Cutaneous Basement Membrane Zone Research Articles

Overview of epidermolysis bullosa

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.

Molecular therapies for heritable blistering diseases

Tremendous progress has been made over the past two decades in understanding the molecular genetics of heritable skin diseases. The paradigm for such conditions is epidermolysis bullosa (EB), which comprises a group of heritable blistering disorders caused by mutations in ten genes expressed in the cutaneous basement membrane zone and has high morbidity and mortality. Identification of distinct mutations has improved the diagnosis and subclassification of EB, leading to improvements in disease prognosis, and has provided a basis for prenatal and pre-implantation genetic diagnosis for this disorder. Nevertheless, there is no cure or effective treatment for EB. Here, we review recent exciting developments in the areas of molecular therapies, including gene therapy, protein replacement therapy and bone-marrow-derived stem cell transfer, as potential new avenues to treat EB and other currently intractable heritable skin diseases.

Epidermolysis bullosa acquisita: concise review and practical considerations

Introduction Epidermolysis bullosa acquisita (EBA) is a cutaneous subepi-dermal autoimmune blistering disorder (CSEAIBD) that resultsfrom the effects of IgG autoantibodies directed against the145-kDa noncollagenous amino-terminal (NC-1) domain ofcollagen VII, a major component of anchoring fibrils (Fig. 1).

Progress in Heritable Skin Diseases: Translational Implications of Mutation Analysis and Prospects of Molecular Therapies*

Epidermolysis bullosa, a group of blistering disorders, serves as the paradigm of the tremendous progress made in understanding the molecular genetics of heritable skin diseases. Mutations in 10 distinct genes have been disclosed in the classic forms of epidermolysis bullosa, and the level of expression of the mutated genes within the cutaneous basement membrane zone, the types and combinations of mutations and their consequences at the mRNA and protein levels, when placed in the context of the individual's genetic background and exposure to environmental trauma, all determine the subtype and the phenotypic severity in each case. The translational implications of mutation analysis include improved diagnosis and subclassification, refined genetic counseling of families at risk, and development of DNA-based pre natal and preimplantation genetic diagnosis. The prospects of molecular therapies for epidermolysis bullosa include further development of strategies for gene therapy, protein replacement therapy and cell-based therapies, including stem cell therapy and bone marrow transfer. Collectively, advances in the molecular genetics of heritable skin diseases clearly emphasize the value of basic research for improved diagnostics and patient care for genetic skin diseases.

Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.

Epidermolysis Bullosa: Prospects for Cell-Based Therapies

Heritable forms of epidermolysis bullosa (EB) are characterized by chronic, lifelong blistering and erosions due to mutations in 10 distinct genes expressed at the cutaneous basement membrane zone. No specific treatment for this group of intractable diseases is currently available. Recent progress in molecular therapies has indicated that cell-based approaches may potentially offer amelioration--and perhaps even cure--for afflicted individuals. In this issue, Wong et al. (2008) demonstrate the feasibility of direct intradermal injection of allogeneic fibroblasts to the lesional skin of patients with recessive dystrophic EB, with improvement in skin fragility.

Bone Marrow Cell Transfer into Fetal Circulation Can Ameliorate Genetic Skin Diseases by Providing Fibroblasts to the Skin and Inducing Immune Tolerance

Recent studies have shown that skin injury recruits bone marrow-derived fibroblasts (BMDFs) to the site of injury to accelerate tissue repair. However, whether uninjured skin can recruit BMDFs to maintain skin homeostasis remains uncertain. Here, we investigated the appearance of BMDFs in normal mouse skin after embryonic bone marrow cell transplantation (E-BMT) with green fluorescent protein-transgenic bone marrow cells (GFP-BMCs) via the vitelline vein, which traverses the uterine wall and is connected to the fetal circulation. At 12 weeks of age, mice treated with E-BMT were observed to have successful engraftment of GFP-BMCs in hematopoietic tissues accompanied by induction of immune tolerance against GFP. We then investigated BMDFs in the skin of the same mice without prior injury and found that a significant number of BMDFs, which generate matrix proteins both in vitro and in vivo, were recruited and maintained after birth. Next, we performed E-BMT in a dystrophic epidermolysis bullosa mouse model (col7a1(-/-)) lacking type VII collagen in the cutaneous basement membrane zone. E-BMT significantly ameliorated the severity of the dystrophic epidermolysis bullosa phenotype in neonatal mice. Type VII collagen was deposited primarily in the follicular basement membrane zone in the vicinity of the BMDFs. Thus, gene therapy using E-BMT into the fetal circulation may offer a potential treatment option to ameliorate genetic skin diseases that are characterized by fibroblast dysfunction through the introduction of immune-tolerated BMDFs.

