Frasier 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. To 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. Splice 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). This splicing effect map can serve as functional evidence to guide the clinical interpretation of newly observed variants in and around WT1 exon 9.

S.A. is an employee of Invitae. J.O.K. serves as a scientific advisor to MyOme, Inc. The authors declare that there are no further competing interests.

This work was supported by the National Institute of General Medical Sciences (R01GM129123 to J.O.K.) and a National Institute of Child Health & Human Development Clinical Scientist Institutional Career Development Program Award (5K12HD028820 to J.L.Y.).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

WCG IRB gave ethical approval for this work (protocol #1167406).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present work are contained in the manuscript.