Extensive DNA methylation in normal colorectal
mucosa in hyperplastic polyposis
P Minoo, K Baker, R Goswami, G Chong, W D Foulkes, A R Ruszkiewicz, M Barker,
D Buchanan, J Young, J R Jass
............................................................... ............................................................... .
See end of article for
Professor J R Jass,
Department of Pathology,
McGill University, Duff
Medical Building, 3775
University Street, Montreal,
Quebec H3A 2B4,
Revised version received
18 January 2006
Accepted for publication
31 January 2006
Published online first
9 February 2006
Gut 2006;55:1467–1474. doi: 10.1136/gut.2005.082859
Background: Hyperplastic polyposis of the colorectum is a precancerous condition that has been linked
with DNA methylation. The polyps in this condition have been distinguished from typical small hyperplastic
polyps and renamed sessile serrated adenomas. Sessile serrated adenomas also occur sporadically and
appear to be indistinguishable from their counterparts in hyperplastic polyposis.
Aims and methods: The existence of distinguishing molecular features was explored in a series of serrated
polyps and matched normal mucosa from patients with and without hyperplastic polyposis by assessing
mutation of BRAF, DNA methylation in 14 markers (MINTs 1, 2 and 31, p16, MGMT, MLH1, RASSF1,
RASSF2, NORE1 (RASSF5), RKIP, MST1, DAPK, FAS, and CHFR), and immunoexpression of MLH1.
Results: There was more extensive methylation in sessile serrated adenomas from subjects with
hyperplastic polyposis (p,0.0001). A more clearcut difference in patients with hyperplastic polyposis was
the finding of extensive DNA methylation in normal mucosa from the proximal colon.
Conclusions: A genetic predisposition may underlie at least some forms of hyperplastic polyposis in which
the earliest manifestation may be hypermethylation of multiple gene promoters in normal colorectal
mucosa. Additionally, some of the heterogeneity within hyperplastic polyposis may be explained by
different propensities for MLH1 inactivation within polyps.
mas and have been classified as non-neoplastic lesions that
are lacking in malignant potential.1While an individual HP
may have extremely limited potential for malignant trans-
formation, there are reports linking the condition hyperplas-
tic polyposis (HPP) with colorectal cancer (CRC).2–4This
raises the question of whether HPs occurring in the condition
HPP are qualitatively different from their sporadic counter-
The definition of HPP allows for two major phenotypes: (1)
multiple small and mainly distal polyps; and (2) small
numbers of large and mainly proximal polyps.5
morphological heterogeneity may explain why some studies
have failed to show a convincing link with CRC.6 7It is
pertinent that investigations that have focused on right sided
HPs have suggested that these may be the precursors of the
subset of CRC with high level DNA microsatellite instability
(MSI-H) whether presenting in HPP8or sporadically.9–12The
first detailed morphological characterisation of large and
proximal HPs occurred in the context of HPP.2The authors
argued that the polyps in question were distinct from
traditional HPs and that the condition they were describing
was more aptly termed serrated adenomatous polyposis. The
same authors then went on to demonstrate that identical
‘‘sessile serrated adenomas’’ (SSA) could occur sporadically
and described in considerable detail the morphological
features that distinguished these lesions from typical HPs.13
Parallel studies focusing on immunohistochemical and
molecular changes also showed differences between proximal
and distal lesions, whether these were sporadic or presented
in the context of HPP. Specifically, proximal polyps and/or
polyps with the features of SSA showed more aberrant
proliferation,14and high frequencies of BRAF mutation15 16
and DNA methylation.15 17In contrast, small and distal HPs
had frequent KRAS mutation3 15or alterations implicating
olorectal hyperplastic polyps (HPs) with their charac-
teristic serratedglandular architecture are easily
distinguished on morphological grounds from adeno-
chromosome 1p3and low frequencies of both BRAF muta-
tion16and DNA methylation.3 15 17
It is not known if the serrated polyps presenting in HPP are
qualitatively different from their sporadic counterparts that
are large, proximal, and show the morphological features of
SSA. Based on findings with respect to BRAF mutation and
DNA methylation, it would appear that the serrated polyps
occurring in these two clinical scenarios do not differ
qualitatively and the link between HPP and colorectal cancer
is observed merely because the polyps are very numerous.
