Junctional Epidermolysis Bullosa Patients Research Articles

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.

Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer

Sustainable correction of severe human genetic disorders of self-renewing tissues, such as the blistering skin disease junctional epidermolysis bullosa (JEB), is facilitated by stable genomic integration of therapeutic genes into somatic tissue stem cells. While integrating viral vectors can achieve this, they suffer from logistical and biosafety concerns. To circumvent these limitations, we used the Sleeping Beauty transposable element to integrate the LAMB3 cDNA into genomes of epidermal holoclones from six unrelated JEB patients. These cells regenerate human JEB skin that is normalized at the level of laminin 5 protein expression, hemidesmosome formation and blistering. Transposon-mediated gene delivery therefore affords an opportunity for stable gene delivery in JEB and other human diseases.

PhiC31 integrase-mediated nonviral genetic correction of junctional epidermolysis bullosa.

Patients afflicted with severe laminin 5-deficient junctional epidermolysis bullosa (JEB) often die in infancy with massive cutaneous blistering. Prior approaches to genetically correct this disorder have relied on stable integration of wild-type LAMB3 sequences, using retroviral vectors. To develop a nonviral approach to JEB gene therapy, we used the phiC31 integrase, which mediates unidirectional genomic integration of plasmids containing a specific attB site. An attB-containing laminin 5 beta3 expression plasmid was integrated into the genomes of primary keratinocytes from four unrelated, genetically characterized JEB patients. phiC31 integrase supported genomic integration into epidermal progenitor cells. Regeneration of human skin on immunedeficient mice, using these cells, produced human skin tissue with restored laminin 5 expression. Furthermore, corrected JEB tissue restored hemidesmosome formation and abolished histologic evidence of subepidermal blistering. These findings provide an approach to durable nonviral correction of JEB.

Animal Models for Skin Blistering Conditions: Absence of Laminin 5 Causes Hereditary Junctional Mechanobullous Disease in the Belgian Horse

Recent achievements in the genetic correction of keratinocytes isolated from patients with junctional epidermolysis bullosa have paved the way to a gene therapy approach for the disease. Because gene therapy protocols require preclinical validation in animals, we have characterized spontaneous animal models of junctional epidermolysis bullosa. In this study we have elucidated the genetic basis of the hereditary junctional mechanobullous disease in the Belgian horse, a condition characterized by blistering of the skin and mouth epithelia, and exungulation (loss of the hoof). Immunofluorescence analysis associated the condition to the absent expression of the gamma2 chain of laminin 5 and designated Lamc2 as the candidate gene. Comparative analysis of the nucleotide sequence of the full-length gamma2 cDNA isolated by reverse transcription polymerase chain reaction amplification of total RNA purified from the epithelium of a junctional epidermolysis bullosa foal and a healthy control disclosed a homozygous basepair insertion (1368insC) in the affected animal. Mutation 1368insC results in a downstream premature termination codon and is predicted to cause absent expression of the laminin gamma2 polypeptide. Our results also show that: (i) the horse junctional epidermolysis bullosa genetically corresponds to the severe Herlitz form of junctional epidermolysis bullosa in man; (ii) the amino acid sequence and structure of the horse laminin gamma2 chain are virtually identical to the human counterpart; (iii) the moderate eruption of skin blisters in the affected animals with respect to the human Herlitz junctional epidermolysis bullosa patients correlates with the protection provided by hair. Our observations suggest that the affected foals are a convenient source of epithelial cells from tissues that cannot be obtained from human junctional epidermolysis bullosa patients, and imply that hairless strains of animals with recessive skin disorders would be the best models for in vivo gene therapy approaches to skin blistering diseases.

Novel Mutations in the LAMC2 Gene in Non-Herlitz Junctional Epidermolysis Bullosa: Effects on Laminin-5 Assembly, Secretion, and Deposition

