Supplemental methods
Sequencing the MLH1 promoter
In brief, the entire promoter region and 5’UTR from c.-906 through exon 1 was PCRamplified using primers 5’-ctggctcggttaaaaagctg-3’ and 5’-actccctccgtaccagttct-3’.
The purified PCR products were sequenced using the same primers and an
additional internal primer 5’-gaaaactagagcctcgtcgactt-3’. Where a frameshift in the
sequence was identified indicative of an indel, the two alleles were separated and
sequenced individually by cloning the amplicons into the pGEMTeasy vector
(Promega).
Methylation screening analysis
For methylation screening, genomic DNA (500ng) was treated with sodium bisulphite
using the EZ Methylation Gold Kit (Zymo Research) and 50ng used for qMSP.
Positive qMSP products with the melt profile consistent with the presence of
methylation were electrophoresed and correct bands visualised on agarose gels. In
addition, selected qMSP products were cloned into the pGEMTeasy vector and the
inserts from individual colonies sequenced to confirm the presence of methylation at
individual CpG sites.
Confirmatory methylation assays and allelic methylation analysis
Samples were then further analysed according to their level of methylation by
pyrosequencing, and whether an informative SNP was present within the promoter to
determine allelic methylation patterns. Where sufficient sample was available,
COBRA and allelic methylation analyses, using primer sets unbiased with respect to
methylation status, were performed on those showing methylation levels of 5% or
above by pyrosequencing. The previously described MLH1 C-region COBRA assay,
encompassing both the c.-269C>G (rs35032294) and c.-93G>A (rs1800734) SNPs,
was used to determine allelic methylation patterns in individuals heterozygous for
either of these SNPs 8. For methylation-positive individuals who were heterozygous
for a single nucleotide variant within the 5’UTR or coding region of exon 1, allelic
methylation analyses were performed on downstream fragments that encompassed
these variants, as previously described by our group
15.
Amplicons were cloned in
the pGEMTEasy vector and plasmid inserts from at least 16 individual clones were
sequenced using vector primers.
Luciferase Promoter reporter assays
A promoter reporter construct containing the entire wild-type MLH1 promoter and
5’UTR (from c.-513 to c.-1 with respect to the ATG start codon) inserted upstream of
the Firefly luciferase reporter in the pGL3-Basic vector (Promega) were subjected to
site-directed mutagenesis to create constructs containing the promoter sequence
variants identified in this study. The wild-type and variant constructs were transiently
co-transfected into HEK293 and HCT116 cells with the pRL-TK Renilla luciferase
reporter to normalise for transfection efficiency. Transfections were performed in
triplicate on multiple (6-9) separate occasions alongside transfections of the pGL3Control luciferase reporter under the SV40 promoter, and empty pGL3-Basic vector.
Luciferase outputs from both Firefly and Renilla luciferase were measured step-wise
by luminometry on the Glomax luminometer (Promega) 24 hours post-transfection
using the Dual-Glo luciferase assay system (Promega). The background from
measurements of Fugene carrier alone was subtracted from each read. Firely
luciferase values were normalised to the Renilla luciferase value for each data point,
and then expressed as a percentage of the positive control vector (pGL3-Control
under the SV40 promoter). A average of the three replicates within each
independent experiment provided a single value. The mean values and standard
deviation from experiments performed on multiple independent occasions were
plotted on histograms. A Student’s T test was used to compare the set of mean
values from each promoter sequence variant to those of the wild-type sequence. The
level of statistical significance was stratified as *P<0.05, **P<0.001 and ***P<0.0001
and reflected the fold-change in levels of promoter reporter activity.