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162 A keratinocyte culture model for epidermodysplasia verruciformis
ABSTRACTS | Genetics and Cell Based Therapy 158
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Pharmacogenetics of ustekinumab in patients with moderate-to-severe plaque psoriasis E Dauden1, R Prieto-Perez2, M Llamas-Velasco1, T Cabaleiro2 and F Abad-Santos2 1 Dermatology, Hospital Universitario la Princesa, Madrid, Spain and 2 Pharmacology, Hospital Universitario la Princesa, Madrid, Spain Ustekinumab is an effective drug against moderate-to-severe psoriasis, but not all patients are responders to therapy and some patients may develop adverse effects. Only two studies have evaluated the influence of pharmacogenetics in psoriatic patients treated with ustekinumab. We evaluated 121 polymorphisms to study a possible association between these SNPs and the response to ustekinumab measured by PASI75 at 4 months of treatment (N¼69). The weight was evaluated for response to treatment (PASI75) and a higher weight was obtained in nonresponders than responders (p¼0.018). In addition, the adjusted results (FDR) showed an association between 5 SNPs in TNFRSF1A, HTR2A, NFKBIA, ADAM33, and IL13 genes and poor response to ustekinumab. Furthermore, 6 SNPs in CHUK, C17orf51, ZNF816A, STAT4, SLC22A4, and Corf72 genes were associated with better response to ustekinumab. However, there was not significant association between response to ustekinumab and SNPs in HLA-C as has been recently described.
PASH syndrome is not a genetically distinct entity but belongs to the spectrum of autoinflammatory skin phenotypes J Uitto1, M Faraji Zonooz2, F Sabbagh-Kermani3, Z Fattahi2, M Fadaee2, M Reza Akbari4, R Amiri3, H Vahidnezhad1, H Najmabadi2 and A Kariminejad2 1 Thomas Jefferson University, Philadelphia, PA, 2 Kariminejad-Najmabadi Pathology & Genetics Center, Tehran, Iran (the Islamic Republic of), 3 Kerman University of Medical Sciences, Kerman, Iran (the Islamic Republic of) and 4 University of Toronto, Toronto, ON, Canada PASH syndrome, comprising pyoderma gangrenosum (PG) in association with acne and hidradenitis suppurative (HS), was initially suggested to be associated with increased number of CCTG microsatellite repeats in the promoter region of PSTPIP1. In this study we characterized an extended multigenerational Iranian family, including 24 affected and 32 unaffected individuals with manifestations of HS, acne, and PG in an autosomal dominant pattern of inheritance. Mutation analysis was first performed by PCR amplification of the reporter region as well as all 50 exons and flanking intronic sequences of PSTPIP1. No pathogenic variants were detected, and sequencing of the promoter region showed (CCTG)5 repeat as the normal allele size in this gene. Next, whole genome single nucleotide polymorphism genotyping was applied which resulted in identification of genomic intervals with LOD scores >2 on chromosomes 1, 11, and 20. Whole exome sequencing identified a total of 20 rare sequence variants in these chromosomal regions, and filtering prioritization and cosegregation analyses identified a mutation, c.1635C/G (p.Tyr545*) in the NCSTN gene, recently shown to harbor mutations in cases with HS. This mutation perfectly co-segregated with the disease phenotype in the family. NCSTN encodes nicastrin, one of the three subunits of g-secretase, a transmembrane endoprotease complex that catalyzes cleavage of many transmembrane proteins, such as Notch receptors. Thus, PG may be part of the autoinflammatory spectrum of skin diseases, together with HS and acne. Considering the phenotypic and genotypic heterogeneity of these autoinflammatory skin phenotypes, we conclude that PASH syndrome is not a genetically distinct entity, but rather belongs to the spectrum of g-secretase-related disorders.
