Chapter 62. Inherited Epidermolysis Bullosa

Inherited epidermolysis bullosa (EB), is a family of diseases, with the common feature of blistering in response to mild trauma. Patients with EB can show blistering in the form of small vesicles or larger bullae, which can occur on both the cutaneous surfaces as well as on the mucosal tissues. The fragility of the skin and mucosa and the traumatic production of painful blisters are what all EB cases share in common. However, the distribution of the involvement, the depth of blister formation, any associated extracutaneous involvement and the severity of the blistering process vary with the different EB subtypes and depend on the underlying heritable molecular defect. Different EB subtypes also vary in the way in which blistered areas heal. The wound repair responses are often abnormal and can eventuate into chronic erosions, hypertrophic granulation tissue, scarring, or even invasive carcinoma. While the milder EB subtypes are associated with a normal lifespan and little or no internal involvement, the most severe recessively inherited forms are mutilating, multiorgan disorders that threaten both the quality and length of life.

A number of early studies identified the major subtypes of EB. Studies of von Hebra1–3 were the first to distinguish pemphigus from inherited blistering and the term epidermolysis bullosa hereditaria was first suggested by Koebner.4 Hallopeau was the first to distinguish between simplex (nonscarring) and dystrophic (scarring) forms of the disease5 while Weber6 and Cockayne,7 Dowling and Meara8 and Koebner4 each described unique forms of epidermolysis bullosa simplex. Hoffman,9 Cockayne,10 Touraine,11 Pasini,12 and Bart13 provided much of the information about subtypes of dystrophic epidermolysis bullosa. Herlitz described epidermolysis bullosa letalis,14 which was later found to be a part of the third major category of epidermolysis bullosa: the junctional form. The application of electron microscopy toward diagnosis of epidermolysis bullosa led to the studies of Pearson15 and collaborators who classified the patients not only on the basis of clinical findings but also on the existence of ultrastructural changes. A comprehensive classification of epidermolysis bullosa based on a combination of ultrastructural and clinical findings was completed in an early landmark treatise by Gedde-Dahl.16 Recent major advances have led to the identification of protein and genetic abnormalities in most types of epidermolysis bullosa patients. These studies have led to an improved understanding of the biological basis of epidermolysis bullosa and, finally, a classification of epidermolysis bullosa based on genetic/protein defects, which provides a rational approach to specific molecular therapy.

Overview

Epidermolysis bullosa arises from defects of attachment of basal keratinocytes to the underlying dermis. These defects can arise from inside the keratinocyte plasma membrane or extracellularly in the dermal–epidermal basement membrane zone (BMZ). Many tissues such as the skin and cornea, which are subjected to external disruptive forces, contain a complex BMZ composed of a group of specialized components that combine together to form anchoring complexes (Fig. 62-1). At the most superior aspect of the BMZ, keratin-containing intermediate filaments of the basal cell cytoskeleton insert upon electron dense condensations of the basal cell plasma membrane termed hemidesmosomes. Anchoring filaments span the lamina lucida connecting hemidesmosomes with the lamina densa and anchoring filaments. At the most inferior aspect of the BMZ, collagen VII-containing anchoring fibrils extend from the lamina densa into the papillary dermis and combine with the lamina densa and anchoring plaques, trapping interstitial collagen fibrils. Thus, the cutaneous BMZ connects the extensive basal cell cytoskeletal network with the abundant network of interstitial collagen fibrils in the dermis.17,18

Keratin Filaments

Keratins are obligate heteropolymers that are composed of pairs of acidic and basic monomers. The keratin pair 5 and 14 assembles together to form the extensive intermediate filament network of the basal cell cytoskeleton.19 Keratins contain a central α-helical rod with several nonhelical interruptions as well as nonhelical carboxyl and amino-terminal regions. The regions of highest conservation between the keratins are located on the ends of the keratin rod in the helix boundary motifs. Extensive mutagenesis studies suggest that helical regions near the ends of the central rod are important in keratin filament elongation, whereas the nonhelical domains may be important in forming lateral associations.20

Hemidesmosomes

Hemidesmosomes contain intracellular proteins, including plectin and BP230. Plectin is a 500-kDa protein, which acts as an intermediate filament-binding protein. It is possible that plectin also interacts with microfilaments, as plectin contains a domain with similarity to the actin-binding domain of spectrin.21,22 BP230, also known as BPAG1, is a 230-kDa protein which has homology both to desmoplakin23 and to plectin. Several splicing variants of BP230 are of vital importance in the nervous system.24–26 BP230 localizes to a region referred to as the inner plate on the cytoplasmic surface of the hemidesmosome and like plectin, functions in the connection between hemidesmosomes and intermediate filaments. BP230 negative transgenic mice lack a hemidesmosomal inner plate and the connection between hemidesmosomes and intermediate filaments is severed, creating a cytoplasmic zone of mechanical fragility just above the hemidesmosomes.

Anchoring Filaments

Hemidesmosomes also contain the transmembrane proteins collagen XVII (also termed BPAG2 and BP180)27 and α6β4 integrin.28 The cytoplasmic portions of these molecules make up part of the hemidesmosome dense plaque. The extracellular portions of these molecules make up portions of the anchoring filament that probably contribute to the structure known as the subbasal dense plate, which underlies hemidesmosomes in the lamina lucida region. β4 integrin only pairs with the α6 subunit, whereas the α6 subunit can combine either with the β4 integrin or with the β1 integrin. Both the α6β1 or α6β4 integrin combinations have been shown to act as receptors for laminins, but only α6β4 integrin acts as a specific receptor for laminin-332. α6β4 integrin plays a central role in organization of the hemidesmosome. The β4 integrin contains an especially large cytoplasmic domain, which functions in the interaction with other proteins of the hemidesmosomal plaque, including collagen XVII and plectin.29 Skin from transgenic mice lacking β4 integrin is devoid of hemidesmosomes and shows severe deficits in cell adhesion.30 Interactions between plectin and α6β4 integrin appear to be critical both in the assembly as well as the disassembly of hemidesmosomes.31

Collagen XVII (BPAG2, BP180) is a collagenous protein with a type II transmembrane orientation. Based on electron microscopy and cross-linking studies, collagen XVII assembles into a triple-helical homotrimer and contains three main regions: (1) an intracellular amino-terminal globular head, (2) a central rod, and (3) an extracellular flexible tail.32 Collagen XVII associates with laminin-332 and α6β4 integrin in adhesion structures termed stable anchoring contacts. These stable anchoring contacts are formed by keratinocytes in vivo, and are thought to represent prehemidesmosomes.33 The autoantigen in linear immunoglobulin (Ig) A bullous dermatosis, LAD-134,35 is a 120-kD protein which has been shown by peptide sequencing to be the cleaved exodomain of collagen XVII.36 Collagen XVII undergoes processing in keratinocyte cultures and in skin through the action of sheddases, membrane-associated proteases that solubilize cell surface receptors.37–39