Nevertheless, there are grounds for suspecting that the polyps
in HPP may differ. A key rate limiting step in the evolution of
sporadic CRC with MSI-H is methylation of the promoter
region and subsequent inactivation of the DNA mismatch
repair gene MLH1.18Using immunohistochemistry to test for
loss of expression of MLH1 in 44 sporadic serrated polyps that
were all resected from the proximal colon, none of the polyps
showed even focal loss of MLH1.14It would therefore appear
that loss of expression of MLH1 is an uncommon event in
sporadic serrated polyps, although such loss has been
described in a selected series of sporadic mixed polyps.19In
contrast, loss of MLH1 has been observed in a high
proportion of serrated polyps occurring within the condition
In the present study, we have identified three patients with
HPP in which the phenotype was particularly severe, as
manifested by the presence of at least one CRC, serrated
polyps with adenomatous dysplasia (mixed polyps or serrated
adenomas), as well as very numerous polyps with the
features of SSA. We compared patterns of DNA methylation
Abbreviations: HP, hyperplastic polyp; HPP, hyperplastic polyposis;
SSA, sessile serrated adenoma; TSA, traditional serrated adenoma;
HNPCC, hereditary non-polyposis colorectal cancer; CRC, colorectal
cancer; MSI, microsatellite instability; CIMP, CpG island methylator
phenotype; PCR, polymerase chain reaction; MSP, methylation specific
polymerase chain reaction; MAPK, mitogen activated protein kinase
and expression of MLH1 in polyps and normal mucosa from
these patients with samples obtained from patients with only
small numbers of right sided serrated polyps. As inhibition of
apoptosis has been linked to the aetiology of serrated
polyps,20 21the marker panel for DNA methylation included
multiple genes with a proapoptotic function.
MATERIALS AND METHODS
Patients and tissues
Patient Nos 1–3 met one or more of the diagnostic criteria for
HPP, each having multiple serrated polyps in the proximal
colon of which at least two exceeded 10 mm in diameter.5
The numbers and types of polyps are shown in table 1. In
patient No 2, over 100 polyps were counted and 54 of these
were diagnosed histologically. Patient No 4 and 5 presented
with proximal CRC and small numbers of proximal serrated
polyps that did not meet the criteria for HPP. The serrated
polyps in patient No 1–5 were classified as HPs, mixed polyps,
traditional serrated adenomas (TSA), and the variant HP
described as SSA13
(table 1, fig 1). Diffuse mucosal
hyperplasia was present in the appendix in patient No 1, 4,
and 5. Patient No 1 and No 3–5 were treated by right
hemicolectomy and patient No 2 by proctocolectomy. Patient
No 6–12 had a single SSA of the proximal colon that was
removed endoscopically. Patient No 13–15 had a right sided
CRC and at least one right sided HP but only DNA obtained
from normal mucosa was tested.
Seven of the CRCs shown in table 1 for patient No 1–5
presented in the proximal colon. Patient No 2 had synchro-
nous rectal cancer arising in a tubulovillous adenoma that
was not subjected to further analysis. CRCs in patient No 2, 4,
and 5 were contiguous with residual SSA (not included in
totals shown in table 1). None of the patients had
inflammatory bowel disease. The study was approved by
the institutional review board of McGill University.
Immunohistochemical staining for the mismatch repair
protein MLH1 was undertaken for 18, 54, and four serrated
polyps from patient No 1–3, respectively, and seven CRCs
(including any contiguous polyps) from patient No 1–5.
Sections (4 mm) of formalin fixed paraffin embedded tissues
were cut, mounted onto charged slides, and prepared for
incubation with the primary mouse monoclonal antibody to
MLH1 (1:100 clone G168-15; BD Pharmingen, Bedford,
Massachusetts, USA), as described previously.14Loss of
MLH1 expression was scored only when there was complete
loss of nuclear expression within a distinct subclone or an
entire colorectal crypt, including the crypt base cells.
