Impaired Mitochondrial and Metabolic Function of Fibroblasts Derived from Patients with Recessive Dystrophic and Junctional Epidermolysis Bullosa

Background: Recessive dystrophic epidermolysis bullosa (RDEB) and junctional EB (JEB) are inherited disorders characterised by fragility and blistering of epithelial tissues leading to pain, pruritus, and adherent scarring. The severity and chronic nature of the resultant skin wounds significantly reduces quality and length of life. Current therapies primarily consist of protective bandaging and nutritional supplementation; there is no cure for these disorders. Although the skin fragility results from a lack of C7 protein (RDEB) and laminin-332 (JEB), other serious aspects of these disorders, such as inflammation that interferes with healing and aggressive squamous cell carcinoma, have not been completely elucidated. Recent research has suggested that mitochondrial function plays a significant role in skin healing. Objective: To evaluate how mitochondrial function differs in patients with RDEB and JEB. Method: The energy status of RDEB and JEB patient-derived fibroblasts was determined by Seahorse analysis and metabolite production. The energetics and overall morphology of RDEB and JEB patient-derived fibroblasts were assayed as a measure of metabolic stress. Results: EB patient-derived fibroblasts showed impaired oxidative phosphorylation with concomitant compensation by glycolysis. Morphological parameters were altered in RDEB and JEB fibroblasts compared with controls. Conclusion: This is the first study to describe changes in mitochondrial energy metabolism, metabolic profile, and mitochondrial morphology of EB patients.

Download Full-text

A Novel Phenotype of Junctional Epidermolysis Bullosa with Transient Skin Fragility and Predominant Ocular Involvement Responsive to Human Amniotic Membrane Eyedrops

Genes ◽

10.3390/genes12050716 ◽

2021 ◽

Vol 12 (5) ◽

pp. 716

Author(s):

Daniele Castiglia ◽

Paola Fortugno ◽

Angelo Giuseppe Condorelli ◽

Sabina Barresi ◽

Naomi De Luca ◽

...

Keyword(s):

Amniotic Membrane ◽

Epidermolysis Bullosa ◽

Ocular Involvement ◽

Human Amniotic Membrane ◽

Vitro Assay ◽

Junctional Epidermolysis Bullosa ◽

Mucosal Involvement ◽

Skin Fragility ◽

Laminin 332

Junctional epidermolysis bullosa (JEB) is a clinically and genetically heterogeneous skin fragility disorder frequently caused by mutations in genes encoding the epithelial laminin isoform, laminin-332. JEB patients also present mucosal involvement, including painful corneal lesions. Recurrent corneal abrasions may lead to corneal opacities and visual impairment. Current treatments are merely supportive. We report a novel JEB phenotype distinguished by the complete resolution of skin fragility in infancy and persistent ocular involvement with unremitting and painful corneal abrasions. Biallelic LAMB3 mutations c.3052-5C>G and c.3492_3493delCG were identified as the molecular basis for this phenotype, with one mutation being a hypomorphic splice variant that allows residual wild-type laminin-332 production. The reduced laminin-332 level was associated with impaired keratinocyte adhesion. Then, we also investigated the therapeutic power of a human amniotic membrane (AM) eyedrop preparation for corneal lesions. AM were isolated from placenta donors, according to a procedure preserving the AM biological characteristics as a tissue, and confirmed to contain laminin-332. We found that AM eyedrop preparation could restore keratinocyte adhesion in an in vitro assay. Of note, AM eyedrop administration to the patient resulted in long-lasting remission of her ocular manifestations. Our findings suggest that AM eyedrops could represent an effective, non-invasive, simple-to-handle treatment for corneal lesions in patients with JEB and possibly other EB forms.

Download Full-text

Phenotype and Variant Spectrum in the LAMB3 Form of Amelogenesis Imperfecta

Journal of Dental Research ◽

10.1177/0022034519835205 ◽

2019 ◽

Vol 98 (6) ◽

pp. 698-704 ◽

Cited By ~ 4

Author(s):

C.E.L. Smith ◽

J.A. Poulter ◽

S.J. Brookes ◽

G. Murillo ◽

S. Silva ◽

...

