Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing

Yuya Aoto; Tomoko Horinouchi; Tomohiko Yamamura; Atsushi Kondo; Sadayuki Nagai; Shinya Ishiko; Eri Okada; Rini Rossanti; Nana Sakakibara; China Nagano; Hiroyuki Awano; Hiroaki Nagase; Yuko Shima; Koichi Nakanishi; Masafumi Matsuo; Kazumoto Iijima; Kandai Nozu

doi:10.1016/j.ekir.2021.10.012

Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing

Kidney Int Rep. 2021 Oct 21;7(1):108-116. doi: 10.1016/j.ekir.2021.10.012. eCollection 2022 Jan.

Authors

Yuya Aoto¹, Tomoko Horinouchi¹, Tomohiko Yamamura¹, Atsushi Kondo¹, Sadayuki Nagai¹, Shinya Ishiko¹, Eri Okada¹, Rini Rossanti¹, Nana Sakakibara¹, China Nagano¹, Hiroyuki Awano¹, Hiroaki Nagase¹, Yuko Shima², Koichi Nakanishi³, Masafumi Matsuo⁴, Kazumoto Iijima^{5

6}, Kandai Nozu¹

Affiliations

¹ Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.
² Department of Pediatrics, Wakayama Medical University, Wakayama, Japan.
³ Department of Child Health and Welfare (Pediatrics), Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan.
⁴ Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Kobe, Japan.
⁵ Hyogo Prefectural Kobe Children's Hospital, Hyogo, Japan.
⁶ Department of Advanced Pediatric Medicine, Kobe University Graduate School of Medicine, Hyogo, Japan.

Abstract

Introduction: COL4A5 is a causative gene of X-linked Alport syndrome (XLAS). Male patients with XLAS with nonsense variants have the most severe phenotypes of early onset end-stage kidney disease (ESKD); those with splicing variants have middle phenotypes and those with missense variants have the mildest phenotypes. Therefore, genotyping for male patients with XLAS can be used to predict kidney prognosis. Single-base substitutions at the last nucleotide position in each exon are known to affect splicing patterns and could be splicing variants. Nevertheless, in XLAS, these variants are generally considered to be missense variants, without conducting a transcript analysis, which underestimates some patients as having mild phenotypes. This study aimed to investigate whether single-base substitutions at the last nucleotide position of COL4A5 exons cause aberrant splicing.

Methods: In total, 20 variants were found in the Human Gene Mutation Database (n = 14) and our cohort (n = 6). We performed functional splicing assays using a hybrid minigene analysis and in vivo transcript analyses of patients' samples when available. Then, we investigated genotype-phenotype correlations for patients with splicing variants detected in this study by comparing data from our previous studies.

Results: Among the 20 variants, 17 (85%) caused aberrant splicing. Male patients with splicing variants had more severe phenotypes when compared with those with missense variants. Findings from the in vivo analyses for 3 variants were identical to those from the minigene assay.

Conclusion: Our study revealed that most single-base substitutions at the last nucleotide position of COL4A5 exons result in splicing variants, rather than missense variants, thereby leading to more severe phenotypes.

Keywords: COL4A5; genotype–phenotype correlation; last nucleotide position; missense variants; single-base substitutions; splicing.