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528 Anacardic acid reduces lipogenesis in human differentiated adipocytes via inhibition of histone acetylation
Genetic Disease, Gene Regulation and Gene Therapy | ABSTRACTS 528
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Anacardic acid reduces lipogenesis in human differentiated adipocytes via inhibition of histone acetylation M Kim1, E Kim2, Y Kim3, Y Lee2, D Lee2 and J Chung4 1 Department of Dermatology, Seoul National University Hospital, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 2 Department of Dermatology, Seoul National University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 3 Seoul National University Hospital, Seoul, Seoul-t’ukpyolsi, Republic of Korea and 4 Seoul National University, Seoul, Seoul-t’ukpyolsi, Republic of Korea Here we investigated effects of anacardic acid (AA) on the regulation of lipogenesis in differentiated human adipocytes, and elucidated possible epigenetic mechanisms via p300 histone acetyltransferase activity. To investigate the role of histone acetylation in lipogenesis regulation, we evaluated triglyceride (TG) contents, expression of key lipogenic enzyme acetyl-CoA carboxylase (ACC) and sterol regulatory element binding protein 1c (SREBP-1c) in primary cultured adipocytes isolated from subcutaneous adipose tissues. Treatment of AA or knockdown of p300 by using transient transfection of p300 siRNA led to significant reduction of TG contents, SREBP-1c and ACC expression, indicating that p300 mediates SREBP-1, ACC expression, and corollary lipid production. While p300 overexpression by p300WT was associated with significantly enhanced activity of SREBP1-908luc promoter, the SREBP1908luc promoter activity was significantly reduced in the presence of p300DHAT. In addition, we performed a promoter assay using HEK293T cells treated with AA or TSA. While the SREBP1-908luc promoter activity was significantly decreased by AA, but significantly increased by TSA treatment. These findings suggest that histone acetyltransferase activity of p300, not a p300 expression per se, is critical for the transcriptional regulation of SREBP-1 and p300HAT inhibitors such as AA could be employed as anti-obesity modalities.
Novel CYP26 inhibitors potentiate the effects of all-trans-retinoic acid on phenotype of normal and Darier disease keratinocytes in reconstructed human epidermis J Veit1, N Guilloteau1, D Mendes2, V De Glas3, B Balau3, F Astruc-Diaz2, Y Poumay3 and P Diaz2 1 University of Montana, Missoula, MT, 2 DermaXon, Missoula, MT and 3 Universite´ de Namur, Namur, Belgium Darier disease (DD) is a rare hereditary dominant human disorder characterized by warty papules and plaques in seborrheic areas of the skin. DD has a chronic course with frequent relapses while treatments often remain unsatisfactory. Retinoic Acid (RA) and its derivatives (retinoids) cause substantial clinical improvements in patients with DD. However, their use remains limited due to significant adverse effects. The endogenous concentration of RA is predominantly controlled via RA-inducible cytochrome P450 family 26 enzymes (CYP26), which inactivate RA by hydroxylation. As an alternative approach to therapeutic administration of retinoids, inhibition of CYP26 enzymes and thus of RA clearance represents an attractive strategy due to resulting elevated concentrations of endogenous RA. We previously designed and synthesized a CYP26A1/B1 dual inhibitor and inhibitors selective for either CYP26A1 or CYP26B1. In this study, we investigated the effects of RA alone or in combination with these CYP26 inhibitors on gene expression and on epidermal barrier using transepithelial electrical resistance (TEER), in reconstructed human epidermis (RHE) made of primary keratinocytes from either normal or DD patients. When the dual CYP26A1/B1 inhibitor was combined with RA (10-9 M), the TEER and the expression of differentiation markers were modulated similarly to the effects obtained with higher concentrations of RA. Our data revealed that dual selective inhibition of CYP26A1/B1 with our new compound DX308 potentiates the effect of RA at low concentration on RHE. Hence, the result of this study indicates that dual inhibition of CYP26A1 and B1 by DX308 may represent a promising therapeutic strategy for the treatment of DD with reduced retinoid-induced adverse effects.
