Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

Gene-Modified Blister Fluid-Derived Mesenchymal Stromal Cells for Treating Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genodermatosis caused by variants in COL7A1-encoded type VII collagen, a major component of anchoring fibrils. In this study, we developed an ex vivo gene therapy for RDEB using autologous mesenchymal stromal cells (MSCs). On the basis of our previous studies, we first attempted to isolate MSCs from the blister fluid of patients with RDEB and succeeded in obtaining cells with a set of MSC characteristics from all 10 patients. We termed these cells blister fluid-derived MSCs. Blister fluid-derived MSCs were genetically modified and injected into skins of type VII collagen-deficient neonatal mice transplanted onto immunodeficient mice, resulting in continuous and widespread expression of type VII collagen at the dermal-epidermal junction, particularly when administered into blisters. When injected intradermally, the efforts were not successful. The gene-modified blister fluid-derived MSCs could be cultured as cell sheets and applied to the dermis with an efficacy equivalent to that of intrablister administration. In conclusion, we successfully developed a minimally invasive and highly efficient ex vivo gene therapy for RDEB. This study shows the successful application of gene therapy in the RDEB mouse model for both early blistering skin and advanced ulcerative lesions.

First report of the c.1676G>A homozygous variant in a family with Kindler syndrome.

Kindler syndrome (KS) was first described by Theresa Kindler in 1954, and since then > 60 pathogenic variants have been identified in the FERMT1 gene for KS. Most FERMT1 variants associated with KS are null variants. We present the case of a child with poikilodermic changes on the forehead and cheeks, who was found to have a homozygous c.1676G>A mutation. To our knowledge, this is the first report of this mutation in a family with KS.

The Immunogenetics of Autoimmune Blistering Diseases.

Dermatological conditions constituting the group of autoimmune blistering diseases (AIBD) are characterized by loss of immunotolerance and humoral, as well as cellular, autoimmune responses that result in the development of bullae and erosions on the skin and mucous membranes. AIBDs are broadly categorized into pemphigus and pemphigoid classes with several distinct subtypes amongst them. Advances in genetics have allowed for the study and identification of alleles, and even single nucleotide polymorphisms, that harbor increased susceptibility or confer protection for the development of these conditions. The focus of this chapter pertains to a comprehensive review of the known genetic associations with AIBDs, including HLA class I-III, as well as non-HLA genes and non-coding sequences that influence cellular processes and signaling pathways.

A novel likely pathogenic PLCG2 variant in a patient with a recurrent skin blistering disease and B-cell lymphopenia.

Pathogenic variants of PLCG2 encoding phospholipase C gamma 2 (PLCγ2) were first reported in 2012 and their clinical manifestations vary widely. PLCG2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) are representative examples of PLCG2 pathogenic variants. In this report, we describe a 17-year-old male with recurrent blistering skin lesions, B-cell lymphopenia, and asthma. Distinct from the patients in previous reports, this patient had the heterozygous de novo c.2119T > C missense variant (NM_002661.4) resulting in a serine to proline amino acid substitution (p.Ser707Pro). The variant located to the PLCγ2 C-terminal Src homology 2 (cSH2) domain, which is a critical site for the restriction of intrinsic enzyme activity. This variant could be classified as "likely pathogenic" according to American College of Medical Genetics and Genomics guidelines. Laboratory results showed a reduction in circulating B cells without a decrease of serum IgG and IgA. Our findings expand the variety of clinical phenotypes for PLCG2 missense variants.

Genetic and acquired blistering disorders of pediatric age group: An experience from Eastern India.

INTRODUCTION: Blistering or vesiculobullous disorders in pediatric population are either immunobullous or mechanobullous. Spectrum was analyzed using demographic details, clinical features, histopathology, direct immunofluorescence (DIF) and Immunofluorescence mapping (IFM). METHODOLOGY: This was a single institution based observational study in children below 18 years. The demographic details were collected using proforma containing particulars of the patient, history, complaints, and other parameters. Punch biopsy of the skin lesion was done. Biopsy samples were examined under light microscope followed by DIF using fluorescent conjugated polyclonal antibody against immunoglobulins IgG, IgM, IgA, and complement C3. The salt-split technique was also used in particular cases. IFM was done using anticytokeratin (CK) 5 & 14, antilaminin 332, anticollagen VII, and anticollagen IV antibodies. RESULTS: Out of total 50 cases, linear IgA bullous dermatosis (LABD) was the commonest. The average concordance between clinical and final diagnosis (histopathological examination + DIF) was 87.5% and discordance was 12.5%. The agreement between histopathological examination and DIF was found to be substantially significant (κ = 0.6892). IFM depicted epidermolysis bullosa simplex with reduced CK 14 expression, dystrophic epidermolysis bullosa with reduced Collagen VII expression and junctional epidermolysis bullosa with absent laminin 5 expression. CONCLUSION: The spectrum of bullous lesions in childhood was properly delineated and subcategorization of EB was done. Histopathological examination showed the hallmarks that were conclusive in most of the cases except in LABD and EB. DIF and IFM proved indispensable in those cases. Thus, DIF is not a substitute for histopathology but complementary to it.

