Suzanne de Bruijn

272 Chapter 5 SUPPLEMENTARY FIGURES B SLC085 Wildtype (624 bp) c.471C>T (542 bp) RHO exon 3 SLC26A4 exon 5 SLC26A4 exon 6 RHO exon 5 RHO exon 3 e5 SLC26A4 exon 6 RHO exon 5 GAPDH 100bp WT MT 500 bp 600 bp PEI NT -RT MQ A SLC048 Wildtype (555 bp) c.1342-2A>C (542 bp) RHO exon 3 SLC26A4 exon 11 SLC26A4 exon 12 SLC26A4 exon 13 RHO exon 3 SLC26A4 exon 11 e12 SLC26A4 exon 13 RHO exon 5 RHO exon 5 100bp WT MT PEI NT -RT MQ 500 bp 600 bp RHO exon 3 SLC26A4 exon 5 r.416_497del T T C A C C G T C A A G G A G C A A T G G A A C T G T A T T SLC26A4 exon 11 SLC26A4 exon 12 r.1342_1355del G A A C C C T T G C A G A A G C T G T T G T A A T T G C C A Figure S1. Results of in vitro splice assays for variants in SLC048 and SLC085. In vitro splice assays were performed in HEK293T cells to validate predicted splice defects. (A) In SLC048, a canonical splice site SLC26A4 variant (c.1342-2A>C) was detected. According to SpliceAI predictions, the splice variant (MT) weakens the canonical splice acceptor site. Splice assay results revealed usage of an alternative splice acceptor site located 13 nucleotides downstream. This leads to the formation of a truncated out-of-frame exon 12 (NM_000441.1:r.1342_1355del; p.Ser448Leufs*3). (B) In SLC085, a synonymous variant was detected (c.471C>T, p.(Pro157=)). According to SpliceAI, the variant (MT) potentially strengthens an alternative splice acceptor site. The in vitro splice assay confirmed that the alternative splice acceptor site (located 27 nucleotides downstream of the variant) is used, which leads to partial exon 5 skipping (NM_000441.1:r.416_497del; p.Gly139Alafs*6,=). Bp, base pair; wt, wildtype; mt, mutant; PEI, transfection reagent-only; RT, reverse transcriptase control; MQ, water control.