DNA extraction and bisulphite modification
Samples were microdissected from paraffin embedded tissue
using two 8 mm thick sections that included 23 SSAs, two
mixed polyps, one TSA, two conventional adenomas, and
seven CRCs. Care was taken to exclude as much normal
mucosa as possible. In the case of polyps, no more than 5% of
crypts would have been derived from normal mucosa.
Normal mucosa was obtained from two separate samples in
patient No 1, 2, 3, and 5, and one sample in patient No 4, 6,
and 13–15. Meticulous care was taken to exclude any non-
normal tissue. Samples of normal mucosa were all obtained
from the proximal colon. Cell lysis and DNA extraction were
performed using a QIAamp DNA mini kit (QlAGEN,
Mississauga, Ontario, Canada) according to the manufac-
turer’s protocol. Extracted genomic DNA was diluted in 40 ml
of distilled water and denatured by adding 6 ml of 2N NaOH
and incubation at 75˚C for 20 minutes. Freshly prepared
4.8 M sodium bisulphite (500 ml) and 28 ml of 10 mM
hydroquinone were added to the denatured genomic DNA
and the reaction was carried out overnight in the dark at
55˚C. DNA was then purified using a Wizard DNA clean-up
(Promega, Madison, Wisconsin, USA) and then ethanol
precipitated after five minutes of alkali treatment with
8.8 ml of 2 N NaOH at room temperature.
Methylation specific polymerase chain reaction (PCR)
Methylation of promoter region of 11 genes and three MINT
loci (see below) was examined by methylation specific
polymerase chain reaction (MSP) using AmpliTaq Gold kit
(Roche, Branchburg, New Jersey, USA). MINT1, MINT2,
MINT31, p16 (CDKN2A), MLH1, and O-6-methylguanine DNA
methyltransferase (MGMT) have been employed in several
previous studies defining CpG island methylator phenotype
(CIMP) in colorectal polyps and cancers.22–26
RASSF2, and NORE1 (RASSF5) are members of the RAS
association domain family (RASSF) of RAS effectors, known
to link RAS activation to cell cycle arrest and apoptosis.27–31
Mammalian sterile20-like 1 (MST1) is a ubiquitously
expressed proapoptotic protein kinase that mediates the
apoptotic effect of RAS by forming a complex with RASSF1/
NORE1 and induction of the caspase cascade.31–33The cell
surface receptor FAS and death associated protein kinase
subjects with colorectal cancer (CRC)?
Clinical features and polyp diagnoses in
(y)Sex HP SSAMP TSAAD CRC
*A synchronous rectal cancer was not subjected to molecular analysis.
HP, hyperplastic polyp; SSA, sessile serrated adenoma; MP, mixed polyp
(all were part SSA, part AD); TSA, traditional serrated adenoma; AD,
adenoma; CRC, colorectal cancer.
SSAs contiguous with CRCs from patient No 2, 5, and 6 are not included
in the polyp count but the contiguous SSA from patient No 4 was
analysed for DNA methylation.
?Respective age and sex for patient No 6–15 with diagnoses presented
under methods are: 75 M; 63 M; 65 F; 72 M; 74 M; 64 F; 48 F; 73 F; 74
F; 72 F.
adenoma from patient No 2. The crypts are dilated and mucin filled,
show exaggerated serration, and increased branching, and extend
horizontally along the muscularis mucosae. Haematoxylin and eosin
Typical histological appearances of a sessile serrated
1468 Minoo, Baker, Goswami, et al
(DAPK) are known mediators of the extrinsic (death receptor
initiated) apoptotic pathway.34 35Checkpoint gene with fork-
head and RING finger domains (CHFR) functions as a
mitotic checkpoint to delay entry into metaphase.36Raf
kinase inhibitor protein (RKIP) negatively regulates mitogen
activated protein kinase (MAPK) by direct binding and
inhibition of Raf kinase.37 38The primer sequences of these
Primer sequences and methylation specific polymerase chain reaction conditions
nameSense primer (59-39) Antisense primer (59-39)
temp (˚ C)Reference
dysplastic clone (upper right) within a sessile serrated adenoma (giving a
mixed polyp) from patient No 1. ABC technique.
