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1.  A homozygous mutation in the integrin alpha6 gene in junctional epidermolysis bullosa with pyloric atresia. 
Journal of Clinical Investigation  1997;99(12):2826-2831.
The alpha6 integrin subunit participates in the formation of both alpha6beta1 and alpha6beta4 laminin receptors, which have been reported to play an important role in cell adhesion and migration and in morphogenesis. In squamous epithelia, the alpha6beta4 heterodimer is the crucial component for the assembly and stability of hemidesmosomes. These anchoring structures are ultrastructurally abnormal in patients affected with junctional epidermolysis bullosa with pyloric atresia (PA-JEB), a recessively inherited blistering disease of skin and mucosae characterized by an altered immunoreactivity with antibodies specific to integrin alpha6beta4. In this report, we describe the first mutation in the alpha6 integrin gene in a PA-JEB patient presenting with generalized skin blistering, aplasia cutis, and defective expression of integrin alpha6beta4. The mutation (791delC) is a homozygous deletion of a single base (C) leading to a frameshift and a premature termination codon that results in a complete absence of alpha6 polypeptide. We also describe the DNA-based prenatal exclusion of the disease in this family at risk for recurrence of PA-JEB. Our results demonstrate that, despite the widespread distribution of the alpha6 integrin subunit, lack of expression of the alpha6 integrin chain is compatible with fetal development, and results in a phenotype indistinguishable from that caused by mutations in the beta4 chain, which is expressed in a more limited number of tissues.
PMCID: PMC508131  PMID: 9185503
2.  Rat Model for Dominant Dystrophic Epidermolysis Bullosa: Glycine Substitution Reduces Collagen VII Stability and Shows Gene-Dosage Effect 
PLoS ONE  2013;8(5):e64243.
Dystrophic epidermolysis bullosa, a severely disabling hereditary skin fragility disorder, is caused by mutations in the gene coding for collagen VII, a specialized adhesion component of the dermal-epidermal junction zone. Both recessive and dominant forms are known; the latter account for about 40% of cases. Patients with dominant dystrophic epidermolysis bullosa exhibit a spectrum of symptoms ranging from mild localized to generalized skin manifestations. Individuals with the same mutation can display substantial phenotypic variance, emphasizing the role of modifying genes in this disorder. The etiology of dystrophic epidermolysis bullosa has been known for around two decades; however, important pathogenetic questions such as involvement of modifier genes remain unanswered and a causative therapy has yet to be developed. Much of the failure to make progress in these areas is due to the lack of suitable animal models that capture all aspects of this complex monogenetic disorder. Here, we report the first rat model of dominant dystrophic epidermolysis bullosa. Affected rats carry a spontaneous glycine to aspartic acid substitution, p.G1867D, within the main structural domain of collagen VII. This confers dominant-negative interference of protein folding and decreases the stability of mutant collagen VII molecules and their polymers, the anchoring fibrils. The phenotype comprises fragile and blister-prone skin, scarring and nail dystrophy. The model recapitulates all signs of the human disease with complete penetrance. Homozygous carriers of the mutation are more severely affected than heterozygous ones, demonstrating for the first time a gene-dosage effect of mutated alleles in dystrophic epidermolysis bullosa. This novel viable and workable animal model for dominant dystrophic epidermolysis bullosa will be valuable for addressing molecular disease mechanisms, effects of modifying genes, and development of novel molecular therapies for patients with dominantly transmitted skin disease.
PMCID: PMC3662756  PMID: 23717576
3.  Gain of glycosylation in integrin α3 causes lung disease and nephrotic syndrome 
The Journal of Clinical Investigation  2012;122(12):4375-4387.
Integrins are transmembrane αβ glycoproteins that connect the extracellular matrix to the cytoskeleton. The laminin-binding integrin α3β1 is expressed at high levels in lung epithelium and in kidney podocytes. In podocytes, α3β1 associates with the tetraspanin CD151 to maintain a functional filtration barrier. Here, we report on a patient homozygous for a novel missense mutation in the human ITGA3 gene, causing fatal interstitial lung disease and congenital nephrotic syndrome. The mutation caused an alanine-to-serine substitution in the integrin α3 subunit, thereby introducing an N-glycosylation motif at amino acid position 349. We expressed this mutant form of ITGA3 in murine podocytes and found that hyperglycosylation of the α3 precursor prevented its heterodimerization with β1, whereas CD151 association with the α3 subunit occurred normally. Consequently, the β1 precursor accumulated in the ER, and the mutant α3 precursor was degraded by the ubiquitin-proteasome system. Thus, these findings uncover a gain-of-glycosylation mutation in ITGA3 that prevents the biosynthesis of functional α3β1, causing a fatal multiorgan disorder.
