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Keywords:

  • basement membrane;
  • blister;
  • bone marrow transplantation;
  • diagnosis;
  • epidermolysis bullosa

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References

Epidermolysis bullosa (EB) is classified into major types – EB simplex (EBS), junctional EB (JEB) and dystrophic EB (DEB) – on the basis of the level of tissue separation within the cutaneous basement membrane zone. Recent advances in research on EB have led to the identification of 10 genes responsible for EB. The Japanese Ministry of Health, Labor and Welfare has designated JEB and DEB, but not EBS, as specified diseases. However, EBS has a lethal variant and should also be registered as a specified disease. In the Third Consensus Meeting on the Diagnosis and Classification of EB held in Vienna in 2007, it was recommended that Kindler syndrome should be classified as a subtype of EB. Corrective gene therapy is the most ideal therapy for EB, but much more research is required before it can be developed and used in clinical practice. Cell-based therapies using fibroblasts and bone marrow cells have recently attracted considerable attention.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References

Epidermolysis bullosa (EB) constitutes a group of phenotypically diverse genodermatoses, which manifest with blistering and erosion of the skin and mucous membranes (Fig. 1).1 Recent advances in research on EB have led to the identification of mutations in 10 different genes, which account for the clinical heterogeneity in EB.2,3 EB is classified into three main types on the basis of the level of tissue separation within the cutaneous basement membrane zone (BMZ); tissue separation occurs in the basal keratinocytes, dermis and lamina lucida of the basement membrane in EB simplex (EBS), dystrophic EB (DEB) and junctional EB (JEB), respectively (Fig. 2, Table 1).

image

Figure 1.  Epidermolysis bullosa constitutes a group of phenotypically diverse genodermatoses, which manifest with blistering and erosion of the skin and mucous membranes.

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image

Figure 2.  Recent advances in research on epidermolysis bullosa have led to the identification of mutations in 10 different genes, which encode basement membrane proteins and account for the clinical heterogeneity in epidermolysis bullosa.

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Table 1.   Major categories of epidermolysis bullosa classification and their related underlying defects
Major typeMajor subtypeProtein/gene
Simplex (EBS)Dowling–Meara EBS Koebner EBS Weber–Cockayne EBSKeratin 5, 14
 EBS with muscular dystrophy EBS with pyloric atresiaPlectin
Junctional (JEB)Herlitz JEB Non-Herlitz JEB JEB with pyloric atresiaLaminin 332 BP180, laminin 332 α6β4 integrins
Dystrophic (DEB)Dominant DEB Hallopeau–Siemens Recessive DEB Non-Hallopeau–Siemens Recessive DEBType VII collagen

Eb simplex

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References

In all forms of EBS, blister formation first occurs within the basal keratinocytes. Dominantly inherited EBS is caused by mutations in the basal keratinocyte-specific keratin 5 and keratin 14 genes and is divided into three subtypes: the Weber–Cockayne, Koebner and Dowling–Meara (Table 1). The most severe subtype is Dowling–Meara EBS; Weber–Cockayne EBS has a milder phenotype than that of Koebner EBS. The position of the mutation in the keratin 5 and keratin 14 genes is closely related to the resulting phenotypes. Dowling–Meara EBS is usually caused by mutations in the initiation or termination peptides of the rod domains, whereas the mutations in other parts of the rod domain or in the linker region result in Weber–Cockayne EBS.4,5

Recessively inherited EBS is caused by mutations in the plectin gene. The majority of these cases are characterized by generalized blistering and muscular dystrophy; muscle weakness is first observed during the latter part of the first decade of life. Recent studies have revealed the existence of a lethal subtype of EBS that is caused by a mutation in the plectin gene; this subtype is characterized by cutaneous blisters and pyloric atresia (Table 1).6,7

Junctional EB

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References

All JEB subtypes are inherited in an autosomal recessive manner and are characterized by blister formation in the lamina lucida. JEB is divided into three subtypes: the Herlitz subtype, the non-Herlitz subtype and the subtype with pyloric atresia (Table 1). The Herlitz subtype is a severe phenotype, and many of the infants affected by it have intractable erosion and blisters and die early in infancy owing to overwhelming infection. The clinical phenotype of the non-Herlitz subtype is much milder than that of the Herlitz subtype, and many patients affected by the non-Herlitz subtype survive to adulthood. Herlitz JEB is caused by mutations in any of the three genes encoding laminin 332: these mutations are homozygous or compound heterozygous premature termination codon (PTC) mutations (Fig. 2, Table 1).8,9 Missense mutations in one or both alleles of the genes encoding laminin 332 are associated with non-Herlitz JEB.10 Non-Herlitz JEB is also caused by mutations in the BPAG2 gene, which encodes the BP180 polypeptide.11,12 There is a strong similarity in the phenotypes of the non-Herlitz subtype associated with mutations in the laminin 332 gene and that associated with mutations in the BP180 gene.