Annual Meeting of the Society for Cutaneous Ultrastructure Research (34th Annual Meeting of SCUR)

Kindler syndrome is an autosomal recessive genodermatosis characterized by trauma-induced acral skin blistering from infancy, photosensitivity, generalized poikiloderma, and diffuse cutaneous atrophy. Mutations in the KIND1 gene that encodes for the focal contact-associated protein kindlin-1 have been identified in several patients although the spectrum of clinical abnormalities is protean and genetic heterogeneity is plausible. A 32-year-old Japanese male, born to consanguineous parents, presented with acral skin blistering, cutaneous atrophy, palmoplantar thickening, finger contractures, absence of dermatoglyphics, and nail dystrophy. He also had leukoplakia of the tongue but did not report photosensitivity or show signs of poikiloderma. A biopsy taken from bullous atrophic skin on the dorsal surface of his left hand showed sub-epidermal blistering, vacuoles within basal keratinocytes and immunohistochemical evidence of basement membrane reduplication. Intensity of Kindlin-1 expression in patient skin was indistinguishable from that in normal skin. Electron microscopy demonstrated multiple tubulo-vesicular structures and electron dense amorphous keratinocyte-derived material immediately below the dermal-epidermal junction. Mutational analysis of the KIND1 gene failed to identify any pathogenic mutation(s). Although this patient’s clinical and skin biopsy features appear to match the clinico-pathological criteria for a diagnosis of Kindler syndrome, no mutation in KIND1 was detected. This suggests genetic heterogeneity in this genodermatosis or perhaps the existence of a subset of patients that have a distinct but currently unclassified related inherited disorder. Identification of similar patients and characterization of this Kindler-like syndrome is expected to provide new insight into clinical disorders associated with structural pathology within the cutaneous basement membrane zone.

Molecular pathology of the cutaneous basement membrane zone

Molecular pathology of the cutaneous basement membrane zone

Immunity to Extracellular Matrix Antigens is Associated with Ultrastructural Alterations of the Stroma and Stratified Epithelium Basement Membrane in the Skin of Hashimoto's Thyroiditis Patients

Employing purified extracellular matrix (ECM) proteins, i.e. type I, III, IV and V collagens (CI, CIII, CIV, CV), laminin (LM) and fibronectin (FN), as antigen sources we detected autoantibodies to conformational and/or denatured ECM antigens among 34 of 50 sera obtained from Hashimotos thyroiditis (HT) patients and 6 of 51 control sera obtained from non-autoimmune thyroid disease patients and healthy donors (HT sera vs. control sera p=4 x 10-9). Reactivity to conformational antigens, mostly due to autoantibodies of the IgG isotype, was observed in 30/50 HT sera and in 6/51 control sera (p=3.5 x 10-7) and was not always concomitant with that to linear antigens, found in 23/50 HT and in 6/51 control sera (p=1.6 x 10-4). Ultrastructural analysis of skin biopsies obtained from 18 HT patients without symptomatic cutaneous diseases revealed defects of the stratified squamous epithelium basement membrane in 11/18, alterations of the stroma in 13/18 and both basement membrane and stromal defects in 9/18. Interestingly, 13/13 (p=0.012) and 9/11 (p=0.012) patients with stromal and basement membrane defects respectively, exhibited serum antibodies to at least one ECM antigen involved in the organization of the altered tissue compartment. Lastly, 10/18 skin biopsies presented immunoglobulin (Ig) and/or complement (C3) deposits along the cutaneous basement membrane zone (BMZ) or in the papillary dermis and 9/10 sera from the same patients simultaneously showed antibodies to at least one ECM antigen involved in the organization of these two skin compartments. Besides, 8/11 HT patients with basement membrane defects exhibited Ig or C3 deposits along the BMZ. Our findings suggest that autoantibodies to ECM molecules might contribute to the development of asymptomatic extra-thyroid skin diseases in HT patients.