PMCID: PMC3533548  PMID: 23114595
4.  An Incompletely Penetrant Novel Mutation in COL7A1 Causes Epidermolysis Bullosa Pruriginosa and Dominant Dystrophic Epidermolysis Bullosa Phenotypes in an Extended Kindred 
Pediatric dermatology  2012;29(6):725-731.
Epidermolysis bullosa pruriginosa (EBP) is a rare subtype of dystrophic epidermolysis bullosa (DEB) characterized by intense pruritus, nodular or lichenoid lesions, and violaceous linear scarring, most prominently on the extensor extremities. Remarkably, identical mutations in COL7A1, which encodes an anchoring fibril protein present at the dermal–epidermal junction, can cause both DEB and EBP with either autosomal dominant or recessive inheritance. We present one family with both dystrophic and pruriginosa phenotypes of epidermolysis bullosa. The proband is a 19-year-old Caucasian woman who initially presented in childhood with lichenoid papules affecting her extensor limbs and intense pruritus consistent with EBP. Her maternal grandmother saw a dermatologist for similar skin lesions that developed without any known triggers at age 47 and mostly resolved spontaneously after approximately 10 years. The proband’s younger brother developed a small crop of pruritic papules on his elbows, dorsal hands, knees, and ankles at age 13. Her second cousin once removed, however, reported a mild blistering disease without pruritus consistent with DEB. Genetic sequencing of the kindred revealed a single dominant novel intron 47 splice site donor G>A mutation, c.4668 + 1 G>A, which we predict leads to exon skipping. Incomplete penetrance is confirmed in her clinically unaffected mother, who carries the same dominant mutation. The wide diversity of clinical phenotypes with one underlying genotype demonstrates that COL7A1 mutations are incompletely penetrant and strongly suggests that other genetic and environmental factors influence clinical presentation.
PMCID: PMC3709244  PMID: 22515571
5.  An Incompletely Penetrant Novel Mutation in COL7A1 Causes Epidermolysis Bullosa Pruriginosa and Dominant Dystrophic Epidermolysis Bullosa Phenotypes in an Extended Kindred 
Pediatric Dermatology  2012;29(6):725-731.
Epidermolysis bullosa pruriginosa (EBP) is a rare subtype of dystrophic epidermolysis bullosa (DEB) characterized by intense pruritus, nodular or lichenoid lesions, and violaceous linear scarring, most prominently on the extensor extremities. Remarkably, identical mutations in COL7A1, which encodes an anchoring fibril protein present at the dermal–epidermal junction, can cause both DEB and EBP with either autosomal dominant or recessive inheritance. We present one family with both dystrophic and pruriginosa phenotypes of epidermolysis bullosa. The proband is a 19-year-old Caucasian woman who initially presented in childhood with lichenoid papules affecting her extensor limbs and intense pruritus consistent with EBP. Her maternal grandmother saw a dermatologist for similar skin lesions that developed without any known triggers at age 47 and mostly resolved spontaneously after approximately 10 years. The proband’s younger brother developed a small crop of pruritic papules on his elbows, dorsal hands, knees, and ankles at age 13. Her second cousin once removed, however, reported a mild blistering disease without pruritus consistent with DEB. Genetic sequencing of the kindred revealed a single dominant novel intron 47 splice site donor G>A mutation, c.4668 + 1 G>A, which we predict leads to exon skipping. Incomplete penetrance is confirmed in her clinically unaffected mother, who carries the same dominant mutation. The wide diversity of clinical phenotypes with one underlying genotype demonstrates that COL7A1 mutations are incompletely penetrant and strongly suggests that other genetic and environmental factors influence clinical presentation.
PMCID: PMC3709244  PMID: 22515571
6.  Multicentre consensus recommendations for skin care in inherited epidermolysis bullosa 
Inherited epidermolysis bullosa (EB) comprises a highly heterogeneous group of rare diseases characterized by fragility and blistering of skin and mucous membranes. Clinical features combined with immunofluorescence antigen mapping and/or electron microscopy examination of a skin biopsy allow to define the EB type and subtype. Molecular diagnosis is nowadays feasible in all EB subtypes and required for prenatal diagnosis. The extent of skin and mucosal lesions varies greatly depending on EB subtype and patient age. In the more severe EB subtypes lifelong generalized blistering, chronic ulcerations and scarring sequelae lead to multiorgan involvement, major morbidity and life-threatening complications. In the absence of a cure, patient management remains based on preventive measures, together with symptomatic treatment of cutaneous and extracutaneous manifestations and complications. The rarity and complexity of EB challenge its appropriate care. Thus, the aim of the present study has been to generate multicentre, multidisciplinary recommendations on global skin care addressed to physicians, nurses and other health professionals dealing with EB, both in centres of expertise and primary care setting.
