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500 A novel Abcc6 knockout rat model of PXE: Perfusion studies revealed the critical role of liver in PXE
ABSTRACTS | Genetic Disease, Gene Regulation and Gene Therapy 498
499
Elevated maternal dietary magnesium prevents ectopic mineralization in offspring in a mouse model of generalized arterial calcification of infancy J Kingman, J Uitto and Q Li TJU, Philadelphia, PA Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder caused by mutations in the ENPP1 gene. It is characterized by early onset mineralization of the arterial blood vessels, often diagnosed prenatally, and characteristically associated with death in early childhood. There is no effective treatment for this devastating disorder. We previously characterized the Enpp1asj mutant mouse as a model of GACI, and we have now explored the effect of elevated dietary magnesium in mothers during pregnancy and nursing and continuing in the offspring for the first 14 weeks of postnatal life. The mothers were kept on either control diet or an experimental diet supplemented with magnesium (five-fold). Upon weaning at 4 weeks of age the pups were placed either on control diet or high magnesium diet. The degree of mineralization in the offspring was assessed at 14 weeks of age by histopathology and chemical assay of calcium in aorta, kidney, and muzzle skin. Mice placed on high magnesium diet showed little, if any, evidence of mineralization when their corresponding mothers were also placed on diet enriched with magnesium during pregnancy and nursing. The reduced ectopic mineralization in these mice was accompanied by increased urinary calcium and magnesium content, suggesting that magnesium competes calciumphosphate binding thereby preventing the mineral deposition. These results have implications for dietary management of pregnancies in which the fetus is suspected of having GACI. Moreover, augmenting the diet with high magnesium may be beneficial for other ectopic mineralization diseases, including nephrocalcinosis and calcinosis cutis.
Atherogenic diet enhances ectopic mineralization in Abcc6-/- mice, a model for pseudoxanthoma elasticum D Wang1, J Zhao2, J Kingman3, J Uitto3 and Q Li3 1 Thomas Jefferson University, Philadelphia, PA, 2 TJU, The First Affiliated Hospital of Zhejiang Chinese Medical University, Philadelphia, PA and 3 TJU, Philadelphia, PA Pseudoxanthoma elasticum (PXE) is a multisystem heritable disorder caused by mutations in the ABCC6 gene. The disease is characterized by ectopic mineralization of connective tissues, including the skin, eyes and the arterial blood vessels. Previous studies have suggested that cardiovascular complications in PXE are caused in part by premature atherosclerosis. To study the effect of atherogenic diet on ectopic mineralization, we used Abcc6-/-mice as a preclinical model of PXE. After 50-58 weeks feeding on atherogenic diet with high-fat, highsucrose, high-cholesterol and high-cholic acid, serum lipid profiles revealed a statistically significant increase in total cholesterol and LDL/VLDL cholesterol levels in Abcc6-/- mice compared to the same mice on control diet. Hypercholesterolemia was accompanied with significantly increased lipid accumulation in the liver, a characteristic feature of hepatic steatosis. Direct assay of calcium demonstrated significantly increased mineralization of the muzzle skin containing the dermal sheath of vibrissae, a reliable biomarker of the ectopic mineralization process in these mice. Oil Red O stains did not demonstrate presence of lipid deposition at sites of ectopic mineralization in the skin, arterial blood vessels and eyes. These observations suggest that challenge with atherogenic diet resulting in hypercholesterolemia can accelerate ectopic mineralization in the Abcc6-/- mouse model, with clinical implications for patients with PXE, a currently intractable disorder with considerable morbidity and occasional mortality.
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A novel Abcc6 knockout rat model of PXE: Perfusion studies revealed the critical role of liver in PXE Q Li1, J Kingman1, K van de Wetering2, S Tannouri2, J Sundberg3 and J Uitto1 1 TJU, Philadelphia, PA, 2 Thomas Jefferson University, Philadelphia, PA and 3 The Jackson Laboratory, Bar Harbor, ME Pseudoxanthoma elasticum (PXE), a heritable multisystem ectopic mineralization disorder, is caused by loss-of-function mutations in the ABCC6 gene primarily expressed in liver and kidney. An open question with respect to the pathogenesis of PXE is whether lack of ABCC6 expression in liver or kidney underlies the pathology seen in peripheral tissues. In this study, we generated Abcc6-/- rats devoid of ABCC6 expression using zinc finger nuclease technology. An advantage of the rat model over the extensively studied mouse models is their larger organ size which allows physiological studies, such as in vivo organ perfusions. Complete necropsy of the homozygous Abcc6-/- rats revealed ectopic mineralization in the skin, eyes, and arteries, features found in human PXE. The plasma PPi level was reduced (Abcc6-/- rats leading to lowered PPi/Pi plasma ratio allowing ectopic mineralization to take place. In situ liver and kidney perfusions were performed to determine the relative contribution of the liver and kidneys to the PPi levels present in the circulation. Results demonstrated significantly reduced PPi levels in both liver and kidney perfusates of Abcc6-/- rats, indicating the critical role of ABCC6 expression in these organs in the generation of circulatory PPi. In addition, PPi levels in the liver perfusates of wild type rats were 10-fold higher than that in the kidney perfusates, indicating the critical role of hepatic ABCC6 in contributing to plasma PPi levels.
