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Molecular Genetics of Heritable Blistering Disorders
Jouni Uitto, MD, PhD;
Leena Pulkkinen, PhD
Arch Dermatol. 2001;137:1458-1461.
INTRODUCTION
Over the past decade, there has been tremendous progress in understanding the genetic basis of different forms of genodermatoses. Specifically, with the advent of technologies in molecular biology in general, an increasingly large number of gene defects have been identified in different genodermatoses, and mutations are now known to occur in more than 100 distinct genes in such a manner that the genetic lesions explain the spectrum of phenotypic manifestations encountered in these diseases.1
THE PARADIGM OF EPIDERMOLYSIS BULLOSA
An example of genodermatoses in which spectacular success has been recently made is epidermolysis bullosa (EB), a heterogeneous group of mechanobullous disorders characterized primarily by blistering and erosions of the skin.2 In addition to the unifying diagnostic feature of skin fragility, a variety of extracutaneous manifestations can be encountered in different variants of EB; these include corneal erosions, enamel hypoplasia, scarring alopecia, erosions in the tracheal epithelium, development of esophagus strictures, congenital pyloric atresia, and late-onset muscular dystrophy, among others. Adding to the complexity of this disorder is the fact that the inheritance can be either autosomal dominant or autosomal recessive.2
The Complexity of the Cutaneous BMZ
Epidermolysis bullosa is a disease of the cutaneous basement membrane zone (BMZ).3 Electron microscopic examination of normal cutaneous BMZ reveals the presence of several critical attachment complexes that are necessary for stable association of epidermis to the underlying basement membrane and for the association of the basement membrane to the underlying dermis4 (Figure 1). Specifically, hemidesmosomes—multiprotein complexes extending from the intracellular milieu of basal keratinocytes to the extracellular compartment—consist of at least 5 gene products: the 180- and 230-kd bullous pemphigoid antigens,  6 4 integrin, and plectin.5 In the extracellular space, hemidesmosomes associate with anchoring filaments, threadlike structures traversing the lamina lucida. A critical protein contributing to the BMZ stability at the lamina lucida–lamina densa interface is laminin 5, which consists of 3 subunit polypeptides—the 3, 3, and  2 chains—encoded by LAMA3, LAMB3, and LAMC2, respectively. On the dermal side, anchoring fibrils, which consist of type VII collagen, extend from the lower portion of the lamina densa to anchoring plaques, basement membrane–containing structures within the papillary dermis (Figure 1). On the basis of detailed ultrastructural and biochemical analyses of the molecular components of the dermoepidermal attachment complexes, a concept of an intricate network extending from the intracellular milieu of basal keratinocytes to the upper papillary dermis has been proposed.3 A perturbation in this network structure, such as the absence of a critical component or altered conformation with an impact on protein-protein interactions, due to genetic lesions, could result in fragility of the cutaneous BMZ and manifest clinically as a form of EB.6
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Schematic representation of the cutaneous basement membrane zone. The illustration depicts the presence of basal keratinocytes overlying the papillary dermis, with the dermoepidermal basement membrane separating these 2 compartments. Ultrastructurally recognizable attachment complexes within this adhesion zone are indicated on the left, and individual protein components composing these attachment structures are shown on the right. The level of tissue separation in the simplex, hemidesmosomal, junctional, and dystrophic forms of epidermolysis bullosa is indicated on the right (adapted from Uitto and Christiano4).