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Demonstration of the critical role of the liver in systemic PPi homeostasis using a novel Abcc6 knockout rat model of PXE Q Li1, K van de Wetering2, J Sundberg3 and J Uitto1 1 Thomas Jefferson University, Philadelphia, PA, 2 Netherlands Cancer Institute, Amsterdam, Netherlands and 3 The Jackson Laboratory, Bar Harbor, ME Pseudoxanthoma elasticum (PXE) is a heritable multisystem ectopic mineralization disorder. It is caused by mutations in the ABCC6 gene that encodes ABCC6, a transmembrane efflux transporter expressed primarily in the liver, and to some extent in the kidneys. The precise pathomechanistic details leading from lack of ABCC6 expression in the liver or kidney to mineralization in peripheral tissues are unknown. We generated Abcc6-/- rats using zinc finger nuclease technology and these rats provide a model for metabolic studies to specifically unravel the role of PPi and Pi homeostasis in PXE. Immunostaining of liver sections using a rat ABCC6 specific antibody showed basolateral plasma membrane localization in hepatocytes of wild type rats and complete absence in the homozygous Abcc6-/- rats. As a consequence of ABCC6 deficiency, complete necropsy revealed ectopic mineralization in the muzzle skin, eyes, and arterial blood vessels in the knockout rats, features mimicking human PXE. Mineralization in the dermal sheath of vibrissae was also monitored by small animal CT scanning, which allows non-invasive evaluation of the progression of mineralization. Plasma PPi level was significantly reduced (<60%) in the Abcc6-/- rats as compared to wild type rats, leading to a significantly lower PPi/Pi ratio in plasma. To assess whether absence of ABCC6 in the liver is responsible for PXE pathology, we performed in vivo liver perfusion experiments followed by quantification of PPi in the perfusate. PPi levels in the perfusates of Abcc6-/- rats were significantly lower (<3%) than perfusates in the wild type rats, pointing to the critical role of hepatic ABCC6 in providing the circulation with PPi. Since PPi is a potent antimineralization factor, the results highlight the role of PPi in the context of the PPi/Pi ratio in the novel Abcc6-/- rat model due to loss-of-function of ABCC6, leading to ectopic mineralization in PXE.
One mutation - different phenotypes: Novel insights into the link between familial pityriasis rubra pilaris and psoriasis I Spoerri1, O Eytan2, O Sarig2, E Sprecher2, P Itin1 and B Burger1 1 Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland and 2 Department of Dermatology, Tel-Aviv Sourasky Medical Centre, Tel-Aviv, Israel The purpose of our study was to clinically and genetically investigate affected as well as unaffected members of a family with pityriasis rubra pilaris (PRP) in order to determine the cause of this severe erythematous inflammatory skin disease in this family. The cause of the familial PRP has been determined as heterozygous activating mutations in CARD14, a known activator of the NF-kB pathway. Different genetic variations in CARD14 have been associated with psoriasis. A father and two of his children with clinical signs of PRP as well as a third child unaffected by PRP were investigated clinically. In addition we extracted genomic DNA from blood of each individual and performed whole exome sequencing as well as direct sequencing of single genes. Clinical investigation confirmed that three family members are affected by familial PRP, with the skin showing the characteristic pattern of PRP, early onset and chronic course. One family member is unaffected by PRP, suffers however from psoriasis. Genetic investigation revealed a heterozygous missense mutation in exon 4 of CARD14, p.[Leu124Pro], present in all investigated individuals and described before in another family with PRP. There were no further variations in CARD14 associated with either PRP or psoriasis. There were however serious genetic variations in other genes involved in the NF-kB as well as the NOTCH pathway, either segregating with PRP or being present in the psoriasis affected individual only. The presence of an individual carrying the same CARD14 mutation as his PRP-affected relatives but suffering from psoriasis instead strengthens the relation between PRP and psoriasis, which has been repeatedly suggested in literature. We propose a balance between familial PRP and psoriasis in the family investigated in this study and present genetic variations, which might influence this balance in addition to variations in CARD14.
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A keratinocyte culture model for epidermodysplasia verruciformis E Imahorn1, M Aushev2, O March3, SJ de Jong4, E Jouanguy5, J Casanova4, P Itin1, J Reichelt3 and B Burger1 1 Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland, 2 Institute of Cellular Medicine, University of Newcastle, Newcastle upon Tyne, United Kingdom, 3 EB House, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 4 St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York City, NY and 5 Imagine Institute, Universite´ Paris Descartes / INSERM, Paris, France Epidermodysplasia verruciformis (EV) is a rare hereditary skin disease leading to a primary immunodeficiency to certain types of cutaneous human papilloma viruses (EV-HPV). Homozygous or compound heterozygous loss of function mutations either in TMC6/EVER1 or in TMC8/EVER2 have been described in several EV patients, but more than one third of patients do not have a mutation in either TMC gene. A third unpublished gene (EVER 3) has been identified to be mutated in EV patients without TMC6/8 deficiency. Investigations on the function of that gene may elucidate pathomechanisms of EV. We aim to generate an EVER3 deficient model keratinocyte line to study the effects of EVER3 despite the scarcity of patient material. For this purpose we used the CRISPR/Cas9 system to delete the EVER3 coding sequence in an immortalized keratinocyte line. After isolation by FACS, single cell clones have been expanded. We screened 41 clones for deletion of the whole gene as well as for the expression of EVER3. Three clones showed no detectable gDNA sequence or expression of the EVER3 gene. We found only one wildtype clone without deletion of EVER3 or alterations near the Cas9 cut sites. All clones have been characterized by a SNP-array as well as sequencing of the knockout site and the most probable offsite targets. These EVER3 deficient keratinocyte lines are the first cell culture model for EV and will be a valuable tool to identify cellular pathomechanisms of the disease.
