The potential of gene therapy for recessive dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) encompasses a heterogeneous group of inherited skin fragility disorders, with mutations in genes encoding the basement membrane zone (BMZ) proteins that normally ensure dermal-epidermal integrity. Of the four main EB types, recessive dystrophic EB (RDEB), especially the severe variant, represents one of the most debilitating clinical entities, with recurrent mucocutaneous blistering and ulceration leading to chronic wounds, infections, inflammation, scarring and ultimately cutaneous squamous cell carcinoma, which leads to premature death. Improved understanding of the molecular genetics of EB over the past three decades and advances in biotechnology have led to rapid progress in developing gene and cell-based regenerative therapies for EB. In particular, RDEB is at the vanguard of advances in human clinical trials of advanced therapeutics. Furthermore, the past decade has witnessed the emergence of a real collective, global effort involving academia and industry, supported by international EB patient organizations such as the Dystrophic Epidermolysis Bullosa Research Association (DEBRA), among others, to develop clinically relevant and marketable targeted therapeutics for EB. Thus, there is an increasing need for the practising dermatologist to become familiar with the concept of gene therapy, fundamental differences between various approaches, and their human applications. This review explains the principles of different approaches of gene therapy, summarizes its journey, and discusses its current and future impact in RDEB.

Mutations in genes encoding desmosomal proteins: spectrum of cutaneous and extracutaneous abnormalities.

The desmosome is a type of intercellular junction found in epithelial cells, cardiomyocytes and other specialized cell types. Composed of a network of transmembranous cadherins and intracellular armadillo, plakin and other proteins, desmosomes contribute to cell-cell adhesion, signalling, development and differentiation. Mutations in genes encoding desmosomal proteins result in a spectrum of erosive skin and mucosal phenotypes that also may affect hair or heart. This review summarizes the molecular pathology and phenotypes associated with desmosomal dysfunction with a focus on inherited disorders that involve the skin/hair, as well as associated extracutaneous pathologies. We reviewed the relevant literature to collate studies of pathogenic human mutations in desmosomes that have been reported over the last 25 years. Mutations in 12 different desmosome genes have been documented, with mutations in nine genes affecting the skin/mucous membranes (DSG1, DSG3, DSC2, DSC3, JUP, PKP1, DSP, CDSN, PERP) and eight resulting in hair abnormalities (DSG4, DSC2, DSC3, JUP, PKP1, DSP, CDSN, PERP). Mutations in three genes can result in cardiocutaneous syndromes (DSC2, JUP, DSP), although mutations have been described in five genes in inherited heart disorders that may lack any dermatological manifestations (DSG2, DSC2, JUP, PKP2, DSP). Understanding the diverse nature of these clinical phenotypes, as well as the desmosome gene mutation(s), has clinical value in managing and counselling patients, as well as demonstrating the biological role and activity of specific components of desmosomes in skin and other tissues.

WNT10A, dermatology and dentistry.

WNTs (Wingless-related integration sites) are secreted glycoproteins that are involved in signalling pathways critical to organ development and tissue regeneration. Of the 19 known WNT ligands, one member of this family, WNT10A, appears to have specific relevance to skin, its appendages and teeth. This review focuses on how variants in the WNT10A gene have been associated with various ectodermal disorders and how such changes may have clinical relevance to dermatologists and dentists. Germline mutations in WNT10A underlie several forms of autosomal recessive ectodermal dysplasia in which heterozygous carriers may also display some lesser ectodermal anomalies. Within the general population, multiple heterozygous variants in WNT10A can cause skin, hair, sweat gland or dental alterations, also known as ectodermal derivative impairments. WNT10A variants have also been implicated in hair thickness, male androgenetic alopecia, hair curl, acne vulgaris, lipodystrophy, keloids, wound healing, tooth size, tooth agenesis, hypodontia, taurodontism and oral clefting. Beyond dermatology and dentistry, WNT10A abnormalities have also been identified in kidney fibrosis, keratoconus, certain malignancies (particularly gastrointestinal) and neuropathic pain pathways. In this review, we detail how WNT10A is implicated as a key physiological and pathological contributor to syndromic and nonsyndromic disorders, as well as population variants, affecting the skin and teeth, and document all reported mutations in WNT10A with genotype-phenotype correlation.