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Molecular Genetics of EB
Traditionally, based on the level of tissue separation within the cutaneous BMZ, EB has been divided into 3 broad categories: (1) the simplex forms, in which tissue separation occurs within the basal keratinocytes on the epidermal side of the basement membrane; (2) the junctional forms, in which blister formation takes place within the lamina lucida at the dermoepidermal basement membrane itself; and (3) the dystrophic forms, in which tissue separation occurs at the dermal side of the cutaneous BMZ, within the upper papillary dermis.2 More recently, we have introduced a fourth broad category—the hemidesmosomal variants of EB—in which tissue separation occurs at the basal cell–lamina lucida interface at the level of hemidesmosomes.7
Cloning and molecular characterization of the candidate genes within the cutaneous BMZ, together with sequence information emanating from the human genome project, have allowed us and others to develop sensitive and specific mutation detection strategies that permit streamlined identification of sequence variants in the candidate genes in families with different forms of EB.8 As a result, it is now well established that as many as 10 different genes of the cutaneous BMZ harbor mutations underlying different forms of EB (Table 1). In fact, mutations in these 10 genes explain the clinical manifestations in all major forms of EB, although additional candidate genes have been recently identified in rare variants of EB as well as in other skin fragility syndromes.9
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Clinical and Molecular Heterogeneity of Epidermolysis Bullosa (EB) and Consequences of the Mutations in the Gene/Protein Systems
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Genotype/Phenotype Correlations
Examination of the existing mutation database has allowed us to begin to develop understanding of the genotype/phenotype correlations, although additional information is clearly required to draw definitive conclusions on the consequences of the mutations at the level of messenger RNA stability, protein folding, and supramolecular assembly of the BMZ. Nevertheless, demonstration of these mutations in EB has increased our understanding of the complexity of the cutaneous BMZ and has provided novel information about the role of individual components in stabilizing the dermoepidermal junction. Collectively, the types and combinations of mutations in the 10 different genes are able to predict, in general terms, the clinical severity, phenotypic constellation, and natural history of the disease.8 It should be noted, however, that the clinical severity of EB represents a continuum in the spectrum of clinical manifestations, and the precise nature of the genetic lesions, their positions along the affected gene, and the dynamic interplay of the mutant alleles on the individual's genetic background will all determine the precise phenotype encountered in a given patient.
CLINICAL IMPLICATIONS OF BASIC RESEARCH ON HERITABLE BLISTERING DISORDERS
The total number of variant alleles containing pathogenetic mutations disclosed thus far in various forms of EB is well in excess of 500. This progress, while it has clearly enhanced our understanding of the complexity of the cutaneous BMZ, raises the critical question, "What are the benefits of this progress in basic research on EB to the patients and their families?" In other words, how can we translate this basic information to improved patient care? Is there anything that the practicing dermatologist can convey to the benefit of patients with EB as a result of improved knowledge of their disease and its molecular basis? The answer is very clear: Significant benefits are already emanating from the basic research on heritable blistering skin diseases, and expansion of the research database will provide additional insights into the clinical perspective of these conditions. The clinical implications of molecular diagnostics for patients with heritable blistering diseases are as follows:
- Improved diagnosis and refined classification with prognostic implications
- Profound consequences for genetic counseling concerning the mode of inheritance and recurrence risk
- DNA-based prenatal testing of families at risk for recurrence
- Development of preimplantation diagnosis by blastomere analysis
- Basis for future development of gene therapy and other novel treatment modalities
Refined Classification With Prognostic Implications
Immediate benefits have already materialized through improved, molecularly based diagnosis with refined classification that allows better prognostication regarding the severity and prediction of the progress of the disease. An example is provided by the junctional variants of EB (JEB), which manifest in 2 clinical forms: (1) the Herlitz variant of JEB, which is usually lethal during the first few months of postnatal period; and (2) the non-Herlitz variants of JEB, which demonstrate persistent blistering tendency but do not significantly compromise the life span of the affected individuals.2 Molecular analysis of the DNA of patients with JEB has revealed that those with the Herlitz variant of JEB harbor, as a general rule, a premature termination codon mutation, ie, a gene defect that predicts synthesis of a truncated and nonfunctional polypeptide, in both alleles of any of the 3 genes encoding laminin 5 subunits (LAMA3, LAMB3, and LAMC2).10 In contrast, patients with the non-Herlitz variants frequently harbor a missense mutation, ie, an amino acid substitution–causing mutation, in one or both alleles of the corresponding genes. Thus, analyzing DNA from a newborn with clinical, histopathological, and ultrastructural evidence suggesting the diagnosis of JEB allows general predictions as to whether the disease is severe—the Herlitz (lethal) variant, resulting in early death—or whether the individual is expected to have a milder, non-Herlitz type of JEB, with a normal life span.