S188 Journal of Investigative Dermatology (2016), Volume 136
Development of a skin care product that makes your face appear “more cheerful” M Shono, Y Saito, A Tada and H Takeuchi POLA Chemical Industries, Inc., Yokohama, Japan Have you ever been told that you look tired? To others, our complexion appears to reflect our psychological and/or physiological condition. Therefore, it came as no surprise that the results of a survey revealed a high percentage of healthy middle-aged Japanese women had a strong desire to look “more cheerful”. Consequently, we sought to determine which skin color tones (SCTs) created a more cheerful impression. 1) To achieve this, we evaluated the relationship between the cheerful impression and SCTs on face. Several pseudo-face photographs (composite images of Japanese women) with a range of red-green-blue (RGB) values were created and subjected to assessment. The assessment revealed pseudo-face photographs with higher all RGB values were considered to be more cheerful by the evaluators. 2) Based on these findings, we sought a biological molecule that could increase all RGB values in the skin. We focused on endogenous fluorescent molecules that can emit light at broad visible wavelengths. Based on the molecular structure, glycosaminoglycans (GAGs) were potential candidates. When GAGs were irradiated with different wavelengths, the fluorescence intensity was measured at various wavelengths. Surprisingly, some GAGs showed fluorescent characteristics. Among them, keratan sulfate (KS) showed the strongest emittance and the broadest emission spectrum in the visible region. Furthermore, measurements of RGB values of KS demonstrated that it could raise all RGB values. These results suggested increasing KS in human skin could make faces appear more cheerful. 3) To improve the impressions elicited by SCTs, plant extracts that increase KS-related mRNA expression were screened and sweet flag extract was found to be more effective. Continuous application of a skin care product including the extract increased all RGB values in Japanese women. In conclusion, the results of this research indicated that increasing KS in human skin could contribute to changes in facial color tone resulting in a more cheerful appearance and that this effect was the result of changes in RGB values.
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Pharmacogenetics of ustekinumab in patients with moderate-to-severe plaque psoriasis E Dauden1, R Prieto-Perez2, M Llamas-Velasco1, T Cabaleiro2 and F Abad-Santos2 1 Dermatology, Hospital Universitario la Princesa, Madrid, Spain and 2 Pharmacology, Hospital Universitario la Princesa, Madrid, Spain Ustekinumab is an effective drug against moderate-to-severe psoriasis, but not all patients are responders to therapy and some patients may develop adverse effects. Only two studies have evaluated the influence of pharmacogenetics in psoriatic patients treated with ustekinumab. We evaluated 121 polymorphisms to study a possible association between these SNPs and the response to ustekinumab measured by PASI75 at 4 months of treatment (N¼69). The weight was evaluated for response to treatment (PASI75) and a higher weight was obtained in nonresponders than responders (p¼0.018). In addition, the adjusted results (FDR) showed an association between 5 SNPs in TNFRSF1A, HTR2A, NFKBIA, ADAM33, and IL13 genes and poor response to ustekinumab. Furthermore, 6 SNPs in CHUK, C17orf51, ZNF816A, STAT4, SLC22A4, and Corf72 genes were associated with better response to ustekinumab. However, there was not significant association between response to ustekinumab and SNPs in HLA-C as has been recently described.
