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PubMed2001

Sucralfate: a help during oral management in patients with epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is a group of genetic disorders that lead to blister formation at variable depths in skin and mucosa. Vesicles may arise spontaneously or be caused by friction or trauma. Oral tissue fragility and blistering is common in all EB types. The majority of patients with mild forms of EB are able to receive dental treatment. The prevention of dental caries is most challenging in subjects with severe mucosal involvement, as they are least able to routinely undergo correct preventive procedures. The aim of this study was to evaluate the effectiveness of a sucralfate suspension in reducing both pain and the number of blisters in patients with EB, and to obtain improved oral hygiene and a lower incidence of caries. METHODS: Five patients with dystrophic EB were treated with sucralfate suspension for the prevention and management of oral blisters. Oral blisters were assessed using a quantitative scale, while pain was assessed using visual analogue scale (VAS), and hygiene was evaluated through plaque and gingival indexes. RESULTS: The number of blisters, oral pain, and plaque decreased in all cases. CONCLUSIONS: Oral prophylaxis with sucralfate prevented oral blisters and oral discomfort. The procedure proved to be cost effective and easy to administer. It did not show significant side effects and may be used routinely in patients with EB.

Anti-Ulcer Agents↗

PubMed1999

Ocular involvement in IgA-epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is an autoimmune bullous disease with frequent ocular involvement, but visual loss is rare. In contrast, EBA patients with predominant IgA autoantibodies more frequently develop severe ocular involvement, which tends to be refractory to therapy. We report two patients with 'IgA-EBA' with ocular involvement. Both initially presented with a generalized bullous disease, and direct immunofluorescence microscopy demonstrated IgA in the basement membrane zone of the skin, and in the conjunctiva and cornea of patient 1. On salt-split patient skin, IgA was found predominantly on the dermal side of the artificial split in both patients. Direct immunoelectron microscopy demonstrated IgA below the lamina densa in close association with the anchoring fibrils in both patients. In patient 1, who had a prolonged course of the disease, the skin disorder responded well to treatment with cyclosporin, but the ocular involvement ended in bilateral blindness despite repeated surgical treatment. In patient 2, the blister formation and scarring conjunctivitis was stopped by a combination of prednisolone and colchicine. These patients show that in subepithelial blistering diseases, early delineation of disease nosology is critical to detect subtypes with severe ocular involvement such as 'IgA-EBA'. In addition, colchicine may be a valuable alternative in the treatment of EBA with ocular involvement.

Adult↗

PubMed2026

An isogenic hiPSC-derived keratinocyte model reveals CXCL10/CXCL11 inflammatory dysregulation in epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a genetic skin disorder driven by dominant pathogenic variants in KRT5 or KRT14 genes, leading to cytoskeletal fragility in basal keratinocytes and intraepidermal blistering. No curative therapies are currently available, and the link between keratin mutations and disease mechanisms remains incompletely understood. To further investigate the inflammatory component of EBS, we used a model of hiPSC-derived keratinocytes carrying dominant KRT5 variants, alongside a genetically corrected isogenic counterpart. This approach established a direct link between the KRT5 variants and keratin aggregation, impaired proliferation, and an inflammatory phenotype. The inflammatory signature was confirmed by increased expression of IL1A and IL1B, consistent with previous observations in EBS, while CXCL10 and CXCL11 emerged as newly identified dysregulated chemokines. Their consistent increase across independent cell lines, elevated secretion, and normalization in the CRISPR-corrected isogenic cells indicate that KRT5 variants trigger a keratinocyte-intrinsic CXCL10/CXCL11 inflammatory response. Pharmacological inhibition of the IFN-γ-JAK1/2-STAT1 pathway suppressed their secretion, supporting JAK inhibition as a potential therapeutic strategy to modulate EBS-associated inflammatory dysregulation. In conclusion, this study shows that beyond structural defects, KRT5 variants establish a keratinocyte-intrinsic inflammatory phenotype in which the CXCL10 and CXCL11 axis emerges as a key disease-associated signature and a promising therapeutic target.