Keyword(s):

Epidermolysis Bullosa ◽

Third Molar ◽

Dental Enamel ◽

Amelogenesis Imperfecta ◽

Splice Acceptor Site ◽

Acceptor Site ◽

Loss Of Function ◽

Inherited Disorders ◽

Junctional Epidermolysis Bullosa ◽

Molar Teeth

Amelogenesis imperfecta (AI) is a heterogeneous group of inherited disorders characterized by abnormal formation of dental enamel, either in isolation or as part of a syndrome. Heterozygous variants in laminin subunit beta 3 ( LAMB3) cause AI with dominant inheritance in the absence of other cosegregating clinical features. In contrast, biallelic loss-of-function variants in LAMB3 cause recessive junctional epidermolysis bullosa, characterized by life-threatening skin fragility. We identified 2 families segregating autosomal dominant AI with variable degrees of a distinctive hypoplastic phenotype due to pathogenic variants in LAMB3. Whole exome sequencing revealed a nonsense variant (c.3340G>T, p.E1114*) within the final exon in family 1, while Sanger sequencing in family 2 revealed a variant (c.3383-1G>A) in the canonical splice acceptor site of the final exon. Analysis of cDNA from family 2 revealed retention of the final intron leading to a premature termination codon. Two unerupted third molar teeth from individual IV:5 in family 2 were subject to computerized tomography and scanning electron microscopy. LAMB3 molar teeth have a multitude of cusps versus matched controls. LAMB3 enamel was well mineralized but pitted. The architecture of the initially secreted enamel was abnormal, with cervical enamel appearing much less severely affected than coronal enamel. This study further defines the variations in phenotype-genotype correlation for AI due to variants in LAMB3, underlines the clustering of nonsense and frameshift variants causing AI in the absence of junctional epidermolysis bullosa, and highlights the shared AI phenotype arising from variants in genes coding for hemidesmosome proteins.

Download Full-text

LAMB3 Missense Variant in Australian Shepherd Dogs with Junctional Epidermolysis Bullosa

Genes ◽

10.3390/genes11091055 ◽

2020 ◽

Vol 11 (9) ◽

pp. 1055 ◽

Cited By ~ 1

Author(s):

Sarah Kiener ◽

Aurore Laprais ◽

Elizabeth A. Mauldin ◽

Vidhya Jagannathan ◽

Thierry Olivry ◽

...

Keyword(s):

Basement Membrane ◽

Epidermolysis Bullosa ◽

Clinical Signs ◽

Autosomal Recessive Inheritance ◽

Missense Variant ◽

Loss Of Function ◽

Junctional Epidermolysis Bullosa ◽

Skin Fragility ◽

Close Relatives ◽

Tentative Diagnosis

In a highly inbred Australian Shepherd litter, three of the five puppies developed widespread ulcers of the skin, footpads, and oral mucosa within the first weeks of life. Histopathological examinations demonstrated clefting of the epidermis from the underlying dermis within or just below the basement membrane, which led to a tentative diagnosis of junctional epidermolysis bullosa (JEB) with autosomal recessive inheritance. Endoscopy in one affected dog also demonstrated separation between the epithelium and underlying tissue in the gastrointestinal tract. As a result of the severity of the clinical signs, all three dogs had to be euthanized. We sequenced the genome of one affected puppy and compared the data to 73 control genomes. A search for private variants in 37 known candidate genes for skin fragility phenotypes revealed a single protein-changing variant, LAMB3:c.1174T>C, or p.Cys392Arg. The variant was predicted to change a conserved cysteine in the laminin β3 subunit of the heterotrimeric laminin-322, which mediates the binding of the epidermal basement membrane to the underlying dermis. Loss-of-function variants in the human LAMB3 gene lead to recessive forms of JEB. We confirmed the expected co-segregation of the genotypes in the Australian Shepherd family. The mutant allele was homozygous in two genotyped cases and heterozygous in three non-affected close relatives. It was not found in 242 other controls from the Australian Shepherd breed, nor in more than 600 other controls. These data suggest that LAMB3:c.1174T>C represents the causative variant. To the best of our knowledge, this study represents the first report of a LAMB3-related JEB in domestic animals.