Laminin-5 is the major adhesion ligand of epithelial cells. Mutations in the three genes (LAMA3, LAMB3, LAMC2) encoding the laminin-5 chains cause junctional epidermolysis bullosa, a clinically and genetically heterogeneous blistering skin disease. Here, we describe a non-Herlitz junctional epidermolysis bullosa patient, compound heterozygote for two novel mutations affecting the LAMC2 gene. The mutation in the paternal allele is a de novo splice site mutation (522-1G-->A) that results in in-frame skipping of exon 4 and synthesis of a mutated gamma2 polypeptide (gamma2Delta4) carrying a 33 amino acid deletion within the N-terminal domain V. The maternal mutation is a one base pair insertion (3511insA) in the 3' terminal exon of LAMC2 resulting in a frameshift and a premature termination codon. Mutation 3511insA is predicted to lead to the synthesis of a gamma2 polypeptide (gamma2t) disrupted in its alpha-helical C-terminal structure and truncated of the last 25 amino acids. Keratinocytes isolated from the patient's skin showed a markedly decreased level of gamma2 chain mRNA and secreted scant amounts of laminin-5, which undergoes physiologic proteolytic processing. To investigate the biologic function of the laminin-5 molecules synthesized by the patient, mutant gamma2 cDNAs were transiently expressed in gamma2-null keratinocytes. Transfection of the gamma2Delta4 cDNA resulted in restoration of laminin-5 deposition onto the culture substrate, which demonstrates that the gamma2 polypeptides carrying a deletion in domain V, upstream of the gamma2 proteolytic cleavage site, are assembled into native laminin-5 that is secreted and extracellularly processed. In contrast, transfection of a mutant cDNA expressing the gamma2t chain failed to restore laminin-5 immunoreactivity, which indicates that integrity of the gamma2 C-terminal amino acid sequences is required for laminin-5 assembly. These results correlate for the first time a functional alteration in a laminin-5 domain with a mild junctional epidermolysis bullosa phenotype.

In vivo restoration of laminin 5 beta 3 expression and function in junctional epidermolysis bullosa.

The blistering disorder, lethal junctional epidermolysis bullosa (JEB), can result from mutations in the LAMB3 gene, which encodes laminin 5 beta3 (beta3). Appropriate expression of LAMbeta3 in JEB skin tissue could potentially ameliorate the symptoms of the underlying disease. To explore the utility of this therapeutic approach, primary keratinocytes from six unrelated JEB patients were transduced with a retroviral vector encoding beta3 and used to regenerate human skin on severe combined immunodeficient (SCID) mice. Tissue regenerated from beta3-transduced JEB keratinocytes produced phenotypically normal skin characterized by sustained beta3 expression and the formation of hemidesmosomes. Additionally, beta3 gene transfer corrected the distribution of a number of important basement membrane zone proteins including BPAG2, integrins beta4/beta1, and laminins alpha3/gamma2. Skin produced from beta3-negative (beta3[-]) JEB cells mimicked the hallmarks of the disease state and did not exhibit any of the aforementioned traits. Therefore, by effecting therapeutic gene transfer to beta3-deficient primary keratinocytes, it is possible to produce healthy, normal skin tissue in vivo. These data support the utility of gene therapy for JEB and highlight the potential for gene delivery in the treatment of human genetic skin disease.

Toward epidermal stem cell-mediated ex vivo gene therapy of junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a group of severe, inherited skin diseases caused by mutations in the genes encoding laminin 5 or other components of the hemidesmosome. Since human epidermis is a self-renewing tissue, gene therapy of JEB requires the stable integration of the transgene into the genome of the epidermal stem cell. Human epidermal stem cells can indeed be cultivated and stably transduced with replication-defective retroviral vectors, allowing full phenotypic correction of the adhesion properties of JEB keratinocytes. Epidermal stem cells generate cohesive sheets of stratified epithelium suitable for the permanent coverage of massive skin defects, and genetically modified epidermal sheets maintain long-term expression of the transgene after transplantation on immunodeficient animals. Moreover, we have developed a clinical procedure that allows transplantation of cultured epidermal sheets on large body areas under local anesthesia and without cicatricial outcomes. Thus, (1) the possibility of cultivating lining epithelia, (2) the availability of noninvasive surgical procedures that allow the grafting of large skin areas, and (3) the demonstration of sustained transgene expression in vitro and in vivo by epidermal stem cells, prompt us to propose the implementation of a phase I/II clinical trial aimed at the ex vivo gene therapy of selected JEB patients. The aim of the trial is to validate the ex vivo procedure in a clinical setting, to prove its overall safety, and to analyze critical issues such as long-term survival of the genetically modified implant, immune response against the transgene product, and persistence of transgene expression at therapeutic levels.