Loss of nuclear expression of MLH1 in adenomatous or
staining) in a single crypt non-dysplastic crypt in a sessile serrated
adenoma from patient No 2. The crypt is not longitudinally sectioned but
loss is clearly occurring near the crypt base. This was the only evidence
of loss of expression of MLH1 in a total of 54 polyps that were tested in this
patient (the colorectal cancer also showed loss of MLH1). ABC technique.
Loss of nuclear expression of MLH1 (possible mild residual
DNA methylation in hyperplastic polyposis1469
genes are listed in table 2. Conditions for amplification were
10 minutes at 95˚C followed by 39 cycles of denaturing at
95˚C for 30 seconds, annealing at certain temperatures
(table 2) for 30 seconds, and 30 seconds of extension at
72˚C. PCR products were subjected to electrophoresis on 8%
acrylamide gels and visualised by SYBR gold nucleic acid gel
stain (Molecular Probes, Eugene, USA). CpGenome Universal
Methylated DNA (Chemicon, Temecula, California, USA) was
used as a positive control for methylation. Gene promoters
were considered methylated if the intensity of methylated
bands was more than 10% of their respective unmethylated
BRAF mutation analysis at codon 600 (V600E; formerly
V599E)39was performed by a real time PCR based allelic
discrimination method, as previously described.40Briefly,
primers were designed to selectively amplify the wild-type
(T1796) and mutant (A1796) BRAF alleles. PCR amplification
and melting curve analysis was performed on a Rotor-gene
3000 (Corbett Research, NSW, Australia). Cycling conditions
were as follows: 50˚C for two minutes, 95˚C for two minutes,
and 40 cycles of 95˚C for 15 seconds and 60˚C for 60 seconds.
After amplification, samples were subjected to a temperature
ramp from 60˚C to 99˚C, rising 1˚C each step. For wild-type
RKIP CHFRMST1 NORE1 RASSF2RASSF1P16 MINT31MINT2 MINT1
Methylation patterns in gene promoters in polyps and cancers. SSA, sessile serrated adenoma; TSA, traditional serrated adenoma; CRC,
1470Minoo, Baker, Goswami, et al
samples, single peaks were observed at 80˚C and samples
containing mutant alleles produced additional peaks at 85˚C.
The x2test was used to compare the frequency of locus
methylation in polyps of patients with or without hyperplas-
tic polyposis. Loci that did not show amplification with
primers for both methylated and unmethylated sequences
were excluded from analysis. A p value of less than 0.05 was
considered statistically significant.
Immunoexpression of MLH1
There was complete loss of expression of MLH1 in the six
proximal CRCs from patient No 1, 2, 3, and 5, and these CRCs
also showed high level DNA MSI (data not shown). Of 18
serrated polyps from patient No 1, seven showed loss of
expression of MLH1. This included the unequivocally
dysplastic subclones in four of four mixed polyps (fig 2)
and clusters of non-dysplastic crypts in three SSAs. Among
54 serrated polyps from patient No 2, only one showed
convincing loss of expression of MLH1 and this was limited
to a single non-dysplastic crypt (fig 3). Of four serrated
polyps from patient No 3, a mixed polyp showed loss of
MLH1 within the adenomatous subclone and multifocal loss
of MLH1 was present in an SSA. A large SSA contiguous with
the CRC from patient No 4 showed normal expression of
CpG island methylation
The methylation status of markers was examined in 23 SSAs,
two mixed polyps, one TSA, two conventional adenomas, and
seven CRCs from 12 patients (fig 4). Markers MGMT, MLH1,
FAS, and DAPK were less specific than the others in showing
relatively high frequencies of methylation in normal mucosa
of three subjects (No 4, 5 and 6) without HPP (data not
shown). The range of locus methylation in the polyps of
patient No 1–3 with HPP was 70–100% (7–10 of 10 markers)
versus 30–100% (3–10 of 10 markers) in the polyps of patient
No 4–12 with sporadic SSAs. The 93% methylated loci (112 of
120) demonstrated in DNA samples derived from 12 SSAs
from patient No 1–3 with HPP was significantly more
frequent than 73% of methylated loci (79 of 108) in 11
sporadic SSAs (patient No 4–12) (p,0.0001) (fig 4).