Download Full-text

Junctional Epidermolysis Bullosa of the Larynx

Annals of Otology Rhinology & Laryngology ◽

10.1177/000348949210101010 ◽

1992 ◽

Vol 101 (10) ◽

pp. 861-865 ◽

Cited By ~ 15

Author(s):

Shelley Berson ◽

Robert F. Ward ◽

Andrew N. Lin ◽

D. Martin Carter

Keyword(s):

Respiratory Distress ◽

Epidermolysis Bullosa ◽

Minor Trauma ◽

Inherited Disorders ◽

Blister Formation ◽

Junctional Epidermolysis Bullosa ◽

Laryngeal Involvement

Epidermolysis bullosa (EB) is a group of rare inherited disorders in which minor trauma causes blister formation in the skin and mucosa, including the esophagus. Morbidity varies with the type of disease and ranges from occasional trivial skin blisters to death in infancy. Laryngeal involvement presenting as hoarseness and respiratory distress has been reported in nine patients, five of whom had junctional EB. We present the sixth case of junctional EB with laryngeal involvement, and offer guidelines for otolaryngologists and anesthesiologists caring for these fragile patients.

Download Full-text

Multiple Skin Squamous Cell Carcinomas in Junctional Epidermolysis Bullosa Due to Altered Laminin-332 Function

International Journal of Molecular Sciences ◽

10.3390/ijms21041426 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1426

Author(s):

Paola Fortugno ◽

Angelo Giuseppe Condorelli ◽

Elena Dellambra ◽

Liliana Guerra ◽

Francesca Cianfarani ◽

...

Keyword(s):

Squamous Cell ◽

Epidermolysis Bullosa ◽

Proteolytic Processing ◽

Disulphide Bond ◽

Structural Element ◽

Junctional Epidermolysis Bullosa ◽

Skin Fragility ◽

Gene Expression Studies ◽

Laminin 332

Variably reduced expression of the basement membrane component laminin-332 (α3aβ3γ2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a β3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to γ2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.

Download Full-text

Late-onset skin fragility in childhood: a case of junctional epidermolysis bullosa of late onset caused by a missense mutation inCOL17A1

British Journal of Dermatology ◽

10.1111/bjd.12353 ◽

2013 ◽

Vol 169 (3) ◽

pp. 714-715 ◽

Cited By ~ 2

Author(s):

S. Vanotti ◽

C. Chiaverini ◽

A. Charlesworth ◽

N. Bonnet ◽

P. Berbis ◽

...

Keyword(s):

Missense Mutation ◽

Epidermolysis Bullosa ◽

Late Onset ◽

Junctional Epidermolysis Bullosa ◽

Skin Fragility

Download Full-text

Epidermolysis bullosa in Greece: the patients’ journey so far

International Journal of Research in Dermatology ◽

10.18203/issn.2455-4529.intjresdermatol20182414 ◽

2018 ◽

Vol 4 (3) ◽

pp. 282

Author(s):

Ioanna Verroiou ◽

Vassiliki N. Tzanetakou ◽

Alexandra Katsarou ◽

Giovanna Zambruno ◽

Daniele Castiglia ◽

...

Keyword(s):