Epidermolysis bullosa. I. Molecular genetics of the junctional and hemidesmosomal variants

Introduction: Epidermolysis bullosa (EB), a group of autosomal heritable blistering diseases, is characterised by extensive phenotypic variability with considerable morbidity and mortality. EB is classified into distinct subtypes depending on...

452. Bone Marrow Stem Cells Are Potent Targets for Gene Therapy of Epidermolysis Bullosa, a Genetic Blistering Skin Disease

Pluripotency of bone marrow stem cell (BMSC) has been intensively investigated, and promoted new concept, i.e. BMSC-gene therapy, for various genetic diseases to regenerate gene-corrected tissues derived from BMSCs. Despite these intensive studies, the plasticity of BMSCs to the epidermal keratinocytes is still under controversial arguments. The rarity of the BMSCs-derived keratinocytes in vivo has led pessimistic view on the roles of BMSCs for maintenance of the epidermal structures. Here, we investigated our hypothesis that follicular and interfollicular epidermal stem/ progenitor cells may migrate, at least in part, from bone marrow to the engrafted new born mouse skin, which will newly develop abundant follicles and epidermal appendages as well as squamous epithelia under regional severe hypoxic condition. Concretely, the skin sheets of newborn and adult mice were surgically engrafted onto the back of 8 week-old mice with prior transplantation of GFP-transgenic bone marrow cells after lethal dose X-ray irradiation, and examined appearance of GFP-positive epidermal cells in the grafts in various time points. In a week after the skin grafting, dense migration of GFP+ mesenchymal cells appeared in the upper dermis of the newborn skin graft, whereas only sparse GFP+ cells in the adult skin graft were detected. In 2 to 4 weeks after engraftment, various regions of newly generated follicular and interfollicular epidermis were significantly composed of GFP+ keratinocytes, and some regions exhibited full thickness of GFP+ epidermis, suggesting existence of BMSCs-derived epidermal stem cells. Recruitment of the GFP-transgenic BMSCs to the skin graft was further enhanced by transplantation of the type VII collagen gene (COL7A1) null newborn mouse skin, which required rapid proliferation of the epidermal keratinocytes due to frequent detachment of the epidermis from the underlying dermis. The COL7A1 null skin graft exhibited more than 50% occupancy of the follicular and interfollicular epidermis with GFP+ keratinocytes at 4 weeks after engraftment. These data suggest that BMSCs may provide epidermal stem cells in the newborn skin, especially of severe epidermolysis bullosa patients. COL7A1 Gene-collected bone marrow cell transplantation in the blisters of dystrophic epidermolysis bullosa patients, who are suffering from defect of type VII collagen at the cutaneous basement membrane zone, would be potential strategy for effective gene therapy of this intractable genetic blistering skin disease.

Progress in epidermolysis bullosa: genetic classification and clinical implications.

Epidermolysis bullosa (EB), a heterogenous group of genodermatoses, is characterized by fragility and blistering of the skin associated with extracutaneous manifestations. Based on clinical severity, constellation of the phenotypic manifestations, and the level of tissue separation within the cutaneous basement membrane zone (BMZ), EB has been divided into distinct subcategories. Traditionally, these include the simplex, junctional, and dystrophic forms of EB, and recently attention has been drawn to hemidesmosomal variants demonstrating tissue separation at the level of the hemidesmosomes. Specific mutations in ten distinct genes expressed within the cutaneous BMZ have been delineated in >500 families with different variants of EB. The types of mutations, their positions along the affected genes, and their consequences at the mRNA and protein levels provide explanation for the phenotypic variability and genetic heterogeneity of this group of genodermatoses. Elucidation of mutations in different forms of EB has direct translational applications for improved diagnosis and molecularly based classification with prognostic implications as well as for genetic counseling and DNA-based prenatal testing in families with EB.

