PDF file - 79K, A: Average array signal intensities SD for the single probe representing COL7A1, n=3-5. B: Western blotting shows the level of type VII collagen varies between sample groups, increased in both UVSCCF samples compared with NHF. Type VII collagen is identified in RDEB fibroblast samples from patients with the following COL7A1 genotypes: x79 6501G>A/unknown and 2470insG/2470insG, and RDEBSCC fibroblasts samples from patients with x31 3832-1G>A/Unknown and x73 6075delC/unknown COL7A1 genotype.

XLS file - 8K, describing KEGG pathway analysis results

PDF file - 382K, Includes Microarray and Expression Analysis, BRB Array Clustering Analysis, and Tumorigenicity assays

PDF file - 190K, A: RDEBSCCF derived matrix promotes adhesion of keratinocytes compared with matrix derived from other fibroblast groups (* = p<0.01). All data normalized to collagen I adhesion, results are the mean of three separate experiments performed in quintuplicate SD, n=3-5 per fibroblast sample group. B-E: Keratinocyte adhesion to cell derived matrix from the indicated fibroblast samples. Each graph combines the results from up to three experiments for each keratinocyte group as indicated, mean SD performed in quintuplicate. NHK = immortalized normal keratinocyte lines (NHK38 spontaneous immortalized foreskin, Thai5 HPV immortalized axilla), UVSCCK = spontaneously immortalized UV cSCC keratinocytes (6), RDEBK = HPV immortalized RDEB keratinocytes, RDEBSCCK = spontaneously immortalized RDEB cSCC keratinocytes (6). Fibroblast sample details are given in Supplementary Data Table 1.

XLS file - 36K, Table describing the patient samples used in this study

PDF file - 56K, Unsupervised clustering of the core serum response gene set is able to cluster NHF but not RDEBSCCF sample groups in culture

PDF file - 409K, Invasion of SCCRDEB4 keratinocytes into RDEB cell derived matrix is increased after 1 week compared with equivalent non-RDEB cell derived matrix. Representative images from pan-keratin stained images of 1-week invasion of tumor cells into fibroblast derived matrix. Magnification 200X.

Background:Mechanisms of pain associated with joint hypermobility are poorly understood and include nociceptive pain from structural joint changes along with soft tissue injuries linked to impaired proprioception; central sensitisation associated with chronic pain and muscle weakness alongside deconditioning. Anxiety and depression are also thought to play a role in patients presenting with pain and hypermobility. We have observed an increase in the rate of orthopaedic surgical procedures undertaken in patients attending the hypermobility clinics compared to those attending the general rheumatology and chronic pain clinics. There is limited published data regarding orthopaedic interventions in patients with hypermobility related disorders especially those with confirmed genetic mutations.Objectives:We aimed to evaluate the characteristics of patients in our hypermobility cohort focusing on those who had received prior surgical intervention in order to understand the underlying mechanism behind their presentations.Methods:A retrospective review of medical records was conducted of patients attending a hypermobility clinic at our tertiary referral centre, University College London Hospital, between January 2018 and December 2018.Results:There were 350 patients (300 females, 50 males) with a mean age of 36 years (range 18-71 years). 63% had a diagnosis of Hypermobility Spectrum Disorder or Hypermobility Syndrome and 37% had a type of Ehlers-Danlos Syndromes (EDS) (hypermobile, classical, vascular or other rare type). 46 patients (13%) had documented genetic mutations. 83 patients (24%) had undergone orthopaedic interventions including 9 who had EDS with confirmed genetic mutations. 54% of patients who had surgical intervention were under the age of 40. The total number of surgical procedures in the cohort was 227 (equating to 0.6485 interventions per patient). Of those requiring operative intervention, the average number of interventions per patient was 2.73. One third of patients had surgery on two or more joint groups, including 8 patients (2%) who had surgery in four or more joint groups. Knees (24%) and hips (23%) were the most common sites for operative intervention with 9% having surgery on their shoulders. 29% of pts had significant hypermobility with a Beighton score of 7 and above but there was no correlation between Beighton score and number of surgical procedures. Only 2% of cases were referred from an orthopaedic team thereby excluding a referral bias.Conclusion:Patients with hypermobility related disorders have a significant number of orthopaedic surgical procedures on multiple sites and at a young age, with indication of mechanical pathology playing an important role in their symptoms. The Beighton score does not appear to be a reliable predictor of surgical intervention. This is not surprising given that the score only covers 5 joint areas and excludes common surgical sites such as the hips and shoulders. Early diagnosis and a holistic non-operative approach combining physiotherapy and chronic pain management is essential to reduce the need for multiple surgical procedures.References:[1]Chopra P, Tinkle B, Hamonet C, Brock I, Gompel A, Bulbena A, et al. Pain management in the Ehlers-Danlos syndromes. Am J Med Genet C Semin Med Genet [Internet]. 2017 [cited 2020 Jan 27];175(1):212–9. Available from:http://www.ncbi.nlm.nih.gov/pubmed/28186390[2]Shirley ED, Demaio M, Bodurtha J. Ehlers-danlos syndrome in orthopaedics: etiology, diagnosis, and treatment implications. Sports Health [Internet]. 2012 Sep [cited 2019 Jan 30];4(5):394–403. Available from:http://www.ncbi.nlm.nih.gov/pubmed/23016112Disclosure of Interests:None declared