In addition to α6β4 integrin and collagen XVII, anchoring filaments contain the molecules laminin-332 and laminin-311. Like all members of the family of laminin proteins,40–42 laminin-332 is a large heterotrimeric molecule, and contains α3, β3, and γ2 chains.43,44 The first laminins to be described contained three short arms and one long arm, forming a cross shape as shown by rotary shadowing analysis. In contrast, laminin-332 contains truncations of each short arm.45–47 Because of these short arm truncations, laminin-332 cannot self-polymerize with other laminins or bind to nidogen. Instead, laminin-332 forms a disulfide-bonded attachment to laminin-311,48 the other known anchoring filament laminin49 which contains α3, β1, and γ1 chains. Laminin-332 also undergoes processing of its γ2 and α3 chains.50 While the rat laminin γ2 chain has been previously been shown to be processed by metalloproteinase-251 and membrane type metalloproteinase type 1,52 the predominant site of cleavage by these enzymes is not conserved in human laminin-332.53 Other studies have shown that processing of laminin γ2 chain takes place through a special class of proteins termed C-proteinases which also process C-terminal domains of procollagen molecules.54 While one member of this class of proteins, bone morphogenic protein 1,55 is capable of performing this action, a splice variant of bone morphogenic protein 1 (mammalian tolloid) is predominantly expressed in keratinocytes and fibroblasts and thus likely performs this function in the skin.53 Mammalian tolloid also processes the laminin α3 chain,53 although other enzymes such as plasmin,56 matrix metalloproteinase 251 or membrane type matrix metalloproteinase 152 are also capable of this function. The γ2 chain short arm appears important in the assembly of laminin-332 into basement membrane.57 The antigen recognized by mAb 19-DEJ-158 also localizes to anchoring filaments but its molecular identity remains unknown.

Anchoring Fibrils

Collagen VII is the major constituent of anchoring fibrils. Analysis of the deduced amino acid sequence of collagen VII59 reveals the presence of a long central collagenous region characterized by repeating GlyXY sequences that contains a number of noncollagenous interruptions, including a 39 amino acid noncollagenous segment in the center of the helix which corresponds to the “hinge region” predicted by biochemical studies.60,61 These interruptions account for the flexibility of the collagen VII molecule, and explain its ability to loop around and entrap dermal matrix molecules, thus stabilizing the basement membrane to the underlying papillary dermis.62 A 50 KDa component of anchoring fibrils has also been identified which appears to localize to the insertion sites of anchoring fibrils to the lamina densa.63

The 145-kDa N-terminal end of collagen VII contains the largest noncollagenous domain, which inserts onto the lamina densa and anchoring plaques. Collagen IV, the most abundant component of these structures, binds to the collagen VII NC-1 domain. A direct interaction between anchoring filaments and anchoring fibrils exists from a specific interaction between the anchoring filament component laminin-332 and the collagen VII NC-1 domain.64,65 Collagen VII binds the β3 chain on laminin-332.64,66–68 This appears to be a critical factor in the maintenance of dermal–epidermal cohesion. Like all collagens, collagen VII assembles into a triple helix. Only one gene and one chain of collagen VII, the α1 chain, have been identified, and thus collagen VII is a homotrimer. Collagen VII triple helices are joined together at their processed NC2 globular domains to form antiparallel dimers.62,69 Processing of the NC-2 domains takes place via the same family of C-proteinases (bone morphogenic protein 1 and/or mammalian tolloid) that are known to process laminin-332, a closely associated molecule. Anchoring fibrils may derive from lateral associations of collagen VII antiparallel dimers.

Epidermolysis bullosa has been traditionally classified according to the level of BMZ separation on transmission electron microscopy into simplex, junctional and dystrophic subtypes,70 (Fig. 62-2). Although this ultrastructural “gold standard” for diagnostic grouping of EB has been electron microscopy, immunomapping of basement membrane antigens as viewed by indirect immunofluorescence can also be quite useful in distinguishing subtypes of EB as well as from other disorders in the differential (see Box 62-1) and is considerably less labor intensive. Within each of these groups there are several distinct types of EB based on clinical, genetic, histologic, and biochemical evaluation.71 These findings are summarized below (Table 62-1).

Epidermolysis Bullosa Simplex

Epidermolysis bullosa simplex (EBS) is a disease group characterized by intraepidermal blistering and most often is associated with keratin gene mutations. The disease phenotypes range from mild to severe among different subgroups.72 The common EBS types are dominantly inherited and include Dowling–Meara (Herpetiformis) EBS, generalized (Koebner) EBS, and localized (Weber–Cockayne) EBS. There are several uncommon varieties that include EBS Ogna, EBS with muscular dystrophy, EBS with mottled pigmentation, EBS associated with BP230/BPAG1 mutation, and a group of suprabasal EBS subtypes. EBS is not usually associated with growth retardation or anemia.

Dowling–Meara EBS

This subtype presents at birth, has a generalized distribution and is regarded as the most severe of the EBS subtypes (Fig. 62-3). However, it differs from the Koebner variant in that the oral mucosa is more often involved, occasionally showing extensive erosions. Milium formation may sometimes occur in infancy in patients with this subtype; however, this postwound phenomenon usually resolves after infancy. The disease can often be associated with spontaneous appearance of grouped or “herpetiform” blisters. These occur on the trunk and proximal extremities and heal without scarring. It should be noted that this herpetiform pattern may not be seen when patients show a generalized blistering. Therefore, its absence should not be used as a basis to exclude this EB subtype. Dowling–Meara EBS often shows nail involvement, with shedding and regrowth with dystrophy or long nails with subungual hyperkeratosis. Hyperkeratosis of the palms and soles often develops beginning in early childhood and can progress to confluent keratoderma of the palms and soles. These can be quite painful and, occasionally, interference with ambulation has led to flexural contractures. Esophageal involvement in Dowling–Meara has occasionally been reported, and ranges ranging from erosions to pyloric atresia.73 The upper respiratory tract can also be affected, including the laryngeal mucosa.74 Natal teeth have been described.

Generalized EBS

The other common form of generalized EBS is also known as the Koebner EBS. This subtype shows an onset of generalized blistering at birth or at latest during early infancy. The hands, feet, and extremities usually show the most involvement. Lesions often heal with postinflammatory hyper- or hypopigmentation. Atrophy and milia can occur, but are much less frequent than in Dowling–Meara EBS. Palmoplantar hyperkeratosis and erosions may be present (Fig. 62-4). Thickening of the soles is common, but often does not present until later childhood. The oral mucosa sometimes shows mild erosive activity but these usually improve with increasing age.

Localized EBS

This is the mildest form of EB and is often referred to as the Weber–Cockayne subtype of EBS (Fig. 62-5). This disease is the most common form of EB and often presents during infancy or childhood. Occasionally, it presents in early adulthood, such as when blisters are noted following marching during military service. It is speculated that there are a number of undiagnosed cases of this form of EB, as it can be mild enough to escape reporting or detection during clinical visits. Hyperhidrosis of the palms and soles is a common association. Blisters can occasionally become secondarily infected. Postinflammatory pigmentary abnormalities occur with this variant, but milia and scarring as a rule are absent. Blistering activity usually follows areas of trauma, with hands and feet being the most common and the scalp being the least common. Mild oral erosions are present only rarely and usually resolve with increasing age. Nail involvement is rare with this EB subtype.