Hair follicle stem cell progeny heal blisters while pausing skin development.

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.

Severe epidermolysis bullosa/Kindler syndrome-like phenotype of an autoinflammatory syndrome in a child.

A 5-year-old boy presented with generalized cutaneous erosions, severe scarring, depigmentation and contractures affecting major joints. The lesions had initially affected his ears, nose, feet, and the genital and ocular mucosa, leading to significant depigmentation, scarring, contractures and mutilation. The whole of the trunk and limbs were involved at the time of presentation, with the exception of some islands of spared skin on the proximal thighs, legs, nipples and external genitalia. Electron microscopy revealed a split in the sublamina densa with the absence of anchoring fibrils, suggestive of dystrophic epidermolysis bullosa (EB). Immunofluorescence antigen mapping demonstrated a broad reticulate pattern of staining with collagen IV, VII, and laminin 332 in the floor of the blister, suggestive of Kindler syndrome. Next-generation sequencing revealed a de novo heterozygous missense mutation (a variant of unknown significance) in exon 22 of the phospholipase-C gamma 2 gene (PLCG2), which resulted in a substitution of serine by asparagine at codon 798 (p.Asp798Ser), a result that was validated using Sanger sequencing. The child was diagnosed with PLCG2-associated antibody deficiency and immune dysregulation (PLAID)/autoinflammation and PLCG2-associated antibody deficiency and immune dysregulation (APLAID) syndrome. The cutaneous and corneal erosions, inflammation and scarring of this magnitude, and the eventual result of death have not been described previously for the PLAID/APLAID spectrum previously. In conclusion, this was an unusual acquired autoinflammatory severe EB-like disease that may be associated with de novo PLCG2 mutation.

Targeting TRPV1-mediated autophagy attenuates nitrogen mustard-induced dermal toxicity.

Nitrogen mustard (NM) causes severe vesicating skin injury, which lacks effective targeted therapies. The major limitation is that the specific mechanism of NM-induced skin injury is not well understood. Recently, autophagy has been found to play important roles in physical and chemical exposure-caused cutaneous injuries. However, whether autophagy contributes to NM-induced dermal toxicity is unclear. Herein, we initially confirmed that NM dose-dependently caused cell death and induced autophagy in keratinocytes. Suppression of autophagy by 3-methyladenine, chloroquine, and bafilomycin A1 or ATG5 siRNA attenuated NM-induced keratinocyte cell death. Furthermore, NM increased transient receptor potential vanilloid 1 (TRPV1) expression, intracellular Ca2+ content, and the activities of Ca2+/calmodulin-dependent kinase kinase ß (CaMKKß), AMP-activated protein kinase (AMPK), unc-51-like kinase 1 (ULK1), and mammalian target of rapamycin (mTOR). NM-induced autophagy in keratinocytes was abolished by treatment with inhibitors of TRPV1 (capsazepine), CaMKKß (STO-609), AMPK (compound C), and ULK1 (SBI-0206965) as well as TRPV1, CaMKKß, and AMPK siRNA transfection. In addition, an mTOR inhibitor (rapamycin) had no significant effect on NM-stimulated autophagy or cell death of keratinocytes. Finally, the results of the in vivo experiment in NM-treated skin tissues were consistent with the findings of the in vitro experiment. In conclusion, NM-caused dermal toxicity by overactivating autophagy partially through the activation of TRPV1-Ca2+-CaMKKß-AMPK-ULK1 signaling pathway. These results suggest that blocking TRPV1-dependent autophagy could be a potential treatment strategy for NM-caused cutaneous injury.

