Molecular Genetics of Epidermolysis Bullosa

Abstracts

New data regarding the molecular aspects of the heterogeneous group of epidermolysis bullosa has brought some important information about its pathogenesis. In epidermolysis bullosa simplex the majority 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 (gene PLEC1), a protein found in the inner hemidesmosomal plaque, leads also to intraepidermal blisters. In junctional epidermolysis bullosa many proteins from the basal membrane zone are involved, such as laminin 5 (genes LAMA3, LAMB3 and LAMC2), integrin alpha6beta4 (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 they alter the structure of collagen VII, the principal compound of anchoring fibrils, splitting the skin under the lamina densa. This information can also be used in the prenatal diagnosis of epidermolysis bullosa, with future perspectives of gene therapy.

prenatal diagnosis; epidermolysis bullosa; genetics; biochemical; mutation; polymerase chain reaction

O estudo das alterações moleculares das epidermólises bolhosas tem contribuído para que se compreenda melhor essas enfermidades. Na epidermólise bolhosa simples a maioria dos casos está associada com alteração nas citoqueratinas basais 5 (gen KRT5) e 14 (gen KRT14), o que modifica o citoesqueleto na camada basal da epiderme, levando à degeneração dessa camada, formando bolha intra-epidérmica. Mutações na plectina (gen PLEC1), componente da placa interna do hemidesmossoma, levam também à clivagem intra-epidérmica. Na epidermólise bolhosa juncional vários gens estão envolvidos, em decorrência da complexidade da zona da membrana basal, todos levando ao descolamento dos queratinócitos basais na lâmina lúcida, pela disfunção da aderência entre esses e a lâmina densa. Alterações na laminina 5 (gens LAMA3, LAMB3 e LAMC2), integrina alfa6beta4 (gens ITGA6 e ITGB4) e colágeno XVII (gen COL17A1) foram descritas. Por fim, na epidermólise bolhosa distrófica apenas um gen está mutado, alterando o colágeno VII (gen COL7A1), principal componente das fibrilas ancorantes, produzindo clivagem abaixo da lâmina densa, variando fenotipicamente de acordo com a conseqüência da mutação. Outra aplicação importante dessas informações refere-se ao diagnóstico pré-natal, com a perspectiva no futuro da terapia gênica.

Diagnóstico pré-natal; epidermólise bolhosa; genética bioquímica; mutação; reação em cadeia da polimerase

CONTINUING MEDICAL EDUCATION

Molecular Genetics of Epidermolysis Bullosa^*

Hiram Larangeira de Almeida Jr.

Adjunct Professor of Dermatology, Federal University of Pelotas

^{Correspondence}

SUMMARY

New data regarding the molecular aspects of the heterogeneous group of epidermolysis bullosa has brought some important information about its pathogenesis. In epidermolysis bullosa simplex the majority 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 (gene PLEC1), a protein found in the inner hemidesmosomal plaque, leads also to intraepidermal blisters. In junctional epidermolysis bullosa many proteins from the basal membrane zone are involved, such as laminin 5 (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 they alter the structure of collagen VII, the principal compound of anchoring fibrils, splitting the skin under the lamina densa. This information can also be used in the prenatal diagnosis of epidermolysis bullosa, with future perspectives of gene therapy.

Key-words: prenatal diagnosis; epidermolysis bullosa; genetics, biochemical; mutation; polymerase chain reaction.

INTRODUCTION

Before the discovery and standardization of polymerase chain reaction (PCR), gene sequencing was an arduous task, requiring a long time to analyze small segments. PCR allows the fast amplification of DNA segments, which are then sequenced, thereby contributing to enormous progress in this field. To date, inputting PCR as a key word in the Medline database, lists over 150,000 publications 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 is to isolate the DNA, for instance taking blood and making use of its insolubility and precipitation in certain solvents and its water solubility, several commercial kits are available for this function.