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Discovery of novel somatic mutations in patients with lymphatic malformations MN Pyles1, JH Tu1, W Donahue2, P Toydemir2, K Sarin1, K Rieger1 and J Teng1 1 Stanford Dermatology, Stanford, CA and 2 Department of Pathology, University of Utah, Salt Lake City, UT Recent studies have demonstrated that a significant number of patients with lymphatic (LM) or venolymphatic malformations (VLM) have associated PIK3CA mutations. Our goal of the study was to understand the prevalence of somatic PIK3CA mutation among patients with LM or VLM; and to discover other possible somatic mutations in affected patients. Highthroughput sequencing was performed on archived tissue samples from 24 patients; blood samples were obtained as controls. Of these 24 patients, 14 (58%) patients had PIK3CA mutations ranging from 1-9% in affected tissue. Eight of the 14 (57%) patients were found to have mutations in functional domain p.E542K of PIK3CA, and 4/14 (29%) patients have mutations in functional domain p.H1047R. Other somatic mutations in patients with PIK3CA mutation were point mutations in TEK, encoding endothelial cell tyrosine kinase receptor TIE2, found in 5 patients at R918C, at R915C, in T1106T, A1045T, and R1099X. Other somatic mutations in this cohort include point mutations in TSC2 at P1282L, E793D, A1537T, R208Q, V692I and in TSC1 at R1062W and P513S; in GJC2 at R367C and R342C; in AKT2 at R357C. Point mutations in the mTOR protein complex pathway were discovered in RICTOR R380C, RPTOR R1148Q, and MTOR R2060Q. In patients without somatic PIK3CA mutations, somatic mutations were found in TEK, 2 at R918C and R915C and 1 inC1118Y and Y897C. Mutations in AKT2 p.P375P and MTOR p.R454C were similar to those found in patients with PIK3CA somatic mutations. Mutations in TEK have been reported to cause venous malformations and GJC has been associated with lymphedema. Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent TEK mutations in LMs with potential significance of effective specific drug therapy.
Identification of a heterozygous p.Gly568Val missense mutation in the TRPV3 gene in a patient with Olmsted syndrome: In silico analysis of TRPV3 H Nagai1, Y Takaoka2, A Sugano2, Y Nakamachi3, S Kawano4 and C Nishigori1 1 Division of Dermatology, Department of Internal Related, Kobe University Graduate School of Medicine, Kobe, Japan, 2 Division of Medical Informatics and Bioinformatics, Kobe University Graduate School of Medicine, Kobe, Japan, 3 Department of Clinical Laboratory, Kobe University Hospital, Kobe, Japan and 4 Division of Laboratory Medicine, Department of Internal Related, Kobe University Graduate School of Medicine, Kobe, Japan Olmsted syndrome is a very rare congenital disorder, characterized by palmoplantar keratoderma and periorificial keratotic lesions. Recently, TRPV3 was reported to be a causative gene of Olmsted syndrome. We identified a heterozygous missense mutation of TRPV3, c.1703G>T, p.Gly568Val, in a Japanese patient with severe palmoplantar keratodermas caused by Olmsted syndrome. We conducted in silico analysis of TRPV3 to evaluate whether the p.Gly568Val leads to structural changes in the TRPV3 selectivity filter. The selectivity filter was shown to become dilated as a result of genetic mutation (p.Gly573Ser, p.Tr692Gly, or p.Gly568Val) as well as after a change in temperature (300K to 310K). Although further analyses are required, in silico analysis of TRPV3 could be a useful approach in predicting mutation-induced activated states of ion channels, and thus enrich our understanding of the pathogenesis of Olmsted syndrome.
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siRNA based non-viral gene therapy for the treatment of epidermolysis bullosa simplex(EBS) J O’Keeffe Ahern1, D Zhou1, L Cutlar1, I Lara-Sa´ez1 and W Wang2 1 Charles Institute of Dermatology UCD, Dublin, Ireland and 2 University College Dublin (UCD), Ireland, Dublin, Ireland EBS is an inherited, skin fragility disorder predominantly caused by dominant-negative mutations in genes encoding for the cytoskeletal proteins, Keratin5 and Keratin14 within the basal cell layer. To date, EBS is incurable with only symptomatic therapies currently available. The downregulation of these mutant genes would provide an excellent curative therapy for treating EBS. siRNA therapeutics have been identified as an attractive therapy option for EBS given the highly accessible nature of skin tissue. The biggest hurdle thus far has been the identification of an effective system to deliver siRNA’s into the skin. Among non-viral delivery vector systems, polymers, in particular cationic polymers are some of the most extensively studied delivery systems for RNA payloads. Cationic polymers have the ability to electrostatically bind to the negatively charged RNA. The ideal delivery system would be an amalgamation of the structural flexibility and stability of highly branched cationic polymers with the membrane interaction profile of lipids to yield a delivery system that will be both safe and effective. Utilizing the tunable nature of our novel highly branched polymer based delivery system provides an excellent platform to screen and assess these siRNA delivery vectors. Thus far we have successfully encapsulated a siRNA into a lipid-like nanoparticle through conjugation with a highly branched polymer with promising preliminary data indicating a safe and efficient knockdown of a target gene of interest. Further comprehensive analysis of the delivery system shall involve assaying physicochemical properties identified as key indicators of in-vitro transfection success.
Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants C Wong1, S Denil2, J Foo3, H Chen2, A Tay2, R Haines2, M Tang4, I McLean5, B Lane2, J Liu6 and J Common2 1 Institute of Medical Biology, Singapore, 2 Institute of Medical Biology, Singapore, Singapore, 3 Nanyang Technological University, Singapore, 4 National Skin Centre, Singapore, 5 University of Dundee, Dundee, United Kingdom and 6 Genome Institute of Singapore, Singapore The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation, and has a highly repetitive nucleotide sequence containing very limited stretches of unique nucleotides for precise mapping to reference genomes. Sequencing strategies using PCR and conventional Sanger sequencing have been successful for complete FLG coding DNA sequence amplification to identify pathogenic mutations but this time-consuming, labour intensive method has restricted utility. Next-generation sequencing (NGS) offers obvious benefits to accelerate FLG analysis but standard re-sequencing techniques can be expensive, especially for a single target gene of interest. We therefore designed a protocol to improve FLG sequencing throughput using a set of FLG-specific PCR primer assays compatible with microfluidic amplification, multiplexing and current NGS protocols. Using DNA reference samples with known FLG genotypes for benchmarking, this protocol is shown to be concordant for variant detection. Analyzing cohorts from ethnicities previously not studied for FLG variants demonstrates usefulness for discovery projects. This comprehensive sequencing protocol is labour-efficient and offers an affordable solution to scale up FLG sequencing for larger cohorts. Robust and rapid FLG sequencing can improve patient stratification for research projects and provide a framework for gene specific diagnosis in the future.
www.jidonline.org S91
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Anacardic acid reduces lipogenesis in human differentiated adipocytes via inhibition of histone acetylation M Kim1, E Kim2, Y Kim3, Y Lee2, D Lee2 and J Chung4 1 Department of Dermatology, Seoul National University Hospital, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 2 Department of Dermatology, Seoul National University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 3 Seoul National University Hospital, Seoul, Seoul-t’ukpyolsi, Republic of Korea and 4 Seoul National University, Seoul, Seoul-t’ukpyolsi, Republic of Korea Here we investigated effects of anacardic acid (AA) on the regulation of lipogenesis in differentiated human adipocytes, and elucidated possible epigenetic mechanisms via p300 histone acetyltransferase activity. To investigate the role of histone acetylation in lipogenesis regulation, we evaluated triglyceride (TG) contents, expression of key lipogenic enzyme acetyl-CoA carboxylase (ACC) and sterol regulatory element binding protein 1c (SREBP-1c) in primary cultured adipocytes isolated from subcutaneous adipose tissues. Treatment of AA or knockdown of p300 by using transient transfection of p300 siRNA led to significant reduction of TG contents, SREBP-1c and ACC expression, indicating that p300 mediates SREBP-1, ACC expression, and corollary lipid production. While p300 overexpression by p300WT was associated with significantly enhanced activity of SREBP1-908luc promoter, the SREBP1908luc promoter activity was significantly reduced in the presence of p300DHAT. In addition, we performed a promoter assay using HEK293T cells treated with AA or TSA. While the SREBP1-908luc promoter activity was significantly decreased by AA, but significantly increased by TSA treatment. These findings suggest that histone acetyltransferase activity of p300, not a p300 expression per se, is critical for the transcriptional regulation of SREBP-1 and p300HAT inhibitors such as AA could be employed as anti-obesity modalities.