Plasma PPi is the major, but not exclusive, cause of ectopic mineralization in pseudoxanthoma elasticum J Zhao1, J Kingman2, J Uitto2 and Q Li2 1 TJU, The First Affiliated Hospital of Zhejiang Chinese Medical University, Philadelphia, PA and 2 TJU, Philadelphia, PA Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, affects the skin, eyes and the arterial blood vessels. PXE is caused by inactivating mutations in the ABCC6 gene and the disease is characterized by calcium hydroxyapatite deposition on connective tissues. It was recently discovered that absence of ABCC6-mediated ATP release from the liver and consequently reduced plasma PPi levels underlie PXE. In this study, we examined whether elevated circulating levels of PPi, a powerful mineralization inhibitor, is the sole mechanism by which ABCC6 counteracts ectopic mineralization in PXE. Abcc6-/mice, a model of PXE, were crossed with transgenic mice with ubiquitous expression of human ENPP1, an ectonucleotidase which generates PPi from ATP. We generated Abcc6-/-mice either wild type or hemizygous for human ENPP1. The Enpp1asj mice, a model for generalized arterial calcification of infancy caused by ENPP1 mutations, were also crossed with human ENPP1 transgenic mice to compensate for the loss of the endogenous mouse protein. Plasma levels of PPi and the degree of mineralization of connective tissue capsule of vibrissae, a reliable biomarker of the mineralization process in these mice, were determined. Overexpression of human ENPP1 in Enpp1asj mice normalized plasma PPi levels to that of wild type mice, and consequently, completely prevented mineralization. In contrast, significantly reduced mineralization was noted in Abcc6-/- mice with overexpression of human ENPP1, however, with small mineralization foci still evident despite increased plasma PPi levels. These results suggest that PPi is the major mediator of connective tissue mineralization in PXE, but there is a second, as-yet unknown, alternative mechanism other than PPi by which ABCC6 prevents ectopic mineralization under physiologic conditions.
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Sex bias in response to an NRF2 inducer in a pachyonychia congenita model ML Kerns1, J Hakim2, A Zieman2 and PA Coulombe3 1 Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD, 2 Johns Hopkins Bloomberg School of Public Health, Baltimore, MD and 3 Johns Hopkins University, Baltimore, MD Background: Pachyonychia congenita (PC) is a genodermatosis typified by painful and debilitating palmoplantar keratoderma (PPK). Mice genetically null for keratin 16 (Krt16), a gene mutated in PC, develop PPK akin to that seen in PC. In male Krt16 null mice, oxidative stress associated with impaired glutathione (GSH) synthesis and NRF2-dependent gene expression precedes PPK onset, and the latter can be prevented by topical sulforaphane (SF)mediated activation of NRF2. Given that signaling via estrogen receptor (ER)-a and ER-b has been shown to directly inhibit and activate NRF2-mediated transcription, respectively, we tested whether SF treatment is also efficacious in female Krt16 null mice. Methods: We treated female Krt16 null mice with topical SF alone and in combination with intraperitoneal DPN, an ER-b agonist and NRF2 activator. We evaluated PPK onset, NRF2 activation, and redox status using immunohistochemistry, RT-PCR, and GSH measurements. Results: Even though depressed GSH levels, oxidative stress, and hypofunctional NRF2 occur in paw skin of both sexes, treatment with SF completely fails to activate NRF2 and prevent PPK in female Krt16 null mice. Studies reveal a temporal shift in PPK onset between Krt16 null males and females, coinciding with differential onset of puberty and paralleled by sex-specific fluctuations in glutathione levels in footpad epidermis. In contrast to SF alone, dual treatment with SF and DPN successfully resulted in NRF2 activation and prevention of PPK formation in female Krt16 null paws. Conclusions: A sex-independent effect of Krt16 on GSH synthesis along with sex-specific fluctuations in redox balance contribute to the increased levels of oxidative stress in the Krt16 null mouse model. Both components need to be addressed in the development of a successful therapeutic intervention for PC and related conditions.