Hemizygosity for a Glycine Substitution in Collagen XVII: Unfolding and Degradation of the Ectodomain

Defects of collagen XVII, a keratinocyte adhesion protein, are associated with epidermal detachment in junctional epidermolysis bullosa. Although some missense mutations in the collagen XVII gene COL17A1 have been described, the molecular mechanisms leading to disease have remained elusive in these cases. Here we assessed the biologic consequences of a missense mutation by studying the folding and stability of wild-type and mutated recombinant collagen XVII domains. The mutation occurred in a junctional epidermolysis bullosa patient who was compound heterozygous for the novel glycine substitution mutation G633D and the novel nonsense mutation R145X. Collagen XVII mRNA was significantly reduced, indicating nonsense-mediated mRNA degradation and hemizygosity of the patient for the G633D substitution. As glycine residues within the collagen triple helices are important for stable conformation, the thermal stability of the wild-type and mutated eukaryotic recombinant Col15 domain of collagen XVII was assessed. The stability of the mutated fragment was clearly reduced. The midpoint of the helix-to-coil transition, Tm, was 5 degrees C lower than that of wild-type rCol15, indicating abnormal triple-helix folding and susceptibility to proteolysis. Consistently, immunoassays demonstrated reduced amounts of the full-length collagen XVII and absence of the soluble ectodomain in keratinocyte cultures, and lack of the ectodomain from the junctional epidermolysis bullosa skin. These observations show that the glycine substitution G633D in collagen XVII causes abnormal folding and susceptibility to degradation, and thus perturbs the physiologic adhesive functions of collagen XVII in the skin.

Moderation of Phenotypic Severity in Dystrophic and Junctional Forms of Epidermolysis Bullosa Through In-Frame Skipping of Exons Containing Non-Sense or Frameshift Mutations

Non-sense mutations on both alleles of either the type VII collagen gene (COL7A1) or the genes encoding laminin 5 (LAMA3, LAMB3, or LAMC2) usually result in clinically severe forms of recessive dystrophic or junctional epidermolysis bullosa, respectively. In this study we assessed two unrelated families whose mutations in genomic DNA predicted severe recessive dystrophic epidermolysis bullosa or junctional epidermolysis bullosa phenotypes but in whom the manifestations were milder than expected. The recessive dystrophic epidermolysis bullosa patients had a homozygous single base-pair frameshift mutation in exon 19 of COL7A1 (2470insG). Clinically, there was generalized blistering but only mild scarring. Skin biopsy revealed positive type VII collagen immunoreactivity and recognizable anchoring fibrils. The junctional epidermolysis bullosa patients were compound heterozygotes for a frameshift/non-sense combination of mutations in exons 3 and 17 of LAMB3 (29insC/Q834X). These patients did not have the lethal form of junctional epidermolysis bullosa but, as adults, displayed the milder generalized atrophic benign epidermolysis bullosa variant. There was undetectable laminin 5 staining at the dermal-epidermal junction using an antibody to the beta3 chain, but faintly positive alpha3 and gamma2 chain labeling, and there was variable hypoplasia of hemidesmosomes. To explain the milder recessive dystrophic epidermolysis bullosa and junctional epidermolysis bullosa phenotypes in these families, reverse transcription-polymerase chain reaction, using RNA extracted from frozen skin, was able to provide evidence for some rescue of mutant mRNA transcripts with restoration of the open- reading frame. In the recessive dystrophic epidermolysis bullosa patients, transcripts containing in-frame skipping of exon 19 of COL7A1 in the cDNA were detected, and in the junctional epidermolysis bullosa patients transcripts with in-frame skipping of exon 17 of LAMB3 were identified. The truncated proteins encoded by these transcripts are expected to lack certain critical domains involved in cell-matrix attachment, but may still be able to contribute to adhesion thereby moderating the severity of the skin blistering. This study shows the limitations in predicting phenotype in epidermolysis bullosa solely based on mutation analysis of genomic DNA and emphasizes the importance of immunohistochemistry, electron microscopy, and mRNA assessment as parallel investigations.

Extent of laminin-5 assembly and secretion effect junctional epidermolysis bullosa phenotype.

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 alpha3beta3gamma2 heterotrimer assembles intracellularly via a beta3gamma2 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.