Almost no controlled trials for EB treatment have been performed to date. For this reason, recommendations were prepared by a multidisciplinary team of experts from different European EB centres based on available literature and expert opinion. They have been subsequently revised by a panel of external experts, using an online-modified Delphi method to generate consensus.
Recommendations are reported according to the age of the patients. The major topics treated comprise the multidisciplinary approach to EB patients, global skin care including wound care, management of itching and pain, and early diagnosis of squamous cell carcinoma. Aspects of therapeutic patient education, care of disease burden and continuity of care are also developed.
The recommendations are expected to be useful for daily global care of EB patients, in particular in the community setting. An optimal management of patients is also a prerequisite to allow them to benefit from the specific molecular and cell-based treatments currently under development.
PMCID: PMC4110526  PMID: 24884811
Inherited epidermolysis bullosa; Multidisciplinary management recommendations; Skin care; Wound care; Itch; Pain; Therapeutic education; Disease burden; Quality of life; Continuity of care
7.  Molecular Identification of Collagen 17a1 as a Major Genetic Modifier of Laminin Gamma 2 Mutation-Induced Junctional Epidermolysis Bullosa in Mice 
PLoS Genetics  2014;10(2):e1004068.
Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic jeb allele of laminin gamma 2 (Lamc2). By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the Lamc2jeb mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in Lamc2jeb mice. This modifier is defined by variations in 1–3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the Lamc2jeb mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder.
Author Summary
Epidermolysis bullosa (EB) is a group of rare genetic Mendelian disorders that result in mechanical fragility of the skin and mucosal membranes. Junctional EB is a subset caused by mutations that result in cleavage of the dermal-epidermal junction. All forms of EB demonstrate substantial variability in their clinical phenotype that is not readily explained. The possibility of genetic modifiers as the cause of this variability has been difficult to address in humans. We apply a mouse model carrying a hypomorphic allele of the laminin gamma 2 (Lamc2) gene to address the possibility of genetic modifiers of JEB. We document the potent impact of differing genetic backgrounds on multiple facets of the JEB syndrome expressed in these mice and show that three neighboring amino acid changes within the non-collagenous domain 4 of the collagen XVII protein strongly modify their disease. The study provides a molecular explanation of how a primary mutation that weakens one component of the cutaneous basement membrane is influenced by normally innocuous allelic variants of another component to affect strength of dermal-epidermal adhesion and consequently, the severity of JEB. This approach may guide the genetic prognosis and diagnosis of human EB disorders.
PMCID: PMC3923665  PMID: 24550734
8.  Transplanted Bone Marrow–Derived Circulating PDGFRα+ Cells Restore Type VII Collagen in Recessive Dystrophic Epidermolysis Bullosa Mouse Skin Graft 
Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic blistering skin disease in which the epithelial structure easily separates from the underlying dermis because of genetic loss of functional type VII collagen (Col7) in the cutaneous basement membrane zone. Recent studies have demonstrated that allogeneic bone marrow transplantation (BMT) ameliorates the skin blistering phenotype of RDEB patients by restoring Col7. However, the exact therapeutic mechanism of BMT in RDEB remains unclear. In this study, we investigated the roles of transplanted bone marrow–derived circulating mesenchymal cells in RDEB (Col7-null) mice. In wild-type mice with prior GFP-BMT after lethal irradiation, lineage-negative/GFP-positive (Lin−/GFP+) cells, including platelet-derived growth factor receptor α-positive (PDGFRα+) mesenchymal cells, specifically migrated to skin grafts from RDEB mice and expressed Col7. Vascular endothelial cells and follicular keratinocytes in the deep dermis of the skin grafts expressed SDF-1α, and the bone marrow–derived PDGFRα+ cells expressed CXCR4 on their surface. Systemic administration of the CXCR4 antagonist AMD3100 markedly decreased the migration of bone marrow–derived PDGFRα+ cells into the skin graft, resulting in persistent epidermal detachment with massive necrosis and inflammation in the skin graft of RDEB mice; without AMD3100 administration, Col7 was significantly supplemented to ameliorate the pathogenic blistering phenotype. Collectively, these data suggest that the SDF1α/CXCR4 signaling axis induces transplanted bone marrow–derived circulating PDGFRα+ mesenchymal cells to migrate and supply functional Col7 to regenerate RDEB skin.