S86 Journal of Investigative Dermatology (2017), Volume 137
Genome-wide association study identifies novel susceptibility loci for tanning ability in Japanese population K Shido1, K Kojima2, A Hozawa3, S Ogishima3, N Minegishi3, Y Kawai3, G Tamiya3, K Tanno4, K Yamasaki1, S Aiba1, Y Suzuki3 and M Nagasaki3 1 Department of Dermatology, Tohoku University, Sendai, Japan, 2 Tohoku Medical Megabank Organization (ToMMo), Sendai, Japan, 3 ToMMo, Sendai, Japan and 4 School of Medicine, Iwate Medical University, Iwate, Japan Genome-wide association studies (GWAS) in European population identified genes involved in the melanogenesis pathway such as MC1R, TYR, OCA2, SLC24A5, MATP, and ASIP as those associated with hair, eye and/or skin color. However, these studies could not identify genes related to tanning ability. In this study, we tried to identify genes related to tanning ability in Japanese population by a GWAS study using data from the Tohoku Medical Megabank cohort study. Japanese skin type is classified based on an individual’s susceptibility to sunburn and ability to tan: type I-III: type I, always burns, never tans; type II, moderately burns, moderately tans; type III, never burns, always tans. Single nucleotide polymorphisms (SNPs) were tested for association with 3 subjectively determined tanning abilities among 9,966 Japanese in Miyagi and Iwate prefectures. As a result, we identified two candidate genes at genome-wide significance of at least less than 5 10-8, chr15:28228553 C>T (rs74653330) and chr15: 27951891 T>C (rs1800414) at 15q12-13 (OCA2 gene) and chr9:16808172 C>G (rs10122901) at 9p22.3-22.2 (BNC2 gene). Two SNPs in the OCA2 genes, rs74653330, an OCA2 pathogenic allele, and rs1800414, a benign allele, have been reported to be the causal variants for the skin color association in the Japanese population. This study revealed that two SNPs of OCA2 and one located in a non-coding region of BNC2 gene, were closely linked with subjectively determined tanning ability in the Japanese population.
499
Elevated maternal dietary magnesium prevents ectopic mineralization in offspring in a mouse model of generalized arterial calcification of infancy J Kingman, J Uitto and Q Li TJU, Philadelphia, PA Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder caused by mutations in the ENPP1 gene. It is characterized by early onset mineralization of the arterial blood vessels, often diagnosed prenatally, and characteristically associated with death in early childhood. There is no effective treatment for this devastating disorder. We previously characterized the Enpp1asj mutant mouse as a model of GACI, and we have now explored the effect of elevated dietary magnesium in mothers during pregnancy and nursing and continuing in the offspring for the first 14 weeks of postnatal life. The mothers were kept on either control diet or an experimental diet supplemented with magnesium (five-fold). Upon weaning at 4 weeks of age the pups were placed either on control diet or high magnesium diet. The degree of mineralization in the offspring was assessed at 14 weeks of age by histopathology and chemical assay of calcium in aorta, kidney, and muzzle skin. Mice placed on high magnesium diet showed little, if any, evidence of mineralization when their corresponding mothers were also placed on diet enriched with magnesium during pregnancy and nursing. The reduced ectopic mineralization in these mice was accompanied by increased urinary calcium and magnesium content, suggesting that magnesium competes calciumphosphate binding thereby preventing the mineral deposition. These results have implications for dietary management of pregnancies in which the fetus is suspected of having GACI. Moreover, augmenting the diet with high magnesium may be beneficial for other ectopic mineralization diseases, including nephrocalcinosis and calcinosis cutis.