Although there was more methylation in the polyps of
patients with HPP, there was considerable overlap between
the patient groups (fig 4). A more striking difference was
found with respect to the frequency of methylation in normal
mucosa in patients with and without HPP. While patient No
1–3 with HPP showed methylation rates of 85%, 75%, and
90% in normal colonic mucosa (17, 15, and 18 of 20 loci,
respectively), we detected methylation in only 13% of loci
when testing normal mucosal samples derived from patient
No 4–6 and 13–15 with sporadic serrated polyps of the
proximal colon (fig 5). Examples of MSP on DNA derived
from normal mucosa as well as polyps in patients with and
without HPP are shown in fig 6.
Mutation of BRAF was found in 100% of SSAs (10 of 10) in
patients with HPP and in 87% (7 of 8) of sporadic SSAs
(fig 4). Mutation of KRAS was found in a single mixed polyp
from patient No 1 (data not shown). SSAs from patients with
and without HPP were therefore well matched in terms of
anatomical location and BRAF mutation.
The role of DNA methylation or the CIMP25 44in the evolution
of CRC has been a matter of some controversy. It has been
argued by some that CRCs with CIMP do not have distinct
clinical, pathological, and molecular features and that DNA
methylation is largely an epiphenomenon.45However, this
interpretation ignores three important groups of observa-
tions. Firstly, CIMP positive CRCs do in fact share a number
of features regardless of the presence or absence of DNA MSI-
H status. These include proximal location, female predilec-
tion, mucinous and poor differentiation, and a high
frequency of the serrated pathway specific BRAF muta-
tion.46–49Secondly, CIMP (as well as mutation of BRAF) is
fully established in the putative precursor lesions of CIMP
positive CRCs, namely large and proximal HPs or SSAs (see
above). Thirdly, the observations that: (a) there is extensive
Methylation patterns in gene promoters in normal colonic mucosa.
DNA methylation in hyperplastic polyposis1471
and concordant DNA methylation across all polyps in HPP,22
(b) the condition HPP segregates within families,3 50and (c)
CRCs with BRAF mutation and/or DNA methylation occur
within families,40 49 51
serve as evidence for a genetic
mechanism underlying CIMP although this remains con-
troversial.52 53We have therefore adopted the premise that
CIMP is an early pathogenic change that serves to mould
colorectal tumorigenesis whether this culminates in MSI-H
or non-MSI-H CRCs with CIMP.
The range of DNA methylation markers used in studies of
serrated polyps of the colorectum has been limited to date.
Assuming that a subset of serrated polyps is characterised by
a state known as the CpG island methylator phenotype
(CIMP) then it is desirable to identify an optimum panel that
is both sensitive and specific for CIMP. This study employed
markers that have been used extensively in studies of
colorectal cancers and polyps (MINT1, MINT2, MINT31,
p16, MLH1, and MGMT) as well as less widely used markers.
Inhibition of apoptosis has been regarded as a pathogenic
mechanism in serrated polyps20 21but is regulated by multiple
signalling pathways. We therefore reasoned that more
advanced serrated polyps may be characterised by a more
comprehensive inactivation of apoptosis signalling pathways
through methylation of proapoptotic genes. Oncogenic
activation of BRAF appears to be both proapoptotic and
antiapoptotic.54RASSF1, RASSF2, and NORE1 (RASSF5) are
proapoptotic genes downstream of KRAS that are known to be
methylated as a component of CIMP.43 55–61Silencing of these
genes by methylation could therefore direct MAP kinase
activation towards antiapoptotic signalling. MST1 proapop-
totic kinase is activated by RASSF1/NORE1 complex31 32
although it has not been shown to be methylated previously.
Augmentation of MAPK signalling may occur through
inactivation of RKIP.37 38Two further proapoptotic genes, cell
surface receptor FAS and DAPK, and the mitotic checkpoint
is possible that BRAF mutation initiates a tissue alteration (that
is, a serrated polyp) when antiapoptotic signalling becomes
dominant through silencing of proapoptotic genes. This could
explain the close association between BRAF mutation and
CIMP. However, the present study has not shown that
methylation of proapoptotic genes has functional consequences
with respect to gene silencing.
