- Email: [email protected]
154 Why should transcriptomics and proteomics analyses performed for genodermatoses?
Genetics and Cell Based Therapy | ABSTRACTS 152
153
Predictive phenotyping of inherited ichthyosis by next generation DNA sequencing R Saito1, A Boyce2, C Hsu2, E Rashidghamat2, J Mellerio2, M Hide1 and MA John2 1 Department of Dermatology, Hiroshima University, Hiroshima, Japan and 2 St John’s Institute of Dermatology, King’s College London, Guy’s Hospital, London, United Kingdom Advances in DNA sequencing technologies have had a major impact on disease gene discovery strategies, including for many genodermatoses. Clinically, one of the major challenges in assessing neonates and infants with ichthyosis is to be able to make an early and accurate diagnosis. This is important so that the likely natural history of that individual’s ichthyosis can be predicted and optimal treatment initiated. We report a 7-month-old boy who was noted to be erythrodermic with generalized ichthyosis. Thick adherent scale was also noted on the palms and soles, although these areas of hyperkeratosis almost completely resolved over the first week of life. Aged 3 months, he developed extensive exfoliative changes affecting the whole body with thick scale. Following informed consent, whole exome sequencing (WES) was performed using genomic DNA extracted from peripheral blood of the child, his mother and father. It revealed an acceptor splice site mutation, c.1374-1G>C (p.Ser458Argfs*120), in intron 6 of KRT10 (encoding keratin 10). Using Sanger sequencing, we confirmed that this mutation was only present in the child’s DNA and not in parental DNA. This mutation has been reported previously in three cases of ichthyosis with confetti (IWC; MIM609165). IWC is a severe non-syndromic form of ichthyosis due to heterozygous mutations in KRT10 or KRT1. The disease manifests at birth with erythroderma and scaling, or sometimes a collodion membrane, and is characterized by the gradual development of numerous confetti-like spots of normal skin. Importantly, diagnosis of IWC is frequently delayed until adolescence or adulthood, i.e. after the development of normal skin spots. Prior to our case, the youngest patient diagnosed was 3 years of age, although the diagnosis in that case was prompted by skin biopsy. In contrast, IWC was diagnosed in our case at 7 months of age, before the development of any pathognomonic skin lesions and without skin biopsy.
Characterization of interstitial collagenase MMP1 as a candidate gene for gene therapy in psoriasis A Mezentsev1, A Zolotarenko1, N Starodubtseva3, J Mogulevtseva2, V Sobolev1, A Soboleva1 and S Bruskin1 1 Functional Genomics, Federal Non-profit Research Institution of Russian Academy of Sciences, NI Vavilov Institute of General Genetics, Moscow, Russian Federation, 2 Federal Non-profit Institution of Higher Education, KA Timiryazev Russian State Agrarian University, Moscow, Russian Federation and 3 Department of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation Psoriasis is an immune-mediated skin condition with genetic predisposition. The gene that encodes interstitial collagenase MMP1 is upregulated in lesional psoriatic skin and downregulated in the skin in the period of disease remission. Our aim was to analyze the data on MMP1 expression, secretion and activation to characterize MMP1 as a possible candidate gene for gene therapy of psoriasis. Immunohistochemistry was used to detect MMP1 in psoriatic skin lesions. Changes in gene expression were detected by qPCR. Cell migration was assessed by scratch assay. Cell proliferation was estimated by comparing the cell growth in control cells and cells treated with proinflammatory factors (IL17, TNF, IFNG and S100A7). Our results suggest that MMP1 is among three other matrix metalloproteinases differentially expressed in psoriatic skin. We also found that MMP1 levels decline after a course of PUVA therapy. Moreover, higher MMP1 levels have been observed in psoriatic plaques and the blood serum of psoriatic patients. In addition, we were able to induce MMP1 by treatment of HaCaT with the proinflammatory factors IL17, TNF, IFNG and S100A7 and demonstrate that higher MMP1 levels in cells are associated with faster migration and higher proliferation rates. We conclude that MMP1 could be reconsidered as a treatment option for psoriasis if negative consequences were taken in account and necessary precautions were applied.
