Abstract: PO1595
Elucidation of Molecular Pathogenesis of Lowe Syndrome and Dent Disease Type 2
Session Information
- Genetic Diseases of the Kidneys: Non-Cystic - 1
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1002 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Sakakibara, Nana, 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
- Nozu, Kandai, 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
- Ishiko, Shinya, 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
- Aoto, Yuya, 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
- Nagano, China, 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
- Horinouchi, Tomoko, 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
- Yamamura, Tomohiko, 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
- Ninchoji, Takeshi, 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
- Iijima, Kazumoto, 1. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
Background
Lowe syndrome and Dent disease-2(Dent-2) are both X-linked kidney diseases caused by OCRL gene abnormalities. However, the severity of these diseases are quite different. Genetic studies have shown that patients with truncating mutation in exon 1-7 of OCRL gene were diagnosed with Dent-2, and those with truncating mutations in exon 8-24 were diagnosed with Lowe syndrome. OCRL protein encodes a 5-phosphatase that acts on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and is related to cellular functions by the regulation of inositol phospholipids. The molecular mechanism by which different phenotypes of Dent-2 and Lowe syndrome are caused by the same gene variant has not been clarified until now, but it is suspected that an isoform consisting of exon 8-24 exists and it works partially as a 5-phosphatase. However, such isoform has not been identified yet.
Methods
We extracted mRNA from cultured urine derived cells of healthy controls and Dent-2 patient with truncating mutation in Exon4 of OCRL gene and then examined 5 'end of mRNA of these cells by using rapid amplification of cDNA ends (5’ RACE) method. We also prepared three types of OCRL protein expression vector: wild type model, Dent-2 models harboring truncating mutation in exon 4 and exon 7, and Lowe syndrome models harboring truncating mutation in exon 16 and exon 22. These vectors were transfected into Hela cells and analyzed the protein expression and 5-phosphatase activity.
Results
As a result of 5' RACE, the 5' end starting from Exon 6 was detected in both cells of healthy control and Dent-2 patient. In fluorescent immunostaining of transfected Hela cells, strong protein expression was observed in the wild type model, relatively weak expression was observed in Dent-2 models and no expression was observed in Lowe syndrome models. Western blot analysis detected two bands of 105kDa and 80kDa in the wild type model, single band of 80kDa in Dent-2 models, and no band in Lowe syndrome models. 5-phosphatase activity of Dent-2 models was 50-85% of that of wild type model, whereas that of Lowe syndrome models was less than 20% of that of wild type model.
Conclusion
An isoform OCRL protein with 5-phosphatase activity is synthesized by alternative transcription of OCRL gene. This isoform contributes to the mild clinical phenotype in Dent-2.
Funding
- Government Support - Non-U.S.