PMCID: PMC4319308  PMID: 25601922
9.  A COL7A1 Mutation Causes Dystrophic Epidermolysis Bullosa in Rotes Höhenvieh Cattle 
PLoS ONE  2012;7(6):e38823.
We identified a congenital mechanobullous skin disorder in six calves on a single farm of an endangered German cattle breed in 2010. The condition presented as a large loss of skin distal to the fetlocks and at the mucosa of the muzzle. All affected calves were euthanized on humane grounds due to the severity, extent and progression of the skin and oral lesions. Examination of skin samples under light microscopy revealed detachment of the epidermis from the dermis at the level of the dermo epidermal junction, leading to the diagnosis of a subepidermal bullous dermatosis such as epidermolysis bullosa. The pedigree was consistent with monogenic autosomal recessive inheritance. We localized the causative mutation to an 18 Mb interval on chromosome 22 by homozygosity mapping. The COL7A1 gene encoding collagen type VII alpha 1 is located within this interval and COL7A1 mutations have been shown to cause inherited dystrophic epidermolysis bullosa (DEB) in humans. A SNP in the bovine COL7A1 exon 49 (c.4756C>T) was perfectly associated with the observed disease. The homozygous mutant T/T genotype was exclusively present in affected calves and their parents were heterozygous C/T confirming the assumed recessive mode of inheritance. All known cases and genotyped carriers were related to a single cow, which is supposed to be the founder animal. The mutant T allele was absent in 63 animals from 24 cattle breeds. The identified mutation causes a premature stop codon which leads to a truncated protein representing a complete loss of COL7A1 function (p.R1586*). We thus have identified a candidate causative mutation for this genetic disease using only three cases to unravel its molecular basis. Selection against this mutation can now be used to eliminate the mutant allele from the Rotes Höhenvieh breed.
PMCID: PMC3371016  PMID: 22715415
10.  Prevalence of specific anti-skin autoantibodies in a cohort of patients with inherited epidermolysis bullosa 
Inherited epidermolysis bullosa (EB) is a group of skin diseases characterized by blistering of the skin and mucous membranes.
There are four major types of EB (EB simplex, junctional EB, dystrophic EB and Kindler syndrome) caused by different gene mutations. Dystrophic EB is derived from mutations in the type VII collagen gene (COL7A1), encoding a protein which is the predominant component of the anchoring fibrils at the dermal-epidermal junction.
For the first time in literature, we have evaluated the presence of anti-skin autoantibodies in a wider cohort of patients suffering from inherited EB and ascertained whether they may be a marker of disease activity.
Sera from patients with inherited EB, 17 with recessive dystrophic EB (RDEB), 10 with EB simplex (EBS) were analysed. As much as 20 patients with pemphigus vulgaris, 21 patients with bullous pemphigoid and 20 healthy subjects were used as controls.
Anti-skin autoantibodies were tested in all samples with the Indirect Immunofluorescence (IIF) method and the currently available ELISA method in order to detect anti-type VII collagen, anti-BP180 and anti-BP230 autoantibodies.
The mean concentrations of anti-type VII collagen autoantibodies titres, anti-BP180 and anti-BP230 autoantibodies were statistically higher in RDEB patients than in EBS patients.
The sensitivity and specificity of the anti-type VII collagen ELISA test were 88.2% and 96.7%. The Birmingham Epidermolysis Bullosa Severity score, which is used to evaluate the severity of the disease, correlated with anti-skin autoantibodies titres.
The precise pathogenic role of circulating anti-skin autoantibodies in RDEB is unclear. There is a higher prevalence of both anti-type VII collagen and other autoantibodies in patients with RDEB, but their presence can be interpreted as an epiphenomenon.
PMCID: PMC4015699  PMID: 24007552
Inherited epidermolysis bullosa; Dystrophic epidermolysis bullosa; Anti-skin autoantibodies; Type VII collagen; BP180; BP230; ELISA; Birmingham Epidermolysis Bullosa Severity score
11.  A homozygous nonsense mutation in the PLEC1 gene in patients with epidermolysis bullosa simplex with muscular dystrophy. 
Journal of Clinical Investigation  1996;98(10):2196-2200.