Novel CYP26 inhibitors potentiate the effects of all-trans-retinoic acid on phenotype of normal and Darier disease keratinocytes in reconstructed human epidermis J Veit1, N Guilloteau1, D Mendes2, V De Glas3, B Balau3, F Astruc-Diaz2, Y Poumay3 and P Diaz2 1 University of Montana, Missoula, MT, 2 DermaXon, Missoula, MT and 3 Universite´ de Namur, Namur, Belgium Darier disease (DD) is a rare hereditary dominant human disorder characterized by warty papules and plaques in seborrheic areas of the skin. DD has a chronic course with frequent relapses while treatments often remain unsatisfactory. Retinoic Acid (RA) and its derivatives (retinoids) cause substantial clinical improvements in patients with DD. However, their use remains limited due to significant adverse effects. The endogenous concentration of RA is predominantly controlled via RA-inducible cytochrome P450 family 26 enzymes (CYP26), which inactivate RA by hydroxylation. As an alternative approach to therapeutic administration of retinoids, inhibition of CYP26 enzymes and thus of RA clearance represents an attractive strategy due to resulting elevated concentrations of endogenous RA. We previously designed and synthesized a CYP26A1/B1 dual inhibitor and inhibitors selective for either CYP26A1 or CYP26B1. In this study, we investigated the effects of RA alone or in combination with these CYP26 inhibitors on gene expression and on epidermal barrier using transepithelial electrical resistance (TEER), in reconstructed human epidermis (RHE) made of primary keratinocytes from either normal or DD patients. When the dual CYP26A1/B1 inhibitor was combined with RA (10-9 M), the TEER and the expression of differentiation markers were modulated similarly to the effects obtained with higher concentrations of RA. Our data revealed that dual selective inhibition of CYP26A1/B1 with our new compound DX308 potentiates the effect of RA at low concentration on RHE. Hence, the result of this study indicates that dual inhibition of CYP26A1 and B1 by DX308 may represent a promising therapeutic strategy for the treatment of DD with reduced retinoid-induced adverse effects.
530
531
Discovery of novel somatic mutations in patients with lymphatic malformations MN Pyles1, JH Tu1, W Donahue2, P Toydemir2, K Sarin1, K Rieger1 and J Teng1 1 Stanford Dermatology, Stanford, CA and 2 Department of Pathology, University of Utah, Salt Lake City, UT Recent studies have demonstrated that a significant number of patients with lymphatic (LM) or venolymphatic malformations (VLM) have associated PIK3CA mutations. Our goal of the study was to understand the prevalence of somatic PIK3CA mutation among patients with LM or VLM; and to discover other possible somatic mutations in affected patients. Highthroughput sequencing was performed on archived tissue samples from 24 patients; blood samples were obtained as controls. Of these 24 patients, 14 (58%) patients had PIK3CA mutations ranging from 1-9% in affected tissue. Eight of the 14 (57%) patients were found to have mutations in functional domain p.E542K of PIK3CA, and 4/14 (29%) patients have mutations in functional domain p.H1047R. Other somatic mutations in patients with PIK3CA mutation were point mutations in TEK, encoding endothelial cell tyrosine kinase receptor TIE2, found in 5 patients at R918C, at R915C, in T1106T, A1045T, and R1099X. Other somatic mutations in this cohort include point mutations in TSC2 at P1282L, E793D, A1537T, R208Q, V692I and in TSC1 at R1062W and P513S; in GJC2 at R367C and R342C; in AKT2 at R357C. Point mutations in the mTOR protein complex pathway were discovered in RICTOR R380C, RPTOR R1148Q, and MTOR R2060Q. In patients without somatic PIK3CA mutations, somatic mutations were found in TEK, 2 at R918C and R915C and 1 inC1118Y and Y897C. Mutations in AKT2 p.P375P and MTOR p.R454C were similar to those found in patients with PIK3CA somatic mutations. Mutations in TEK have been reported to cause venous malformations and GJC has been associated with lymphedema. Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent TEK mutations in LMs with potential significance of effective specific drug therapy.