Additional Variants of EBS

EBS of Ogna

Onset in infancy is common with seasonal blistering (summer) on the acral areas. Small hemorrhagic and serous blisters occur primarily on the extremities. Healing occurs without scarring. This disease was originally reported in patients from Norway. These patients also show a characteristic onychogryphosis of the great toenails.

EBS with Muscular Dystrophy

This rare clinical entity is an autosomal recessive disorder which consists of generalized blistering of the skin at birth or shortly thereafter. This is accompanied by a progressive muscular dystrophy.75 It presents with generalized blistering similar to EBS of Koebner clinically. These patients have been shown to harbor mutations in the gene coding for HD1/plectin.76–78 Mutations in plectin can also cause EB with pyloric atresia (see Junctional EB with Pyloric Atresia) and be difficult to distinguish clinically from disease resulting from integrin mutations.

EBS with Mottled Pigmentation

This form of EB, as its name implies, is characterized by mottled hyperpigmentation of the trunk and proximal extremities. Blistering tends to be mild, and begins at birth or early infancy. Pigmentary alterations appear reticulated and increase with advancing age, as blistering may improve. Mild oral mucosal involvement may be present in infancy. This progressive pigmentation is distinct from the large melanocytic nevi, which can be seen in all three EB types.79

Suprabasal EBS Subtypes

EBS superficialis is an uncommon form of EBS, named after the subcorneal separation that produces the blisters in this disease.80 Erosions and crusts, rather than intact bullae, are usually seen in these patients, and heal with postinflammatory pigmentary changes. Despite the superficial cleavage plane, nail involvement. Atrophic scarring and milia have been observed in this disease.

Lethal acantholytic EBS81 is a rare recessively inherited and lethal disorder characterized by generalized erosions at birth. As in the superficialis subtype, intact blisters are not normally seen, due to the very superficial level of epidermal separation, which appears sheet-like. Nails are dystrophic to absent. Complete alopecia, neonatal teeth, oral erosions, and respiratory involvement distinguish this disorder from other superficial EBS subtypes.

Ectodermal dysplasia skin fragility syndrome is another inherited disorder of suprabasilar epidermal separation, characterized by generalized erosions and sometimes superficial blisters at birth.82 Alopecia is characteristic, as are palmoplantar keratoderma, painful fissures and nail dystrophy. Patients may sometimes demonstrate failure to thrive, cheilitis, hypohidrosis, and pruritus as well. Like the other superficial EBS subtypes, this disorder is associated with dystrophic nails.

Molecular Pathology of EBS

Most of the patients with EB simplex analyzed at the genetic level have been found to be associated with mutations of the genes coding for keratins 5 and 14.20,72 The level of separation of the skin in these patients is at the midbasal cell, shown in Fig. 62-2A, associated with variable intermediate filament clumping. Hemidesmosomes and other BMZ structures are normal by electron microscopy. The majority of keratin gene mutations associated with epidermolysis bullosa simplex are dominantly inherited due to abnormalities in the multimeric assembly of keratin filaments. There is a smaller subset of patients with recessively inherited disease of varying severity.83,84

Mutations coding for the most highly conserved regions of keratins 5 and 14, the helix boundary domains,85 correlate with the most severe form of EBS, the Dowling–Meara subtype, which exhibits intermediate filament clumping seen by transmission electron microscopy. On the other hand, milder types of disease, such as the Weber–Cockayne subtype, are associated with mutations coding for regions of keratins 5 and 14 that are less conserved. Mutations that code for a specific region of the amino-terminus of keratin 5 are present in patients having EBS with mottled pigmentation.86 Although significance of this type of mutation and its association with pigmentary abnormalities remains unclear,87,88 it has been suggested that the keratin 5 globular head domain is responsible for keratin filament insertion onto melanosomes. Recent studies have shown that some mutations of the keratin 5 gene may produce protein which is unstable under increased temperatures.89 This could help to explain the well-observed exacerbation of some subtypes of EBS to warm temperatures.

The mutations associated with EBS with muscular dystrophy produce premature termination codons, splice site, or other mutations which result in lack of expression or defective expression of plectin.90 Although the form of EB associated with plectin abnormalities is classified as simplex, it has an identical level of skin separation to that seen in junctional epidermolysis bullosa (JEB) with pyloric atresia. Specifically the separation is present just above the level of the hemidesmosome in the intracellular part of the BMZ. This separation of EBS with muscular dystrophy and JEB with pyloric atresia, diseases with identical levels of separation, into two distinct EB categories illustrates the limitations of the current EB classification system. Plectin defects, like α6β4 integrin defects, can also be associated with pyloric atresia.90

Plectin is normally expressed in a wide range of tissues, including muscle.21 While the mechanism of muscular dystrophy in plectin-deficient patients is unknown, it has been observed that disorganization of muscle sarcomeres occurs in the absence of plectin. It is possible that absence of plectin's spectrin-like domain, which may normally interact with actin filaments in muscle, may be a key factor in the muscle pathology.91 Interestingly, a case report of autosomal recessive EBS has been recently identified to be associated with both muscular dystrophy and pyloric atresia.92 This case was associated with homozygous premature termination codon mutations in the plectin gene causing a significant loss of plectin expression in the skin.92

Recently, another unique case of recessive dominant EBS has been identified, this one associated with null mutations of the dystonin gene coding for BP230/BPAG1.93 Inherited blistering/skin fragility described with this defect was associated ultrastructurally by loss of hemidesmosomal inner dense plate and a concomitant reduction of other hemidesmosomal proteins, presumably by loss of BP230 stabilization. This patient's blistering was also accompanied by a neuropathy, which may be part of the inherited defect, since a splice variant of this molecule has a neural distribution.

The underlying molecular defect of ectodermal dysplasia skin fragility syndrome has been shown to be loss of function of the desmosomal protein plakophilin 1, encoded by PKP1.94,95–100 Plakophilin is expressed mainly in suprabasilar keratinocytes and outer root sheath cells. Microscopic findings in this disease are usually intraepidermal acantholysis, located in the areas where plakophilin 1 is normally expressed.

Junctional EB

All patients with JEB share the common histopathologic feature of blister formation within the lamina lucida of the BMZ due to defects of anchoring filaments located in the lamina lucida and superior lamina densa. This group of diseases is inherited in an autosomal recessive manner and there is considerable variation of the individual clinical phenotypes depending on the molecular defect. Three principal forms of JEB are most common. Herlitz disease, JEB gravis or lethal JEB are each used to describe patients presenting with the most severe and most common phenotype.14,101

Herlitz JEB

Patients with Herlitz JEB, also known as JEB letalis, is one of the most severe EB subtypes and is lethal in most affected children during infancy or early childhood.102 This disorder is characterized by generalized and often extensive blistering at birth (Fig. 62-6). Later during infancy, a distinctive periorificial granulation tissue begins to manifest, primarily around the mouth, eyes, and nares. The scalp, periauricular areas and, less frequently, locations outside of the head and neck may also be affected. This hypertrophic granulation tissue much less commonly occurs in nonlethal subtypes. Nails are usually severely affected and often are lost during infancy. The presence of nail involvement with periungual hypertrophic granulation tissue during the neonatal period can be a clue to this diagnosis. When nails are still present, they are usually dystrophic. Tooth enamel pitting is characteristic in both primary and secondary teeth, and can progress after tooth eruption. Oropharyngeal mucosal erosions are usually present and may be widespread. Erosions of all stratified squamous epithelial tissues, including nasal, conjunctival, esophageal, tracheal, laryngeal, rectal, and urethral mucosa can be affected. Associated systemic findings in severe cases are important factors in the lethality of this disease. Involvement of the large airways, including tracheolaryngeal stenosis or obstruction, is commonly associated with Herlitz JEB disease and hoarseness in early infancy is an ominous sign. There is a characteristic failure to thrive and growth retardation in this disease, often with a mixed anemia. Sepsis is a common and often lethal complication.

