Evaluation of COL4A5 Non-canonical Splicing Variants in Two Families

Chee Teck Koh^1,2Tina Si Ting Lim^1,2Alwin Hwai Liang Loh³Yan Fei Ng³Jia Liang Kwek⁴Perry Yew Weng Lau^1,2Hui-Lin Chin^1,2Jun Li Ng^1,2Mya Than^1,2Irene Yanjia Mok⁴Cynthia Ciwei Lim⁴Kay Yuan Chong⁵Jason Chon Jun Choo⁴Hui Kim Yap^1,2Kar Hui Ng^1,2*Yao Chun Zhang^1,2*

• ¹Department of Paediatrics, National University of Singapore, Singapore, Singapore
• ²Khoo Teck Puat, National University Children’s Medical Institute (KTP-NUCMI), Singapore, Singapore
• ³Department of Anatomical Pathology, Singapore General Hospital, Singapore, Singapore
• ⁴Department of Renal Medicine, Singapore General Hospital, Singapore, Singapore, Singapore
• ⁵Medicine Academic Clinical Programme, Singapore General Hospital, Singapore, Singapore, Singapore

Alport syndrome is one of the most prevalent monogenic kidney diseases, resulting from defects in the COL4A3, COL4A4, and/or COL4A5 genes. Interpretation of non-canonical splicing variants can be challenging. This study aims to resolve two variants at non-canonical splice sites in the COL4A5 gene using multiple modalities.Exome sequencing was conducted in two families with suspected 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, familial co-segregation analyses in relation to comprehensive phenotyping of family members, immunofluorescence on kidney biopsy specimens to evaluate collagen IV α5 staining patterns, and minigene splicing assays to assess the impact on pre-messenger RNA (mRNA) splicing were performed.Family studies demonstrated co-segregation of the variants in members with characteristic phenotypic features. Immunofluorescence analysis of kidney biopsy samples displayed aberrant collagen IV α5 staining patterns. Minigene splicing assays revealed that both variants caused exon 18 skipping in the COL4A5 gene, resulting in truncated transcripts.The study illustrated a multi-faceted approach in improving the diagnostic accuracy and clinical utility of genetic testing for Alport syndrome.