Background: Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term ‘axon guidance’. Conclusions: This work provides further evidence for keratinocyte-specific mechanisms contributing to immune and neurological, as well as structural, aspects of skin barrier dysfunction. Individuals with filaggrin deficiency may derive benefit from future therapies targeting keratinocyte-immune crosstalk and neurogenic pruritus.

Purpose: Squamous cell carcinoma (SCC) of the skin is the leading cause of death in patients with the severe generalized form of the genetic disease recessive dystrophic epidermolysis bullosa (RDEB). Although emerging data are identifying why patients suffer this fatal complication, therapies for treatment of RDEB SCC are in urgent need. Experimental Design: We previously identified polo-like kinase 1 (PLK1) as a therapeutic target in skin SCC, including RDEB SCC. Here, we undertake a screen of 6 compounds originally designated as PLK1 inhibitors, and detail the efficacy of the lead compound, the multipathway allosteric inhibitor ON-01910, for targeting RDEB SCC in vitro and in vivo. Results: ON-01910 (or rigosertib) exhibited significant specificity for RDEB SCC: in culture rigosertib induced apoptosis in 10 of 10 RDEB SCC keratinocyte populations while only slowing the growth of normal primary skin cells at doses 2 orders of magnitude higher. Furthermore, rigosertib significantly inhibited the growth of two RDEB SCC in murine xenograft studies with no apparent toxicity. Mechanistically, rigosertib has been shown to inhibit multiple signaling pathways. Comparison of PLK1 siRNA with MEK inhibition, AKT inhibition, and the microtubule-disrupting agent vinblastine in RDEB SCC shows that only PLK1 reduction exhibits a similar sensitivity profile to rigosertib. Conclusions: These data support a "first in RDEB" phase II clinical trial of rigosertib to assess tumor targeting in patients with late stage, metastatic, and/or unresectable SCC.

BACKGROUND:Atopic dermatitis (AD) is a common, complex, and highly heritable inflammatory skin disease. Genome-wide association studies offer opportunities to identify molecular targets for drug development. A risk locus on chromosome 11q13.5 lies between 2 candidate genes, EMSY and LRRC32 (leucine-rich repeat-containing 32) but the functional mechanisms affecting risk of AD remain unclear. OBJECTIVES:We sought to apply a combination of genomic and molecular analytic techniques to investigate which genes are responsible for genetic risk at this locus and to define mechanisms contributing to atopic skin disease. METHODS:We used interrogation of available genomic and chromosome conformation data in keratinocytes, small interfering RNA (siRNA)-mediated knockdown in skin organotypic culture and functional assessment of barrier parameters, mass spectrometric global proteomic analysis and quantitative lipid analysis, electron microscopy of organotypic skin, and immunohistochemistry of human skin samples. RESULTS:Genomic data indicate active promoters in the genome-wide association study locus and upstream of EMSY; EMSY, LRRC32, and intergenic variants all appear to be within a single topologically associating domain. siRNA-knockdown of EMSY in organotypic culture leads to enhanced development of barrier function, reflecting increased expression of structural and functional proteins, including filaggrin and filaggrin-2, as well as long-chain ceramides. Conversely, overexpression of EMSY in keratinocytes leads to a reduction in markers of barrier formation. Skin biopsy samples from patients with AD show greater EMSY staining in the nucleus, which is consistent with an increased functional effect of this transcriptional control protein. CONCLUSION:Our findings demonstrate an important role for EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach.

