Abstract
Mutations in COL4A3, COL4A4 and COL4A5 genes lead to Alport syndrome (AS). However, pathogenic variants in some AS patients are not detected by exome sequencing. The aim of this study was to identify the underlying genetic causes of five unrelated AS probands with negative next-generation sequencing (NGS) test results. Urine COL4A3–5 mRNAs were analyzed in the probands with an uncertain inherited mode of AS, and COL4A5 mRNA of skin fibroblasts was analyzed in the probands with X-linked AS. RT-PCR and direct sequencing were performed to detect mRNA abnormalities. PCR and direct sequencing were used to analyze the exons with flanking intronic sequences corresponding to mRNA abnormalities. Six novel deep intronic splicing variants in COL4A4 and COL4A5 genes that cannot be captured by exome sequencing were identified in the four AS probands. Skipping of an exon was caused by an intronic variant, and retention of an intron fragment caused by five variants. In the remaining AS proband, COL4A5 variants c.2677 + 646 C > T and r.2678_r.2767del were detected at the DNA and RNA level, respectively, whereas it is unclear whether c.2677 + 646 C > T may not lead to r.2678_r.2767del. Our results reveal that mRNA analysis for AS genes from either urine or skin fibroblasts can resolve genetic diagnosis in AS patients with negative NGS results. We recommend analyzing COL4A3–5 mRNA from urine as the first choice for these patients because it is feasible and non-invasive.
Highlights
Mutations in COL4A3, COL4A4 and COL4A5 genes lead to Alport syndrome (AS)
X-linked AS (XLAS) is caused by pathogenic variants in the COL4A5 gene, while autosomal recessive AS (ARAS) and autosomal dominant AS (ADAS) are caused by pathogenic variants in the COL4A3 or COL4A4 gene[4]
The sizes of all ten overlapping fragments covering the entire coding sequence of either COL4A3, COL4A4, or COL4A5 mRNA were the same as originally conceived (Fig. 1C), and sequencing of these RT–PCR products confirmed that the amplified sequences mapped precisely to the published COL4A3, COL4A4, and COL4A5 mRNA sequences (NM_000091.5, NM_000092.5, and NM_000495.5), respectively
Summary
Mutations in COL4A3, COL4A4 and COL4A5 genes lead to Alport syndrome (AS). pathogenic variants in some AS patients are not detected by exome sequencing. Since 2000, simultaneous examinations of α5(IV) staining in skin and COL4A5 mutation screening using mRNA extracted from cultured skin fibroblasts have been routinely performed in patients with suspected XLAS This approach cannot be applied for Alport syndrome patients with autosomal inherited patterns, since α3(IV) and α4(IV) are not expressed in skin. We used our developed approach for analysis of the entire coding regions of COL4A3, COL4A4, and COL4A5 mRNAs isolated from urine and COL4A5 mRNA extracted from cultured skin fibroblasts and identified deep intronic splicing variants in the enrolled patients.
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