajudado a esclarecer a patogenia das diversas formas de EBd) Na EB no ocorrem mutaes de novoe) O sequenciamento gnico substitui outros exames no diagnstico da EB

14. Na EBD a clivagem ocorre:a) intraepidrmicab) na lmina lcidac) acima da lmina densad) na placa interna do hemidesmossomae) abaixo da lmina densa

15. Assinale a alternativa correta com relao EBD.a) Vrias protenas encontra-se mutadasb) Apenas uma protena est mutada, o que leva somentea um quadro clnicoc) A herana pode ser autossmica dominante ou recessivad) As fibrilas ancorantes esto sempre ausentese) A mutao leva sempre a PTC

16. Com relao EBD recessiva Halloupeau-Siemens qualalternativa falsa?

a) A microscopia eletrnica mostra ausncia das fibrilas ancorantesb) A imuno-histoqumica com anticorpos contra o colgeno 7 negativac) Ocorre uma PTC no gen COL7A1 com conseqente interrupo na sntese do colgeno VIId) O quadro clnico leve com leses nos joelhos e cotovelose) Ocorre sinquia nas extremidades

17. Assinale a alternativa corretaa) Na EBD-D no so encontradas mutaes recorrentesb) Na EBD-Dominante a substituio de glicina esclarecea herana autossmica dominantec) Nas diferentes formas de EB as mutaes do tipo PTClevam a quadros clnicos mais leves

Gabarito:Eritema Nodoso Hansnico: atualizao clnica eteraputica.2002;77 (4):389-407

1.e2.e3.a4.d5.b6.c7.d8.e9.b10.e

11.e12.a13.d14.a15.d16.c17.a18.c19.a20.b

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