Kidney Week

Abstract: FR-PO0119

Integrated Evaluation of COL4A5 Noncanonical 3′ Splice Site Variants in Alport Syndrome

Session Information

• Hereditary Glomerular and Tubulointerstitial Kidney Diseases
  October 23, 2026 | Location: Exhibit Hall A, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1202 Genetic Diseases of the Kidneys: Non-Cystic (Complex and Non-Cystic Monogenic)

Authors

• Yamamoto, Asahi, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Asahi Yamamoto, MD

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

Nana Sakakibara, MD, PhD

• Aoto, Yuya, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Yuya Aoto, MD, PhD

• Nishimura, Ryo, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Ryo Nishimura

• Fujiwara, Ayako, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Ayako Fujiwara

• Baba, Minato, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Minato Baba

• Aoyama, Shuhei, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Shuhei Aoyama, MD

• Kimura, Yuka, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Yuka Kimura, MD

• Ichikawa, Yuta, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Yuta Ichikawa

• Inoki, Yuta, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Yuta Inoki, MD, MEd

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

China Nagano, MD, PhD

• Horinouchi, Tomoko, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Tomoko Horinouchi, MD, PhD

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

Tomohiko Yamamura, MD, PhD

• Ishimori, Shingo, Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan

Shingo Ishimori, MD, PhD

• Suzuki, Ryota, Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Ryota Suzuki, MD

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

Kandai Nozu, MD, PhD

Background

COL4A5 causes X-linked Alport syndrome, for which genetic diagnosis is increasingly important for diagnosis, prognosis, treatment selection, and donor evaluation. Canonical splice-site variants at invariant ±1/2 positions are established causes of aberrant splicing, whereas variants outside these sites may be overlooked. We previously evaluated COL4A5 non-canonical 5′ splice site variants. In this study, we assessed the pathogenicity of non-canonical 3′ splice site variants by integrating in silico prediction, minigene analysis, and patient-derived mRNA analysis.

Methods

Non-canonical 3′ splice site variants in COL4A5 (NM_000495.5) were identified from the Human Gene Mutation Database Professional (HGMD) and our Alport syndrome cohort (Jan 2006–Feb 2026). These variants were defined as intronic variants located from −14 to −3 upstream of the exon acceptor site. Splicing was analyzed using an in vitro minigene assay, in which variant-containing genomic fragments were cloned into an expression vector and expressed as mRNA in cultured cells. Results were compared with SpliceAI and Pangolin predictions using a cutoff score of ≥0.2. Patient lymphocyte mRNA was analyzed when available.

Results

We identified 30 variants from HGMD and 10 distinct variants from our cohort, which included 29 patients from 16 families with typical Alport syndrome. Three cohort-derived variants were novel. After excluding overlaps, 33 variants were analyzed. Minigene assays showed aberrant splicing in 31/33 variants: exon skipping in 10, cryptic acceptor activation with intronic insertion in 14, both in 7, and no abnormality in 2. Among these 31 variants, 26 created a novel AG dinucleotide upstream of the original acceptor site; 21/26 AG-gain variants caused intronic insertion. SpliceAI or Pangolin predicted the minigene-confirmed aberrant splicing patterns. Patient lymphocyte mRNA was available for 7/10 cohort variants, all concordant with minigene results.

Conclusion

COL4A5 non-canonical 3′ splice site variants located from −14 to −3 represent an important variant class associated with aberrant splicing in Alport syndrome. In particular, AG gain upstream of the original acceptor site was strongly suggestive of cryptic acceptor activation and intronic insertion. These findings support the pathogenicity of such variants when the phenotype is consistent with Alport syndrome and SpliceAI or Pangolin predicts aberrant splicing.