Atherogenic diet enhances ectopic mineralization in Abcc6-/- mice, a model for pseudoxanthoma elasticum D Wang1, J Zhao2, J Kingman3, J Uitto3 and Q Li3 1 Thomas Jefferson University, Philadelphia, PA, 2 TJU, The First Affiliated Hospital of Zhejiang Chinese Medical University, Philadelphia, PA and 3 TJU, Philadelphia, PA Pseudoxanthoma elasticum (PXE) is a multisystem heritable disorder caused by mutations in the ABCC6 gene. The disease is characterized by ectopic mineralization of connective tissues, including the skin, eyes and the arterial blood vessels. Previous studies have suggested that cardiovascular complications in PXE are caused in part by premature atherosclerosis. To study the effect of atherogenic diet on ectopic mineralization, we used Abcc6-/-mice as a preclinical model of PXE. After 50-58 weeks feeding on atherogenic diet with high-fat, highsucrose, high-cholesterol and high-cholic acid, serum lipid profiles revealed a statistically significant increase in total cholesterol and LDL/VLDL cholesterol levels in Abcc6-/- mice compared to the same mice on control diet. Hypercholesterolemia was accompanied with significantly increased lipid accumulation in the liver, a characteristic feature of hepatic steatosis. Direct assay of calcium demonstrated significantly increased mineralization of the muzzle skin containing the dermal sheath of vibrissae, a reliable biomarker of the ectopic mineralization process in these mice. Oil Red O stains did not demonstrate presence of lipid deposition at sites of ectopic mineralization in the skin, arterial blood vessels and eyes. These observations suggest that challenge with atherogenic diet resulting in hypercholesterolemia can accelerate ectopic mineralization in the Abcc6-/- mouse model, with clinical implications for patients with PXE, a currently intractable disorder with considerable morbidity and occasional mortality.
500
501
A novel Abcc6 knockout rat model of PXE: Perfusion studies revealed the critical role of liver in PXE Q Li1, J Kingman1, K van de Wetering2, S Tannouri2, J Sundberg3 and J Uitto1 1 TJU, Philadelphia, PA, 2 Thomas Jefferson University, Philadelphia, PA and 3 The Jackson Laboratory, Bar Harbor, ME Pseudoxanthoma elasticum (PXE), a heritable multisystem ectopic mineralization disorder, is caused by loss-of-function mutations in the ABCC6 gene primarily expressed in liver and kidney. An open question with respect to the pathogenesis of PXE is whether lack of ABCC6 expression in liver or kidney underlies the pathology seen in peripheral tissues. In this study, we generated Abcc6-/- rats devoid of ABCC6 expression using zinc finger nuclease technology. An advantage of the rat model over the extensively studied mouse models is their larger organ size which allows physiological studies, such as in vivo organ perfusions. Complete necropsy of the homozygous Abcc6-/- rats revealed ectopic mineralization in the skin, eyes, and arteries, features found in human PXE. The plasma PPi level was reduced (Abcc6-/- rats leading to lowered PPi/Pi plasma ratio allowing ectopic mineralization to take place. In situ liver and kidney perfusions were performed to determine the relative contribution of the liver and kidneys to the PPi levels present in the circulation. Results demonstrated significantly reduced PPi levels in both liver and kidney perfusates of Abcc6-/- rats, indicating the critical role of ABCC6 expression in these organs in the generation of circulatory PPi. In addition, PPi levels in the liver perfusates of wild type rats were 10-fold higher than that in the kidney perfusates, indicating the critical role of hepatic ABCC6 in contributing to plasma PPi levels.
Plasma PPi is the major, but not exclusive, cause of ectopic mineralization in pseudoxanthoma elasticum J Zhao1, J Kingman2, J Uitto2 and Q Li2 1 TJU, The First Affiliated Hospital of Zhejiang Chinese Medical University, Philadelphia, PA and 2 TJU, Philadelphia, PA Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, affects the skin, eyes and the arterial blood vessels. PXE is caused by inactivating mutations in the ABCC6 gene and the disease is characterized by calcium hydroxyapatite deposition on connective tissues. It was recently discovered that absence of ABCC6-mediated ATP release from the liver and consequently reduced plasma PPi levels underlie PXE. In this study, we examined whether elevated circulating levels of PPi, a powerful mineralization inhibitor, is the sole mechanism by which ABCC6 counteracts ectopic mineralization in PXE. Abcc6-/mice, a model of PXE, were crossed with transgenic mice with ubiquitous expression of human ENPP1, an ectonucleotidase which generates PPi from ATP. We generated Abcc6-/-mice either wild type or hemizygous for human ENPP1. The Enpp1asj mice, a model for generalized arterial calcification of infancy caused by ENPP1 mutations, were also crossed with human ENPP1 transgenic mice to compensate for the loss of the endogenous mouse protein. Plasma levels of PPi and the degree of mineralization of connective tissue capsule of vibrissae, a reliable biomarker of the mineralization process in these mice, were determined. Overexpression of human ENPP1 in Enpp1asj mice normalized plasma PPi levels to that of wild type mice, and consequently, completely prevented mineralization. In contrast, significantly reduced mineralization was noted in Abcc6-/- mice with overexpression of human ENPP1, however, with small mineralization foci still evident despite increased plasma PPi levels. These results suggest that PPi is the major mediator of connective tissue mineralization in PXE, but there is a second, as-yet unknown, alternative mechanism other than PPi by which ABCC6 prevents ectopic mineralization under physiologic conditions.
