Mai Hattori1, Osamu Ishikawa2, Daisuke Oikawa3, Hiroo Amano4, Masahito Yasuda2, Kyoichi Kaira5, Akemi Ishida-Yamamoto6, Hajime Nakano7, Daisuke Sawamura7, Shin-Ichi Terawaki8, Kaori Wakamatsu8, Fuminori Tokunaga3, Akira Shimizu9. 1. Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan; Department of Dermatology, Japan Red Cross Maebashi Hospital, Maebashi, Japan. 2. Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan. 3. Department of Pathobiochemistry, Graduate School of Medicine, Osaka City University, Osaka, Japan. 4. Department of Dermatology, Iwate Medical University, Morioka, Iwate, Japan. 5. Department of Oncology Clinical Development, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan. 6. Department of Dermatology, Asahikawa Medical University, Asahikawa, Hokkaido, Japan. 7. Department of Dermatology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori, Japan. 8. Graduate School of Science and Technology, Gunma University, Kiryu, Gunma, Japan. 9. Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan. Electronic address: shimizuakira@gunma-u.ac.jp.
Abstract
BACKGROUND: Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. OBJECTIVE: The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. METHODS: We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. RESULTS: A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val216 and Ser217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF); however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. CONCLUSION: These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism.
BACKGROUND:Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. OBJECTIVE: The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. METHODS: We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. RESULTS: A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val216 and Ser217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF); however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. CONCLUSION: These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism.