Abnormal mRNA Splicing Effect of to Unclassified Variants.

INTRODUCTION

Genetic diagnosis of Alport syndrome (AS), which results from pathogenic variants in , , or genes, is hindered by large numbers of unclassified variants detected using next-generation sequencing (NGS). We examined the impact on splicing of variants of uncertain significance in to .

METHODS

Nine unrelated patients with clinical diagnosis or suspicion of AS were enrolled according to the criteria. Their clinical and genetic data were collected. Blood and urine samples were obtained from the patients and their family members. Sanger sequencing was used to confirm the 9 to unclassified variants identified by NGS. to mRNAs from urine were analyzed using targeted reverse transcription polymerase chain reaction and direct sequencing.

RESULTS

Nine to unclassified variants were found to alter mRNAs splicing. Skipping of an exon or an exon fragment was induced by variants c.828+5G>A; c.3506-13_3528del; and c.451A>G (p. [Ile151Val]), c.2042-9 T>G, c.2689 G>C (p. [Glu897Gln]) and c.1033-10_1033-2delGGTAATAAA. Retention of an intron fragment was caused by variants c.3211-30G＞T, and c.4316-20T>A and c.1033-10 G>A, respectively. The 9 families in this study obtained genetic diagnosis of AS, including 3 with autosomal recessive AS and 6 with X-linked AS.

CONCLUSIONS

Our findings demonstrate that urine mRNA analysis facilitates the identification of abnormal splicing of unclassified variants in Alport genes, which provides evidence of routine use of RNA analysis to improve genetic diagnosis of AS.