Identification of a heterozygous p.Gly568Val missense mutation in the TRPV3 gene in a patient with Olmsted syndrome: In silico analysis of TRPV3 H Nagai1, Y Takaoka2, A Sugano2, Y Nakamachi3, S Kawano4 and C Nishigori1 1 Division of Dermatology, Department of Internal Related, Kobe University Graduate School of Medicine, Kobe, Japan, 2 Division of Medical Informatics and Bioinformatics, Kobe University Graduate School of Medicine, Kobe, Japan, 3 Department of Clinical Laboratory, Kobe University Hospital, Kobe, Japan and 4 Division of Laboratory Medicine, Department of Internal Related, Kobe University Graduate School of Medicine, Kobe, Japan Olmsted syndrome is a very rare congenital disorder, characterized by palmoplantar keratoderma and periorificial keratotic lesions. Recently, TRPV3 was reported to be a causative gene of Olmsted syndrome. We identified a heterozygous missense mutation of TRPV3, c.1703G>T, p.Gly568Val, in a Japanese patient with severe palmoplantar keratodermas caused by Olmsted syndrome. We conducted in silico analysis of TRPV3 to evaluate whether the p.Gly568Val leads to structural changes in the TRPV3 selectivity filter. The selectivity filter was shown to become dilated as a result of genetic mutation (p.Gly573Ser, p.Tr692Gly, or p.Gly568Val) as well as after a change in temperature (300K to 310K). Although further analyses are required, in silico analysis of TRPV3 could be a useful approach in predicting mutation-induced activated states of ion channels, and thus enrich our understanding of the pathogenesis of Olmsted syndrome.
532
533
siRNA based non-viral gene therapy for the treatment of epidermolysis bullosa simplex(EBS) J O’Keeffe Ahern1, D Zhou1, L Cutlar1, I Lara-Sa´ez1 and W Wang2 1 Charles Institute of Dermatology UCD, Dublin, Ireland and 2 University College Dublin (UCD), Ireland, Dublin, Ireland EBS is an inherited, skin fragility disorder predominantly caused by dominant-negative mutations in genes encoding for the cytoskeletal proteins, Keratin5 and Keratin14 within the basal cell layer. To date, EBS is incurable with only symptomatic therapies currently available. The downregulation of these mutant genes would provide an excellent curative therapy for treating EBS. siRNA therapeutics have been identified as an attractive therapy option for EBS given the highly accessible nature of skin tissue. The biggest hurdle thus far has been the identification of an effective system to deliver siRNA’s into the skin. Among non-viral delivery vector systems, polymers, in particular cationic polymers are some of the most extensively studied delivery systems for RNA payloads. Cationic polymers have the ability to electrostatically bind to the negatively charged RNA. The ideal delivery system would be an amalgamation of the structural flexibility and stability of highly branched cationic polymers with the membrane interaction profile of lipids to yield a delivery system that will be both safe and effective. Utilizing the tunable nature of our novel highly branched polymer based delivery system provides an excellent platform to screen and assess these siRNA delivery vectors. Thus far we have successfully encapsulated a siRNA into a lipid-like nanoparticle through conjugation with a highly branched polymer with promising preliminary data indicating a safe and efficient knockdown of a target gene of interest. Further comprehensive analysis of the delivery system shall involve assaying physicochemical properties identified as key indicators of in-vitro transfection success.
Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants C Wong1, S Denil2, J Foo3, H Chen2, A Tay2, R Haines2, M Tang4, I McLean5, B Lane2, J Liu6 and J Common2 1 Institute of Medical Biology, Singapore, 2 Institute of Medical Biology, Singapore, Singapore, 3 Nanyang Technological University, Singapore, 4 National Skin Centre, Singapore, 5 University of Dundee, Dundee, United Kingdom and 6 Genome Institute of Singapore, Singapore The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation, and has a highly repetitive nucleotide sequence containing very limited stretches of unique nucleotides for precise mapping to reference genomes. Sequencing strategies using PCR and conventional Sanger sequencing have been successful for complete FLG coding DNA sequence amplification to identify pathogenic mutations but this time-consuming, labour intensive method has restricted utility. Next-generation sequencing (NGS) offers obvious benefits to accelerate FLG analysis but standard re-sequencing techniques can be expensive, especially for a single target gene of interest. We therefore designed a protocol to improve FLG sequencing throughput using a set of FLG-specific PCR primer assays compatible with microfluidic amplification, multiplexing and current NGS protocols. Using DNA reference samples with known FLG genotypes for benchmarking, this protocol is shown to be concordant for variant detection. Analyzing cohorts from ethnicities previously not studied for FLG variants demonstrates usefulness for discovery projects. This comprehensive sequencing protocol is labour-efficient and offers an affordable solution to scale up FLG sequencing for larger cohorts. Robust and rapid FLG sequencing can improve patient stratification for research projects and provide a framework for gene specific diagnosis in the future.
www.jidonline.org S91