154
155
Why should transcriptomics and proteomics analyses performed for genodermatoses? L Tanrikulu Dermatology, Zekai Tahir Burak Women’s Health Hospital, Ankara, Turkey Genotype-phenotype correlations in genodermatoses are variable due to allelic heterogenity (mutations in a single gene causing more than one disorders) and locus heterogenity (mutations in different genes causing the same disorder). It is wiser to focus on not only mutations but also its effects on the organism. In addition to performing mutation analyses, comparative gene expression analyses (transcriptomics) and its translation to protein synthesis can not only unveil genotype-phenotype correlations but also indicate potential therapeutical targets for genodermatoses. Due to negative regulation of mTOR signaling by mutated hamartin and tuberin genes in tuberous sclerosis, topical Rapamycin shows promising results in facial angiofibromas in tuberous sclerosis patients. Recently, comparative transcriptomic and proteomic analyses of involved and uninvolved plantar skin of pachyonychia congenita reveals a new therapeutic target for troublesome plantar pain by demonstrating that increased SPRR1A expression, along with other neuropathy-related genes, directly affects plantar pain. These scarce but meaningful examples of functional genomics in genodermatoses should encourage further similar studies.
Identification of isomiRs in recessive dystrophic epidermolysis bullosa R Zauner1, M Wimmer1, T Lettner1, N Niklas2, S Atzmu¨ller2, J Reichelt1, JW Bauer3 and V Wally1 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria, 2 Red Cross Transfusion Service for Upper Austria, Linz, Austria, Linz, Austria and 3 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria The monogenic disorder recessive dystrophic epidermolysis bullosa (RDEB) belongs to the family of rare genodermatoses. It is characterized by mutations in the COL7A1 gene, resulting in absent or dysfunctional anchoring fibrils, which are made of type VII collagen. Missing or defective type VII collagen causes severe skin blistering even upon minor trauma and comes along with an increased risk of developing particularly aggressive squamous cell carcinomas (SCC). MicroRNAs are small members of the non-coding RNA world acting as mediators of post-trancriptional regulation. Various pathologies have been reported to be associated with altered expression levels of canonical microRNAs. Recent research indicates a potential biological role for sequence variants of microRNAs. To investigate biologically relevant microRNA isomer expression patterns in RDEB, RNAseq was performed on RDEB-SCC, nonSCC RDEB and healthy control keratinocyte cell lines. An average two-fold decrease of overall isomiR to canonical counts indicates a trend of general variant reduction in SCC compared to healthy control, such as potential SCC relevant microRNAs like miR-10b. However, a 5’ trimming event in a variant of miR-21 demonstrates a two-fold increase in expression level comparing SCC versus healthy control. The resulting seed shift in isomiR-21 mimics the canonical seed sequence of a different microRNA, miR-5579-3p, which is reported to target the neoplastic transformation inhibitor protein PDCD4 mRNA. The observed adaption of the microRNA-transcriptome comprising a high number of variants, suggests that isomiRs may be regulated during disease progression in RDEB, indicating a putative functional contribution of these non-canonical microRNA variants.
156
157
First report of digenic inheritance in pustular psoriasis S Twelves1, D Burden2, S Ma´rta3, Z Bata-Cso¨rgo¨3, S Choon4, M Mockenhaupt5, CH Smith1, F Capon1 and J Barker1 1 Division of Genetics and Molecular Medicine, King’s College London, London, United Kingdom, 2 Department of Dermatology, University of Glasgow, Glasgow, United Kingdom, 3 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 4 Department of Dermatology, Hospital Sultanah Aminah, Johor Bahru, Malaysia and 5 Department of Dermatology, Medical Center-University of Freiburg, Freiburg, Germany The term pustular psoriasis (PP) refers to a heterogeneous group of inflammatory skin disorders presenting with recurrent pustular eruptions. These can be chronic and localised (acral pustular psoriasis) or manifest during acute disease flares that are often accompanied by systemic upset (generalised pustular psoriasis). Genetic investigations revealed that a proportion of PP cases are the result of uncontrolled IL-36 signalling, caused by mutations of the interleukin-36 receptor antagonist gene (IL36RN). While IL36RN alleles are enriched in individuals with early-onset generalised pustular psoriasis, the clinical phenotypes that are associated with recently discovered PP loci (AP1S3 and CARD14) have yet to be defined, so that the genetic basis of varying disease severity remains poorly understood. To address this issue, we screened AP1S3 in 85 unrelated and newly ascertained patients suffering from generalised (n¼43) or acral pustular psoriasis (n¼42). We found that five of the 52 European cases (9.6%) carried a previously described AP1S3 mutation (p.Phe4Cys or p.Arg33Trp). Interestingly, the two patients harbouring the AP1S3 p.Phe4Cys change also carried an IL36RN disease allele (p.Ser113Leu). Both individuals suffered from generalised pustular psoriasis; one had a sister who had experienced less severe clinical symptoms and only carried the IL36RN mutation. These findings highlight digenic inheritance as a molecular mechanism underlying the more severe forms of pustular psoriasis. The observation that AP1S3 mutations can exacerbate the effect of IL36RN alleles also suggests that the two genes affect the same molecular pathway, highlighting abnormal IL-36 signalling as a fundamental pathogenic process in PP.