Mutations in the genes encoding the β4 integrin and α6 integrin chains can cause JEB with pyloric atresia (Table 1).13,14 Clinically, both lethal and non-lethal variants of this condition have been reported. The lethal variants are associated with severe clinical features and high mortality in early infancy, similar to the Herlitz subtype of JEB. In the non-lethal variants, the clinical severity of skin involvement often tends to decrease with age. Mutation database analysis suggests that the occurrence of PTC predominantly results in the lethal variants of JEB, whereas missense mutations are frequently associated with the non-lethal variants.15

Dystrophic EB

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References

Dystrophic EB, in which tissue separation occurs in the dermis, manifests as blistering, scarring and milia formation. It has both autosomal dominant and autosomal recessive forms; however, all DEB subtypes are caused by mutations in COL7A1, which encodes type VII collagen, a major component of anchoring fibrils (Fig. 2, Table 1). In general, the phenotype of dominant DEB is milder than that of recessive DEB. Over 300 mutations have been reported, and DEB is strongly associated with the glycine substitution mutation in one allele of the gene encoding the collagenous domain of type VII collagen.16 Glycine substitutions probably have a dominant negative effect on the formation and assembly of type VII collagen. PTC are present on both alleles of the COL7A1 gene in the case of the most severe form of DEB: Hallopeau–Siemens recessive DEB.17 Therefore, no or low expression of type VII collagen is observed in the basement membrane in these patients (Fig. 3). In the milder form of recessive DEB, non-Hallopeau–Siemens recessive DEB, PTC mutations are present in one allele and a missense mutation or in-frame mutations are present in the other allele.18 Some level of type VII collagen expression is seen in patients with this form of DEB.

image

Figure 3.  Hallopeau–Siemens recessive dystrophic epidermolysis bullosa show severe clinical phenotype and no or low expression of type VII collagen is observed in the basement membrane in the patients.

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Diagnosis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References

Epidermolysis bullosa is a monogenic disease, and the final diagnosis mainly depends on DNA-based mutation analysis. However, it is not possible to sequence all of the 10 genes involved in the etiology of this disease in every patient. Thus, the initial diagnosis is predominantly based on careful examination of the clinical manifestations and elicitation of the patients’ personal and family medical history. For example, in DEB, hearing lesions result in scarring and milia formation because blisters occur in the dermis. Some forms of EB are associated with extracutaneous features, including pyloric atresia and muscular dystrophy. Autosomal dominant inheritance indicates EBS caused by mutations in the genes encoding keratin 5 and keratin 14 or DEB caused by mutations in the gene encoding type VII collagen.

Following the initial diagnosis, a skin biopsy should be taken to determine the level of tissue separation and to classify the disease into one of the three major categories: EBS, JEB and DEB. For accurate diagnosis, the skin biopsy should be taken from a new blister. It is also preferable to perform a skin biopsy just after gently rubbing non-blistered skin. Both electron microscopic and direct immunofluorescence microscopic studies are effective methods for determining the level of tissue separation, but electron microscopic studies require expensive equipment and have to be carried out by experienced people with excellent technical skills. Therefore, direct immunofluorescence microscopy using specific antibodies against basement membrane proteins is a much easier option for inexperienced dermatologists to diagnose blistering diseases. With this technique, the level of tissue separation can be determined by observing the position of the specific antigen on the roof or the floor of the split skin. For example, in biopsy specimens obtained from patients with EBS or JEB, type IV collagen is found to be located in the lamina densa, and antibody staining is seen in the floor. In contrast, in specimens obtained from patients with DEB, antibody staining is observed in the roof. In addition, absent or reduced expression of specific antigens may indicate the presence of the corresponding EB subtype. No or low expression of BP180 and type VII collagen indicates non-Herlitz JEB and Hallopeau–Siemens recessive DEB, respectively. Currently, many antibodies to basement membrane proteins are commercially available and can also be obtained from other researchers.