Dominant pretibial dystrophic epidermolysis bullosa in an Italian family.

We describe a case of pretibial dystrophic epidermolysis bullosa in a 5-year-old girl, her mother, and maternal great aunt. All subjects had trauma-induced blisters and erosions, with scarring, on the knees and lower legs, and nail dystrophy of variable severity. Genetic analysis in all three patients showed a 6849del18 mutation in the COL7A1 gene, causing the production of shortened collagen VII polypeptides and resulting in a mild phenotype, with localized acral blisters and nail involvement.

A novel pathogenic FERMT1 variant in four families with Kindler syndrome in Argentina.

BACKGROUND: Kindler syndrome is a rare genodermatosis. Major clinical criteria include acral blistering in infancy and childhood, progressive poikiloderma, skin atrophy, abnormal photosensitivity, and gingival fragility. METHODS: FERMT1 gene was sequenced in 5 patients with a clinical diagnosis of Kindler syndrome. RESULTS: We report a novel pathogenic variant detected in four unrelated families of Paraguayan origin, where one nucleotide deletion in FERMT1 gene (c.450delG) is predicted to cause a frameshift mutation leading to loss of function. Haplotype analysis revealed the propagation of an ancestral allele through this population. CONCLUSIONS: The identification of this recurrent pathogenic variant enables optimization of molecular detection strategies in our patients, reducing the cost of diagnosis.

Assessment of the risk and characterization of non-melanoma skin cancer in Kindler syndrome: study of a series of 91 patients.

BACKGROUND: Kindler Syndrome (KS) is a rare genodermatosis characterized by skin fragility, skin atrophy, premature aging and poikiloderma. It is caused by mutations in the FERMT1 gene, which encodes kindlin-1, a protein involved in integrin signalling and the formation of focal adhesions. Several reports have shown the presence of non-melanoma skin cancers in KS patients but a systematic study evaluating the risk of these tumors at different ages and their potential outcome has not yet been published. We have here addressed this condition in a retrospective study of 91 adult KS patients, characterizing frequency, metastatic potential and body distribution of squamous cell carcinoma (SCC) in these patients. SCC developed in 13 of the 91 patients. RESULTS: The youngest case arose in a 29-year-old patient; however, the cumulative risk of SCC increased to 66.7% in patients over 60 years of age. The highly aggressive nature of SCCs in KS was confirmed showing that 53.8% of the patients bearing SCCs develop metastatic disease. Our data also showed there are no specific mutations that correlate directly with the development of SCC; however, the mutational distribution along the gene appears to be different in patients bearing SCC from SCC-free patients. The body distribution of the tumor appearance was also unique and different from other bullous diseases, being concentrated in the hands and around the oral cavity, which are areas of high inflammation in this disease. CONCLUSIONS: This study characterizes SCCs in the largest series of KS patients reported so far, showing the high frequency and aggressiveness of these tumors. It also describes their particular body distribution and their relationship with mutations in the FERMT-1 gene. These data reinforce the need for close monitoring of premalignant or malignant lesions in KS patients.

Fibroblast activation and abnormal extracellular matrix remodelling as common hallmarks in three cancer-prone genodermatoses.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three cancer-prone genodermatoses whose causal genetic mutations cannot fully explain, on their own, the array of associated phenotypic manifestations. Recent evidence highlights the role of the stromal microenvironment in the pathology of these disorders. OBJECTIVES: To investigate, by means of comparative gene expression analysis, the role played by dermal fibroblasts in the pathogenesis of RDEB, KS and XPC. METHODS: We conducted RNA-Seq analysis, which included a thorough examination of the differentially expressed genes, a functional enrichment analysis and a description of affected signalling circuits. Transcriptomic data were validated at the protein level in cell cultures, serum samples and skin biopsies. RESULTS: Interdisease comparisons against control fibroblasts revealed a unifying signature of 186 differentially expressed genes and four signalling pathways in the three genodermatoses. Remarkably, some of the uncovered expression changes suggest a synthetic fibroblast phenotype characterized by the aberrant expression of extracellular matrix (ECM) proteins. Western blot and immunofluorescence in situ analyses validated the RNA-Seq data. In addition, enzyme-linked immunosorbent assay revealed increased circulating levels of periostin in patients with RDEB. CONCLUSIONS: Our results suggest that the different causal genetic defects converge into common changes in gene expression, possibly due to injury-sensitive events. These, in turn, trigger a cascade of reactions involving abnormal ECM deposition and underexpression of antioxidant enzymes. The elucidated expression signature provides new potential biomarkers and common therapeutic targets in RDEB, XPC and KS. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three genodermatoses with high predisposition to cancer development. Although their causal genetic mutations mainly affect epithelia, the dermal microenvironment likely contributes to the physiopathology of these disorders. What does this study add? We disclose a large overlapping transcription profile between XPC, KS and RDEB fibroblasts that points towards an activated phenotype with high matrix-synthetic capacity. This common signature seems to be independent of the primary causal deficiency, but reflects an underlying derangement of the extracellular matrix via transforming growth factor-ß signalling activation and oxidative state imbalance. What is the translational message? This study broadens the current knowledge about the pathology of these diseases and highlights new targets and biomarkers for effective therapeutic intervention. It is suggested that high levels of circulating periostin could represent a potential biomarker in RDEB.