Epidermolysis Bullosa: The Expanding Mutation Database

Epidermolysis bullosa (EB) is a group of heritable mechanobullous disorders characterized by fragility of the skin within the cutaneous basement membrane zone (BMZ) (Fine et al, 2000). The severity of skin manifestations can be highly variable, so that in one end of the spectrum the skin findings may be relatively minor blistering tendency primarily on the hands and feet, whereas at the other end of the spectrum the compromised integrity of skin can result in early postnatal demise of the affected individual. Prior to identification of the gene/protein systems harboring mutations in different variants of EB, this phenotypic variability was quite puzzling, compounded by the fact that there are a number of extracutaneous manifestations associated with the skin findings. This phenotypic complexity, coupled with classifications riddled with eponyms, has led to a suggestion that there are 30 different subtypes of EB (Anton-Lamprecht and Gedde-Dahl, 2002). Traditionally, EB has been divided into three broad categories: (a) the simplex forms demonstrate tissue separation within the basal keratinocytes; (b) the junctional forms depict tissue separation within the lamina lucida of the dermal–epidermal basement membrane; and (c) the dystrophic forms show tissue cleavage below the lamina densa within the upper papillary dermis (Fig 1). We have recently proposed a fourth subtype, the hemidesmosomal variants of EB, in which the tissue separation is at the basal cell/lamina lucida interface at the level of hemidesmosomes (Fig 1) (Pulkkinen and Uitto, 1998). In fact, the hemidesmosomal variants include the following clinical entities: (a) Generalized atrophic benign EB (GABEB) that has been previously included in the category of non-Herlitz junctional EB (JEB); (b) EB with pyloric atresia where tissue separation is either just outside of the plasma membrane of the basal cells or in the low intracellular compartment and has occasionally been referred as ‘‘pseudo-junctional’’ EB; (c) EB with muscular dystrophy which shows low intracellular cytolysis occasionally suggested to belong to the simplex category of EB (Pulkkinen and Uitto, 1998). With the advent of molecular genetics in general and essential completion of the human genome project, specific mutations have now been identified in ten distinct genes expressed within the cutaneous BMZ (Table I) (Uitto et al, 2002). The compartmentalized expression of these genes within the cutaneous BMZ, the types and combinations of the mutations, and their consequences at the mRNA and protein levels, when superimposed on the individuals’ genetic background and combined with environmental factors, explain the spectrum of severity in EB. Considerable clinical and molecular heterogeneity is recognized particularly in the JEB, which has been traditionally divided into Herlitz (lethal) and non-Herlitz variants depending on the severity and outcome of the disease. Specifically, most children with the Herlitz variant die within the first year of life, whereas non-Herlitz patients have life-long blistering but their lifespan is not significantly compromised. The Herlitz JEB is due to absence of laminin 5, and mutations have been identified in three genes (LAMA3, LAMB3, and LAMC2), which encode the subunit polypeptides a3, b3, and g2 of laminin 5, respectively. Most of these mutations result in premature termination codon for translation (PTC) (Nakano et al, 2000). In non-Herlitz variants, missense mutations have been identified also in the laminin 5 genes, but in addition, mutations can reside in the 180 kDa bullous pemphigoid antigen/type XVII collagen gene, BPAG2/ COL17A1 (Nakano et al, 2002). In this issue, Posteraro et al (2004) have analyzed an Italian cohort of 19 patients with JEB, 11 being affected with the severe Herlitz type and eight with milder non-Herlitz type. The authors concentrated their analysis on LAMA3, LAMB3, and LAMC2 and identified 18 mutations, seven of which were previously unpublished. In accordance with previous observations, PTC mutations in both alleles of the laminin 5 genes were predominant in Herlitz JEB patients, whereas splicing or missense mutations resulting in reduced synthesis of partially functional laminin 5 were predominantly found in non-Herlitz variants. The authors also disclosed a number of recurrent mutations, including R635X in the LAMB3 gene, a mutation frequent in Caucasian Herlitz JEB patients, particularly in Northern European

Transcriptional control of the mouse Col7a1 gene in keratinocytes: basal and transforming growth factor-beta regulated expression.

