AUTHOR=Koh Chee Teck , Lim Tina Si Ting , Loh Alwin Hwai Liang , Ng Yan Fei , Kwek Jia Liang , Lau Perry Yew Weng , Chin Hui-lin , Ng Jun Li , Than Mya , Mok Irene Yanjia , Lim Cynthia Ciwei , Chong Kay Yuan , Choo Jason Chon Jun , Yap Hui Kim , Ng Kar Hui , Zhang Yaochun 

TITLE=Case Report: Evaluation of COL4A5 non-canonical splicing variants in two families

JOURNAL=Frontiers in Medicine

VOLUME=Volume 12 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/medicine/articles/10.3389/fmed.2025.1611334

DOI=10.3389/fmed.2025.1611334

ISSN=2296-858X

ABSTRACT=IntroductionAlport syndrome is one of the most prevalent monogenic kidney diseases, resulting from the defects in COL4A3, COL4A4, and/or COL4A5 genes. Interpretation of non-canonical splicing variants can be challenging. This study aimed to resolve two variants at non-canonical splice sites in the COL4A5 gene using multiple modalities.MethodsExome sequencing was performed on two families suspected of having Alport syndrome. Intronic splice site variants in COL4A5, which have been reported in the literature, were identified: c.1032 + 4A > G in one family and a four-nucleotide deletion, c.1032 + 3_1032 + 6delAAGT, in the other. To clarify the pathogenicity of these variants, several analyses were performed: familial co-segregation analyses in relation to comprehensive phenotyping of family members, immunofluorescence analysis of kidney biopsy specimens to evaluate collagen IV α5 staining patterns, and minigene splicing assay to assess the impact on pre-messenger RNA (mRNA) splicing.ResultsThe family studies demonstrated co-segregation of the variants among members with characteristic phenotypic features. The immunofluorescence analysis of the kidney biopsy samples displayed aberrant collagen IV α5 staining patterns. The minigene splicing assay revealed that both variants caused exon 18 skipping in the COL4A5 gene, resulting in truncated transcripts.ConclusionThe study demonstrated a multi-faceted approach to improve the diagnostic accuracy and clinical utility of genetic testing for Alport syndrome.