502
503
Sex bias in response to an NRF2 inducer in a pachyonychia congenita model ML Kerns1, J Hakim2, A Zieman2 and PA Coulombe3 1 Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD, 2 Johns Hopkins Bloomberg School of Public Health, Baltimore, MD and 3 Johns Hopkins University, Baltimore, MD Background: Pachyonychia congenita (PC) is a genodermatosis typified by painful and debilitating palmoplantar keratoderma (PPK). Mice genetically null for keratin 16 (Krt16), a gene mutated in PC, develop PPK akin to that seen in PC. In male Krt16 null mice, oxidative stress associated with impaired glutathione (GSH) synthesis and NRF2-dependent gene expression precedes PPK onset, and the latter can be prevented by topical sulforaphane (SF)mediated activation of NRF2. Given that signaling via estrogen receptor (ER)-a and ER-b has been shown to directly inhibit and activate NRF2-mediated transcription, respectively, we tested whether SF treatment is also efficacious in female Krt16 null mice. Methods: We treated female Krt16 null mice with topical SF alone and in combination with intraperitoneal DPN, an ER-b agonist and NRF2 activator. We evaluated PPK onset, NRF2 activation, and redox status using immunohistochemistry, RT-PCR, and GSH measurements. Results: Even though depressed GSH levels, oxidative stress, and hypofunctional NRF2 occur in paw skin of both sexes, treatment with SF completely fails to activate NRF2 and prevent PPK in female Krt16 null mice. Studies reveal a temporal shift in PPK onset between Krt16 null males and females, coinciding with differential onset of puberty and paralleled by sex-specific fluctuations in glutathione levels in footpad epidermis. In contrast to SF alone, dual treatment with SF and DPN successfully resulted in NRF2 activation and prevention of PPK formation in female Krt16 null paws. Conclusions: A sex-independent effect of Krt16 on GSH synthesis along with sex-specific fluctuations in redox balance contribute to the increased levels of oxidative stress in the Krt16 null mouse model. Both components need to be addressed in the development of a successful therapeutic intervention for PC and related conditions.
S86 Journal of Investigative Dermatology (2017), Volume 137
Genome-wide association study identifies novel susceptibility loci for tanning ability in Japanese population K Shido1, K Kojima2, A Hozawa3, S Ogishima3, N Minegishi3, Y Kawai3, G Tamiya3, K Tanno4, K Yamasaki1, S Aiba1, Y Suzuki3 and M Nagasaki3 1 Department of Dermatology, Tohoku University, Sendai, Japan, 2 Tohoku Medical Megabank Organization (ToMMo), Sendai, Japan, 3 ToMMo, Sendai, Japan and 4 School of Medicine, Iwate Medical University, Iwate, Japan Genome-wide association studies (GWAS) in European population identified genes involved in the melanogenesis pathway such as MC1R, TYR, OCA2, SLC24A5, MATP, and ASIP as those associated with hair, eye and/or skin color. However, these studies could not identify genes related to tanning ability. In this study, we tried to identify genes related to tanning ability in Japanese population by a GWAS study using data from the Tohoku Medical Megabank cohort study. Japanese skin type is classified based on an individual’s susceptibility to sunburn and ability to tan: type I-III: type I, always burns, never tans; type II, moderately burns, moderately tans; type III, never burns, always tans. Single nucleotide polymorphisms (SNPs) were tested for association with 3 subjectively determined tanning abilities among 9,966 Japanese in Miyagi and Iwate prefectures. As a result, we identified two candidate genes at genome-wide significance of at least less than 5 10-8, chr15:28228553 C>T (rs74653330) and chr15: 27951891 T>C (rs1800414) at 15q12-13 (OCA2 gene) and chr9:16808172 C>G (rs10122901) at 9p22.3-22.2 (BNC2 gene). Two SNPs in the OCA2 genes, rs74653330, an OCA2 pathogenic allele, and rs1800414, a benign allele, have been reported to be the causal variants for the skin color association in the Japanese population. This study revealed that two SNPs of OCA2 and one located in a non-coding region of BNC2 gene, were closely linked with subjectively determined tanning ability in the Japanese population.