Then, part of the DNA obtained is incubated with a thermoresistant polymerase (since the DNA is heated in order to separate the double chain) together with known sequences of DNA, the so-called primers If the DNA in question has the same sequence as that of the primer, the polymerase will amplify this segment of DNA. The nucleotides (adenine, thymidine, cytosine and guanine) are part of the reaction, such that obviously the enzyme has the necessary raw material for the polymerization. Heating and cooling cycles are repeated innumerous times, thereby increasing the product of PCR more and more. After which, electrophoresis is performed to identify the presence of a band of DNA, demonstrating the positivity or otherwise of the reaction.

In a subsequent stage the product obtained by PCR is sequenced using a variant of PCR. The sequencing is now automated with laser readings providing polychromatic graphs, with blue representing cytosine, red thymidine, black guanine and green adenine (Figure 1). Comparison of the result obtained in the investigated patient and their progenitors with the normal gene sequence can demonstrate mutation and an inherited pattern.

Each set of three bases of DNA codifies an amino acid for the protein synthesis in the ribosome; leading to a mutation, or in other words, the change of a base, during the protein synthesis there will be an insertion of another amino acid, thus altering the structure of the protein and resultant consequences.

Countless genes have already been sequenced and their composition is available in digital databases. These can be accessed at the Genbank of the National Center of Biotechnology Information of the United States: www.ncbi.nlm.nih.gov.

The mutations are described by citing the amino acids or bases involved. Firstly the aminoacid/base is given that should constitute the protein/gene, followed by a number, wich corresponds to the location of the same in the protein/gene under investigation and finally the aminoacid/base inserted in the place of the first, such as, for instance, Glu20Arg, in other words, the twentieth amino acid should be a glutamine, but mutation leads to an insertion in the protein of an arginine, wich alters its structure. Some sequences of bases codify the end of the protein synthesis; a mutation can lead to this interruption, the so-called premature termination codon, wich is described in the following manner: Lys472Stop, in other words, instead of the insertion of a lysine, at the 472 position, the protein synthesis was interrupted. Abbreviated descriptions with only a single letter can be found, for example such an interruption of this synthesis is denoted by an X; in the above example this woul then be L472X.

The bullous dermatoses make a fascinating chapter in dermatology, they comprise acquired or congenital defects of the intraepidermal or dermoepidermal adhesion, leading to blisters which can be spontaneous or provoked by minimal trauma.

In epidermolysis bullosa (EB) these defects are congenital and can be identified by gene sequencing, which affords a greater understanding of their molecular base,^1,2complements the clinicohistological diagnosis and maybe in the medium term will partly modify the classification of these dermatoses

Three subgroups of EB are recognized:² epidermolysis bullosa simplex (EBS), in the which the cleaving occurs inside the epidermis; junctional epidermolysis bullosa (EBJ), with subepidermal cleaving in the lamina lucida; and epidermolysis bullosa dystrophica (EBD), also subepidermal, but below the lamina densa. EBJ and EBD cannot be differentiated by optical microscopy alone. There are several classifications, but in this work the second international consensus has been adopted regarding the diagnosis and classification of epidermolysis bullosa.³

Epidermolysis bullosa simplex

The group of EBS has several subtypes, according to the intensity and location of the blisters, all of which with autosomal dominant inheritance;⁴ these are also called epidermolytic EB, since the defect is intraepidermal.³The histological aspect most commonly found is degeneration of the basal layer, in the absence of any inflammatory infiltration and without deposit of antibodies in the tissue.

The most serious form of EBS is Dowling-Meara syndrome (EBS-DM),⁵ in which disseminated blisters that also involve the mucous membranes, are accompanied by palmoplantar hyperkeratosis. The mildest form is Weber-Cockayne syndrome (EBS-WC) with lesions restricted to the palmar and plantar regions.⁵ An intermediate form exists, again with disseminated blisters, but with a less intense picture than that of EBS-DM, denominated EBS-Koebner (EBS-K), although certain authors consider this to be a mild variant of EBS-DM.⁶

Some cytokeratins are expressed in the epithelial cells in pairs,⁷ which form heterodimers, in other words, the union of two molecules, configuring the cytoskeleton of the epithelia, with specificity according to the epithelium involved.⁷ The basal layer differs from other epithelia and suprabasal segments of the epidermis by the expression of cytokeratins 5 and 14.

