Evaluation of suspected autosomal Alport Syndrome synonymous variants

【Background】 Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in autosomal Alport syndrome cases, synonymous mutations in these genes have rarely been identified. 【Methods】 We conducted in silico splicing analysis using the Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of 4 synonymous variants detected in 4 patients with suspected autosomal dominant Alport syndrome (COL4A3 (c.693G>A (p.Val231=) and COL4A4 (c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=))). 【Results】 Both in vivo and in vitro splicing assays showed exon skipping in 2 out of the 4 synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using the HSF and Alamut suggested that these 2 variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other 2 variants did not induce aberrant splicing. 【Conclusions】 This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing as shown in 2 of our cases. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in autosomal dominant Alport syndrome cases.