Plectin is a widely expressed cytomatrix component involved in the attachment of the cytoskeleton to the plasma membrane. We have recently reported that the skin and muscles of three patients affected by epidermolysis bullosa simplex with muscular dystrophy (MD-EBS), a genetic disorder characterized by skin blistering associated with muscle involvement, are not reactive with antibodies specific to plectin. We demonstrated that in the skin, lack of plectin leads to failure of keratin filaments to connect to the plasma membrane via the hemidesmosomes, whereas in the muscle the deficient expression of the molecule correlates with an aberrant localization of desmin in the muscle fibers. In this study we demonstrate that in a MD-EBS kindred with two affected members, the disease results from a homozygous nonsense mutation in the plectin (PLEC1) gene leading to a premature stop codon (CGA to TGA) and decay of the aberrant plectin messenger RNA. The segregation of the mutated allele implicates the mutation in the pathology of the disorder. These results confirm the critical role of plectin in providing cell resistance to mechanical stresses both in the skin and the muscle.
PMCID: PMC507667  PMID: 8941634
12.  Genetic linkage of recessive dystrophic epidermolysis bullosa to the type VII collagen gene. 
Journal of Clinical Investigation  1992;90(3):1032-1036.
Generalized mutilating recessive dystrophic epidermolysis bullosa (RDEB) is characterized by extreme skin fragility owing to loss of dermal-epidermal adherence. Immunohistochemical studies have implicated type VII collagen, the major component of anchoring fibrils, in the etiology of RDEB. In this study, we demonstrate genetic linkage of the type VII collagen gene and the generalized mutilating RDEB phenotype. We first identified a Pvull polymorphic site by digestion of an amplified product of the type VII collagen gene, which was shown to reside within the coding region. Genetic linkage analysis between this marker and the RDEB phenotype in 19 affected families which were informative for this polymorphism showed no recombination events, and gave a maximum lod score of 3.97 at a recombination fraction (theta) of 0, demonstrating that this DNA region is involved in this form of RDEB. These data provide strong evidence that the type VII collagen gene, which has also been linked with the dominant form of the disease, harbors the mutation(s) causing the generalized mutilating form of RDEB in these families, thus underscoring the major functional importance of type VII collagen in basement membrane zone stability.
PMCID: PMC329960  PMID: 1355776
13.  Premature termination codons on both alleles of the type VII collagen gene (COL7A1) in three brothers with recessive dystrophic epidermolysis bullosa. 
Journal of Clinical Investigation  1995;95(3):1328-1334.
Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. In the most severe, dystrophic (scarring) forms of EB, blisters form below the cutaneous basement membrane at the level of the anchoring fibrils, which are composed of type VII collagen. Ultrastructural observations of altered anchoring fibrils and genetic linkage to the type VII collagen locus (COL7A1) have implicated COL7A1 as the candidate gene in the dystrophic forms of EB. We have recently cloned the entire cDNA and the gene for human COL7A1. In this study, we describe distinct mutations in both COL7A1 alleles in three brothers with severe, mutilating recessive dystrophic EB (the Hallopeau-Siemens type, HS-RDEB). The patients are compound heterozygotes for two different mutations, both of which result in a premature termination codon in COL7A1, and the parents were shown to be clinically heterozygous carries of the respective mutations. Premature termination codons in both alleles of COL7A1 appear to be the underlying cause of severe, recessive dystrophic EB in this family.
PMCID: PMC441472  PMID: 7883979
14.  Dystrophic Epidermolysis Bullosa in Pregnancy: A Case Report of the Autosomal Dominant Subtype and Review of the Literature 
Case Reports in Medicine  2014;2014:242046.
Epidermolysis bullosa (EB) is a group of inherited blistering skin diseases that vary widely in their pathogenesis and severity. There are three main categories of EB: simplex, junctional, and dystrophic. This classification is based on the level of tissue separation within the basement membrane zone and this is attributed to abnormalities of individual or several anchoring proteins that form the interlocking network spanning from the epidermis to the dermis underneath. Dystrophic EB results from mutations in COL7A1 gene coding for type VII collagen leading to blister formation within the dermis. Diagnosis ultimately depends on the patient's specific genetic mutation, but initial diagnosis can be made from careful examination and history taking. We present a pregnant patient known to have autosomal dominant dystrophic EB and discuss the obstetrical and neonatal outcome. The paper also reviews the current English literature on this rare skin disorder.
PMCID: PMC4017779  PMID: 24864146
15.  A Direct Method to Determine the Strength of the Dermal – Epidermal Junction in a Mouse Model for Epidermolysis Bullosa 
Experimental Dermatology  2012;21(6):453-455.
