High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency

Using a high throughput splicing reporter assay, we tested 1,080 single nucleotide variants in POU1F1, a key transcription factor essential for pituitary development. Our saturation splicing effect map identifies 96 splice disruptive variants, including 14 synonymous variants, of which 8 were found in unrelated patients diagnosed with hypopituitarism. Saturation mutagenesis was used to generate libraries comprising all SNVs within POU1F1 exon 2 and flanking introns (-85 bp to +178 bp). These libraries, comprising 1,080 variants, were transferred to the exon trap construct pSPL3. Degenerate 20mer barcodes were added to the downstream 3'UTR, and paired end sequecning was used to associate each barcode with the POU1F1 variant(s) present on the same plasmid copy. COS-7 cells were transfected with the pooled minigene library, and targeted RNA-seq of the spliced minigene transcripts were measure the splicing outcome associated with each barcode/variant.