Tomohiko Inoue1,2, China Nagano3, Masafumi Matsuo4, Tomohiko Yamamura1, Nana Sakakibara1, Tomoko Horinouchi1, Yugo Shibagaki2, Daisuke Ichikawa2, Yuya Aoto1, Shinya Ishiko1, Shingo Ishimori1, Rini Rossanti1, Kazumoto Iijima1, Kandai Nozu1. 1. Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan. 2. Division of Nephrology and Hypertension, St. Marianna University Graduate School of Medicine, 2-16-1 Sugao, Kawasaki City, Kanagawa, 216-8511, Japan. 3. Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan. china@med.kobe-u.ac.jp. 4. Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe, Hyogo, 651-2180, Japan.
Abstract
BACKGROUND: In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. METHODS: We conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106-17T>G, c.393+4A>G, c.517-8A>G, c.517-3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results. RESULTS: We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106-17T>G). CONCLUSION: We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.
BACKGROUND: In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. METHODS: We conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106-17T>G, c.393+4A>G, c.517-8A>G, c.517-3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results. RESULTS: We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106-17T>G). CONCLUSION: We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.
Entities:
Keywords:
CLCN5; In silico; Minigene; Splicing; Variant
Authors: E Tosetto; M Ceol; F Mezzabotta; A Ammenti; L Peruzzi; M R Caruso; G Barbano; G Vezzoli; G Colussi; G Vergine; M Giordano; N Glorioso; S Degortes; L Soldati; J Sayer; A D'Angelo; F Anglani Journal: Clin Genet Date: 2009-08-10 Impact factor: 4.438
Authors: J P Cox; K Yamamoto; P T Christie; C Wooding; T Feest; F A Flinter; P R Goodyer; E Leumann; T Neuhaus; C Reid; P F Williams; O Wrong; R V Thakker Journal: J Bone Miner Res Date: 1999-09 Impact factor: 6.741