CXCL10/CXCL11↗

PubMed2003

Acellular dermal matrix allograft used to gain attached gingiva in a case of epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is an acquired disease or inherited as either autosomal dominant or recessive with an incidence of 1/50,000. The prominent clinical characteristic of the disease is the development of bullae or vesicles in mucosa or skin in response to minor trauma. AIM: A female patient with a dystrophic type of EB had been put in a maintenance regimen after completion of the initial phase of periodontal therapy and followed for 7 years. The purpose of this report is to document acellular dermal matrix allograft application to increase the width of the attached gingiva in this patient experiencing difficulty in chewing and performing plaque control due to the dramatic loss of attached gingiva after 7 years of supportive periodontal therapy. METHODS: Under local anaesthesia and antibiotic coverage, the acellular dermal matrix allograft was applied in the anterior region of the upper jaw in order to increase the width of attached gingiva, thereby improving patient comfort. RESULTS: The healing was uneventful and a significant gain in attached gingiva dimensions was observed 9 months after the periodontal surgery. The procedure avoided a second surgical site, provided satisfactory results from an aesthetic point of view, and improved patient comfort. CONCLUSION: Acellular dermal matrix allograft may be regarded as an alternative in the treatment of EB cases to increase the width of attached gingiva and facilitate maintenance of the dentition.

Adolescent↗

PubMed2000

Development and characterization of a recombinant truncated type VII collagen "minigene". Implication for gene therapy of dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder of skin caused by mutations in the type VII collagen gene. The lack of therapy for DEB provides an impetus to develop gene therapy strategies. However, the full-length 9-kilobase type VII collagen cDNA exceeds the cloning capacity of current viral delivery vectors. In this study, we produced a recombinant type VII minicollagen containing the intact noncollagenous domains, NC1 and NC2, and part of the central collagenous domain using stably transfected human 293 cell clones and purified large quantities of the recombinant minicollagen VII from culture media. Minicollagen VII was secreted as correctly-folded, disulfide-bonded, helical trimers resistant to protease degradation. Purified minicollagen VII bound to fibronectin, laminin-5, type I collagen, and type IV collagen. Furthermore, retroviral-mediated transduction of the minigene construct into DEB keratinocytes (in which type VII collagen was absent) resulted in persistent synthesis and secretion of a 230-kDa recombinant minicollagen VII. In comparison with parent DEB keratinocytes, the gene-corrected DEB keratinocytes demonstrated enhanced cell-substratum adhesion, increased proliferative potential, and reduced cell motility, features that reversed the DEB phenotype toward normal. We conclude that the use of the minicollagen VII may provide a strategy to correct the cellular manifestations of gene defects in DEB.

Cell Line↗

PubMed1993

The genetic basis of Weber-Cockayne epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical-stress-induced degeneration of basal epidermal cells. Recently, it was discovered that the more severe types, Dowling-Meara and Koebner, are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here, we show that the mildest type of EBS, Weber-Cockayne, is also a disorder of these keratins. Affected members of two unrelated families with Weber-Cockayne EBS had a T-->G point mutation in the second base position of codon 161 of one of two K5 alleles, leading to an Ile-->Ser mutation. This mutation was not present in unaffected members or in 156 alleles from normal individuals. Linkage analyses mapped the defect to the type II keratin gene cluster on chromosome 12q11-q13 (peak logarithm of odds score at theta = 0 of 3.0), providing strong additional evidence that this mutation is responsible for the Weber-Cockayne EBS phenotype. Conserved among type II keratins, Ile-161 is in the nonhelical head domain of K5, a region previously shown to be important for 10-nm filament assembly. The mutation generates a potential substrate site for protein kinase C, which could influence intermediate filament architecture, perhaps leading to the intrafilament association seen ultrastructurally in patients with the mutation.

Amino Acid Sequence↗

PubMed2004

Laminin-5 mutational analysis in an Italian cohort of patients with junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a rare genodermatosis characterized by dermal-epidermal separation that is caused by mutations in the genes encoding hemidesmosomal components and laminin-5, the major epithelial adhesion ligand. Here, we report on the mutational analysis of LAMA3, LAMB3, and LAMC2 genes encoding laminin-5 chains in 19 Italian patients, 11 affected with the severe Herlitz (H JEB) and eight with the mild non-Herlitz variant of JEB (non-H JEB). Eighteen mutations, seven of which were novel, were identified and their consequences analyzed at the mRNA and protein level. Premature termination codon mutations in both alleles of LAMB3 or LAMC2 genes were found in nine of the 11 H JEB patients, with a prevalence of mutations in LAMC2. In one case, a homozygous frameshift mutation in LAMB3 was associated to illegitimate splicing leading to non-H JEB. One H JEB patient showed a large intragenic duplication within LAMC2, a genetic defect so far uncovered in laminin-5 genes. Splicing or missense mutations, were prevalent in non-H JEB patients. Collectively, five mutations appeared to be frequent in laminin-5 JEB patients: R635X, 29insC, E210K, W143X in LAMB3 and R95X in LAMC2. These recurrent mutations account for approximately 44% of laminin-5 JEB alleles in Italian patients.