A decade of next-generation sequencing in genodermatoses: the impact on gene discovery and clinical diagnostics.

BACKGROUND: Discovering the genetic basis of inherited skin diseases is fundamental to improving diagnostic accuracy and genetic counselling. In the 1990s and 2000s, genetic linkage and candidate gene approaches led to the molecular characterization of several dozen genodermatoses, but over the past decade the advent of next-generation sequencing (NGS) technologies has accelerated diagnostic discovery and precision. OBJECTIVES: This review examines the application of NGS technologies from 2009 to 2019 that have (i) led to the initial discovery of gene mutations in known or new genodermatoses and (ii) identified involvement of more than one contributing pathogenic gene in individuals with complex Mendelian skin disorder phenotypes. METHODS: A comprehensive review of the PubMed database and dermatology conference abstracts was undertaken between January 2009 and December 2019. The results were collated and cross-referenced with OMIM. RESULTS: We identified 166 new disease-gene associations in inherited skin diseases discovered by NGS. Of these, 131 were previously recognized, while 35 were brand new disorders. Eighty-five were autosomal dominant (with 43 of 85 mutations occurring de novo), 78 were autosomal recessive and three were X-linked. We also identified 63 cases harbouring multiple pathogenic mutations, either involving two coexisting genodermatoses (n = 13) or an inherited skin disorder in conjunction with other organ system phenotypes (n = 50). CONCLUSIONS: NGS technologies have accelerated disease-gene discoveries in dermatology over the last decade. Moreover, the era of NGS has enabled clinicians to split complex Mendelian phenotypes into separate diseases. These genetic data improve diagnostic precision and make feasible accurate prenatal testing and better-targeted translational research.

Prevalence, pathophysiology and management of itch in epidermolysis bullosa.

Epidermolysis bullosa (EB) is a highly diverse group of inherited skin disorders, resulting from mutations in genes encoding proteins of the dermoepidermal junction. Itch (pruritus) is one of the most common symptoms across all EB subtypes. It occurs in blistered or wounded sites, or manifests as a generalized phenomenon, thereby affecting both intact skin and healing wounds. The mechanism of pruritus in EB is unclear. It is likely that skin inflammation secondary to barrier disruption, wound healing cascades and dysregulated activation of epidermal sensory nerve endings are all involved in its pathophysiology on the molecular and cellular level. Understanding these mechanisms in depth is crucial in developing optimized treatments for people with EB and improving quality of life. This review summarizes current evidence on the prevalence, mechanisms and management of itch in EB.

Heterogeneous addiction to transforming growth factor-beta signalling in recessive dystrophic epidermolysis bullosa-associated cutaneous squamous cell carcinoma.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is associated with a high mortality rate due to the development of life-threatening, metastatic cutaneous squamous cell carcinoma (cSCC). Elevated transforming growth factor-beta (TGF-ß) signalling is implicated in cSCC development and progression in patients with RDEB. OBJECTIVES: To determine the effect of exogenous and endogenous TGF-ß signalling in RDEB cSCC with a view to assessing the potential of targeting TGF-ß signalling for RDEB cSCC therapy. METHODS: A panel of 11 patient-derived RDEB cSCC primary tumour keratinocyte cell lines (SCCRDEBs) were tested for their signalling and proliferation responses to exogenous TGF-ß. Their responses to TGF-ß receptor type-1 (TGFBR1) kinase inhibitors [SB-431542 and AZ12601011 (AZA01)] were tested using in vitro proliferation, clonogenicity, migration and three-dimensional invasion assays, and in vivo tumour xenograft assays. RESULTS: All SCCRDEBs responded to exogenous TGF-ß by activation of canonical SMAD signalling and proliferative arrest. Blocking endogenous signalling by treatment with SB-431542 and AZ12601011 significantly inhibited proliferation (seven of 11), clonogenicity (six of 11), migration (eight of 11) and invasion (six of 11) of SCCRDEBs. However, these TGFBR1 kinase inhibitors also promoted proliferation and clonogenicity in two of 11 SCCRDEB cell lines. Pretreatment of in vitro TGFBR1-addicted SCCRDEB70 cells with SB-431542 enhanced overall survival and reduced tumour volume in subcutaneous xenografts but had no effect on nonaddicted SCCRDEB2 cells in these assays. CONCLUSIONS: Targeting TGFBR1 kinase activity may have therapeutic benefit in the majority of RDEB cSCCs. However, the potential tumour suppressive role of TGF-ß signalling in a subset of RDEB cSCCs necessitates biomarker identification to enable patient stratification before clinical intervention.

