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).