Base Sequence↗

PubMed2002

Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders caused by mutations in the human type VII collagen gene (COL7A1). Individuals with DEB lack type VII collagen and anchoring fibrils, structures that attach epidermis and dermis. The current lack of treatment for DEB is an impetus to develop gene therapy strategies that efficiently transfer and stably express genes delivered to skin cells in vivo. In this study, we delivered and expressed full-length type VII collagen using a self-inactivating minimal lentivirus-based vector. Transduction of lentiviral vectors containing the COL7A1 transgene into recessive DEB (RDEB) keratinocytes and fibroblasts (in which type VII collagen was absent) resulted in persistent synthesis and secretion of type VII collagen. Unlike RDEB parent cells, the gene-corrected cells had normal morphology, proliferative potential, matrix attachment and motility. We used these gene-corrected cells to regenerate human skin on immune-deficient mice. Human skin regenerated by gene-corrected RDEB cells had restored expression of type VII collagen and formation of anchoring fibrils at the dermal-epidermal junction in vivo. These studies demonstrate that it is possible to restore type VII collagen gene expression in RDEB skin in vivo.

Cell Adhesion↗

PubMed1993

Epidermolysis bullosa simplex.

Epidermolysis Bullosa Simplex (EBS) is a genetic disorder usually characterized by an autosomal dominant mode of transmission in which the skin blisters in response to trivial mechanical trauma. There are several clinical variants of EBS, ranging from clinically mild to very severe and even lethal, but in all cases the primary lesion responsible for the blistering is trauma-induced lysis of the epidermal basal layer. Epidermal basal cells normally feature an extensive cytoplasmic network of 10 nm filaments made of keratins K5 and K14, and the architecture of this network is often perturbed in the epidermis of EBS patients. The recent advent of a variety of molecular genetic techniques has allowed us to study the effects of perturbing the keratin filament network in epidermal cells in situ, and test the possible implications for EBS. Thus, targeted expression of K14 mutants which disrupt 10 nm-filament assembly in the epidermal basal layer of transgenic mice causes a phenotype mimicking EBS remarkably well, suggesting that at least some cases of EBS might arise as a result of mutations in basal-specific keratin genes. Indeed, point mutations in either the K5 or K14 coding sequence have recently been discovered in several incidences of EBS, and compelling evidence that these mutations are indeed responsible for the disease has been provided. These recent findings and their implication for the function of 10 nm keratin filaments in epidermis are discussed in this article.

Animals↗

PubMed1997

Maternal uniparental disomy of chromosome 1 with reduction to homozygosity of the LAMB3 locus in a patient with Herlitz junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive disorder characterized by blister formation at the level of the lamina lucida within the cutaneous basement-membrane zone. Classic lethal JEB (Herlitz type [H-JEB]; OMIM 226700) is frequently associated with premature-termination-codon mutations in both alleles of one of the three genes (LAMA3, LAMC2, or LAMB3) encoding the subunit polypeptides (alpha3, beta3, and gamma2) of laminin 5. In this study, we describe a unique patient with H-JEB, who was homozygous for a nonsense mutation, Q243X, in the LAMB3 gene on chromosome 1 and who had normal karyotype 46,XY. The mother was found to be a carrier of the Q243X mutation, whereas the father had two normal LAMB3 alleles. Nonpaternity was excluded by use of 11 microsatellite markers from six different chromosomes. The use of 17 partly or fully informative microsatellite markers spanning the entire chromosome 1 revealed that the patient had both maternal uniparental meroisodisomy of a 35-cM region on 1q containing the maternal LAMB3 mutation and maternal uniparental heterodisomy of other regions of chromosome 1. Thus, the results suggested that reduction to homozygosity of the 1q region containing the maternal LAMB3 mutation caused the H-JEB phenotype. The patient was normally developed at term and did not show overt dysmorphisms or malformations. This is the first description of uniparental disomy of human chromosome 1.