Immune tolerance of allogeneic haematopoietic cell transplantation supports donor epidermal grafting of recessive dystrophic epidermolysis bullosa chronic wounds.

BACKGROUND: Chronic wounds, a common morbidity in recessive dystrophic epidermolysis bullosa (RDEB), lack definitive therapies. OBJECTIVES: To assess allogeneic epidermal skin grafts in terms of wound healing and durability over time. METHODS: In a prospective, open-label clinical trial for postallogeneic haematopoietic cell transplantation (post-alloHCT) patients with RDEB, up to nine chronic wounds per patient were grafted over 1 year. Epidermal grafts measuring 5 cm2 were obtained from related alloHCT donors in the outpatient setting using the CELLUTOMETM Epidermal Harvesting System. Wounds were photographed and symptom inventories completed at baseline and 6, 12 and 52 weeks after grafting. The trial was registered at ClinicalTrials.gov (NCT02670837). RESULTS: Between August 2016 and January 2019, eight patients with RDEB received a total of 35 epidermal allografts at a median of 1157 days (range 548-2884) post-alloHCT. The median (interquartile range) percentage reductions in wound surface area were 75% (52-94), 95% (72-100) and 100% (97-100) at 6, 12 and 52 weeks postgraft, respectively, each significantly reduced from baseline (P < 0·001). Donor harvest sites healed quickly without scarring. Biopsy evaluation at 1 year of an epidermal allograft site revealed wildtype type VII collagen (immunofluorescence), anchoring fibrils (electron microscopy), and full-thickness skin whole-DNA donor chimerism of 42% (compared with 16% in concurrently biopsied native skin). This strategy subsequently supported release of RDEB pseudosyndactyly. CONCLUSIONS: The immune tolerance established by alloHCT supports successful adoptive transfer of donor epidermal grafts. Persistence of donor grafts in a single patient beyond 1 year and observed migration of donor-grafted cells into adjacent wound suggest that epidermal allografts include nonterminally differentiated cells and/or trigger recruitment of donor bone-marrow-derived cells to mediate wound healing.

Autosomal recessive hypotrichosis with loose anagen hairs associated with TKFC mutations.

BACKGROUND: Loose anagen hair is a rare form of impaired hair anchorage in which anagen hairs that lack inner and outer root sheaths can be gently and painlessly plucked from the scalp. This condition usually occurs in children and is often self-limiting. A genetic basis for the disorder has been suggested but not proven. A better understanding the aetiology of loose anagen hair may improve prevention and treatment strategies. OBJECTIVES: To identify a possible genetic basis of loose anagen hair using next-generation DNA sequencing and functional analysis of variants identified. METHODS: In this case study, whole-exome sequencing analysis of a pedigree with one affected individual with features of loose anagen hair was performed. RESULTS: The patient was found to be compound heterozygous for two single-nucleotide substitutions in TKFC resulting in the following missense mutations: c.574G> C (p.Gly192Arg) and c.682C> T (p.Arg228Trp). Structural analysis of human TKFC showed that both mutations are located near the active site cavity. Kinetic assays of recombinant proteins bearing either of these amino acid substitutions showed almost no dihydroxyacetone kinase or D-glyceraldehyde kinase activity, and FMN cyclase activity reduced to just 10% of wildtype catalytic activity. CONCLUSIONS: TKFC missense mutations may predispose to the development of loose anagen hairs. Identification of this new biochemical pathobiology expands the metabolic and genetic basis of hypotrichosis.

Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility.

BACKGROUND: Several new genes and clinical subtypes have been identified since the publication in 2014 of the report of the last International Consensus Meeting on Epidermolysis Bullosa (EB). OBJECTIVES: We sought to reclassify disorders with skin fragility, with a focus on EB, based on new clinical and molecular data. METHODS: This was a consensus expert review. RESULTS: In this latest consensus report, we introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Other disorders with skin fragility, where blisters are a minor part of the clinical picture or are not seen because skin cleavage is very superficial, are classified as separate categories. These include peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility. Because of the common manifestation of skin fragility, these 'EB-related' disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. CONCLUSIONS: The proposed classification scheme should be of value both to clinicians and researchers, emphasizing both clinical and genetic features of EB. What is already known about this topic? Epidermolysis bullosa (EB) is a group of genetic disorders with skin blistering. The last updated recommendations on diagnosis and classification were published in 2014. What does this study add? We introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Clinical and genetic aspects, genotype-phenotype correlations, disease-modifying factors and natural history of EB are reviewed. Other disorders with skin fragility, e.g. peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility are classified as separate categories; these 'EB-related' disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. Linked Comment: Pope. Br J Dermatol 2020; 183:603.

Genotype-phenotype correlation in a large English cohort of patients with autosomal recessive ichthyosis.

BACKGROUND: Recessive forms of congenital ichthyosis encompass a group of rare inherited disorders of keratinization leading to dry, scaly skin. So far, 13 genes have been implicated, but there is a paucity of data on genotype-phenotype correlation in some populations. OBJECTIVES: We compiled an English cohort of 146 individuals with recessive ichthyosis and assessed genotype-phenotype correlation. METHODS: Deep phenotyping was undertaken by history-taking and clinical examination. DNA was screened for mutations using a next-generation sequencing ichthyosis gene panel and Sanger sequencing. RESULTS: Cases were recruited from 13 National Health Service sites in England, with 65% of patients aged < 16 years at enrolment. Pathogenic biallelic mutations were found in 83% of cases, with the candidate gene spread as follows: TGM1 29%, NIPAL4 12%, ABCA12 12%, ALOX12B 9%, ALOXE3 7%, SLC27A4 5%, CERS3 3%, CYP4F22 3%, PNPLA1 2%, SDR9C7 1%. Clinically, a new sign, an anteriorly overfolded ear at birth, was noted in 43% of patients with ALOX12B mutations. The need for intensive care stay (P = 0·004), and hand deformities (P < 0·001), were associated with ABCA12 mutations. Self-improving collodion ichthyosis occurred in 8% of the cases (mostly TGM1 and ALOX12B mutations) but could not be predicted precisely from neonatal phenotype or genotype. CONCLUSIONS: These data refine genotype-phenotype correlation for recessive forms of ichthyosis in England, demonstrating the spectrum of disease features and comorbidities, as well as the gene pathologies therein. Collectively, the data from these patients provide a valuable resource for further clinical assessment, improving clinical care and the possibility of future stratified management. What's already known about this topic? Recessive forms of ichthyosis are rare but often difficult to diagnose. Mutations in 13 genes are known to cause recessive forms of ichthyosis: ABCA12, ALOX12B, ALOXE3, CERS3, CYP4F22, LIPN, NIPAL4, PNPLA1, SDR9C7, SLC27A4, SULT2B1, ST14 and TGM1. Some phenotypic features may associate with certain gene mutations, but paradigms for genotype-phenotype correlation need refining. What does this study add? The genotypic spectrum of recessive ichthyosis in England (based on 146 cases) comprises TGM1 (29%), NIPAL4 (12%), ABCA12 (12%), ALOX12B (9%), ALOXE3 (7%), SLC27A4 (5%), CERS3 (3%), CYP4F22 (3%), PNPLA1 (2%) and SDR9C7 (1%). New or particular phenotypic clues were defined for mutations in ALOX12B, ABCA12, CYP4F22, NIPAL4, SDR9C7 and TGM1, either in neonates or in later life, which allow for greater diagnostic precision. In around 17% of cases, the molecular basis of recessive ichthyosis remains unknown.