Cytokeratins 5 and 14 are regulated by the genes KRT5 and KRT14, located in chromosomes 17 and 12, respectively. It is interesting to note that different genetic defects in EBS, one affecting cytokeratin 5 and the other^14,6,8,9lead to the same histological alteration, because all these defects produce structural alterations in one or another cytokeratin,¹⁰ impeding their structural function in the cytoskeleton11 - i.e. the formation of the heterodimers, responsible for the three-dimensional configuration of the cell. This alteration is easily seen in the histology and culminates with the formation of blisters, making this the only subgroup of EB due to cytolysis and not to an adhesion defect.

The cytokeratins are constituted by four helical segments, 1A, 1B, 2A and 2B,¹²the majority of the mutations of EBS-DM are described in the beginning of segment 1A and at the end of segment 2B (Figure 2)^13,14 of the basal cytokeratins. The mutations of EBS-K have a similar location,¹⁵ reinforcing the hypothesis that it is a variant of EBS-DM. In EBS-WC most of the mutations are located in the non-helical segment between 1B and 2A of the same cytokeratins, though this does not explain the palmoplantar location of the lesions.

A fourth type of EBS is described, in which cytolysis in the basal layer does not occur. It is EBS with tardive muscular dystrophy, due to alteration of the plectin, present in the internal plaque of the hemidesmosome (Figure 3). Since the cleaving occurs within the epidermis, it is included in this group. The plectin is regulated by the gene PLEC11 and is also involved in the cytoskeleton of the smooth musculature,¹⁶ hence the associated myopathy.^1,17 Another component of the internal plaque of the hemidesmosome is the bullous pemphigoid antigen with 230 KD molecular weight. To date mutation in the gene that regulates this has not been described.¹

Junctional Epidermolysis Bullosa

Given the complexity of the basal membrane zone, alterations in several proteins involved in the dermoepidermal adhesion can lead to the various clinical pictures of EBJ; for these molecular alterations to be understood, it is important to be familiar with the substances responsible for the adhesion between the basal keratinocytes and the collagen IV - the lamina densa (Figure 3).

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

The antigen of bullous pemphigoid (180 KD) is in reality a transmembranous collagen, denominated collagen XVII, and is regulated by the gene COL17A1.¹Each segment of the integrin a6b4 is regulated by two different genes, ITGA6 and ITGB4, which are also expressed in the skin and digestive tract.^1,18

Finally, some substances present in the lamina lucida complement this molecular net,¹ of which the most important is laminin 5. The laminins are heterotrimers, or that is, they are constituted by three distinct classes of polypeptides a, b and g,^18-20 and hence regulated by three genes. Laminin 5 is composed of one a3, one b3 and one g2, regulated by the genes LAMA3, LAMB3 and LAMC2, respectively.

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

Several genophenotype correlations have already been made. Such as integrin a6b4 is expressed in the skin and intestine, mutations of which lead to forms of EBJ with atresia pilori, the clinical picture varies according to whether or not it is associated to PTC.

Regarding generalized, benign and atrophic EBJ, characterized by disseminated blisters with nail dystrophy, in which the immunohistochemistry with antibody against collagen XVII is negative, PTC has been demonstrated in the gene COL17A,^1,21 which correlates with the tissular absence of collagen 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 hemidesmosome described above, mutations in the laminin also provoke dislocation of the epidermis. Most of the mutations of the genes of laminin 5 lead to PTC, provoking absence of the protein and intense clinical picture,^23,24 characterized by disseminated lesions also affecting the mucous membranes,²³ with low survival in function of bacterial complications, denominated Herlitz syndrome or epidermolysis bullosa lethalis.