In order to highlight pictorially any differences among the
serrated polyps with respect to DNA methylation in the
preceding panel of 14 markers, we elected to exclude all
markers that were methylated in normal mucosa in patients
with sporadic serrated polyps. On this basis, we excluded
MLH1, MGMT, FAS, and DAPK, leaving a panel of 10 markers.
We and others have shown previously that some of the
preceding genes, notably MGMT and MLH1, may show non-
specific methylation in normal colorectal samples.17 66There
was considerable overlap with respect to methylation in SSAs
from subjects with and without HPP (fig 4). Nevertheless,
there were more instances of marker methylation in the 12
SSAs in HPP patient No 1–3 compared with 11 sporadic SSAs
from patient No 4–12 (p,0.0001). This difference is not
surprising given that the polyps in subjects with HPP arose
within an environment of normal colonic mucosa that
showed very extensive DNA methylation. However, there
was no significant difference between the sets of polyps from
the two groups of patients when the markers RASSF1, p16,
and RKIP were excluded (p=0.1) (fig 4). It is possible that
more time is required for some markers to become
methylated than others. Very extensive DNA methylation
was seen in all three relatively large sporadic polyps from
elderly patient No 5 and in the largest (15 mm) sporadic SSA
from patient No 11 (fig 4).
The normal mucosa in patients with HPP showed very
extensive DNA methylation (fig 5). Two previous reports have
noted the finding of high level CIMP in the normal mucosa of
subjects with hyperplastic polyposis.17 22The present study
extends these observations by showing concordant methyla-
tion across a large marker panel. CIMP markers generally
show little or no methylation in normal colorectal mucosa and
this was confirmed in the DNA samples derived from the
normal colorectal mucosa of six subjects with small numbers
of proximally located serrated polyps. The finding of extensive
DNA methylation in normal colorectal mucosa may serve as a
useful diagnostic biomarker for high cancer risk forms of HPP.
However, there is a need to confirm these findings in
additional subjects with HPP and this should include the
detailed mapping of the extent of methylation in different
regions of the colon. Given the predilection for CIMP positive
CRC to occur in the proximal colon, it is possible that DNA
hypermethylation would be more extensive proximally. The
present findings should be distinguished from the increased
a field change in normal colorectal mucosa from subjects in
whom CRCs show MGMT methylation.67
or without hyperplastic polyposis. Methylation of MINT1, 2, 31 markers, and p16, RASSF1, RASSF2, NORE1, MST1, CHFR, and RKIP was examined
by methylation specific polymerase chain reaction using primers for unmethylated (U) or methylated (M) loci. (1) Normal colonic mucosa from patient
No 3 (sample 2) with hyperplastic polyposis; (2) normal colon mucosa from patient No 5 (sample 2) with sporadic sessile serrated adenoma (SSA); (3)
SSA from patient No 1 (sample 1) with hyperplastic polyposis; (4) SSA from patient No 9 with sporadic SSA; and (5) control methylated DNA (see
materials and methods).
Representative examples of methylation specific polymerase chain reaction at different loci in normal mucosa and polyps from patients with
1472 Minoo, Baker, Goswami, et al
An explanation for the finding of extensive DNA methyla-
tion in normal mucosa in HPP can only be speculative but
nevertheless warrants consideration. As noted above, CRCs
with CIMP have been linked with family history in two
cancer family clinic based studies40 51and one large popula-
tion based study.49The findings in these three studies support
the possibility of genetic predisposition to aberrant DNA
methylation. The population based study was large (911
subjects) and bias free. Patients were stratified on the basis of
BRAF mutation but the authors demonstrated a very strong
correlation between BRAF mutation and CIMP, regardless of
whether CRCs were DNA microsatellite stable or MSI-H.49In
subjects with microsatellite stable CRCs, the odds ratio for a
positive family history when comparing BRAF mutation
positive and BRAF wild-type groups was 4.23 (95% con-
fidence interval (CI) 1.65–10.84). In the case of MSI-H CRCs,
the odds ratio for a positive family history was only 0.64 (95%
CI 0.18–2.19) for subjects with BRAF mutation positive versus
BRAF wild-type CRCs. However, one third of subjects with
BRAF wild-type/MSI-H CRCs were aged less than 55 years
and a proportion would be expected to have Lynch
In a separate study using the same population, the authors
demonstrated a weaker association between CIMP and
family history.53However, they employed a very broad
definition of CIMP (two or more of five markers methylated)
to the extent that less than one third of CIMP positive cancers
had BRAF mutation. Using a similarly broad definition of
CIMP, a hospital based study found no link between CIMP
positive CRC and family history.52However, this study is
difficult to evaluate because it excluded families with
‘‘hereditary non-polyposis colorectal cancer (HNPCC)’’ with-
out stating how HNPCC was defined. Families may meet the
Amsterdam criteria without having HNPCC/Lynch syndrome.