Alleles↗

PubMed2006

Fibroblasts show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-transferred cells can most efficiently express collagen VII in the skin. We demonstrated that gene transfer using a combination of G protein of vesicular stomatitis virus-pseudotyped retroviral vector and retronectin introduced COL7A1 into keratinocytes and fibroblasts from a DEB patient with the lack of COL7A1 expression. Real-time polymerase chain reaction analysis of the normal human skin demonstrated that the quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-transferred fibroblasts supplied higher amount of collagen VII to the new dermal-epidermal junction than the gene-transferred keratinocytes. An ultrastructural study revealed that collagen VII from gene-transferred cells formed proper anchoring fibrils. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes.

Animals↗

PubMed2000

DNA based prenatal testing for the skin blistering disorder epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a skin fragility disorder in which mild physical trauma leads to blistering. The phenotype of the disorder is variable, from relatively mild affecting only the hands and/or feet, to very severe with widespread blistering. For the severest forms of EBS there is a demand for prenatal diagnosis which until now has involved a fetal skin biopsy in the second trimester. The identification of mutations in the genes encoding keratins K5 and K14 as the cause of EBS opens up the possibility of much earlier diagnosis of the disease. We report here four cases in which prenatal testing was performed. In three of the cases the genetic lesions were unknown at the start of the pregnancy, requiring the identification of the causative mutation prior to testing fetal DNA. In two of the four cases novel mutations were identified in K14 and in the two remaining families, a previously identified type of mutation was found. Fetal DNA, obtained by chorionic villus sampling or amniocentesis, was analysed for the identified mutations. Three of the DNA samples were found to be normal; a mutant K14 allele was identified in the fourth case and the pregnancy was terminated. These results demonstrate the feasibility of DNA-based prenatal testing for EBS in families where causative mutations can be found.

Amino Acid Sequence↗

PubMed2003

An autocrine/paracrine loop linking keratin 14 aggregates to tumor necrosis factor alpha-mediated cytotoxicity in a keratinocyte model of epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a blistering cutaneous disease featuring protein aggregates. Here we investigate the molecular mechanisms linking protein aggregates to cell death in a cellular model of EBS in which HaCaT keratinocytes are transfected with plasmids expressing various mutant forms of keratin 14 (K14). In HaCaT cells, mutant K14 was found to form ubiquitinated protein aggregates that suppressed 20 S proteasome function instead of being degraded by 20 S proteasome. Keratinocytes with mutant K14-induced phosphorylation of the stress-activated kinase c-Jun, as well as up-regulation of unfolding protein Bip, indicates induction of endoplasmic reticulum stress. HaCaT cells were susceptible to apoptosis by activation of caspases-3, and -8, but not caspase-9 or -12. Tumor necrosis factor-alpha (TNFalpha) in the culture medium was increased in keratinocytes with mutant K14 compared with wild K14, and the addition of neutralizing anti-TNFalpha antibody to the culture medium rescued keratinocytes from cell death. Thus, TNFalpha release and the subsequent activation of the TNFalpha receptor by an autocrine/paracrine pathway links protein aggregates to cell death in this keratinocyte EBS cellular model. Furthermore, mutation in K14 reduced its affinity to TNFalpha receptor-associated death domain (TRADD), suggesting that the susceptibility of keratinocytes to caspase-8-mediated apoptosis is increased in mutated K14 because of impairment of the cytoprotective mechanism mediated by K14-TRADD interaction.

Annexin A5↗

PubMed1997

Identification of a glycine substitution and a splice site mutation in the type VII collagen gene in a proband with mitis recessive dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a genodermatosis characterized by fragility of the skin and mucous membranes. Underlying mutations in the DEB phenotype have been detected in the gene encoding type VII collagen (COL7A1), both in the dominant and recessive forms of DEB. In this study, we searched for mutations in a proband with a mild form of DEB by PCR amplification of segments of COL7A1, followed by heteroduplex analysis. Examination of PCR fragments corresponding to exons 3-4 and exons 51-53 revealed heteroduplexes. Direct sequencing of the PCR fragment containing exon 3 revealed a previously reported A-to-G transition in the 5' donor splice site of exon 3 in the proband and in the clinically unaffected father, while direct sequencing of the PCR fragment containing exon 53 revealed a novel glycine substitution G1652R in the proband and in the clinically unaffected mother. Patients with relatively mild DEB and no family history are frequently diagnosed as a de novo case of dominant DEB, although a mild case of RDEB cannot be excluded on the basis of clinical and ultrastructural examination. We proved this case to be a recessively inherited disease. This information had a profound impact on the genetic counselling, because if the disease of the patient were to have had a new dominant mutation, he would have been counselled that the risk of his offspring being affected was one in two, but he could be accurately counselled that the risk of this offspring being affected was as low as the general population.