For DNA-based diagnosis, we selected the target genes on the basis of the above-mentioned observations. Because this technique is accurate and is also useful for prenatal diagnosis and genetic counseling, it is becoming increasingly important in clinical practice. Molecular diagnosis of EB is difficult because the genes involved in its etiology are relatively long and the number of hot spot mutations is low.

Problems associated with designating EB as a rare intractable disease

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References

The Japanese Ministry of Health, Labor and Welfare has designated some intractable diseases as specified diseases. These intractable diseases are chronic and may have serious consequences that make it very difficult or even impossible for patients to return to school or work. Treatment of these diseases is very expensive because of the high cost of long-term care and medicine, which causes great financial and mental stress. Therefore, the treatment of 45 such diseases has been subsidized for the public.

Junctional EB and DEB, not EBS, have been designated as specified diseases. This is because the clinical manifestations of EBS are much milder than those of DEB and JEB. Mutations in the plectin gene are generally thought to cause EBS associated with muscular dystrophy. Muscle weakness is first observed during the latter part of the first decade of life. However, we and other authors have recently shown that lethal EBS cases with pyloric atresia also result from mutations in the plectin gene (PLEC1).6,7 These patients manifested with cutaneous blisters, aplasia cutis congenita (severe localized absence of skin) and pyloric atresia; these symptoms often caused the patient’s demise soon after birth. Therefore, EBS, including the lethal variants, should be designated as a specified disease.

Kindler syndrome

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References

Kindler syndrome is inherited in an autosomal recessive manner and is characterized by trauma-induced blistering, poikiloderma, skin atrophy, mucosal inflammation and varying degrees of photosensitivity. The pathogenesis of Kindler syndrome involves loss-of-function mutations in a newly recognized actin cytoskeleton-associated protein, now known as fermitin family homolog 1 and encoded by the gene FERMT1.19 This protein mediates anchorage between the actin cytoskeleton and the extracellular matrix through focal adhesion. EB is caused by abnormalities in hemidesmosome attachment, whereas Kindler syndrome is caused by destruction of focal adhesion; thus, these diseases show distinct clinicopathological and molecular abnormalities. According to some researchers, Kindler syndrome should be classified as a subtype of EB. In the Third Consensus Meeting on the Diagnosis and Classification of EB held in Vienna in 2007, it was recommended that Kindler syndrome should be classified as a subtype of EB.

Challenges for developing new treatment modalities

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References

No specific treatment is available for EB, and symptomatic therapy is the mainstay of clinical management for EB. Because EB is a monogenic disease, corrective gene therapy is the ideal option; however, much more research is required before such a therapy can be used in clinical practice. Cell-based therapies using fibroblasts and bone marrow cells have lately attracted considerable attention.

Allogeneic bone marrow transplantation has been used in the treatment of leukemias, lymphomas and immune deficiency disorders. The defective basement membrane proteins seen in EB are produced by epidermal keratinocytes. Recently, it has been shown that bone marrow stem cells are capable of differentiation into epidermal keratinocytes. Bone marrow transplantation can ameliorate the symptoms of knockout mice lacking type VII collagen and showing DEB.20,21 Furthermore, clinical trials for the treatment of recessive DEB by using cord blood and bone marrow transplantation have already started, and these treatments have been shown to have good results.

Type VII collagen has been reported to be mainly synthesized and secreted by keratinocytes and to a lesser extent by fibroblasts. In fact, animal experiments have shown that the introduction of gene-transferred DEB fibroblasts into the skin restores collagen VII expression in the dermal–epidermal junction.22 Fibroblasts are much more robust and easier to culture than keratinocytes; therefore, fibroblasts may be better targets for cell-based or ex vivo gene therapy. A single i.d. injection of allogeneic fibroblasts was given to five subjects with recessive DEB. Increased type VII collagen expression was seen in these patients, without any adverse clinical or immunopathological effects.23 Thus, it was shown that i.d. injections of allogeneic fibroblasts have therapeutic potential in human subjects with recessive DEB.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Eb simplex
  5. Junctional EB
  6. Dystrophic EB
  7. Diagnosis
  8. Problems associated with designating EB as a rare intractable disease
  9. Kindler syndrome
  10. Challenges for developing new treatment modalities
  11. References