The alterations have already been demonstrated in the three genes that codify laminin 5,²⁵ without a phenotype difference according to the segment involved,²⁶ suggesting that all are important for its adhesion function.^26,27 Eighty percent of the mutations reside in the gene LAMB3,^22,24,28-30 30 two of these are recurrent (R635X and R42X),^26,28 which amount to half of the mutations in LAMB3.¹ In this gene alone 35 different mutations have already been reported.²²

There are reports of mutation in laminin 5 among patients in which the immunohistochemistry demonstrated a reduction in laminin 5, but not a complete absence, as in Herlitz Syndrome, and consequently the clinical picture was not so serious.^31,32 These forms have also been denominated non-Herlitz EBJ,³³ which are clinically similar to the forms arising from mutation of collagen XVII.³³

All forms of EBJ are inherited as an autosomal recessive trait.²³

Epidermolysis bullosa dystrophica

The main clinical characteristic of EBD is scarring after tissue loss, since the separation occurs below the lamina densa.³⁴ As in the other groups, there are variants reflecting the clinical picture; in spite of these variants, the genetic defect is located in a single gene, COL7A1.³⁵ This gene is responsible for codifying collagen VII, the main representative of the anchoring fibrils,³⁵ which participate in the adherence of the lamina densa to the dermis (Figure 3), hence it is also denominated dermolytic EB. In this group there are forms inherited as autosomal dominant and recessive traits.

In order to understand the correlation between genotype and phenotype in EBD it is necessary to also understand the role of collagen VII in the dermoepidermal adhesion.

This is produced by the keratinocytes and has a triple helix configuration of collagen, preceded and followed by non-collagen segments (NC-1 and NC-2, respectively).^36,37 In the center of the triple helix there is small non-collagen segment, which probably provides flexibility to the protein. Later, at the extracellular level, a fusion occurs between two of these molecules with loss of the NC-2 segment, forming antiparallel dimers. The union of several dimers forms the anchoring fibrils^34,36,37 (Figure 4).

Three subtypes of EBD are well characterized: recessive EBD Halloupeau-Siemens (EBD-RHS), with an intense clinical picture, producing acral retractions with synechiae of the digits and involvement of the digestive tract;³⁷recessive EBD mitis (EBD-RM), in which the clinical picture is much less intense in comparison with that of EBD-RHS, with localized lesions in the areas of greatest trauma, such as the knees and extremities; and the dominant form (EBD-D), with a similar picture to that of EBD-RM,³⁷ associated to nail dystrophy and, in some cases, with white papular lesions.

Electron microscopy and immunohistochemical characterization with antibodies against collagen VII show alteration in the anchoring fibrils to the extent of their absence in EBD-RHS and reduction in the milder forms, EBD-RM and EBD-D.³⁸ In some cases the immunohistochemistry is positive but without anchoring fibrils revealed by electron microscopy, which demonstrates the presence of part of the molecule, but with structural alteration.³⁸

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

In EBD-RHS the genetic alteration is a PTC, with consequent interruption in the synthesis of collagen VII,³⁹ which correlates with the intensity of the clinical picture and with the findings of electron microscopy and immunohistochemistry, in which the anchoring fibrils are not detected.^34,37

In EBD-RM the greater part of the mutations occur at the end of the collagen segment and in NC-2, interfering in the formation of the antiparallel dimers and altering the compliance of the protein, though these continue present, giving rise to a milder clinical picture and the presence of anchoring fibrils in the electron microscopy.^37,40

In EBD-D, the characteristic alteration is the substitution of a glycine in the collagen segment,^41,42 altering its stability and maybe propitiating its degradation.^36,37,43 As in EBD-RM, the anchorage fibrils are present, but their function is impaired. Most of the mutations are located immediately after the non-collagen segment of the center of the triple helix;⁴² the G2043R mutation is the most commonly described.^36,41 Likewise it has already been demonstrated that the functional alteration of the anchoring fibrils depends on the location in which the glycine is substituted,^34,44 which in turn contributes to the clinical variability. As yet, there is no convincing explanation as to why the glycine substitution is an inherited dominant trait.