Improved Genetic Counseling
The identification of specific mutations in EB also has profound implications for genetic counseling of families at risk for recurrence of the disease in the same and subsequent generations. An example is provided by a "sporadic" patient with a relatively mild dystrophic form of EB (DEB), no family history, and clinically normal parents. The clinical manifestations in the affected individual could either result from a de novo dominant mutation in one of the COL7A1 alleles or manifest with a mild, mitis recessive DEB owing to mutations in both COL7A1 alleles. These 2 possibilities are indistinguishable on clinical examination, as well as by histopathological, immunohistochemical, and ultrastructural analyses, resulting in a diagnostic dilemma.11 However, identification of a single dominant mutation in one of the alleles of the affected individual, in the absence of the corresponding mutation in the parents' DNA isolated from peripheral blood leukocytes, indicates de novo dominant mutation. In contrast, identification of 2 mutant alleles in the proband's DNA and demonstration of their inheritance from the respective parents imply mitis recessive DEB.12 The implications are, of course, that the risk of an affected individual in case of de novo dominant DEB of having an affected child is 1 in 2, or 50%, while the risk of the individual with recessive DEB having an affected offspring is very low owing to relatively low carrier frequency of the corresponding gene mutations in the general population.13 At the same time, the risk for the parents of the patient with a de novo dominant mutation of having another affected child is very low, while the risk for the parents of a patient with recessive DEB of having another affected offspring is 1 in 4, or 25%.11
Prenatal Testing and Preimplantation Genetic Diagnosis
A significant consequence of the identification of specific mutations in EB is the development of DNA-based prenatal testing in families at risk for recurrence of severe forms of the disease. Such testing can be performed on chorionic villus samples obtained as early as the 10th week of gestation or on amniotic fluid samples obtained at the 12th week. In fact, such prenatal testing has already been established and is readily available to families at risk for severe forms of recessive DEB or for the Herlitz type of JEB during the first trimester of pregnancy.14-15 Prenatal predictions have also been made in cases of EB with congenital pyloric atresia, a frequently lethal variant of EB.16-17 DNA-based analysis has essentially replaced the previously used fetal skin biopsy, which is performed during the second trimester, as a prenatal diagnostic tool for EB.
An extension of prenatal testing is the development of preimplantation genetic diagnosis, a technique that has been successfully applied to a variety of other genetic diseases.18-19 Preimplantation genetic diagnosis is performed in conjunction with in vitro fertilization, and the fertilized embryos are allowed to grow in vitro to the 8-cell stage level, at which time 1 cell is removed for mutation analysis. Embryos lacking the mutation can then be implanted into the uterus to establish pregnancy, which is routinely performed as part of the in vitro fertilization procedure. The problems with preimplantation genetic diagnosis relate to the relatively low efficiency of in vitro fertilization procedures in general, technical difficulties in identification of the mutations from a single cell, and the overall cost of the procedure.20 Nevertheless, couples with a child who has a severe form of EB can now initiate the next pregnancy knowing that there are ways to find out the EB genotype of the fetus through DNA-based prenatal testing in the early stages of the pregnancy or, with the use of preimplantation genetic diagnosis, even before the pregnancy is established.
Prospects of Gene Therapy
Identification of the underlying molecular defects in different variants of EB is a prerequisite for the development of successful therapies in the future. In particular, the development of gene therapy requires the knowledge of the underlying mutations in the affected genes and their consequences at the messenger RNA and protein levels.1 Gene therapy for EB and related conditions could use 2 complementary strategies. The first possibility could revolve around ex vivo manipulation of cells in a manner that a mutation is corrected in cells cultured from the affected individuals, followed by grafting of the cells back to the eroded areas of the skin.21 The second possibility relates to direct application of DNA into the skin in an attempt to elicit genetic reversal of the underlying mutation.22 Despite the fact that skin, owing to its direct accessibility, is a potential target organ for gene therapy, issues relating to the efficiency of gene therapy, feasibility of targeting the epidermal stem cell populations, maintenance of sustained expression of the transgene, and concerns about long-term safety have been raised. Although successful application of gene therapy for treatment of EB may still be several years away, the rapid development of new technologies holds promise for breakthroughs that will lead to durable gene therapy for these devastating skin diseases in the future.
AUTHOR INFORMATION
Accepted for publication July 18, 2001.
The original studies by the authors were supported by the US Public Health Service, National Institutes of Health grant P01 AR38923, the Dermatology Foundation, and the Dystrophic Epidermolysis Bullosa Research Association of America (DebRA).
The authors thank Carol Kelly for excellent assistance.
Corresponding author: Jouni Uitto, MD, PhD, Department of Dermatology and Cutaneous Biology, Jefferson Medical College, 233 S 10th St, Suite 450 BLSB, Philadelphia, PA 19107 (e-mail: Jouni.Uitto{at}mail.tju.edu).
From the Departments of Dermatology and Cutaneous Biology, and Biochemistry and Molecular Pharmacology, Jefferson Medical College, and Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pa.
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