Anchoring fibrils at the cutaneous basement membrane zone of the stratified squamous epithelia are essential to maintaining skin integrity, as absence of these structures leads to the chronic blistering disease, dystrophic epidermolysis bullosa. Type VII collagen, the major component of anchoring fibrils, is synthesized primarily by basal keratinocytes and to a lesser degree by dermal fibroblasts. To elucidate the transcriptional control elements of the type VII collagen gene (Col7a1), 3 kb of 5' flanking sequence of the mouse gene was cloned, sequenced, and fused to the chloramphenicol acetyltransferase reporter gene. Promoter deletion analyses revealed that 560 bp of Col7a1 5' flanking sequence was sufficient and necessary for basal level of transcription in cultured murine keratinocytes. Mutagenesis of DNA sequences with similarity to consensus binding sites for transcription factors, including Sp1/Sp3, AP2, AP1, and Smads, within the p-560Col7a1 promoter/chloramphenicol acetyltransferase construct, coupled with DNA binding assays, revealed the importance of these sites for basal Col7a1 expression. The effect of transforming growth factor beta, an activator of Col7a1 expression in keratinocytes and dermal fibroblasts, was examined using the same Col7a1 promoter/chloramphenicol acetyltransferase constructs. These analyses demonstrated that transforming growth factor beta1 stimulation of Col7a1 transcription is dependent on a putative interaction between Smads and AP1. Interestingly, the Smad-like binding site was essential for both basal and transforming growth factor beta1 stimulated Col7a1 transcription. Collectively, these findings attest to the complex regulation of Col7a1 transcription in epidermal keratinocytes.

Targeted Inactivation of Murine Laminin γ2-Chain Gene Recapitulates Human Junctional Epidermolysis Bullosa

Junctional forms of epidermolysis bullosa (JEB) are associated with mutations in six distinct genes expressed in the cutaneous basement membrane zone; these include LAMA3, LAMB3, and LAMC2, which encode laminin 5 subunit polypeptides, the alpha3-, beta3-, and gamma2-chains, respectively. Here we generated a mouse model for JEB by inactivating the laminin gamma2-chain gene by targeted frameshift deletion of exon 8 in Lamc2. Heterozygous mice were phenotypically normal, whereas the majority of Lamc2-/- mice showed blistering phenotype on days 1 to 2 and died within 5 days of birth. The Lamc2-/- mice demonstrated absent expression of laminin gamma2-chain on the basement membrane zone as well as attenuated expression of alpha3- and beta3-chains of laminin. Transmission electron microscopy revealed rudimentary, poorly developed hemidesmosomes. The epidermis of the Lamc2-/- mice revealed induced apoptosis in the basal cells of the blistered skin, suggesting that cell-matrix adhesion provided by laminin 5 plays a role in cell survival in vivo. Cultured Lamc2-/- keratinocytes demonstrated slightly positive staining with gamma2-chain-specific antibodies, which could be explained by the presence of a transcript with partial restoration of the reading frame owing to alternative splicing in vitro. These cells proliferated in different matrices and attached to type IV collagen and Matrigel as efficiently as the wild-type keratinocytes, whereas their attachment on plastic and laminin was significantly weaker. In summary, Lamc2-/- mouse recapitulates human JEB and provides novel insight into the role of laminin 5 in keratinocyte biology.

A missense mutation (G1506E) in the adhesion G domain of laminin-5 causes mild junctional epidermolysis bullosa

Laminin-5 is the major adhesion ligand for epithelial cells. Mutations in the genes encoding laminin-5 cause junctional epidermolysis bullosa (JEB), a recessive inherited disease characterized by extensive epithelial–mesenchymal disadhesion. We describe a JEB patient compound heterozygote for two novel mutations in the gene (LAMA3) encoding the laminin α3 chain. The maternal mutation (1644delG) generates mRNA transcripts that undergo nonsense-mediated decay. The paternal mutation results in the Gly1506 → Glu substitution (G1506E) within the C-terminal globular region of the α3 chain (G domain). Mutation G1506E affects the proper folding of the fourth module of the G domain and results in the retention of most of the mutated polypeptide within the endoplasmic reticulum (ER). However, scant amounts of the mutated laminin-5 are secreted, undergo physiologic extracellular maturation, and correctly localize within the cutaneous basement membrane zone in patient’s skin. Our findings represent the first demonstration of an ER-retained mutant laminin-5 leading to a mild JEB phenotype.