Epidermolysis bullosa (EB) describes a spectrum of rare, incurable, inherited mechanobullous disorders unified by the fact that they are caused by structural defects in the basement membrane zone which disrupt adhesion between the epidermis and dermis. Mouse models provide valuable tools to define the molecular basis of these diseases and to test novel therapeutic approaches. There is need for rapid, quantitative tests that measure the integrity of dermal-epidermal adhesions in such models. To address this need, we describe a novel quantitative method to determine the mechanical strength of the adhesion between tail skin epidermis and dermis. We show that this test reliably measures the force required to cause dermal-epidermal separation in tails of mice that are genetically predisposed to a form of non-Herlitz Junctional EB which develops as the result of a hypomorphic mutation in the laminin gamma 2 gene (Lamc2jeb). This simple, quantitative method of directly measuring the tensile strength of dermal-epidermal adhesion provides a novel dimension to the pathophysiological screening, evaluation, and therapeutic treatment of mice that may develop progressive forms of EB and potentially other disorders that compromise cutaneous integrity.
PMCID: PMC3360816  PMID: 22507638
junctional epidermolysis bullosa; laminin gamma 2; tail tension test; dermis; epidermis; push-pull force gauge
16.  Inherited epidermolysis bullosa 
Inherited epidermolysis bullosa (EB) encompasses a number of disorders characterized by recurrent blister formation as the result of structural fragility within the skin and selected other tissues. All types and subtypes of EB are rare; the overall incidence and prevalence of the disease within the United States is approximately 19 per one million live births and 8 per one million population, respectively. Clinical manifestations range widely, from localized blistering of the hands and feet to generalized blistering of the skin and oral cavity, and injury to many internal organs. Each EB subtype is known to arise from mutations within the genes encoding for several different proteins, each of which is intimately involved in the maintenance of keratinocyte structural stability or adhesion of the keratinocyte to the underlying dermis. EB is best diagnosed and subclassified by the collective findings obtained via detailed personal and family history, in concert with the results of immunofluorescence antigenic mapping, transmission electron microscopy, and in some cases, by DNA analysis. Optimal patient management requires a multidisciplinary approach, and revolves around the protection of susceptible tissues against trauma, use of sophisticated wound care dressings, aggressive nutritional support, and early medical or surgical interventions to correct whenever possible the extracutaneous complications. Prognosis varies considerably and is based on both EB subtype and the overall health of the patient.
PMCID: PMC2892432  PMID: 20507631
17.  Extent of Laminin-5 Assembly and Secretion Effect Junctional Epidermolysis Bullosa Phenotype  
The Journal of Experimental Medicine  1998;187(8):1273-1283.
Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin blistering disease with both lethal and nonlethal forms, with most patients shown to have defects in laminin-5. We analyzed the location of mutations, gene expression levels, and protein chain assembly of the laminin-5 heterotrimer in six JEB patients to determine how the type of genetic lesion influences the pathophysiology of JEB. Mutations within laminin-5 genes were diversely located, with the most severe forms of JEB correlating best with premature termination codons, rather than mapping to any particular protein domain. In all six JEB patients, the laminin-5 assembly intermediates we observed were as predicted by our previous work indicating that the α3β3γ2 heterotrimer assembles intracellularly via a β3γ2 heterodimer intermediate. Since assembly precedes secretion, mutations that disrupt protein–protein interactions needed for assembly are predicted to limit the secretion of laminin-5, and likely to interfere with function. However, our data indicate that typically the most severe mutations diminish mRNA stability, and serve as functional null alleles that block chain assembly by resulting in either a deficiency (in the nonlethal mitis variety) or a complete absence (in lethal Herlitz-JEB) of one of the chains needed for laminin-5 heterotrimer assembly.
PMCID: PMC2212220  PMID: 9547338
18.  A Human Integrin-α3 Mutation Confers Major Renal Developmental Defects 
PLoS ONE  2014;9(3):e90879.
