72 - Prospectively identify splice-disruptive variants (SDVs) of WT1 using a multiplex splice minigene assay

Poster Number: 72
Publication Number: 72.351

Jennifer Yee, University of Michigan Medical School, Ann Arbor, MI, United States; Cathy Smith, University of Michigan Medical School, Ann Arbor, MI, United States; Bala Bharathi Burugula, University of Michigan, Rochester Hills, MI, United States; Jacob Kitzman, University of Michigan Medical School, Ann Arbor, MI, United States

Background: Variants that disrupt mRNA splicing contribute to pathogenesis in nearly every human genetic disorder. This includes Mendelian forms of nephrotic syndrome (NS), such as Frasier Syndrome (FS), which is caused by splicing disruptive variants (SDVs) near WT1 exon 9 splice donors resulting in decreased ratio of two natural splice isoforms, KTS+ and KTS-. However, beyond the few specific FS SDVs reported from case reports, accurately predicting other SDVs in WT1 remains a challenge.

Objective: In vitro splice minigene assay provides one means to test variant’s splicing effect either one at a time or in highly multiplexed fashion. Therefore, we applied multiplex splice minigene assay across WT1 exon 9 to prospectively identify WT1 SDVs in a high-throughput manner.

Design/Methods: WT1 exon 9 plus 200 bases of the flanking introns were cloned into an established minigene plasmid, in between constant synthetic exons. Large scale mutagenesis was performed to generate a variant library including every single nucleotide variant across the cloned region, each associated with a unique “barcode” sequence in the constant downstream exon. This variant library was then transfected into HEK293T and COS7 cells with multiple replicates. RNA was harvested after 24 hours of transfection and spliced transcripts from the minigene library were analyzed by target RNA-seq. The splicing patterns associated with each variant were quantified from the aligned reads.

Results: Nearly every possible single nucleotide variant was represented (518/519; 99.8%) with a high degree of internal replication (mean=79.7 barcodes per variant). The splicing disruption was heavily concentrated near the canonical splice sites, the alternate KTS+ and KTS- donors. We successfully identified 8 known FS variants dramatically lowered log2 (KTS+/KTS-) to -2.1 or lower, and 16 additional SDVs which disrupted KTS+/KTS- comparably to the known FS variants. We also identified 19 variants that increased KTS+/KTS-, with two have been observed in patients with disorder of sex development (DSD).

Conclusion(s): The pooled minigene assay is highly sensitive and specific for identification of pathogenic WT1 exon 9 splice disruption. Our multiplex screen identifies all known FS SDVs in WT1 exon 9. We also nominate an additional 16 possible yet unseen FS variants with similarly decreased KTS+/KTS-. A set of variants significantly increase KTS+ expression, which might be related to DSD. In summary, multiplex functional analyses can prospectively score genetic variants in NS and guide the clinical decision.