Kindlin-1 Regulates Epidermal Growth Factor Receptor Signaling.

Kindler syndrome is an autosomal recessive genodermatosis that results from mutations in the FERMT1 gene encoding t kindlin-1. Kindlin-1 localizes to focal adhesion and is known to contribute to the activation of integrin receptors. Most cases of Kindler syndrome show a reduction or complete absence of kindlin-1 in keratinocytes, resulting in defective integrin activation, cell adhesion, and migration. However, roles for kindlin-1 beyond integrin activation remain poorly defined. In this study we show that skin and keratinocytes from Kindler syndrome patients have significantly reduced expression levels of the EGFR, resulting in defective EGF-dependent signaling and cell migration. Mechanistically, we show that kindlin-1 can associate directly with EGFR in vitro and in keratinocytes in an EGF-dependent, integrin-independent manner and that formation of this complex is required for EGF-dependent migration. We further show that kindlin-1 acts to protect EGFR from lysosomal-mediated degradation. This shows a new role for kindlin-1 that has implications for understanding Kindler syndrome disease pathology.

Chromosomal Instability Induces Cellular Invasion in Epithelial Tissues.

Most sporadic carcinomas with high metastatic activity show an increased rate of changes in chromosome structure and number, known as chromosomal instability (CIN). However, the role of CIN in driving invasiveness remains unclear. Using an epithelial model in Drosophila, we present evidence that CIN promotes a rapid and general invasive behavior. Cells with an abnormal number of chromosomes delaminate from the epithelium, extend actin-based cellular protrusions, form membrane blebs, and invade neighboring tissues. This behavior is governed by the activation of non-muscle Myosin II by Rho kinase and by the expression of the secreted EGF/Spitz ligand. We unravel fundamental roles of the mitogen-activated protein kinase pathways mediated by the Fos proto-oncogene and the Capicua tumor suppressor gene in the invasive behavior of CIN-induced aneuploid cells. Our results support the proposal that the simple production of unbalanced karyotypes contributes to CIN-induced metastatic progression.

Kindler syndrome in a patient with colitis and primary sclerosing cholangitis: coincidence or association?

Kindler syndrome is a rare, autosomal recessive genodermatosis, caused by mutations in the FERMT1 gene. It is thought to be primarily a skin disease, but other organs may also be involved. We report a case of a novel mutation of FERMT1 gene in a patient with a probable new phenotype of Kindler syndrome, including colitis and primary sclerosing cholangitis. A 42-year-old man, born to first cousin parents, was referred to our outpatient dermatology clinic with an unknown dermatosis since birth. He presented with neonatal blistering and developed photosensitivity and changes in skin pigmentation during childhood. Since the age of 20, he has had regular follow-up in the gastroenterology clinic, owing to esophageal stenosis, ulcerative colitis, and primary sclerosing cholangitis. Clinical examination revealed jaundice, poikiloderma, diffuse cigarette paper-like atrophy on dorsal surfaces of the hands, and palmoplantar hyperkeratosis. Skin biopsy showed epidermal atrophy covered by orthokeratotic hyperkeratosis. DNA molecular analysis revealed FERMT1 homozygous mutation c.1179G>A, p.W393X, which has not been reported before. The intestinal phenotype of Kindler syndrome has already been defined previously. However, to the best of our knowledge, no other case of primary sclerosing cholangitis in a patient with Kindler syndrome has been reported.