Efficacy and mechanism of amlexanox for potential treatment of recessive dystrophic epidermolysis bullosa VS Atanasova1, C Gruber2, M Chen3, J Uitto1 and A South1 1 Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, 2 Dermatology, EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Paracelsus Medical University, Salzburg, Austria and 3 Dermatology, University of Southern California, Los Angeles, CA Recessive dystrophic epidermolysis bullosa (RDEB), a rare skin blistering disorder, is caused by loss-of-function mutations in the gene encoding type VII collagen (C7), COL7A1. The main phenotype in RDEB is compromised skin architecture and fragility, resulting in constant wounding and excessive scarring. Nonfunctional C7 results in defective anchoring fibrils, which provide adhesion between the epidermis and dermis. In this study we are interested in premature termination codon (PTC) mutations in COL7A1. PTCs are associated with mRNA instability and nonsense mediated mRNA decay (NMD) as well as early termination of protein translation, thus leading to non-functional protein. We evaluate a read-through approach for overcoming PTC mutations in COL7A1 as potential therapy for RDEB. Reading-through PTCs allows for insertion of an amino acid at the mutation site and synthesis of full-length protein. Several drugs have been reported for their read-through ability but their efficacy is gene dependent. Here we evaluate an FDA approved drug - amlexanox - and demonstrate that it induces full-length C7 synthesis in patient derived RDEB keratinocytes and fibroblasts in a codon sequence specific manner. From six mutant alleles tested, four responded to amlexanox treatment. Treated RDEB cells showed 3- to 14-fold increase of C7 synthesis by western blot and 2- to 5-fold upregulation of COL7A1 mRNA levels, measured by qPCR. In our system read-through correlated with UPF1 phosphorylation state e the main factor in NMD. In conclusion, our study shows that amlexanox induces PTC read-through and synthesis of fulllength C7 in RDEB cells and upregulates COL7A1 transcript levels. Our results suggest amlexanox as potential therapy for RDEB patients harboring PTC mutations.
www.jidonline.org S187
153
Predictive phenotyping of inherited ichthyosis by next generation DNA sequencing R Saito1, A Boyce2, C Hsu2, E Rashidghamat2, J Mellerio2, M Hide1 and MA John2 1 Department of Dermatology, Hiroshima University, Hiroshima, Japan and 2 St John’s Institute of Dermatology, King’s College London, Guy’s Hospital, London, United Kingdom Advances in DNA sequencing technologies have had a major impact on disease gene discovery strategies, including for many genodermatoses. Clinically, one of the major challenges in assessing neonates and infants with ichthyosis is to be able to make an early and accurate diagnosis. This is important so that the likely natural history of that individual’s ichthyosis can be predicted and optimal treatment initiated. We report a 7-month-old boy who was noted to be erythrodermic with generalized ichthyosis. Thick adherent scale was also noted on the palms and soles, although these areas of hyperkeratosis almost completely resolved over the first week of life. Aged 3 months, he developed extensive exfoliative changes affecting the whole body with thick scale. Following informed consent, whole exome sequencing (WES) was performed using genomic DNA extracted from peripheral blood of the child, his mother and father. It revealed an acceptor splice site mutation, c.1374-1G>C (p.Ser458Argfs*120), in intron 6 of KRT10 (encoding keratin 10). Using Sanger sequencing, we confirmed that this mutation was only present in the child’s DNA and not in parental DNA. This mutation has been reported previously in three cases of ichthyosis with confetti (IWC; MIM609165). IWC is a severe non-syndromic form of ichthyosis due to heterozygous mutations in KRT10 or KRT1. The disease manifests at birth with erythroderma and scaling, or sometimes a collodion membrane, and is characterized by the gradual development of numerous confetti-like spots of normal skin. Importantly, diagnosis of IWC is frequently delayed until adolescence or adulthood, i.e. after the development of normal skin spots. Prior to our case, the youngest patient diagnosed was 3 years of age, although the diagnosis in that case was prompted by skin biopsy. In contrast, IWC was diagnosed in our case at 7 months of age, before the development of any pathognomonic skin lesions and without skin biopsy.
