Kidney Week

Abstract: FR-PO1012

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

Session Information

• Genetic Diseases of the Kidneys: Non-Cystic - II
  October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

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

Tomoko Horinouchi,

• Nozu, Kandai, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Kandai Nozu,

• Yamamura, Tomohiko, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Tomohiko Yamamura,

• Sakakibara, Nana, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Nana Sakakibara,

• Nagano, China, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

China Nagano,

• Rossanti, Rini, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Rini Rossanti,

• Nakanishi, Keita, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Keita Nakanishi,

• Fujimura, Junya, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Junya Fujimura,

• Minamikawa, Shogo, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Shogo Minamikawa,

• Ninchoji, Takeshi, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Takeshi Ninchoji,

• Kaito, Hiroshi, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Hiroshi Kaito,

• Shima, Yuko, Department of Pediatrics, Wakayama Medical University, Wakayama, Japan, Wakayama, Japan

Yuko Shima,

• Nakanishi, Koichi, Department of Pediatrics, Graduate School of Medicine, University of the Ryukyus, Nishihara-cho, Japan, Nishihara-cho, Japan

Koichi Nakanishi,

• Iijima, Kazumoto, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan, Kobe, Japan

Kazumoto Iijima,

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.