Background: Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term ‘axon guidance’. Conclusions: This work provides further evidence for keratinocyte-specific mechanisms contributing to immune and neurological, as well as structural, aspects of skin barrier dysfunction. Individuals with filaggrin deficiency may derive benefit from future therapies targeting keratinocyte-immune crosstalk and neurogenic pruritus.

Atopic eczema (AE) is a common inflammatory skin disease with strong heritability. Genome-wide association studies have identified multiple loci affecting AE risk but many are intergenic and functional mechanisms remain undefined. A locus on chromosome 11q13.5 lies in an intergenic region between two candidate genes: EMSY and LRRC32. Analysis of genome-wide chromosome conformation capture data generated from differentiating human keratinocytes indicates that EMSY, LRRC32 and 11q13.5 variants may all reside within a single topologically associating domain. siRNA-knockdown of EMSY in skin organotypic culture using primary human keratinocytes reduces EMSY mRNA (n=7, mean 36% reduction p<0.0001) and protein expression (n=7, mean 45% reduction p<0.001) and enhances barrier function defined by water content, water loss and dye penetration. Global mass-spec proteomic analysis detected >8000 proteins per sample with increased expression (fold-change ≥2.5 p<0.05) of 154 proteins, notably filaggrin and filaggrin-2. Mass spec lipidomics showed an increase in longer chain ceramides known to be downregulated in AE. Ultrastructural changes including increased desmosome size and corneodesmosome abundance were also observed by electron microscopy. In contrast, over-expression of EMSY in primary keratinocytes leads to a reduction in markers of differentiation and barrier formation. In keeping with these in vitro observations, skin biopsy samples from patients show greater staining of EMSY in the nucleus (14 AE cases and 18 controls), consistent with an increased functional effect of this transcriptional control protein. Taken together our findings demonstrate an important role for EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach for AE.

Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cellsin vitroprovides the opportunity for selected genetic effects to be investigated in detail.Methods:Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown ofFLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements.Results:FLGknockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed evidence of barrier impairment withFLGknockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term ‘axon guidance’. Conclusions:This work provides further evidence for keratinocyte-specific mechanisms contributing to immune and neurological, as well as structural, aspects of skin barrier dysfunction. Individuals with filaggrin deficiency may derive benefit from future therapies targeting keratinocyte-immune crosstalk and neurogenic pruritus.

Atopic eczema (AE or atopic dermatitis) is a common inflammatory skin disease with strong heritability. Genome-wide association studies (GWAS) have identified multiple loci affecting AE risk but many loci are intergenic and the functional mechanisms remain undefined. A locus on chromosome 11q13.5 has been widely replicated in GWAS; it lies between two candidate genes: EMSY and LRRC32. Analysis of genome-wide chromosome conformation capture data generated from differentiating human keratinocytes indicates that the AE risk locus interacts in three-dimensional space with EMSY, bringing an enhancer and promoter into proximity. siRNA-knockdown of EMSY in skin organotypic culture using primary human keratinocytes reduces EMSY mRNA (n=7, mean 36% reduction p<0.0001) and protein expression (n=7, mean 45% reduction p<0.001) and enhances barrier function defined by water content, water loss and dye penetration. Global mass-spec proteomic analysis detected >8000 proteins per sample with increased expression (fold-change ≥2.5 p<0.05) of 154 proteins, notably filaggrin and filaggrin-2, and mass spec lipidomics showed an increase (p<10-7) in longer chain ceramides known to be down-regulated in AE. Ultrastructural changes include increased desmosome size and corneodesmosome abundance. In contrast, over-expression of EMSY in primary keratinocytes leads to a reduction in markers of differentiation and barrier formation. In keeping with these in vitro observations, skin biopsy samples from patients show greater staining of EMSY in the nucleus (14 AE cases and 18 controls), consistent with an increased functional effect of this transcriptional control protein. Taken together our findings demonstrate an important role for EMSY expression in the pathogenesis of AE.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.