Revertant mosaic fibroblasts in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Revertant mosaicism has been described previously in recessive dystrophic epidermolysis bullosa (RDEB), manifesting as regions of skin with normal mechanical and biological characteristics. Here we report the discovery of revertant dermal fibroblasts, unique in that all other documented cases of revertant mosaicism occur in epidermal keratinocytes. OBJECTIVES: To determine the cause of revertant mosaicism found in a patient with RDEB from isolated epidermal keratinocytes and dermal fibroblasts in blister and mosaic skin regions. METHODS: Skin biopsies were taken from blister and mosaic skin regions of a patient with RDEB. Allele identification was confirmed and the type VII collagen (C7) content and COL7A1 expression profile of isolated keratinocytes and fibroblasts was determined. RESULTS: Keratinocytes isolated from the mosaic area had a slight increase in C7, although overall expression of COL7A1 was unchanged between blister and mosaic fibroblasts. Differential allele expression was identified in blister and mosaic fibroblasts using targeted RNA sequencing (TREx), where the allele harbouring a point mutation was preferentially expressed over that containing a frameshift mutation. A crossing over event was identified in mosaic fibroblasts that was not present in blister fibroblasts, yielding a functional COL7A1 allele in a subset of cells. CONCLUSIONS: In documenting a novel case of revertant mosaicism in RDEB, we have identified dermal fibroblasts as having the capacity to correct blistering functionally. We have also pioneered the use of TREx in quantifying allele-specific expression. Using fibroblasts instead of keratinocytes for RDEB therapies offers advantages in the local and systemic therapy of RDEB. What's already known about this topic? Revertant mosaicism has been previously documented in patients with recessive dystrophic epidermolysis bullosa (RDEB), however, it has only been found in epidermal keratinocytes. What does this study add? We have demonstrated that COL7A1 gene reversion in dermal fibroblasts occurs and is able to form functional skin in a patient with RDEB. Additionally, we have pioneered a new application for targeted RNA sequencing in quantifying allele-specific expression in fibroblasts and keratinocytes. What is the translational message? This opens up possibilities for using fibroblasts as local and systemic therapy for patients with RDEB.

Bone marrow transplant with post-transplant cyclophosphamide for recessive dystrophic epidermolysis bullosa expands the related donor pool and permits tolerance of nonhaematopoietic cellular grafts.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe systemic genodermatosis lacking therapies beyond supportive care for its extensive, life-limiting manifestations. OBJECTIVES: To report the safety and preliminary responses of 10 patients with RDEB to bone marrow transplant (BMT) with post-transplant cyclophosphamide (PTCy BMT) after reduced-intensity conditioning with infusions of immunomodulatory donor-derived mesenchymal stromal cells (median follow-up 16 months). METHODS: BMT toxicities, donor blood and skin engraftment, skin biopsies, photographic and dynamic assessments of RDEB disease activity were obtained at intervals from pre-BMT to 1 year post-BMT. RESULTS: Related donors varied from haploidentical (n = 6) to human leucocyte antigen (HLA)-matched (n = 3), with one HLA-matched unrelated donor. Transplant complications included graft failure (n = 3; two pursued a second PTCy BMT), veno-occlusive disease (n = 2), posterior reversible encephalopathy (n = 1) and chronic graft-versus-host disease (n = 1; this patient died). In the nine ultimately engrafted patients, median donor chimerism at 180 days after transplant was 100% in peripheral blood and 27% in skin. Skin biopsies showed stable (n = 7) to improved (n = 2) type VII collagen protein expression by immunofluorescence and gain of anchoring fibril components (n = 3) by transmission electron microscopy. Early signs of clinical response include trends toward reduced body surface area of blisters/erosions from a median of 49·5% to 27·5% at 100 days after BMT (P = 0·05), with parental measures indicating stable quality of life. CONCLUSIONS: PTCy BMT in RDEB provides a means of attaining immunotolerance for future donor-derived cellular grafts (ClinicalTrials.gov identifier NCT02582775). What's already known about this topic? Severe, generalized recessive dystrophic epidermolysis bullosa (RDEB) is marked by great morbidity and early death. No cure currently exists for RDEB. Bone marrow transplant (BMT) is the only described systemic therapy for RDEB. What does this study add? The first description of post-transplant cyclophosphamide (PTCy) BMT for RDEB. PTCy was well tolerated and provided excellent graft-versus-host disease prophylaxis, replacing long courses of calcineurin inhibitors in patients receiving human leucocyte antigen-matched sibling BMT. What is the translational message? The PTCy BMT platform permits identification of a suitable related donor for most patients and for subsequent adoptive transfer of donor nonhaematopoietic cells after establishment of immunological tolerance.