Detection of Novel LAMC2 Mutations in Herlitz Junctional Epidermolysis Bullosa

Laminin 5, an anchoring filament attachment protein within the lamina lucida of the basement membrane zone involved in the pathogenesis of junctional epidermolysis bullosa (JEB), consists of three polypeptide subunits, the alpha 3, beta 3, and gamma 2 chains which are encoded by the LAMA3, LAMB3, and LAMC2 genes, respectively. To facilitate identification of pathogenetic mutations in LAMC2, a strategy based on direct amplification of genomic DNA by PCR or mRNA by RT-PCR, followed by heteroduplex analysis of the PCR products, was developed. Primer pairs for amplification of the complete cDNA as well as the 23 individual exons in the genomic DNA, which encode the entire gamma 2 chain of laminin 5, were established. The primers for amplification of exons from genomic DNA were positioned at least 24 bp away from the intron-exon borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of the entire coding sequence of LAMC2 mRNA were used. The amplified sequences were scanned for pathogenetic mutations and sequence variations in JEB patients and unrelated control individuals by heteroduplex analysis by means of conformation sensitive gel electrophoresis (CSGE). Utilizing the strategy developed in this study, we identified pathogenetic mutations in three patients with the Herlitz (lethal) variant of JEB, and eight intragenic normal polymorphisms, which are useful for linkage analysis, in the LAMC2 gene. The methodology described in this study is capable of detecting single-base substitutions or small insertions and deletions in the LAMC2 gene. Demonstration of mutations in this gene in JEB patients further emphasizes the role of laminin 5 in providing integrity to the cutaneous basement membrane zone.

Defective Integrin α6β4 Expression in the Skin of Patients With Junctional Epidermolysis Bullosa and Pyloric Atresia

Junctional epidermolysis bullosa (JEB) is a heterogeneous group of rare inherited skin disorders in which defects in cell adhesion components cause incomplete formation of hemidesmosomes. We have immunohistochemically examined the skin from ten JEB patients (JEB gravis, n = 4; JEB mitis, n = 3; JEB plus pyloric atresia [JEB/PA], n = 3) using monoclonal antibodies specific for the integrin adhesion receptors alpha 3 beta 1 and alpha 6 beta 4, and for the alpha 3, beta 3, and gamma 2 subunits of the basement membrane ligand, laminin 5. Consistent with our previous reports, only the JEB gravis patients without associated pyloric atresia expressed reduced or absent epitopes for laminin-5 subunits in their epidermal basement membrane. In contrast, all three JEB/PA cases showed abnormalities in integrin alpha 6 beta 4, but not in laminin 5, expression in their basal epidermal cells. Integrin beta 4 subunit was undetectable in the biopsied epidermis of these JEB/PA individuals using five different monoclonal antibodies that recognize both intra- and extracellular epitopes. The absence of the beta 4 subunit in the epidermis of JEB/PA specimens was confirmed by Western blot analysis of tissue extracts. Epidermal expression of the integrin alpha 6 subunit was variable in the JEB/PA patients. Abnormal integrin alpha 6 beta 4 expression may define the subset of JEB cases with pyloric atresia. These results strongly implicate a functional role for alpha 6 beta 4 in the formation of complete hemidesmosomes and in stable adhesion of basal keratinocytes to the basement membrane in vivo.

180-kD bullous pemphigoid antigen (BP180) is deficient in generalized atrophic benign epidermolysis bullosa.

Generalized atrophic benign epidermolysis bullosa (GABEB) is a form of nonlethal junctional epidermolysis bullosa characterized by universal alopecia and atrophy of the skin. We report a deficiency of the 180-kD bullous pemphigoid antigen in three patients with GABEB from unrelated families. We screened specimens of clinically normal skin from nine junctional epidermolysis bullosa patients (3 GABEB, 4 lethal, 1 cicatricial, 1 pretibial) by immunofluorescence using monoclonal antibodies to the 180-kD and 230-kD bullous pemphigoid antigens (BP180 and BP230). In the skin of the three GABEB patients there was no reactivity with antibodies to BP180, whereas staining for BP230 was normal. In the skin of the other six, non-GABEB patients, included in this study the expression of BP180 and BP230 was normal. Immunoblot analysis of cultured keratinocytes from one of the GABEB patients also failed to detect BP180 antigen, whereas BP230 was present in normal amounts. The deficient expression of BP180 is reflected in the RNA message, as in Northern blot analysis a reduced amount of BP180 transcripts, although of normal length, were detected. Interestingly, in another GABEB patient there were not-involved areas of skin, in which blistering could not be induced by rubbing. Biopsy material from these areas showed interrupted staining for BP180. There was no staining for BP180 in areas of clinically normal but involved skin of this patient. In conclusion, this study reveals that the BP180 antigen is deficient and the BP180 mRNA is reduced in generalized atrophic benign epidermolysis bullosa.