Adult↗

PubMed2000

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.

Cells, Cultured↗

PubMed1999

A recurrent frameshift mutation in exon 19 of the type VII collagen gene (COL7A1) in Mexican patients with recessive dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder caused by mutations in the type VII collagen gene (COL7A1). In this study, we determined the molecular basis of autosomal recessive DEB in a 19-year-old Hispanic Mexican woman by PCR amplification of genomic DNA, heteroduplex analysis, and automated sequencing of heteroduplex bandshifts. This approach revealed a homozygous frameshift mutation, 2470insG, in exon 19 of COL7A1 and resulted in attenuated basement membrane zone expression of type VII collagen, a reduced number of anchoring fibrils at the dermal-epidermal junction, and a sub-lamina densa level of blister formation. Clinically, the patient had widespread trauma-induced skin fragility and complete loss of the nails, but had less pseudosyndactyly of the fingers and toes and milder mucosal involvement compared to most patients with the generalized form of this genodermatosis. We also screened 7 other Hispanic-Mexican patients with recessive DEB, none of whom were known to be related to this individual, for the mutation 2470insG using heteroduplex analysis and direct sequencing and detected this mutation on 7/14 alleles. Haplotype analysis using intragenic COL7A1 and flanking polymorphisms and microsatellite markers revealed that all the mutant alleles had arisen on similar allelic backgrounds, consistent with propagation of a common Hispanic Mexican ancestral haplotype. In view of the high allelic frequency of the mutation 2470insG in the patients studied, we recommend initial screening for this mutation when attempting to identify the molecular pathology of recessive DEB in Hispanic Mexican patients.

Adult↗

PubMed1998

Frameshift mutations in the type VII collagen gene (COL7A1) in five Mexican cousins with recessive dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the type VII collagen gene (COL7A1). In this study, we assessed the molecular basis of recessive DEB in five affected individuals from two Mexican families. Both fathers of the affected children were first cousins. Genomic DNA was extracted from peripheral blood samples and assessed for COL7A1 mutations by polymerase chain reaction (PCR) amplification, heteroduplex analysis and direct automated sequencing of PCR products displaying heteroduplex bandshifts. In one family, we identified a homozygous 1 bp insertion of a G nucleotide in exon 19 of COL7A1, designated 2470insG, in three affected sisters. This mutation causes a frameshift and a premature termination codon on both alleles 178 bp downstream from the insertion; both parents were shown to be heterozygous carriers of this mutation. In the second family, the father of the other two affected children was also found to be a heterozygous carrier of this frameshift mutation. In addition, his unrelated partner was shown to be a heterozygous carrier of a different COL7A1 frameshift mutation, an insertion of a T nucleotide in exon 32, designated 3948insT. This mutation also results in a premature termination codon, 126 bp downstream from the insertion. Both affected children were compound heterozygotes for the 2470insG/3948insT mutations in COL7A1. Overall, these molecular findings offer a genetic explanation for the skin fragility in these related Mexican patients with recessive DEB. Immediate benefits from elucidation of the mutations include assessment of carrier status in other members of the family and the feasibility of DNA-based prenatal testing in subsequent pregnancies.

Adolescent↗

PubMed1999

Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) arises from mutations within the keratin 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocytes cytoskeleton. The majority of these defects are missense mutations in the rod domain, whose locations influence the disease severity. We investigated a large family dominantly affected with the Dowling-Meara form of EBS (EBS-DM). Sequencing of amplified and cloned K5 cDNA from cultured keratinocytes revealed a 66 nucleotide deletion in one allele corresponding to the last 22 amino acid residues encoded by exon 1 (Val164 to Lys185). Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygous G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5. This mutation leads to the use of an exonic GT cryptic donor splice site, located 66 nucleotides upstream from the normal donor splice site of intron 1. The corresponding peptide deletion includes the last five amino acids of the H1 head domain and the first 17 amino acids of the conserved amino terminal end of the 1A rod domain, including the first two heptad repeats and the helix initiation peptide. The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein. This is the first splice site mutation to be reported as a cause of EBS-DM. Owing to the functional importance of the removed region, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.

Alternative Splicing↗