Fig. 4. Case 2 patient’s pedigree and confirmation of a splice effect of MLH1 c.306G>A.

a The patient’s pedigree. The circles denote females and the squares denote males. Filled symbols indicate the individual had a tumour, with the age at tumour diagnosis being given in years (y) if known. Deceased individuals are indicated with a strike-through. The patient is denoted by the arrow. b Transcript analysis by PCR amplification of cDNA generated from RNA extracted from PHA-stimulated and puromycin-treated short-term cultures of peripheral blood leukocytes. The PCR primers used are located in MLH1 exon 1 and exon 4. The expected amplicon sizes are 333 bp from full-length MLH1 transcript and 234 bp from MLH1 transcript lacking exon 3 (99 bp). C1-C3: control (MLH1 wild type) leukocyte cDNA. P: patient (MLH1 c.306G>A homozygous) leukocyte cDNA. Ø: negative-template control. c Analysis of minigene transcripts by PCR amplification of cDNA from RTB minigene-transfected cell lines (HEC-155, HEC-1B, HRT-18, and HEK-293) using primers located in RTB minigene exon 1 and exon 4 (Supplementary Figure S7). The expected amplicon sizes are 267 bp from RTB minigene transcript containing MLH1 exon 3 and 168 bp from transcript lacking MLH1 exon 3 (99 bp). WT: cDNA amplicons from cells transfected with RTB minigene containing wild type MLH1 exon 3. V: cDNA amplicons from cells transfected with RTB minigene containing variant (c.306G>A) MLH1 exon 3. Ø: negative-template control. Note: Amplification of cDNA from the RTB minigene containing variant c.306G>A MLH1 exon 3 produces an unexpected third, larger amplicon that appears to be a heteroduplex of the other products based on Sanger sequencing data (Supplementary Fig. S5).