PASH syndrome is not a genetically distinct entity but belongs to the spectrum of autoinflammatory skin phenotypes J Uitto1, M Faraji Zonooz2, F Sabbagh-Kermani3, Z Fattahi2, M Fadaee2, M Reza Akbari4, R Amiri3, H Vahidnezhad1, H Najmabadi2 and A Kariminejad2 1 Thomas Jefferson University, Philadelphia, PA, 2 Kariminejad-Najmabadi Pathology & Genetics Center, Tehran, Iran (the Islamic Republic of), 3 Kerman University of Medical Sciences, Kerman, Iran (the Islamic Republic of) and 4 University of Toronto, Toronto, ON, Canada PASH syndrome, comprising pyoderma gangrenosum (PG) in association with acne and hidradenitis suppurative (HS), was initially suggested to be associated with increased number of CCTG microsatellite repeats in the promoter region of PSTPIP1. In this study we characterized an extended multigenerational Iranian family, including 24 affected and 32 unaffected individuals with manifestations of HS, acne, and PG in an autosomal dominant pattern of inheritance. Mutation analysis was first performed by PCR amplification of the reporter region as well as all 50 exons and flanking intronic sequences of PSTPIP1. No pathogenic variants were detected, and sequencing of the promoter region showed (CCTG)5 repeat as the normal allele size in this gene. Next, whole genome single nucleotide polymorphism genotyping was applied which resulted in identification of genomic intervals with LOD scores >2 on chromosomes 1, 11, and 20. Whole exome sequencing identified a total of 20 rare sequence variants in these chromosomal regions, and filtering prioritization and cosegregation analyses identified a mutation, c.1635C/G (p.Tyr545*) in the NCSTN gene, recently shown to harbor mutations in cases with HS. This mutation perfectly co-segregated with the disease phenotype in the family. NCSTN encodes nicastrin, one of the three subunits of g-secretase, a transmembrane endoprotease complex that catalyzes cleavage of many transmembrane proteins, such as Notch receptors. Thus, PG may be part of the autoinflammatory spectrum of skin diseases, together with HS and acne. Considering the phenotypic and genotypic heterogeneity of these autoinflammatory skin phenotypes, we conclude that PASH syndrome is not a genetically distinct entity, but rather belongs to the spectrum of g-secretase-related disorders.
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Demonstration of the critical role of the liver in systemic PPi homeostasis using a novel Abcc6 knockout rat model of PXE Q Li1, K van de Wetering2, J Sundberg3 and J Uitto1 1 Thomas Jefferson University, Philadelphia, PA, 2 Netherlands Cancer Institute, Amsterdam, Netherlands and 3 The Jackson Laboratory, Bar Harbor, ME Pseudoxanthoma elasticum (PXE) is a heritable multisystem ectopic mineralization disorder. It is caused by mutations in the ABCC6 gene that encodes ABCC6, a transmembrane efflux transporter expressed primarily in the liver, and to some extent in the kidneys. The precise pathomechanistic details leading from lack of ABCC6 expression in the liver or kidney to mineralization in peripheral tissues are unknown. We generated Abcc6-/- rats using zinc finger nuclease technology and these rats provide a model for metabolic studies to specifically unravel the role of PPi and Pi homeostasis in PXE. Immunostaining of liver sections using a rat ABCC6 specific antibody showed basolateral plasma membrane localization in hepatocytes of wild type rats and complete absence in the homozygous Abcc6-/- rats. As a consequence of ABCC6 deficiency, complete necropsy revealed ectopic mineralization in the muzzle skin, eyes, and arterial blood vessels in the knockout rats, features mimicking human PXE. Mineralization in the dermal sheath of vibrissae was also monitored by small animal CT scanning, which allows non-invasive evaluation of the progression of mineralization. Plasma PPi level was significantly reduced (<60%) in the Abcc6-/- rats as compared to wild type rats, leading to a significantly lower PPi/Pi ratio in plasma. To assess whether absence of ABCC6 in the liver is responsible for PXE pathology, we performed in vivo liver perfusion experiments followed by quantification of PPi in the perfusate. PPi levels in the perfusates of Abcc6-/- rats were significantly lower (<3%) than perfusates in the wild type rats, pointing to the critical role of hepatic ABCC6 in providing the circulation with PPi. Since PPi is a potent antimineralization factor, the results highlight the role of PPi in the context of the PPi/Pi ratio in the novel Abcc6-/- rat model due to loss-of-function of ABCC6, leading to ectopic mineralization in PXE.
One mutation - different phenotypes: Novel insights into the link between familial pityriasis rubra pilaris and psoriasis I Spoerri1, O Eytan2, O Sarig2, E Sprecher2, P Itin1 and B Burger1 1 Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland and 2 Department of Dermatology, Tel-Aviv Sourasky Medical Centre, Tel-Aviv, Israel The purpose of our study was to clinically and genetically investigate affected as well as unaffected members of a family with pityriasis rubra pilaris (PRP) in order to determine the cause of this severe erythematous inflammatory skin disease in this family. The cause of the familial PRP has been determined as heterozygous activating mutations in CARD14, a known activator of the NF-kB pathway. Different genetic variations in CARD14 have been associated with psoriasis. A father and two of his children with clinical signs of PRP as well as a third child unaffected by PRP were investigated clinically. In addition we extracted genomic DNA from blood of each individual and performed whole exome sequencing as well as direct sequencing of single genes. Clinical investigation confirmed that three family members are affected by familial PRP, with the skin showing the characteristic pattern of PRP, early onset and chronic course. One family member is unaffected by PRP, suffers however from psoriasis. Genetic investigation revealed a heterozygous missense mutation in exon 4 of CARD14, p.[Leu124Pro], present in all investigated individuals and described before in another family with PRP. There were no further variations in CARD14 associated with either PRP or psoriasis. There were however serious genetic variations in other genes involved in the NF-kB as well as the NOTCH pathway, either segregating with PRP or being present in the psoriasis affected individual only. The presence of an individual carrying the same CARD14 mutation as his PRP-affected relatives but suffering from psoriasis instead strengthens the relation between PRP and psoriasis, which has been repeatedly suggested in literature. We propose a balance between familial PRP and psoriasis in the family investigated in this study and present genetic variations, which might influence this balance in addition to variations in CARD14.