Non-Herlitz JEB

(Figs. 62-7 and 62-8).

Sometimes patients initially presenting with a Herlitz-like phenotype will survive infancy and clinically improve with age. These patients eventually prove to have a severity of blistering and oral erosions less than in the lethal form. In particular, a lack of significant hoarseness is regarded as a favorable prognostic sign, indicative of less severe internal disease manifestations. Scalp and nail lesions, as well as periorificial nonhealing erosions with exuberant granulation tissue are among the most common findings in these patients during childhood. Despite the lack of lethality in infancy, these patients nonetheless can have severe epithelial adhesion abnormalities. Tracheostomies or gastrostomy tubes may help in patient survival. The nonlethal status of these patients distinguishes them from the Herlitz group and the terms nonlethal JEB or JEB mitis have been used in the past to describe these patients. They are much less common than Herlitz EB patients. There are other rare variants of nonlethal JEB that present with localized junctional blistering of the extremities or intertriginous areas.

Interestingly, the association of hypertrophic granulation tissue and laminin-332 defects has been noted in another group of patients with mutations of the laminin α3 (LAMA3) gene leading to a small truncation of the N-terminal IIIa domain of the laminin α3 chain. This disease, termed laryngo-onycho-cutaneous syndrome, is characterized by a triad of cutaneous erosions, nail dystrophy, and exuberant mucocutaneous granulation tissue, especially in the conjunctiva and larynx.103 An experimental animal model of engineered laminin-332 deletions has been recently described which recapitulates this hypergranulation tissue phenotype.104

Generalized atrophic benign EB (GABEB) is a distinct subset of non-Herlitz JEB that also presents at birth with generalized cutaneous involvement.105 Despite the widespread cutaneous blistering, there is a relative paucity of oral erosions or other mucosal disease. While enamel pitting is present, resulting in extensive dental caries, and nail dystrophy can often be severe, there is little other extracutaenous involvement noted in these patients. The blisters in these patients, which heal with a characteristic atrophic scarring, are debilitating and can be widespread, but nonetheless these patients generally have a normal lifespan. Blistering improves with age, growth is normal and anemia is uncommon. Some patients with this disease have undergone normal uncomplicated pregnancies and deliveries. One peculiar characteristic of these patients is a progressive alopecia of the scalp and terminal hairs elsewhere in the body. The hair loss starts to become severe after the onset of puberty. While patchy hair loss associated with atrophic scarring has been described, often the alopecia is quite diffuse and scarring is subtle or nonexistent.

Localized JEB

A very rare subtype of JEB is the localized variant, also known as minimus JEB. These patients generally show mild disease that can be accentuated in localized areas, most often the hands, feet and pretibial regions. Nails can sometimes be shed or become dystrophic, and enamel pitting can occur. Oral or nasal erosions can also occur; however, there is absence of any internal involvement. These patients generally have a favorable prognosis and a normal lifespan.

JEB with Pyloric Atresia

These patients exhibit extreme mucosal and cutaneous fragility and may also have various urological abnormalities, including hydronephrosis, and nephritis, and rudimentary ears. Mutations of the genes coding for the β4 and α6 integrin are associated with EB106 and in which pyloric atresia. Plectin mutations can also be associated with EB and pyloric atresia, although the the integrin mutations are more reliably associated with pyloric atresia. In these patients, hemidesmosomes are usually absent or rudimentary, and the level of separation is intraepidermal at the level of the hemidesmosome as seen in EBS with muscular dystrophy described above. The skin ultrastructure in patients with α6β4 defects is remarkably similar to the β4 integrin knockout mice described above; however, the mice do not show pyloric atresia. These studies illustrate well that there are limitations in the utility of animal models to study human epidermolysis bullosa. Most cases of this disease are quite severe and lethal in infancy, due to extensive extracutaneous epithelial sloughing, in addition to widespread blistering of the skin and mucosa. Rare nonlethal cases of this disease have been characterized which appear to result from a partial loss of function of β4 integrin.107 Interestingly, nonlethal JEB can sometimes ameliorate itself through alterations in mRNA splicing.108,109

Molecular Pathology of JEB

JEB can be associated with mutations of the genes coding for any of the α3, β3, or γ2 subunits of laminin-332.110,111 Absence of any of the three chains results in a lack of trimeric laminin-332 assembly and secretion, which results in a similar blistering phenotype. Patients with mutations of genes coding for the α3 or γ2 laminin subunits will still show normal expression of laminin-311, which contains α3, β1, and γ1 chains.44 Therefore, positive linear BMZ staining of JEB skin for the α3 chain on immunofluorescence mapping studies with absent expression of the other chains is an indication of either a α3 or γ2 chain defect. Conversely, absence of α3 staining on immunofluorescence can be indicative of a mutation of the α3 gene. About 80% of laminin-332 mutations can be traced to one of two recurrent nonsense mutations in the LAMB3 gene, making prenatal testing for laminin-332 lesions easier than other EB candidate genes.112 In the Herlitz patients, all of the mutations so far detected have been those producing premature termination codons, resulting in absence of expression of laminin-332. While Herlitz cases generally show a complete lack of expression of laminin-332, non-Herlitz patients with abnormal granulation tissue and significant mucosal involvement often show reduced expression of laminin-332. This is due to deletions or missense mutations that result in partial loss of laminin-332 function.113–115 In some cases, spontaneous amelioration of blistering in severe junctional epidermolysis bullosa cases has taken place, leading to the expression of laminin-332.109

In the GABEB variant of non-Herlitz JEB, blistering occurs in the lamina lucida region and abnormalities of hemidesmosomes/anchoring filaments are usually present (Fig. 62-3). While laminin-332 mutations underlie a subset of GABEB patients, the majority of these patients have abnormalities of the hemidesmosomal protein collagen XVII (also known as BP180 or BPAG2). A number of mutations of the gene coding for collagen XVII have been described in GABEB patients, including premature termination codon mutations, missense mutations, splice site mutations, truncations, and a glycine substitution mutation.111,116 While intralamina lucida/junctional skin separation has been shown in all patients with this disease, one patient was described with a cytoplasmic deletion of collagen XVII who showed intrabasal epidermal skin separation.117 Localized junctional EB has been shown to be associated with COL17A1 mutations.118

Of interest, mosaic GABEB patients have been identified who demonstrate well-defined areas of blistering associated with absence of collagen XVII expression as well as areas of nonblistering skin associated with normal collagen XVII expression. Careful analysis of these patients’ keratinocytes revealed reversion of one of the two alleles of the mutation, most likely due to a mitotic gene conversion involving nonreciprocal exchange of parental allele DNA.119,120 It is possible that this phenomenon of mutational rescue of genes affecting recessive cases of EB is more widespread that currently reported and more careful examination of involved and uninvolved regions of EB skin undoubtedly will reveal more cases. Understanding how epidermolysis bullosa can undergo spontaneous molecular correction (revertant mosaicism) in these cases could help in the design of future molecular therapeutic strategies.