Identification of COL7A1 Alternative Splicing Inserting 9 Amino Acid Residues Into the Fibronectin Type III Linker Domain

Type VII collagen is the major component of anchoring fibrils within the cutaneous basement membrane zone. The large amino-terminal noncollagenous domain of type VII collagen interacts with various extracellular matrix proteins and contributes to the dermal-epidermal attachment. The purpose of this study was to detect alternative splicing of COL7A1 transcript encoding the noncollagenous 1 domain. The alternative splicing in this region may affect interactions of the noncollagenous 1 domain with extracellular matrix proteins and also dermal-epidermal adhesion. Thus we examined expression of the alternative splicing in situations relating to wound healing and skin remodeling that required dermal-epidermal binding and detachment. Amplification of overlapping cDNA from keratinocytes using reverse transcription-polymerase chain reaction identified alternative splicing, which was generated by a different exon 18 acceptor site 27 bp upstream from the common acceptor site. Expression of this alternatively spliced transcript differed among several cell types. The nine amino acid residues GPLTLPLSP from the 27 bp nucleotides were inserted into the linker of fibronectin type III domains. This insertion was suggested to contribute to flexibility of the linker of fibronectin type III domains and may affect the interactions between the noncollagenous 1 domain and extracellular matrix proteins. Treatment with transforming growth factor-beta 1, which is known to promote wound healing and skin remodeling, enhanced the expression of this 27 bp transcript. Furthermore, keratinocyte biopsies from the wound edge of patients with epithelizing skin ulcers showed a significant increase in the 27 bp transcript expression compared with normal keratinocytes from steady-state body sites. These results suggest that amino acid variation of this alternative splicing may have some role in dermal-epidermal adhesion, wound healing, and skin remodeling. To the best of our knowledge, this is the first evidence of alternative splice insertion of a small peptide into the linker region of the fibronectin type III domains, a common motif within modular proteins.

Prenatal diagnosis for epidermolysis bullosa: a study of 144 consecutive pregnancies at risk.

Epidermolysis bullosa (EB) is a group of inherited disorders characterized by increased skin fragility, resulting in blisters and erosions after minor trauma. Mutations in 10 structural genes expressed in the cutaneous basement membrane zone have been reported. The DebRA Molecular Diagnostics Laboratory at Jefferson Medical College has performed 144 DNA-based prenatal diagnoses since 1993 in families at risk for recurrence of the most severe forms of EB, including the recessive dystrophic EB (RDEB), junctional EB (JEB), EB with pyloric atresia (EB-PA), and EB simplex (EBS). A mutation-detection strategy using either conformation-sensitive gel electrophoresis (CSGE) or denaturing high-performance liquid chromatography (dHPLC) scanning analysis, followed by nucleotide sequencing, was applied to most cases with DEB and to all JEB, EB-PA, and EBS families. For some RDEB families, linkage analysis was performed, either alone when the inheritance pattern was clear or in combination with one mutation. Among the 144 prenatal diagnoses, 63 were for RDEB, 69 for JEB, 6 for EB-PA, and 6 for EBS. Twenty-eight normal, 73 heterozygous carrier, and 28 affected RDEB, JEB, and EB-PA pregnancies were reported in these recessively inherited diseases. Two affected and four normal pregnancies were predicted in dominantly inherited EBS. Among the 144 pregnancies, 9 were terminated without confirmation, 13 cases were lost to follow-up, and 6 pregnancies are ongoing. There were 6 families with inconclusive results due either to recombination events between flanking markers, absence of informative markers for one allele, or lack of sample from the previously affected child. There were three discordant results, one that was explained by maternal contamination of the chorionic villus sample and two that were unresolved. Overall, the availability, relative ease, and over 98% success rate make molecular DNA-based prenatal diagnosis a viable option for EB families at risk.

Drug-induced epidermolysis bullosa acquisita with antibodies to type VII collagen

We describe a 73-year-old patient who had a subepidermal bullous eruption develop after a course of antibiotics, including vancomycin. The patient had deposits of IgA and IgG in the cutaneous basement membrane zone that were located on the dermal side of 1 M NaCl-treated autologous skin. By an enzyme-linked immunosorbent assay, the patient was found to have circulating IgG antibodies directed against type VII collagen, the target antigen of epidermolysis bullosa acquisita. Our observation expands the spectrum of immune-mediated subepidermal bullous skin eruptions precipitated by drugs and lends support to the idea that a subset of these cases represents an unusual variant of drug-triggered epidermolysis bullosa acquisita. (J Am Acad Dermatol 2002;46:S161-4.)