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Kidney Week

Abstract: FR-PO1012

Splicing Assay with Hybrid Minigene: Assessing Pathogenicities in COL4A5 Intronic Mutations

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Horinouchi, Tomoko, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Nozu, Kandai, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Yamamura, Tomohiko, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Sakakibara, Nana, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Nagano, China, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Rossanti, Rini, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Nakanishi, Keita, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Fujimura, Junya, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Minamikawa, Shogo, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Ninchoji, Takeshi, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Kaito, Hiroshi, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
  • Shima, Yuko, Department of Pediatrics, Wakayama Medical University, Wakayama, Japan, Wakayama, Japan
  • Nakanishi, Koichi, Department of Pediatrics, Graduate School of Medicine, University of the Ryukyus, Nishihara-cho, Japan, Nishihara-cho, Japan
  • Iijima, Kazumoto, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan
Background

X-linked Alport syndrome is a congenital renal disease caused by mutations in COL4A5. In recent years, splicing is focused on as the origin of pathogenicity and the target of treatment. But the effect and mechanism of aberrant splicing caused by intronic mutations of COL4A5 are not fully established. To assess the splicing abnormalities arising from intronic mutations of COL4A5, we conducted this research.

Methods

We conducted functional splicing assay with hybrid minigene for 7 intronic mutations in COL4A5 (1 was found in our case and 6 were in reported cases shown on the Human Gene Mutation Database). In addition, we conducted in silico analysis using HSF (http://www.umd.be/HSF3/HSF.shtml) and SVM-BP finder (http://regulatorygenomics.upf.edu/Software/SVM_BP/) to predict and assess the mechanisms for aberrant splicing.

Results

The minigene assay showed exon skipping by 4 variants, exon skipping+10bp insertion by 1 variant and no change by 1 variant. For 1 variant, it was difficult to assess the splicing pattern by some reason and our assay did not work. Among 3 of them, the patients’ transcript analyses were conducted and the results were completely consistent with our in vitro assay results. In silico analysis revealed that in 3 variants, polypyrimidine tracts were weakened and in 2 variants, dramatic change in splicing regulation elements binding sites were observed.

Conclusion

Our splicing assay with hybrid minigene make it possible to assess whether the mutation cause aberrant splicing. In addition, in silico tools can assess the mechanisms for causing aberrant splicing. Our findings may help the discovery of treatment strategy.