The development of the mammalian kidney is a highly complex process dependent upon the interplay of various cell types, secreted morphogens, and the extra-cellular matrix (ECM). Although integrins are the most important receptors for ECM proteins and are ubiquitously expressed during kidney development, mice lacking expression of integrin α3 (Itga3) do not demonstrate a reduced number of nephrons, but mostly a disorganized GBM (glomerular basement membrane) leading to proteinuria. Thus, ITGA3 is considered mostly a passive GBM stabilizer and not an active player in nephrogenesis. Recently, mutations in the human ITGA3 were shown to cause congenital nephrotic syndrome, epidermolysis bullosa and interstitial lung disease, otherwise termed NEP syndrome (Nephrotic syndrome, Epidermolysis bullosa and Pulmonary disease). Herein, we performed histological and molecular analysis on the kidneys of a single patient from the initial cohort harboring an ITGA3 mutation, to illuminate the role of ITGA3 in human renal development. We show the patient to harbor a unique phenotype at birth, including severe unilateral renal hypodysplasia. Interrogation of global gene expression in the hypodysplastic kidney versus three controls (fetal, child and adult kidneys) revealed perturbed expression in several renal developmental pathways implicated in hypodysplasia, including the Wnt, BMP (bone morphogenetic protein) and TGF (transforming growth factor) pathways. Moreover, the affected kidney showed upregulation of early embryonic genes (e.g. OCT4 and PAX8) concomitant with downregulated kidney differentiation markers, implying a defect in proper renal differentiation. In conclusion, we show for the first time that ITGA3 is not merely a passive anchor for renal ECM proteins, as predicted by mouse models. Instead, our results may suggest it plays a central role in the interplay of cells, morphogens and ECM, required for proper nephrogenesis, thus adding ITGA3 to the list of CAKUT (congenital anomalies of the kidney and urinary tract)-causing genes.
PMCID: PMC3951280  PMID: 24621570
19.  Somatic Correction of Junctional Epidermolysis Bullosa by a Highly Recombinogenic AAV Variant 
Molecular Therapy  2014;22(4):725-733.
Definitive correction of disease causing mutations in somatic cells by homologous recombination (HR) is an attractive therapeutic approach for the treatment of genetic diseases. However, HR-based somatic gene therapy is limited by the low efficiency of gene targeting in mammalian cells and replicative senescence of primary cells ex vivo, forcing investigators to explore alternative strategies such as retro- and lentiviral gene transfer, or genome editing in induced pluripotent stem cells. Here, we report correction of mutations at the LAMA3 locus in primary keratinocytes derived from a patient affected by recessive inherited Herlitz junctional epidermolysis bullosa (H-JEB) disorder using recombinant adenoassociated virus (rAAV)-mediated HR. We identified a highly recombinogenic AAV serotype, AAV-DJ, that mediates efficient gene targeting in keratinocytes at clinically relevant frequencies with a low rate of random integration. Targeted H-JEB patient cells were selected based on restoration of adhesion phenotype, which eliminated the need for foreign sequences in repaired cells, enhancing the clinical use and safety profile of our approach. Corrected pools of primary cells assembled functional laminin-332 heterotrimer and fully reversed the blistering phenotype both in vitro and in skin grafts. The efficient targeting of the LAMA3 locus by AAV-DJ using phenotypic selection, together with the observed low frequency of off-target events, makes AAV-DJ based somatic cell targeting a promising strategy for ex vivo therapy for this severe and often lethal epithelial disorder.
PMCID: PMC3982486  PMID: 24390279
20.  Laminin 332 in junctional epidermolysis bullosa 
Cell Adhesion & Migration  2013;7(1):135-141.
Laminin 332 is an essential component of the dermal-epidermal junction, a highly specialized basement membrane zone that attaches the epidermis to the dermis and thereby provides skin integrity and resistance to external mechanical forces. Mutations in the LAMA3, LAMB3 and LAMC2 genes that encode the three constituent polypeptide chains, α3, β3 and γ2, abrogate or perturb the functions of laminin 332. The phenotypic consequences are diminished dermal-epidermal adhesion and, as clinical symptoms, skin fragility and mechanically induced blistering. The disorder is designated as junctional epidermolysis bullosa (JEB). This article delineates the signs and symptoms of the different forms of JEB, the mutational spectrum, genotype-phenotype correlations as well as perspectives for future molecular therapies.
PMCID: PMC3544777  PMID: 23076207
skin fragility; blistering; hemidesmosome; granulation tissue; basement membrane
21.  Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy. 
Journal of Clinical Investigation  1996;97(10):2289-2298.
Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament-associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD-EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD-EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility.
PMCID: PMC507309  PMID: 8636409
22.  Defining keratin protein function in skin epithelia: Epidermolysis Bullosa Simplex and its aftermath 
Epidermolysis bullosa simplex (EBS) is a rare genetic condition typified by superficial bullous lesions following incident frictional trauma to the skin. Most cases of EBS are due to dominantly-acting mutations in keratin 14 (K14) or K5, the type I and II intermediate filament (IF) proteins that co-polymerize to form a pan-cytoplasmic network of 10nm filaments in basal keratinocytes of epidermis and related epithelia. Defects in K5–K14 filament network architecture cause basal keratinocytes to become fragile, and account for their rupture upon exposure to mechanical trauma. The discovery of the etiology and pathophysiology of EBS was intimately linked to the quest for an understanding of the properties and function of keratin filaments in skin epithelia. Since then, continued cross-fertilization between basic science efforts and clinical endeavors has highlighted several additional functional roles for keratin proteins in the skin, suggested new avenues for effective therapies for keratin-based diseases, and expanded our understanding of the remarkable properties of skin as an organ system.
PMCID: PMC3279600  PMID: 22277943
23.  Prenatal diagnosis for recessive dystrophic epidermolysis bullosa in 10 families by mutation and haplotype analysis in the type VII collagen gene (COL7A1). 
Molecular Medicine  1996;2(1):59-76.
BACKGROUND: Epidermolysis bullosa (EB) is a group of heritable diseases that manifest as blistering and erosions of the skin and mucous membranes. In the dystrophic forms of EB (DEB), the diagnostic hallmark is abnormalities in the anchoring fibrils, attachment structures beneath the cutaneous basement membrane zone. The major component of anchoring fibrils is type VII collagen, and DEB has been linked to the type VII collagen gene (COL7A1) at 3p21, with no evidence for locus heterogeneity. Due to life-threatening complications and significant long-term morbidity associated with the severe, mutilating form of recessive dystrophic EB (RDEB), there has been a demand for prenatal diagnosis from families with affected offspring. MATERIALS AND METHODS: Intragenic polymorphisms in COL7A1 and flanking microsatellite markers on chromosome 3p21, as well as detection of pathogenetic mutations in families, were used to perform PCR-based prenatal diagnosis from DNA obtained by chorionic villus sampling at 10-15 weeks or amniocentesis at 12-15 weeks gestation in 10 families at risk for recurrence of RDEB. RESULTS: In nine cases, the fetus was predicted to be normal or a clinically unaffected carrier of a mutation in one allele. These predictions have been validated in nine cases by the birth of a healthy child. In one case, an affected fetus was predicted, and the diagnosis was confirmed by fetal skin biopsy. CONCLUSIONS: DNA-based prenatal diagnosis of RDEB offers an early, expedient method of testing which will largely replace the previously available invasive fetal skin biopsy at 18-20 weeks gestation.
PMCID: PMC2230038  PMID: 8900535
24.  Genetic linkage of type VII collagen (COL7A1) to dominant dystrophic epidermolysis bullosa in families with abnormal anchoring fibrils. 
Journal of Clinical Investigation  1992;89(3):974-980.
Epidermolysis bullosa (EB) in a group of genodermatoses characterized by the fragility of skin. Previous studies on the dystrophic (scarring) forms of EB have suggested abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association of the cutaneous basement membrane to the underlying dermis. Since type VII collagen is the major component of the anchoring fibrils, we examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoring fibrils. Strong genetic linkage of EBDD and COL7A1 loci was demonstrated with the maximum logarithm of odds (LOD) score of 8.77 at theta = 0. This linkage was further confirmed with two additional markers in this region of the short arm of chromosome 3, and these analyses allowed further refinement of the map locus of COL7A1. Since there were no recombinants between the COL7A1 and EBDD loci, our findings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families.
PMCID: PMC442946  PMID: 1347297
25.  Epidermolysis bullosa acquisita antigen is the globular carboxyl terminus of type VII procollagen. 
Journal of Clinical Investigation  1988;81(3):683-687.
Epidermolysis bullosa acquisita (EBA) is a severe, chronic blistering disease of the skin. EBA patients have circulating and tissue-bound autoantibodies to a large (Mr = 290,000) macromolecule that is localized within the basement membrane zone between the epidermis and dermis of skin, the site of blister formation. The "EBA antigen" is known to be distinct from laminin, heparan sulfate proteoglycan, fibronectin, the bullous pemphigoid antigen, elastin, and collagen types I, II, III, IV, and V. Sera from patients with EBA, two monoclonal antibodies to the EBA antigen, and a monoclonal antibody to the carboxyl terminus of type VII procollagen identically label human amnion and skin by immunofluorescent and immunoelectron microscopy. Western immunoblots of the EBA antigen extracted from skin and of type VII procollagen labeled with the above sera and antibodies are identical. None of the sera or antibodies labels Western blots of pepsinized type VII collagen which is missing the globular amino and carboxyl terminal domains. These data show that the EBA antigen is the carboxyl terminus of type VII procollagen.
PMCID: PMC442515  PMID: 3278005

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