While there is no consensus in the literature, there is good
evidence for the heritability of CIMP and the contrary
evidence can be explained by limitations in study design.
HPP is associated with CIMP positive polyps and CRCs but
instances of HPP have been documented within the setting
of CRC families in which cancers show BRAF mutation and/or
DNA methylation40and in siblings.50It is conceivable that the
usual CIMP positive CRCs develop through a ‘‘two hit’’
mechanism whereby one mutant copy of a gene that is
implicated in the regulation of DNA methylation is inherited,
while the wild-type allele is inactivated at the somatic level.
The widespread methylation of normal mucosa found in HPP
might then occur in subjects who have inherited two copies
of such a mutated gene. On the basis of the above, HPP may
serve as an important genetic model for CIMP. These
suggestions remain highly speculative and do not preclude
a role for environmental factors in the aetiology of both DNA
methylation and HPP. Age and inflammatory bowel disease
are known to influence DNA methylation in colorectal
The phenotypes in patient No 1 and 3 are very similar. Both
patients had only moderate numbers of serrated polyps but a
high proportion of these showed dysplasia (mixed polyps or
traditional serrated adenomas) and/or loss of expression of
MLH1. In contrast, patient No 2 had very large numbers of
serrated polyps, including many typical hyperplastic polyps.
Only one polyp (a mixed polyp) showed mild dysplasia. In
addition, loss of MLH1 expression was confined to a single
non-dysplastic crypt in a sessile serrated adenoma. While all
three subjects showed extensive DNA methylation in normal
colorectal mucosa, it is possible that methylation leading to
loss of expression of MLH1 is influenced by an additional
partly independent mechanism. This would explain why
some patients with hyperplastic polyposis, such as patient No
3 and others reported in the literature,8 69develop multiple
synchronous CRCs that may be MSI-H.
In summary, we have documented the finding of extensive
DNA methylation in samples of normal mucosa from the
proximal colon of subjects with HPP and suggest that there
may be a genetic basis for this observation. Some of the
observed heterogeneity within HPP may be explained by
differing propensities for MLH1 inactivation that may be due
to mechanisms that are partly independent of a general
predisposition to DNA methylation. Finally, observations
relating to methylation of multiple proapoptotic genes
provide an explanation for the synergy between DNA
methylation and mutation of BRAF although this will need
to be confirmed by functional studies.
The study was funded by the Canadian Institutes of Health Research,
Grant No MOP-67206. The specimen for patient No 4 was kindly
provided by Professor A Paterson, School of Pathology, University of
the Witwatersrand, Johannesburg, South Africa. We thank H
Minassian for technical support.
P Minoo, K Baker, R Goswami, J R Jass, Department of Pathology,
McGill University, Montreal, Canada
G Chong, Department of Human Genetics, SMBD Jewish General
Hospital, Montreal, Canada
W D Foulkes, Departments of Human Genetics and Oncology, McGill
University, Montreal, Canada, and Department of Medical Genetics,
SMBD Jewish General Hospital, Montreal, Canada
A R Ruszkiewicz, Institute of Medical and Veterinary Science, Adelaide,
M Barker, D Buchanan, J Young, Queensland Institute of Medical
Research, Brisbane, Australia
Conflict of interest: None declared.
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