Dystrophic EB

Dystrophic EB (DEB) is characterized by blisters that heal with scarring and milium formation. DEB can be inherited either in an autosomal recessive (RDEB) or dominant fashion (DDEB). One of the most important reasons to distinguish between these two subtypes is the increased prevalence of invasive squamous carcinoma associated primarily with the recessive form. Regardless of the mode of inheritance, DEB is derived from defects of the ultrastructural entity known as the anchoring fibril, which results in sublamina densa separation.

Localized DDEB

The localized subtype of dominant DDEB (sometimes called the Cockayne–Touraine of DDEB) can present at birth, but occasionally it is not appreciated until childhood. While generalized blistering can take place especially early in life, the blistering usually becomes localized to repetitively traumatized areas such as knees, sacrum, and acral surfaces (Fig. 62-9). These areas show a characteristic scarred, dystrophic appearance. Often the scarring is hypertrophic. Milia are common accompanying features of the healing process in these patients. Nail dystrophy or nail loss with atrophic scarring of the distal digits are common. Occasionally, nail abnormalities can be the only presenting abnormality in DDEB. Oral lesions are not common and teeth are usually unaffected. These patients have a good prognosis and a normal lifespan.

Generalized DDEB

The generalized form of DDEB (sometimes referred to as the Pasini subtype of DDEB) presents at birth with a generally more severe and widespread blistering phenotype compared to the localized subtype. Blisters in generalized DDEB heal with scarring plaques and milia, in a fashion similar to other DEB subtypes. In addition, this disease is sometimes distinguished by the spontaneous appearance of distinctive scar-like flesh colored papules on the trunk. These albopapuloid lesions are not pathognomonic, and can also be seen in other EB subtypes. As the patients get older the generalized blistering may eventually localize to the extremities. Patients often show dystrophic or absent nails. Oral erosions can often be present, but usually are not extensive, and enamel defects can be seen in some patients. A rare variant of self-remitting generalized DDEB, termed bullous dermolysis of the newborn, consists of generalized blistering that gradually recedes after infancy.121

Recessive DEB

Recessive DEB (RDEB) can be quite variable in its severity. While the severe subtype is the most common, a localized form can occasionally be seen which has been termed RDEB mitis. Similar to localized DDEB, localized RDEB is usually confined to repetitively traumatized skin surfaces, most often in an acral distribution. Scarring and milium formation accompany the healing of blisters. Mucosal involvement in localized RDEB, if present, is mild.

Severe RDEB, also known by the eponym the Hallopeau–Siemens, is a devastating disease (Fig. 62-10). This disease presents with generalized blistering at birth. Occasionally, there is extensive denudation of an entire region of skin at birth, often involving one of the limbs. This congenital absence of skin is sometimes termed Bart's syndrome, and has been described with all three major subtypes of EB. In RDEB, healing and blistering cycles occurring during infancy can lead to a progressive scarring which can become quite extensive. Pseudosyndactyly, resulting from a closure of the digits in a “mitten” of skin, is extremely common in this disease (Fig. 62-2J). Scarring can lead to flexion contracture of the hands, as well as the limbs. In contrast to the severe JEB patients, these patients do not show significant periorificial involvement. Instead, the scalp is the most commonly affected area on the head and neck of these patients. Alopecia is progressive, often without significant associated blistering.

The oropharynx can be extensively involved in both dominant and recessive DEB (Fig. 62-11) with generalized erosions evolving into a scarring which limits the movement of the tongue and narrows the opening of the oral cavity. The teeth can show significant enamel pitting, and caries can be extensive, leading to loss of teeth. Involvement of the trachea or larynx can lead to a narrowing of the airway, which can require intervention with a tracheostomy. Mucosal erosions of the esophagus can lead to stricture formation and webbing. The combination of oral lesions, dental caries, esophageal strictures, and increased caloric needs from extensive wound healing can lead these patients toward malnutrition and growth retardation. These patients usually have problems with anemia and may show a deficiency of iron absorption.

In the past, most severe RDEB patients died in infancy of sepsis and other complications of extensive blistering. With improved nutritional, infection, and wound support, these patients now usually can survive into their teens or into adulthood. However, after puberty, another devastating complication, squamous cell carcinoma, can, and often does, appear. It is estimated that 50%–80% of HS-RDEB patients eventually develop these carcinomas and many of these die of metastatic disease. RDEB-associated carcinomas are distinct from other cutaneous squamous cell carcinomas in that they are extremely aggressive with strong tendencies for invasion and metastasis.

Molecular Pathology of DEB

Abnormalities of anchoring fibrils are present in DEB patients, ranging from subtle changes in some patients with dominant disease, to absence of anchoring fibrils in patients with RDEB. A sublamina densa plane of blister cleavage is present in all dystrophic blistering (Fig. 62-3). These observations correlate with immunofluorescent microscopic analysis of DEB patients, which demonstrates varying degrees of linear basement membrane staining in dominant patients and total absence of staining in severe recessive patients. In some patients, there is cytoplasmic retention of collagen VII in patient keratinocytes.122

Dystrophic EB has been shown to be associated in all cases thus far with mutations of the gene coding for collagen VII (COL7A1). In the recessive forms, mutations usually cause premature termination codons, leading to lack of collagen VII in tissue. It is known that mRNAs bearing premature stop codons show accelerated turnover.123 In addition, truncated proteins that are not secreted or not assembled into anchoring fibrils may also show accelerated turnover. Either or both of these mechanisms can explain the lack of detectable collagen VII in the tissue of individuals with severe RDEB whose mutations lead to premature termination.62,124,125 Recently, a revertant mosaicism phenotype has also been reported in RDEB.126

Generally, COL7A1 mutations that do not cause premature termination codons produce less severe disease.110,116 In particular, mutations which produce glycine substitutions of the triple helical region interfere with triple helical assembly of the collagen VII molecule. These types of mutations are present in many patients with milder dominant forms of this disease. In these patients, collagen VII molecules may not be able to assume the proper conformation needed to polymerize into anchoring fibrils. One subtype of DEB associated with increased pruritus, EB pruriginosa, has also been associated with glycine mutations.127 Other COL7A1 mutations have been associated with impaired secretion of collagen VII, resulting in intracellular accumulation of this molecule. In one study, DEB patient mutations that involve the area of the gene coding for the collagen VII NC-2 domain were shown to interfere with NC-2 processing and the assembly of anchoring fibrils.128