Kalinin is abnormally expressed in epithelial basement membranes of Herlitz's junctional epidermolysis bullosa patients.

Kalinin is an extracellular adhesion molecule specific to epithelial basement membranes (BM) identified as a component of anchoring filaments of hemidesmosomes. This heterotrimeric protein is synthesized by cultured normal human keratinocytes and is involved in cell attachment. In indirect immunofluorescence studies, the epidermal BM of patients with junctional epidermolysis bullosa (JEB) of Herlitz's type were found not to be reactive with the anti-kalinin monoclonal antibodies ka146 and K140 and displayed a decreased immunoreactivity to two anti-kalinin antibodies cross-reacting with K-laminin, an anchoring filament component recently described. The intrinsic defect of JEB keratinocytes in the synthesis of kalinin was further documented by indirect immunofluorescence on in vitro cultures of these cells. In non-Herlitz JEB patients, staining of BM was constantly detected. Impairment of expression of kalinin correlated with the lack of reactivity to the monoclonal antibody GB3, which detects the BM component nicein/BM600. These results clearly demonstrate a defect of kalinin expression in epithelial basement membranes of Herlitz JEB patients and suggest that kalinin may play a role in the pathogenesis of the disease. Further studies are in progress to define possible relationships between kalinin and nicein.

Southern Analysis of the 230-kD Bullous Pemphigoid Antigen Gene in Normal Humans, Animals, and Patients with Junctional Epidermolysis Bullosa

To begin to characterize the 230-kD bullous pemphigoid antigen (BPA) gene, we performed Southern analysis on genomic DNA with probes derived from 7 kb of cDNA that spans most of the coding region of this hemidesmosomal plaque protein. When hybridized to a 1-kb fragment of this BPA cDNA, normal human genomic DNA digested with EcoRI, BamHI, PstI, HindIII, or EcoRV showed only a single band, which was unique for each enzyme, indicating a single human gene for BPA. To determine if a related gene exists in animals, we used probes covering the full 7 kb of cDNA for Southern analysis of genomic DNA from various vertebrates. A related gene was detected in other mammals (monkey, cow, dog, rabbit, mouse, and rat) but not in chicken, frog, or fish. Under these same hybridization conditions a probe for human beta-actin could detect an actin gene in all these species. Furthermore, immunofluorescence showed that an antibody raised against portions of the 230-kD BPA bound to the epidermal basement membrane of mammals but not that of a bird or amphibian. Finally, because most patients with junctional epidermolysis bullosa (JEB) have defective hemidesmosomes in ultrastructure, and probably function, we analyzed genomic DNA from these patients. No restriction fragment length polymorphisms (RFLP) were detected when the DNA from 11 normals and 8 JEB patients (representing 16 possible defective genes) was digested with BamHI, EcoRI, or PstI and hybridized to any part of the cDNA. These findings indicate that 1) there is a single BPA gene in humans; 2) a closely related gene exists in other mammals but not birds, amphibia, or fish; and 3) gross abnormalities of the BPA gene are not characteristic of JEB patients.

Abnormal expression of hemidesmosome-like structures by junctional epidermolysis bullosa keratinocytes in vitro

Hemidesmosomes are frequently rudimentary in junctional epidermolysis bullosa (JEB), and JEB keratinocytes display abnormal attachment to the substrate in culture. Our aim was to determine whether this abnormality reflects defective hemidesmosome synthesis in vitro. Keratinocytes from five JEB patients were cultured under standard conditions. Control cultures, from four healthy males, three patients with dystrophic EB (DEB), and one patient with the simplex variant (EBS), were also examined. Post-confluent cultures were processed for transmission electron microscopy. Hemidesmosome-like structures were counted in electron micrograph montages. The number of hemidesmosome-like structures in JEB cultures (0.97 +/- 0.57, per 10 microns of basal cell membrane) was approximately 17% of the value for normal controls (5.81 +/- 3.08, P less than 0.02). The values for EBS (3.72) and DEB (3.28 +/- 1.44) were not statistically different from normal controls. In addition hemidesmosome-like structures in JEB cultures were morphologically ill-defined, compared with those from controls. This correlated with the loose apposition between JEB keratinocytes and the substrate, which appeared tight in controls. JEB keratinocytes continue to express in vitro a major phenotypic abnormality which characterizes the disease in vivo. Therefore, this model should prove useful in studies determining the pathogenesis and possible new treatments of JEB.