Figure legends
Fig. 1 Patient pedigree. The patient described here is a 49-year-old man who was diagnosed with colon cancer at 43. Nine family members were affected with colorectal cancer and six members were diagnosed with endometrial cancer in this family.
Fig. 2 RT-PCR and Southern blot analysis. a) Agarose gel electrophoresis of the RT-PCR of MSH2 exon 6-16 on mRNA of short-term cultured lymphocytes treated with (+chx) or without (-chx) cycloheximide. A very faint band, approximately 200 bp larger than the normal RT-PCR product, is visible in patient’s RNA sample treated with CHX. The PCR is not quantitative; the faintness of the larger band is possibly caused by problematic amplification of a GC-rich aberrant transcript, or diminished expression of the mutant allele. b) Southern blot gels of genomic DNA of the patient (two independent DNA samples PAT_D1 and PAT_D2) and two negative controls (CTR1 and CTR2) digested with four different restriction enzymes and hybridized with an MSH2 exon 10-16 cDNA probe (M2-3’b). All enzymes show an approximately 3 kb larger extra fragment in the patient’s DNA in addition to the normal wildtype fragment. c) Restriction fragment analysis and approximate localization of the insertion in the MSH2 gene.
Fig. 3 MSK-IMPACT and MLPA results. a) IGV shows aberrant sequence in exon 12 of MSH2 . b) CNV plots of the patient showed no deletion or duplication in MSH2 exon 12. c) MLPA results confirmed no CNV change in MSH2 of the patient.
Fig. 4 Long-range PCR and sequencing results after gel extraction. a) LR-PCR products run on 1% agarose gel. An extra band were observed in the patient, but not in controls. b) Electropherogram showing the site of insertion. c) Partial inserted sequences in red in anti-sense orientation with respect to the MSH2 gene transcription direction. Target site duplication is highlighted in yellow. DNA sequence in the first row matches with those in reverse direction in b.
Fig. 5 SVA insertion in exon 12 of the MSH2 gene in the anti-sense direction. a) Final inserted sequence identified. Sequences that are not present on Chromosome 3 are highlighted in gray. “ …” indicates sequences that were not deciphered. b) Sequence on chromosome 3 (GRCh38.p12 Primary Assembly from 48,210,600 to 48,213,111.). The sequences that are not present in the SVA insertion in MSH2 are highlighted in yellow. The sequences present in the SVA insertion in MSH2 but not on Chromosome 3 are left in blank. c) Schematic view of full length SVA_F1 (not in scale). d) Schematic representation of the SVA element inserted in MSH2 exon 12 (not in scale).