Kindler Syndrome

New advances in our understanding of the molecular pathology of the skin have brought to light the underlying pathophysiology of a disease related to EB, Kindler syndrome. Kindler syndrome was first described by Theresa Kindler in 1954.129 It is characterized by EB-like trauma-induced blistering at birth and during infancy, with atrophic changes during healing reminiscent of junctional or dystrophic EB.130–136 However, in later childhood, the blistering usually subsides, and gives way to a progressive poikiloderma which distributes to sun-exposed areas. The poikiloderma may show areas of atrophy and hyperkeratosis, as well as hypopigmentation, hyperpigmentation, and telangiectasias. These patients often show photosensitivity. Nail changes and webbing of the toes and fingers are also sometimes present. Internal complications include oral inflammation, esophageal, or ureteral strictures and ectropion. Ultrastructurally, these patients show reduplication of the basement membrane, which is the most consistent feature seen. While there is often a sublamina densa split with anchoring fibril abnormalities, sometimes lamina lucida or intraepidermal separation can be seen associated with the blistering phenotype. Molecular investigation of this disease led to the discovery of a new epidermal protein, kindlin-1, which shows decreased expression by immunofluorescent microscopy in this disease. Kindlin-1 appears to have some homology to signaling proteins such as talin, which suggests a signaling function.137 A number of mutations of the gene coding for kindlin-1, KIND1, have been described.138–140 These include nonsense, frameshift, and splice site mutations which underlie the observed decrease in kindlin-1 expression in affected skin. Why the disease evolves from a blistering to a poikilodermatous phenotype and the exact function of kindlin-1 in epidermal homeostasis remain to be fully elucidated.

The first step toward making the diagnosis of EB begins with a thorough history and physical examination (Fig. 62-12). Useful historical information includes the age of onset of blistering and the presence of blistering in other family members. A review of gastrointestinal, respiratory, ocular, dental, bone, and genitourinary systems is important as is evaluation of general growth and development. Physical examination requires not only a complete skin exam, but a thorough evaluation of mucosal tissues, hair, nails, and teeth. Laboratory measurements of importance in the initial visit include evaluation for anemia, and for measures of nutrition, such as serum albumin.

Skin biopsy is another important diagnostic step. Routine histologic analysis cannot be used to diagnose EB but can be useful for excluding other causes of blistering. The dermal–epidermal BMZ is simply too small to be visualized by light microscopy. To differentiate levels of BMZ separation in skin biopsies, transmission electron microscopy (TEM), and/or indirect immunofluorescent microscopy (IDIF) must be used. While both TEM and IDIF will map the level of skin separation, each test can also provide additional unique diagnostic information, the utility of which depends on the clinical circumstances. For example, in dominant diseases such as EBS and DDEB, there is often not a detectable loss of antigens by IDIF, but ultrastructural abnormalities such as keratin filament clumping or anchoring filament abnormalities can still be seen by TEM. Conversely, in recessive disease, while TEM can detect ultrastructural abnormalities such as rudimentary hemidesmosomes, it cannot distinguish the loss of individual components of hemidesmosomes and other BMZ structures, as IDIF can.

The interior of blisters rapidly reepithelializes, which can obscure correct determination of blister levels. For this reason, it is critical to biopsy a blister that is as fresh as possible. One way to ensure a fresh blister is for the clinician to induce it in the office. This can be accomplished by gently rotating a pencil eraser over an intact area of patient's skin until separation of the epidermis from the dermis can be observed. When actually doing the biopsy, one should place the circular biopsy punch so that only 10% of the punch covers the visible blister with 90% covering intact skin. This is because it is helpful to have both intact and blistered skin on the same biopsy and extension of the blister is likely to occur either during the biopsy process or during shipping.

TEM has been used for decades for determining the level of blistering in EB subtypes. In addition to determining the level of blistering, ultrastructural entities can also be analyzed by TEM for characteristic alterations. For example, clumping of keratin intermediate filaments in basal keratinocyte cytoplasm is a pathognomonic finding for Dowling–Meara EBS. Rudimentary hemidesmosomes can be an important clue to the diagnosis of junctional EB. Absent or altered anchoring fibrils often occur in DEB subtypes especially the recessive forms.

IDDF microscopy has proved increasingly useful in recent years due to the availability of antibody panels directed against all the known EB antigens. In this technique, a panel of antibodies against known BMZ antigens is applied to frozen sections of blistered patient skin. The localization of the antigens to the epidermal or dermal portion of the blister indicates the level of skin separation in the BMZ. In EBS samples, for example, intracellular hemidesmosomal components such as BP230 and a lamina densa protein such as type IV collagen would each localize to the floor of the blister. In JEB cases, BP230 would localize to the roof of the blister while type IV collagen would localize to the floor. In DEB, collagen VII and BP230 would localize to the roof of the blister. The specific absence or presence of staining with a particular antibody in frozen sections of intact portions of patient skin gives important clues to the specific molecular defect. Samples that lack staining with antibodies specific to laminin-332 would further support a JEB diagnosis while a lack of staining for collagen VII would support a DEB diagnosis. Absence of staining for collagen XVII would support a non-Herlitz JEB/generalized atrophic benign EB diagnosis.

A complete panel of antibodies to support IDDF-based diagnosis of EB would include antibodies against laminin-332 (Herlitz and non-Herlitz JEB), as well as antibodies against BP230/BPAG1, BP180/collagen XVII (non-Herlitz JEB/generalized atrophic benign EB), collagen VII (RDEB), α6 and β4 integrins (JEB with pyloric atresia), plectin (EBS with muscular dystrophy), and keratins 5 and 14 (recessive EBS). Antibodies against the individual chains α3, γ2, and β3 chains of laminin-332 are especially helpful.

Its known that laminin-311 (which shares the same laminin α3 chain as laminin-332) is expressed in Herlitz JEB associated with null mutations of genes coding for β3 chain (LAMB3) and γ2 chain (LAMC2), but not in Herliz JEB associated with null mutations of the α3 chain gene (LAMA3).44 Therefore, if laminin β3 and γ2 antibodies are negative and α3 antibody is positive, this could point the genetic analysis to examine the LAMB3 and LAMC2. Conversely, if all three laminin-332 chains are absent by IDIF, this could point the genetic investigation toward LAMA3, saving time and effort in arriving at the final molecular diagnosis.

Gene mutation analysis has revolutionized our understanding of the EB family of diseases and is considered the ultimate final step in arriving at the molecular diagnosis in EB. Concurrent advances in our knowledge of the biochemical structure and supramolecular assembly of BMZ proteins have both facilitated and complemented the molecular biology studies. Thus, EB patient diagnosis requires both clinical and molecular information. Blood samples or buccal swabs are taken from the patient as well as the parents and siblings for genetic analysis.

Genetic Counseling

DNA mutation analyses can be extremely helpful to EB patients. The prognostic benefit to the patient can often be highly significant. For example, milder recessive DEB cases may have equivalent blistering activity compared to the more severe dominant cases, but their risk of invasive SCC is much greater. Through DNA diagnosis, these two groups can be distinguished, thereby identifying patients potentially at risk for invasive SCC.