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A keratinocyte culture model for epidermodysplasia verruciformis E Imahorn1, M Aushev2, O March3, SJ de Jong4, E Jouanguy5, J Casanova4, P Itin1, J Reichelt3 and B Burger1 1 Department of Biomedicine, University Hospital Basel and University of Basel, Basel, Switzerland, 2 Institute of Cellular Medicine, University of Newcastle, Newcastle upon Tyne, United Kingdom, 3 EB House, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 4 St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York City, NY and 5 Imagine Institute, Universite´ Paris Descartes / INSERM, Paris, France Epidermodysplasia verruciformis (EV) is a rare hereditary skin disease leading to a primary immunodeficiency to certain types of cutaneous human papilloma viruses (EV-HPV). Homozygous or compound heterozygous loss of function mutations either in TMC6/EVER1 or in TMC8/EVER2 have been described in several EV patients, but more than one third of patients do not have a mutation in either TMC gene. A third unpublished gene (EVER 3) has been identified to be mutated in EV patients without TMC6/8 deficiency. Investigations on the function of that gene may elucidate pathomechanisms of EV. We aim to generate an EVER3 deficient model keratinocyte line to study the effects of EVER3 despite the scarcity of patient material. For this purpose we used the CRISPR/Cas9 system to delete the EVER3 coding sequence in an immortalized keratinocyte line. After isolation by FACS, single cell clones have been expanded. We screened 41 clones for deletion of the whole gene as well as for the expression of EVER3. Three clones showed no detectable gDNA sequence or expression of the EVER3 gene. We found only one wildtype clone without deletion of EVER3 or alterations near the Cas9 cut sites. All clones have been characterized by a SNP-array as well as sequencing of the knockout site and the most probable offsite targets. These EVER3 deficient keratinocyte lines are the first cell culture model for EV and will be a valuable tool to identify cellular pathomechanisms of the disease.
S188 Journal of Investigative Dermatology (2016), Volume 136
Development of a skin care product that makes your face appear “more cheerful” M Shono, Y Saito, A Tada and H Takeuchi POLA Chemical Industries, Inc., Yokohama, Japan Have you ever been told that you look tired? To others, our complexion appears to reflect our psychological and/or physiological condition. Therefore, it came as no surprise that the results of a survey revealed a high percentage of healthy middle-aged Japanese women had a strong desire to look “more cheerful”. Consequently, we sought to determine which skin color tones (SCTs) created a more cheerful impression. 1) To achieve this, we evaluated the relationship between the cheerful impression and SCTs on face. Several pseudo-face photographs (composite images of Japanese women) with a range of red-green-blue (RGB) values were created and subjected to assessment. The assessment revealed pseudo-face photographs with higher all RGB values were considered to be more cheerful by the evaluators. 2) Based on these findings, we sought a biological molecule that could increase all RGB values in the skin. We focused on endogenous fluorescent molecules that can emit light at broad visible wavelengths. Based on the molecular structure, glycosaminoglycans (GAGs) were potential candidates. When GAGs were irradiated with different wavelengths, the fluorescence intensity was measured at various wavelengths. Surprisingly, some GAGs showed fluorescent characteristics. Among them, keratan sulfate (KS) showed the strongest emittance and the broadest emission spectrum in the visible region. Furthermore, measurements of RGB values of KS demonstrated that it could raise all RGB values. These results suggested increasing KS in human skin could make faces appear more cheerful. 3) To improve the impressions elicited by SCTs, plant extracts that increase KS-related mRNA expression were screened and sweet flag extract was found to be more effective. Continuous application of a skin care product including the extract increased all RGB values in Japanese women. In conclusion, the results of this research indicated that increasing KS in human skin could contribute to changes in facial color tone resulting in a more cheerful appearance and that this effect was the result of changes in RGB values.