Epidermolysis Bullosa ◽

Clinical Characteristics ◽

Laboratory Diagnosis ◽

Published Data ◽

Laboratory Assessment ◽

Inherited Disorders ◽

Mucosal Involvement ◽

Skin Fragility ◽

Diagnostic Management ◽

Adults And Children

Background: Hereditary epidermolysis bullosa (EB) represents a group of rare, inherited disorders with different penetrance patterns characterized by skin fragility and easy inducibility of blisters. Mucosal involvement of internal organs may occur. As no published data on EB in Greece exist, this study aimed to record demographics and clinical characteristics of EB patients. Another objective was to identify the associations among clinical characteristics of different types in connection with immunofluorescence mapping (IMF) findings and molecular analysis (MA) used for the laboratory diagnosis of the disease.Methods: This is a descriptive study conducted at the outpatient clinic of rare diseases of Andreas Sygros Hospital, Athens, Greece from March 2012 until February 2015. Adults and children presenting with EB were enrolled. Patients underwent a thorough clinical and laboratory assessment. Specific laboratory analyses were performed in Rome and two sets of data based on IFM and MA were collected.Results: In total, 41 patients were enrolled. Prevalence rate of EB was 0.024%. The most frequent type was dystrophic EB, as it affected 20 patients (48.8%). Twelve patients (29.3%) were diagnosed with EB simplex, 6 patients (14.6%) with Kindler syndrome and 3 (7.3%) with junctional EB. IFM was performed in 26 patients and MA in 8 patients. The concordance among clinical and laboratory diagnosis was 88.5%.Conclusions: This study is the first report on hereditary EB in Greece. Since there is a lack in diagnostic management of EB, we would strongly encourage an effort to perform the required laboratory tests in Greece.

Download Full-text

Structure, compositon and function of the dermal-epidermal junction: Relevance in cutaneous disease and diagnosis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156328 ◽

1989 ◽

Vol 47 ◽

pp. 868-869

Author(s):

K. A. Holbrook

Keyword(s):

Basement Membrane ◽

Epidermolysis Bullosa ◽

Normal Skin ◽

Lamina Densa ◽

Inherited Disorders ◽

Ultrastructural Studies ◽

Type Iv ◽

Type Vii Collagen ◽

Attachment Structures ◽

Matrix Components

The dermal-epidermal junction (DEJ), or basement membrane rone, is the boundary between the epithelial and mesenchymal compartments of the skin; epidermal and fibroblastic cells in these two regions collaborate to synthesire its components. Ultrastructural studies (TEM and SEM) have defined a series of planes or layers (basal epidermal, lamina lucida, lamina densa, sublamina densa) and the morphology and density of attachment structures (hemidesmosomes, anchoring filaments, anchoring fibrils and anchoring plaques) in this region of normal skin. Change in structure of the DEJ provides information about the history of the tissue; reduplication of the lamina densa, for example, indicates a site of cell detachment or migration, or remodelling that accompanies repair of focal damage. In normal skin the structure of the DEJ is stable; in pathologic conditions it can be compromised by the congenital absence of certain structures or antigens (e.g., in the inherited disorders, epidermolysis bullosa [EB]) or by enzymatic degradation (e.g., in tumor invasion). Dissolution of the DEJ can also occur normally during the formation of epidermal appendages (e.g., hair follicles) and as melanocytes and Langerhans cells migrate into the epidermis during development.Biochemical and immunohisto/cytochemical studies have identified more than 20 molecules at the DEJ. These include well known matrix molecules (e.g., types IV and V collagen, laminin and fibronectin) and skin-specific antigens. The latter have been identified by autoantibodies or specific polyclonal or monoclonal antibodies raised against the skin, cultured cells and other epithelia. Some of the molecules of the DEJ are are present in basement membrane zones of many epithelia and thus are considered ubiquitous components (type IV, V, laminin, fibronectin, nidogen, entactin, HSPG, LDA-1, CSP [3B3]). All of them (that have been investigated in developing skin) appear ontogenetically as early as human embryonic tissue can be obtained and their expression is typically normal in patients with EB. The known properties of many of these molecules (particularly the matrix components) suggest functions they might impart to the DEJ: support of an epithelium (type IV collagen), regulation of permeability (heparan sulfate proteoglycan) or facilitation of cell attachment (fibronectin) and movement (laminin). Another group of matrix components and antigens of the DEJ includes molecules that are skin-specific or characteristic of stratified squamous epithelia (type VII collagen=LH 7:2 antigen, bullous pemphigoid antigen, AA3, GB3, KF-1,19-DEJ-1, epidermolysis bullosa acquisita antigen [EBA], AF-1 and AF-2, cicatricial pemphigoid antigen [CPA]) . These molecules are expressed in the DEJ later in development than the first group of molecules, in conjunction with the morphologic appearance of the structure they represent. Their appearance is also coordinated with specific developmental events (e.g., epidermal stratification) and the expression of molecules of differentiation in the epidermis and dermis. One or more of them is typically absent or reduced in expression in the skin of patients with heritable disorders affecting this region. There is no apparent correlation between the location of molecules in the DEJ and the stability of their expression.

Download Full-text