High-Throughput Splicing Assays Identify Known and Novel WT1 Exon 9 Variants in Nephrotic Syndrome
Cathy Smith, Bala Bharathi Burugula, Ian Dunn, Swaroop Aradhya, Jacob O. Kitzman, Jennifer Lai Yee
Abstract
IntroductionFrasier Syndrome (FS) is a rare Mendelian form of nephrotic syndrome caused by variants which disrupt the proper splicing of WT1. This key transcription factor gene is alternatively spliced at exon 9 to produce two isoforms (“KTS+” and “KTS-”), which are normally expressed in the kidney at a ∼2:1 (KTS+:KTS-) ratio. FS results from variants that reduce this ratio by disrupting the splice donor of the KTS+ isoform. FS is extremely rare, and it is unclear whether any variants beyond the eight already known could cause FS.MethodsTo prospectively identify other splicing-disruptive variants, we leveraged a massively parallel splicing assay. We tested every possible single nucleotide variant (n=519) in and around WT1 exon 9 for effects upon exon inclusion and KTS+/- ratio.ResultsSplice disruptive variants made up 11% of the tested point variants overall and were tightly concentrated near the canonical acceptor and the KTS+/- alternate donors. Our map successfully identified all eight known FS or focal segmental glomerulosclerosis variants and 16 additional novel variants which were comparably disruptive to these known pathogenic variants. We also identified 19 variants that, conversely, increased the KTS+/KTS- ratio, of which two are observed in unrelated individuals with 46,XX ovotesticular disorder of sex development (46,XX OTDSD).ConclusionsThis splicing effect map can serve as functional evidence to guide the clinical interpretation of newly observed variants in and around WT1 exon 9.