Fig. 5: WGS-based detection and characterization of a novel deep-intronic PV in MLH1 in patient #4.

Allele imbalance detected at the MLH1 c.-93G > A heterozygote marker in patient #4 (A). Note: results from the reverse sequencing are presented. Puromycin treatment almost restored the allelic ratios measured at the gDNA-level. Red arrows show the position of MLH1 c.-93G > A. Chromatogram of gDNA-based Sanger sequencing of the MLH1 c.306+1222 A > G variant (B). The arrow points to the heterozygous locus of c.306+1222 A > G. Electrophoretic separation from MLH1 cDNA amplicons incorporating exons 1-9 from patient #4 and controls (C). Puromycin (Puro) treatment inhibits NMD. Note: Uncropped image is demonstrated in Figure S6. Electrophoretic separation of MLH1 cDNA amplicons restricted from alleles containing the reference ’G’ base at MLH1 c.-93 position (D). This allowed the amplification to occur from the allele which carried the detected deep intronic variant, as the alternative ’G’ base at the c.306+1222 position was in phase with the reference ’G’ base at the c.-93 position in patient #4 (as highlighted in A). Note: Uncropped image is demonstrated in Figure S7. Chromatogram of cDNA-based Sanger sequencing restricted from wild type cDNA transcript highlighting the -93G > A position from patient #4 (E). Restriction for the wild-type cDNA transcript was allowed by the reference primer which was specific to the exon 3-4 boundary. Note: results from the reverse sequencing are presented and might be compared to those presented in (A). The red arrow shows the position of MLH1 c.-93G > A. Genomic architecture of altered splicing resulting from the deep intronic variant MLH1 c.306+1222 A > G (F). Pedigree of patient #4 (G). bp base pair, BrainT brain tumor, CRC colorectal cancer, L ladder, NMD nonsense-mediated decay, Puro puromycin.