The majority of cases involving the pretibial form of EBD are autosomal dominant and the substitution of glycine has also been described.⁴⁵ Recessive cases have been published,⁴⁵ these could equally be considered variants of the mild forms of EBD, the reason behind the localized occurrence of the lesions is not known.

About 100 different mutations have already been described in EBD,³⁴ and are found in 80% of the cases examined.³⁷ As in other forms of EB, some mutations are not defined within the above described outline, because, for instance, some substitutions of glycine have been found in EBD-RM;^37,40,41 to date, it has yet to be clarified why in these cases the progenitors that present such glycine substitution may be normal, or in other words, the mutation is not dominant and is only expressed in a recessive manner, with the inheritance of two changed alleles.⁴¹

Various intermediate clinical pictures, presenting difficult clinical classification, have already been described with such uncommon mutations, for instance, recessive EBD with PTC 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 of EB can be (Table 1). In EBS the genetic defects of the basal cytokeratins produce a histological alteration due to the modification of the cytoskeleton in the basal layer of the epidermis, in that alteration of the plectin, a component of the internal plaque of the hemidesmosome, also leads to the intraepidermal separation. In EBJ several genes are involved, due to the complexity of the basal membrane zone, but all lead to the dislocation of the basal keratinocytes of the lamina densa, in other words, the cleaving occurs in the lamina 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 consequence of the mutation.

Despite its important contribution to progress in the understanding of these illnesses, gene sequencing should be used together with clinical, histological, electron microscopy and immunohistochemical findings in the diagnosis of EB.⁴⁷

Another important application for molecular genetics is in prenatal diagnosis (PND),^2,48 examining fetal DNA obtained from the chorion rather than the fetal skin. PND performed on the basis of the lesions requires the collection of a skin specimen, which should be representative of the illness, in order to avoid a false-negative result, one should wait until the eighteenth or twentieth week.⁴³Sequencing has the advantage that it can be performed around the tenth week, which means that a more precocious decision to terminate the gestation can be made in those countries in which this procedure is permitted. Furthermore, complications arising from fetoscopy with biopsy occur in between four to 7% of cases compared to 1% in chorionic biopsy.⁴³

Genetic sequencing has already been used in PND for all forms of EB,^{23,24,43,49-51} and has already been performed before implantation, based on a cell obtained from an embryo with a number of cells varying from five to eight.⁵²

Genetic counseling is another important application of this new information, since it helps to explain the inheritance pattern, especially when dealing with frequent and well-known mutations. Also in the case of de novo mutations, when the DNA exam of the progenitors is normal and the mutation is only found in the patient, it can be affirmed that the risk factor for the next gestation is very low. Regarding the patient's offspring, this will depend on whether 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. There have already been reports of ex vivo manipulation of keratinocytes from patients with EBJ, unable to produce theb3 chain of laminin 5, which, after gene transfer, were demonstrated to be capable - albeit transitorily - of synthesizing it, thereby opening new therapeutic perspectives for this group of genodermatoses.⁵⁴ Animal models using transgenic mice to simulate human disease, has been contributing information relevant to the research of EB.^10,12

Some authors consider that research into the correlation between genotype and phenotype is just at the beginning³⁴ and that the expansion of the databases on gene alterations is of extreme importance, since it will enable an ever increasingly improved correlation and perhaps even a reclassification of some genodermatoses based on molecular aspects.38

REFERENCES

Received in January, 30^th of 2002.

Approved by the Consultive Council and accepted for publication in July, 30^th of 2002.

*Work done with a post-doctorate grant from Capes and the Alexander von Humbold Foundation, in the Genetic Diagnosis Laboratory, University of Cologne, Germany (Service of Prof. Thomas Krieg)

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Publication Dates

• Publication in this collection
  19 May 2006
• Date of issue
  Oct 2002

History

• Received
  30 Jan 2002
• Accepted
  30 July 2002