Characterization of interstitial collagenase MMP1 as a candidate gene for gene therapy in psoriasis A Mezentsev1, A Zolotarenko1, N Starodubtseva3, J Mogulevtseva2, V Sobolev1, A Soboleva1 and S Bruskin1 1 Functional Genomics, Federal Non-profit Research Institution of Russian Academy of Sciences, NI Vavilov Institute of General Genetics, Moscow, Russian Federation, 2 Federal Non-profit Institution of Higher Education, KA Timiryazev Russian State Agrarian University, Moscow, Russian Federation and 3 Department of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation Psoriasis is an immune-mediated skin condition with genetic predisposition. The gene that encodes interstitial collagenase MMP1 is upregulated in lesional psoriatic skin and downregulated in the skin in the period of disease remission. Our aim was to analyze the data on MMP1 expression, secretion and activation to characterize MMP1 as a possible candidate gene for gene therapy of psoriasis. Immunohistochemistry was used to detect MMP1 in psoriatic skin lesions. Changes in gene expression were detected by qPCR. Cell migration was assessed by scratch assay. Cell proliferation was estimated by comparing the cell growth in control cells and cells treated with proinflammatory factors (IL17, TNF, IFNG and S100A7). Our results suggest that MMP1 is among three other matrix metalloproteinases differentially expressed in psoriatic skin. We also found that MMP1 levels decline after a course of PUVA therapy. Moreover, higher MMP1 levels have been observed in psoriatic plaques and the blood serum of psoriatic patients. In addition, we were able to induce MMP1 by treatment of HaCaT with the proinflammatory factors IL17, TNF, IFNG and S100A7 and demonstrate that higher MMP1 levels in cells are associated with faster migration and higher proliferation rates. We conclude that MMP1 could be reconsidered as a treatment option for psoriasis if negative consequences were taken in account and necessary precautions were applied.
154
155
Why should transcriptomics and proteomics analyses performed for genodermatoses? L Tanrikulu Dermatology, Zekai Tahir Burak Women’s Health Hospital, Ankara, Turkey Genotype-phenotype correlations in genodermatoses are variable due to allelic heterogenity (mutations in a single gene causing more than one disorders) and locus heterogenity (mutations in different genes causing the same disorder). It is wiser to focus on not only mutations but also its effects on the organism. In addition to performing mutation analyses, comparative gene expression analyses (transcriptomics) and its translation to protein synthesis can not only unveil genotype-phenotype correlations but also indicate potential therapeutical targets for genodermatoses. Due to negative regulation of mTOR signaling by mutated hamartin and tuberin genes in tuberous sclerosis, topical Rapamycin shows promising results in facial angiofibromas in tuberous sclerosis patients. Recently, comparative transcriptomic and proteomic analyses of involved and uninvolved plantar skin of pachyonychia congenita reveals a new therapeutic target for troublesome plantar pain by demonstrating that increased SPRR1A expression, along with other neuropathy-related genes, directly affects plantar pain. These scarce but meaningful examples of functional genomics in genodermatoses should encourage further similar studies.
Identification of isomiRs in recessive dystrophic epidermolysis bullosa R Zauner1, M Wimmer1, T Lettner1, N Niklas2, S Atzmu¨ller2, J Reichelt1, JW Bauer3 and V Wally1 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria, 2 Red Cross Transfusion Service for Upper Austria, Linz, Austria, Linz, Austria and 3 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria The monogenic disorder recessive dystrophic epidermolysis bullosa (RDEB) belongs to the family of rare genodermatoses. It is characterized by mutations in the COL7A1 gene, resulting in absent or dysfunctional anchoring fibrils, which are made of type VII collagen. Missing or defective type VII collagen causes severe skin blistering even upon minor trauma and comes along with an increased risk of developing particularly aggressive squamous cell carcinomas (SCC). MicroRNAs are small members of the non-coding RNA world acting as mediators of post-trancriptional regulation. Various pathologies have been reported to be associated with altered expression levels of canonical microRNAs. Recent research indicates a potential biological role for sequence variants of microRNAs. To investigate biologically relevant microRNA isomer expression patterns in RDEB, RNAseq was performed on RDEB-SCC, nonSCC RDEB and healthy control keratinocyte cell lines. An average two-fold decrease of overall isomiR to canonical counts indicates a trend of general variant reduction in SCC compared to healthy control, such as potential SCC relevant microRNAs like miR-10b. However, a 5’ trimming event in a variant of miR-21 demonstrates a two-fold increase in expression level comparing SCC versus healthy control. The resulting seed shift in isomiR-21 mimics the canonical seed sequence of a different microRNA, miR-5579-3p, which is reported to target the neoplastic transformation inhibitor protein PDCD4 mRNA. The observed adaption of the microRNA-transcriptome comprising a high number of variants, suggests that isomiRs may be regulated during disease progression in RDEB, indicating a putative functional contribution of these non-canonical microRNA variants.