Prenatal diagnosis of EB in affected families can be an extremely accurate technique, especially if the original proband has previously had mutational analysis or identification of the defective gene. Fetal skin biopsies and fetoscopy with their increased risk of pregnancy loss can now be avoided by analysis of either chorionic villus sampling as early as 8–10 weeks141 or amniocentesis in the second trimester.142 The development of highly informative intragenic and flanking polymorphic DNA markers in EB candidate genes together with rapid screening of genetic “hotspots” makes genetic screening of at risk pregnancies a viable option.112,143 Coupling of the technique of in vitro fertilization with EB prenatal diagnosis, preimplantation diagnosis has now been successfully performed for EB cases.144 Another promising area of prenatal diagnosis with potential future applications to EB is the detection and analysis of fetal cells in the maternal circulation.145

Most therapy for epidermolysis bullosa is supportive. The regimen is tailored to the severity and extent of skin and systemic involvement and usually entails a combination of wound management, infection control, surgical management as needed, and nutritional support. Skin care and supportive care for other organ systems in certain EB subtypes is most optimally coordinated through a multidisciplinary approach. Comprehensive topical therapy is a mainstay of treatment in EB, with avoidance of trauma as a primary goal. Wound healing is impaired by endogenous factors, including foreign bodies, bacteria, nutritional deficiencies, anemia, and repeated trauma. Therefore, optimizing wound healing in EB patients involves control of all of these factors.146

Supportive Skin Care

Extensive areas of denuded skin can result in the loss of the barrier provided by the stratum corneum. Subsequent microbial penetration can result in the accumulation of serum and moisture that further enhances bacterial propagation. The above factors combined with immunosuppressive therapy facilitate development of infections. Prevention of infection is obviously the preferred strategy.

A modified Dakin's solution (0.025% w/v sodium hypochlorite) can be helpful in reducing the bacterial load in patient skin. Soaking wounds in this solution for 20 minutes prior to dressing changes also helps to free adherent bandages that have dried onto the wound bed. After soaking, wounds can be dressed with mupirocin or other topical antibiotics, and covered with semiocclusive nonadhesive dressings. Tape causes further blistering and peeling of the skin; thus, it is essential to use self-adhering (clinging) gauze or self-adherent tape to hold nonadherent dressings in place.

For patients with generalized or localized subtypes of EBS, controlling exposure to heat may prove helpful in controlling blister formation. Advising patients to use soft, well-ventilated shoes is also recommended. Herlitz JEB patients, lacking functional laminin-332, an extracellular matrix protein shown to be involved in keratinocyte adhesion and migration, may have especially difficult problems with wound healing. For DEB patients, use of finger splinting or diligent hand wrapping and appropriate hand protection against trauma are helpful, especially following hand surgery (see below).

Infection

Management of skin infections is a critical part of EB patient care. Large areas of denuded skin provide an inadequate barrier to microbial penetration. Patients with severe EB subtypes such as recessive DEB may also have immunologic abnormalities including decreased lymphocyte production due to poor nutritional status that lower patients’ resistance to infection. Staphylococcus aureus and Streptococcus pyogenes are common infectious agents. Gram-negative infections with Pseudomonas aeruginosa can also occur. Sepsis is a common cause of death in Herlitz JEB patients. Skin cultures and the use of the appropriate systemic antibiotics are indicated for wound infection. To prevent infections in chronic wounds, a regimen entailing regular whirlpool therapy followed by topical antibiotics is the preferred strategy. Rotation of topical antibiotics is also a helpful way to combat resistant bacteria.

Surgical Treatment

Among the EB patient population, those with the severe recessive DEB (Hallopeau–Siemens) variant are generally the most in need of surgical intervention.147 Mitten pseudosyndactyly in these patients can be surgically released; however, this procedure may have to be repeated periodically due to the strong tendency of this condition to recur.148–151 Splinting following surgery is essential to reducing recurrence of hand deformities. Surgery may also be used to correct limb, perioral, and perineal contracture deformities, but a high rate of recurrence is common. Extra care must be taken to minimize trauma to oral mucosa in EB patients during intubation.

Application of allogeneic skin equivalents has recently demonstrated some promise in wound healing and improvement of quality of life152 and thus represents an area of future investigation. While long-term benefit or persistence of allografted cells has not been demonstrated, this therapy nonetheless appears to a helpful short-term therapy to help chronic EB wounds to heal.

Tumors

Squamous cell carcinoma often arises after puberty in patients with recessive DEB. SCC may arise in multiple primary sites, especially in nonhealing areas. Careful surveillance of nonhealing areas is of utmost importance since patients often die from metastatic disease, and the average survival rate after diagnosis of an RDEB-associated squamous cell carcinoma is 5 years. Surgical excision using either Mohs or non-Mohs approaches is an important first-line modality with radiation therapy limited by poor tumor response and impaired site healing.153 Amputation is often used to control local spread of carcinomas detected on the limbs. Isotretinoin has been used for RDEB patients for chemoprevention of SCC. While it appears well tolerated, it is not clear whether in can increase the overall survival rate of these patients.154 The epidermal growth factor receptor antagonist cetuximab has also been reported used in RDEB associated with advanced metastatic disease.155 While the short-term results were positive in the one patient tested, published reports of long-term results and experience with wider groups of RDEB cancer patients are currently lacking.

Care for Extracutaneous Involvement

GI Management

Esophageal lesions are often the most disabling complication found in recessive DEB and JEB of both the Herlitz and non-Herlitz varieties. Esophageal strictures usually respond to dilatation; however, recurrence of strictures after dilation is common.156 Colonic interposition has proven effective in advanced cases. Gastrostomy tube insertion has been effective in providing nutrition to individuals with esophageal strictures.157 Increased fluid and fiber intake and stool softeners may also be of value in EB patients who suffer from constipation.

Eye Lesions

EBS patients, particularly those with the Dowling–Meara subtype can experience recurrent inflammation of the eyelid, with bullous lesions in the conjunctivae. Junctional EB patients and recessive DEB patients can experience corneal ulcerations with scarring, obliteration of tear ducts, and eyelid lesions.158 Cicatricial conjunctivitis can also occur in recessive DEB patients. Corneal erosions are treated supportively with application of antibiotic ointments and use of cycloplegic agents to reduce ciliary spasms and provide comfort. Moisture chambers and ocular lubricants are also commonly used. Severely affected upper eyelids may be surgically managed with full-thickness skin grafting. Complete correction of any eye disorder in EB patients is difficult to achieve. Proper management of eye lesions in EB patients must include the assistance of an ophthalmologist to prevent serious visual compromise.159

Oropharyngeal Lesions

Good dental hygiene is essential for EB patients, and regular visits to the dentist are especially important. Enamel defects in JEB and DEB patients often lead to dental carries.160,161 The softest brush available should be used for regular cleansing. Oral mucosal blistering may also accompany forms of JEB and DEB. Normal saline rinses are effective for gentle cleaning of the mucosal surfaces. Mouthwashes containing alcohol or other harsh agents should be avoided. Erosions and scarring involving the trachea and larynx with resultant narrowing of the airway. In patients with airway involvement, there is danger of pulmonary aspiration.162