Beneficial effect of ustekinumab in familial pityriasis rubra pilaris with a new missense mutation in CARD14.

Pityriasis rubra pilaris (PRP) represents a group of rare chronic inflammatory skin disorders in which around one in 20 affected individuals show autosomal dominant inheritance. In such cases there may be gain-of-function mutations in CARD14, encoding caspase recruitment domain-containing protein 14 (CARD14), which activates the noncanonical nuclear factor (NF)-κB pathway, thereby promoting cutaneous inflammation. Here we report a mother and son with PRP due to a new missense mutation in CARD14 and describe the beneficial clinical effects of ustekinumab, a monoclonal antibody against interleukins 12 and 23, in both patients. A 49-year-old woman and her 20-year-old son had lifelong, generalized, patchy erythematous scale with a few islands of sparing, as well as minor nail ridging and mild palmoplantar keratoderma, features consistent with generalized PRP. Topical steroids, phototherapy and oral retinoids proved ineffective. Following informed consent, Sanger sequencing of CARD14 in both individuals revealed a new heterozygous single-nucleotide transversion in exon 4, c.356T>G, resulting in the missense mutation p.Met119Arg. Ustekinumab, at a dose of 45 mg every 12 weeks, brought about a significant physical and emotional improvement in both the mother and son within a few days of the initial dose, which was sustained on maintenance dosing. This report highlights the therapeutic potential of biologics that downregulate NF-κB signalling in familial PRP with mutations in CARD14.

Management of cutaneous squamous cell carcinoma in patients with epidermolysis bullosa: best clinical practice guidelines.

This article summarizes recommendations reached following a systematic literature review and expert consensus on the diagnosis and management of cutaneous squamous cell carcinomas in people with epidermolysis bullosa. The guidelines are intended to help inform decision making by clinicians dealing with this complex complication of a devastating disease.

Intrafamilial phenotypic heterogeneity of epidermolytic ichthyosis associated with a new missense mutation in keratin 10.

Mutations in the keratin 10 gene (KRT10) have been shown to underlie several forms of epidermolytic ichthyosis (EI), including generalized, annular and naevoid variants. We investigated an autosomal dominant pedigree with ichthyosis in which there was intrafamilial clinical heterogeneity, with the affected individual family members presenting with features of either erythrokeratoderma progressiva, annular EI, localized or superficial EI, or more generalized EI. Sanger sequencing identified a new heterozygous missense mutation (c.457C>A; p.Leu153Met) in KRT10 in all affected individuals. No additional mutations were identified in the genes for keratin 1 (KRT1) keratin 2 (KRT2), connexin 31 (GJB3) or connexin 30.3 (GJB4) that might account for the clinical heterogeneity seen in this family. Our findings illustrate the intrafamilial variability in phenotype and diverse clinical presentations that can occur in EI resulting from a single mutation in KRT10.