156
157
First report of digenic inheritance in pustular psoriasis S Twelves1, D Burden2, S Ma´rta3, Z Bata-Cso¨rgo¨3, S Choon4, M Mockenhaupt5, CH Smith1, F Capon1 and J Barker1 1 Division of Genetics and Molecular Medicine, King’s College London, London, United Kingdom, 2 Department of Dermatology, University of Glasgow, Glasgow, United Kingdom, 3 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 4 Department of Dermatology, Hospital Sultanah Aminah, Johor Bahru, Malaysia and 5 Department of Dermatology, Medical Center-University of Freiburg, Freiburg, Germany The term pustular psoriasis (PP) refers to a heterogeneous group of inflammatory skin disorders presenting with recurrent pustular eruptions. These can be chronic and localised (acral pustular psoriasis) or manifest during acute disease flares that are often accompanied by systemic upset (generalised pustular psoriasis). Genetic investigations revealed that a proportion of PP cases are the result of uncontrolled IL-36 signalling, caused by mutations of the interleukin-36 receptor antagonist gene (IL36RN). While IL36RN alleles are enriched in individuals with early-onset generalised pustular psoriasis, the clinical phenotypes that are associated with recently discovered PP loci (AP1S3 and CARD14) have yet to be defined, so that the genetic basis of varying disease severity remains poorly understood. To address this issue, we screened AP1S3 in 85 unrelated and newly ascertained patients suffering from generalised (n¼43) or acral pustular psoriasis (n¼42). We found that five of the 52 European cases (9.6%) carried a previously described AP1S3 mutation (p.Phe4Cys or p.Arg33Trp). Interestingly, the two patients harbouring the AP1S3 p.Phe4Cys change also carried an IL36RN disease allele (p.Ser113Leu). Both individuals suffered from generalised pustular psoriasis; one had a sister who had experienced less severe clinical symptoms and only carried the IL36RN mutation. These findings highlight digenic inheritance as a molecular mechanism underlying the more severe forms of pustular psoriasis. The observation that AP1S3 mutations can exacerbate the effect of IL36RN alleles also suggests that the two genes affect the same molecular pathway, highlighting abnormal IL-36 signalling as a fundamental pathogenic process in PP.
Efficacy and mechanism of amlexanox for potential treatment of recessive dystrophic epidermolysis bullosa VS Atanasova1, C Gruber2, M Chen3, J Uitto1 and A South1 1 Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, 2 Dermatology, EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Paracelsus Medical University, Salzburg, Austria and 3 Dermatology, University of Southern California, Los Angeles, CA Recessive dystrophic epidermolysis bullosa (RDEB), a rare skin blistering disorder, is caused by loss-of-function mutations in the gene encoding type VII collagen (C7), COL7A1. The main phenotype in RDEB is compromised skin architecture and fragility, resulting in constant wounding and excessive scarring. Nonfunctional C7 results in defective anchoring fibrils, which provide adhesion between the epidermis and dermis. In this study we are interested in premature termination codon (PTC) mutations in COL7A1. PTCs are associated with mRNA instability and nonsense mediated mRNA decay (NMD) as well as early termination of protein translation, thus leading to non-functional protein. We evaluate a read-through approach for overcoming PTC mutations in COL7A1 as potential therapy for RDEB. Reading-through PTCs allows for insertion of an amino acid at the mutation site and synthesis of full-length protein. Several drugs have been reported for their read-through ability but their efficacy is gene dependent. Here we evaluate an FDA approved drug - amlexanox - and demonstrate that it induces full-length C7 synthesis in patient derived RDEB keratinocytes and fibroblasts in a codon sequence specific manner. From six mutant alleles tested, four responded to amlexanox treatment. Treated RDEB cells showed 3- to 14-fold increase of C7 synthesis by western blot and 2- to 5-fold upregulation of COL7A1 mRNA levels, measured by qPCR. In our system read-through correlated with UPF1 phosphorylation state e the main factor in NMD. In conclusion, our study shows that amlexanox induces PTC read-through and synthesis of fulllength C7 in RDEB cells and upregulates COL7A1 transcript levels. Our results suggest amlexanox as potential therapy for RDEB patients harboring PTC mutations.
www.jidonline.org S187