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Abstract: SA-PO562

Aberrant Splicing Affected by Single Nucleotide Variants Positioned at the Second and Third to the End of Exons in COL4A5 Gene

Session Information

  • Genetic Diseases: Diagnosis
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

  • 1102 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Okada, Eri, Kobe Daigaku, Kobe, Hyogo, Japan
  • Aoto, Yuya, Kobe Daigaku, Kobe, Hyogo, Japan
  • Horinouchi, Tomoko, Kobe Daigaku, Kobe, Hyogo, Japan
  • Yamamura, Tomohiko, Kobe Daigaku, Kobe, Hyogo, Japan
  • Nozu, Kandai, Kobe Daigaku, Kobe, Hyogo, Japan
Background

As it has become more evident that male patients with X-linked Alport Syndrome show obvious genotype-phenotype correlation, it has been of great importance to clarify the impact on aberrant splicing caused by identified variants. We previously reported that single nucleotide variants (SNVs) at the last nucleotide of exons in COL4A5 gene highly cause aberrant splicing. It is generally considered that the 2nd and 3rd to the last nucleotides of exons can also play an important role in the first step of splicing process. The aim of the recent study is to investigate aberrant splicing affected by SNVs positioned at 2nd or 3rd to the last nucleotide of exons in COL4A5 gene.

Methods

We selected 8 candidate variants:6 from Human Gene Mutation Database Professional and 2 from our cohort. We performed in vitro splicing reporter assay and reverse transcription polymerase chain reaction (RT-PCR) for messenger RNA obtained from the patients if available.

Results

Initial classification of the candidate variants was as follows: 3 nonsense, 2 missense and 3 synonymous. Splicing reporter assay and RT-PCR for messenger RNA revealed that 6 of 8 variants caused aberrant splicing. Four variants initially assessed as non-truncating variants were revealed to be truncating variants. One variant (No.7, c.685A>T, p.Lys229*) generated not only normal transcript but also aberrant transcript, which resulting in in-frame deletion.

Conclusion

We revealed that exonic SNVs positioned at the 2nd and 3rd to the last nucleotide of exon in COL4A5 gene can highly cause aberrant splicing. Minigene splicing assay is useful to confirm the effect of variants on aberrant splicing especially for genes that genotype-phenotype correlation is evident like COL4A5 gene to predict the patients’ prognosis.

Candidate variants and splicing outcome