Nutrition and Anemia Complications

Nutritional assessment and support can be critical in EB patients163 for several reasons. Extensive cutaneous injury is associated with marked alterations in hemodynamic and metabolic responses, with increased caloric and protein requirements. Nutritional problems such as low selenium, iron overload, and chronic anemia in the setting of severe blistering may predispose the patient to severe internal complications. Congestive heart failure and cardiomyopathy are prime example of this. This uncommon but nonetheless severe and potentially fatal complication can occur both patients with RDEB or non-Herlitz JEB, becoming more frequent as patients get older.164

Oropharyngeal as well as gastrointestinal lesions provide the greatest overall threat to nutritional well-being. These include oral blistering, abnormal esophageal motility, strictures, dysphagia, diarrhea, malabsorption, and dental problems. Nutritional assessment must take into account the above factors in order to develop a supplemental regimen to replenish nutritional deficiencies. Patients are often unable to increase their food intake to balance this increased caloric need. For example, hypoplastic enamel formation in certain subtypes of EB such as GABEB may lead to tooth decay, mucosal blistering, and oral candidiasis. All of these potential complications may compromise patients’ ability to eat. Extensive internal mucosal disadhesion in the gastrointestinal tract may cause abnormal GI motility, strictures, and diarrhea—complications that may lead to malabsorption of iron and other nutrients. Anemia of chronic disease can certainly affect all severe EB subtypes. Recessive DEB patients often show a particularly severe defect of iron absorption from the GI tract. In these patients, an iron deficient anemia can develop, which is not responsive to oral supplementation. For these patients, parenteral iron can be helpful. Furthermore, if a lack of a reticulocyte response to iron supplementation is seen in iron deficient patients, it is helpful to assess the erythropoietin level and to treat with recombinant erythropoietin if necessary.165 Transfusion is also useful in the treatment of anemia in EB, especially when symptoms require a rapid correction. Deficiencies of other trace minerals and other vitamins such as vitamin D, zinc have also been noted in severe RDEB patients.166 Osteoporosis and fractures are also a frequent complication of RDEB patients, and bone scans, vitamin D and calcium supplementation may be helpful. Bisphosphonates can be useful in combating this problem.167

Psychological Aspects of Epidermolysis Bullosa

Patients with EB, especially the severe subtypes, can be plagued with chronic pain and debilitation.168–170 While many, despite extremely adverse conditions, seem to find a way to maintain a surprisingly positive outlook on life, others lapse into depression.171,172 Severe epidermolysis bullosa patients can also create stress for their families and loved ones.173 Thus, it is important to identify the warning signs of depression when they arise and to work in a mutidisciplinary approach with psychiatrists and clinical psychologists as needed. Supportive psychotherapy and patient support group meetings can help patients and their families in this regard. An additional source of support for patients and families include several important patient-based organizations that assist with education and support including Dystrophic Epidermolysis Bullosa Research Association and Epidermolysis Bullosa Medical Research Foundation.

Systemic Therapies

Systemic therapies are not effective in ameliorating the fundamental blistering tendencies in EB patients. Tetracycline and phenytoin have been used in the past for EB but are not currently indicated treatment.174 Corticosteroids, either topical or systemic, have no beneficial long-term use in inherited EB.

Molecular Therapy for Epidermolysis Bullosa

A number of preclinical models have facilitated the study and development of therapy for epidermolysis bullosa. Some of these models utilize mouse genetic approaches to produce entirely murine tissue models.175 These include transgenic mouse models with skin targeted expression of trans-dominant molecules including inducible disease phenotype models.176 These include dominant-negative basal keratins in models of epidermolysis bullosa simplex. Murine knockout models have also been used, including knockout of basal keratin genes in a model of the same disorder and of the α6 and β4 integrins, laminin-332, and types XVII and VII collagens.177–181 These targeted gene disruptions in mice have accurately recapitulated the blistering phenotype of the corresponding human disorders. These murine genetic models have been of great utility in clarifying our understanding of the pathogenesis of this group of disorders and in providing disease models for development of potential future therapies. Other animal models have also been shown to have the potential to study epidermolysis bullosa pathophysiology and therapy in a preclinical setting. Canine, murine, equine, and feline forms of a number of subtypes of epidermolysis bullosa show promise as preclinical models of molecular therapy.

In addition to such pure animal models, human skin/mouse chimeras have been generated in an attempt to produce true human tissue models of epidermolysis bullosa. These chimeras have either used full thickness grafting of EB patient skin or epidermis regenerated on skin composites and sheets from EB keratinocytes grown in culture.182 Immune deficient SCID and nude mice readily accept such xenografts and the grafted EB skin retains the clinical and molecular characteristics of the patient donor. This immunodeficient mouse models can be extended to study injections of human cells or proteins as well. This approach has been reported for recessive dystrophic EB due to mutations in collagen VII and for benign junctional EB due to mutations in collagen XVII. Because human epidermis differs dramatically from mouse in tissue architecture, these in vivo models of human tissue in EB may be of special utility in development of models for gene therapy of these disorders.

Potential therapies for EB include protein and gene therapy. In the former case, the missing or defective protein is produced by recombinant methods and applied or injected directly to intact or blistered skin or given intravenously. Recent preclinical studies of collagen VII protein transfer into RDEB-derived skin equivalents have shown successful incorporation of this molecule into anchoring fibrils combined with clinical improvement of skin separation.183 Collagen VII has a longer in vivo half-life than most molecules. This suggests that protein application or injection could take place at extended intervals, perhaps greater than 3 months, as persistence of exogenous collagen VII protein was detected in animal models at least this long.

In the case of gene therapy, delivery of genes targeted to restore normal protein expression is the goal.184,185 Successful corrective gene delivery has recently been achieved in human EB tissue in the immune deficient model system described below for both laminin-332 and collagen XVII.186,187 More recently both viral and nonviral methods of gene transfer have demonstrated successful expression of collagen VII in RDEB skin equivalents on immunodeficient mice183,188–190 as well as in a canine animal model.191

The approach of retroviral ex vivo gene therapy using autologous keratinocytes was recently performed for one patient with junctional epidermolysis bullosa.192 In this study, a patient with a missense mutation of laminin-332 was grafted with genetically corrected keratinocyte monolayers expressing wild type laminin-332. After over 1 year of post grafting follow up, grafts still showed positive expression of laminin-332 and resistance to blistering. Observations from this study suggested that proper selection of patient skin stem cells were an important part of the success of this study. This latter study was the first to demonstrate that gene therapy for epidermolysis bullosa can be successfully performed in humans, and opens the door for many new clinical studies in other EB subtypes.

Dermal fibroblasts have long been appreciated as contributors of collagen VII to the dermal–epidermal basement membrane.193 The technique of injecting collagen VII expressing fibroblasts have proven successful in restoring collagen VII expression and reversing the blistering phenotype in RDEB skin equivalents on immunodeficient mice189,194 and in a mouse model of RDEB.195 In addition, allogenic fibroblast-based approaches to the treatment of dystrophic EB have recently found their way into clinical trials, showing some success at delivering collagen VII to deficient RDEB patient skin.196,197 Although profound immunosupression against a background of extensive skin erosions is not without significant risks, including sepsis and death, new advances in bone marrow transplantation of collagen VII expressing stem cells represents another novel and potentially effective route of cell-based molecular therapy for RDEB.198–201