G C A T
T A C G
G C A T
genes
Article
Using Comorbidity Pattern Analysis to Detect Reliable
Methylated Genes in Colorectal Cancer Verified by
Stool DNA Test
Yi-Chiao Cheng 1 , Po-Hsien Wu 1 , Yen-Ju Chen 2 , Cing-Han Yang 3 , Jhen-Li Huang 3 , Yu-Ching Chou 4 ,
Pi-Kai Chang 1 , Chia-Cheng Wen 1 , Shu-Wen Jao 1 , Hsin-Hui Huang 5 , Yi-Hsuan Tsai 2 and Tun-Wen Pai 2,3, *






Citation: Cheng, Y.-C.; Wu, P.-H.;
Chen, Y.-J.; Yang, C.-H.; Huang, J.-L.;
Chou, Y.-C.; Chang, P.-K.; Wen, C.-C.;
Jao, S.-W.; Huang, H.-H.; et al. Using
Comorbidity Pattern Analysis to
Detect Reliable Methylated Genes in
Colorectal Cancer Verified by Stool
DNA Test. Genes 2021, 12, 1539.
https://doi.org/10.3390/genes
12101539
Academic Editor: Yutaka Suzuki
Received: 6 September 2021
Accepted: 27 September 2021
Published: 28 September 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Genes 2021, 12, 1539. https://doi.org/10.3390/genes12101539
1 Division of Colon and Rectal Surgery, Department of Surgery, Tri-Service General Hospital,
National Defense Medical Center, Taipei 11490, Taiwan; ndmcjoe@mail.ndmctsgh.edu.tw (Y.-C.C.);
wupohsien@office365.ndmctsgh.edu.tw (P.-H.W.); pencil8850@hotmail.com (P.-K.C.);
wjason@mail2000.com.tw (C.-C.W.); jaosw@ndmctsgh.edu.tw (S.-W.J.)
2 Department of Computer Science and Information Engineering, National Taipei University of Technology,
Taipei 10608, Taiwan; campyenju@gmail.com (Y.-J.C.); t109598090@ntut.edu.tw (Y.-H.T.)
3 Department of Computer Science and Engineering, National Taiwan Ocean University, Keelung 20224,
Taiwan; gblab@email.ntou.edu.tw (C.-H.Y.); 20889002@mail.ntou.edu.tw (J.-L.H.)
4 School of Public Health, National Defense Medical Center, Taipei 11490, Taiwan;
trishow@mail.ndmctsgh.edu.tw
5 Department of Biochemistry and Molecular Cell Biology, School of Medicine, Taipei Medical University,
Taipei 11042, Taiwan; toe3273917@outlook.com
* Correspondence: twp@ntut.edu.tw; Tel.: +886-2-2771-2171 (ext. 4222)
Abstract: Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide in 2020.
Colonoscopy and the fecal immunochemical test (FIT) are commonly used as CRC screening tests, but
both types of tests possess different limitations. Recently, liquid biopsy-based DNA methylation test
has become a powerful tool for cancer screening, and the detection of abnormal DNA methylation
in stool specimens is considered as an effective approach for CRC screening. The aim of this study
was to develop a novel approach in biomarker selection based on integrating primary biomarkers
from genome-wide methylation profiles and secondary biomarkers from CRC comorbidity analytics.
A total of 125 differential methylated probes (DMPs) were identified as primary biomarkers from
352 genome-wide methylation profiles. Among them, 51 biomarkers, including 48 hypermethylated
DMPs and 3 hypomethylated DMPs, were considered as suitable DMP candidates for CRC screening
tests. After comparing with commercial kits, three genes (ADHFE1, SDC2, and PPP2R5C) were
selected as candidate epigenetic biomarkers for CRC screening tests. Methylation levels of these three
biomarkers were significantly higher for patients with CRC than normal subjects. The sensitivity and
specificity of integrating methylated ADHFE1, SDC2, and PPP2R5C for CRC detection achieved 84.6%
and 92.3%, respectively. Through an integrated approach using genome-wide DNA methylation
profiles and electronic medical records, we could design a biomarker panel that allows for early and
accurate noninvasive detection of CRC using stool samples.
Keywords: DNA methylation; electronic medical records; biomarkers; colorectal cancer; stool
DNA test
1. Introduction
Colorectal cancer (CRC) was the third most commonly diagnosed cancer and the
second leading cause of cancer-related death worldwide in 2020 [1]. According to the
Taiwan Cancer Registry Annual Report 2018, more than 16,000 new cases of CRC and
approximately 6000 CRC-related deaths occurred [2]. The 5-year relative survival rate
is greater than 90% for stage I CRC but only 10% for stage IV CRC [3]. Therefore, early
detection of CRC is essential for reducing death from CRC.
https://www.mdpi.com/journal/genes
Genes 2021, 12, 1539 2 of 14

Several screening tools are available for CRC. Although colonoscopy provides the
highest sensitivity and accuracy for the detection of colorectal lesions, some patients
refuse this examination due to it being high cost, its unpleasant bowel preparation, being
time-consuming and an uncomfortable procedure, and the risk of bowel perforation and
bleeding [4]. As a substitute, the fecal immunochemical test (FIT) is the most commonly
used screening method worldwide [5] because it is noninvasive and more economical, can
be performed at home, and does not require bowel preparation. Although the FIT has these
advantages, its sensitivity is relatively low. In studies using colonoscopy as a reference
standard, the sensitivity of FITs for detecting invasive cancer and precancerous lesions only
ranged from 65.8% to 75.0% and from 27.0% to 29.0%, respectively [6–10]. Moreover, the
FIT shows a worse performance for the detection of right-sided advanced adenomas than
left-sided lesions [11].
To overcome these limitations, the technique of detecting abnormal DNA methylation
levels in exfoliated tumor cells from stool specimens has been developed [12]. Unlike the
FIT, stool DNA testing not only detects left- and right-sided colorectal neoplasms equally
well but also has better adherence [13]. However, despite the approval of multitarget stool
DNA testing as an alternative approach for people at average risk for CRC by the U.S. Food
and Drug Administration [9], the selection and validation of appropriate DNA methylation
biomarkers are time-consuming and require large financial investments. Furthermore, race
differences might affect the sensitivity and specificity of the selected biomarkers [14,15].
The Cancer Genome Atlas (TCGA) database provides the most comprehensive and
integrated DNA methylation profiles at the genomic levels [16]. A large number of the
genome-wide methylation profiles were produced and explored through Illumina Hu-
man Methylation 450K Bead Chip Array experiments. Performing quality assessment
and executing standard pipelines could facilitate bioinformaticians to identify effective
biomarkers through in silico analysis. However, due to a paradigm shift in early diagnosis
for CRC screening, a transition from tissue biopsy to noninvasive stool biopsy is expected
to achieve the goals. It was expected that all initially identified biomarkers from differential
methylation analysis on TCGA tissue specimens could be considered as good candidates
for designing a test panel on tissue specimens. However, in this study, only Asian stool
biopsies were used as specimens for candidate biomarker verification. Fewer candidate
biomarkers for designing verification experiments becomes a practical challenge to be
overcome. Under such a circumstance, comorbidity of Asian CRC subjects was proposed to
incorporate the identified biomarkers by single use of TCGA methylated profiles, and the
identified associated disease-genes could be considered as good constraints to effectively
narrow down the number of suitable biomarker candidates.
Taiwan has an internationally well-known National Health Insurance Research Database
(NHIRD) that contains all the medical records of Taiwanese citizens. As a population-level
data source, Taiwan’s NHIRD can serve as a foundation for big data analysis for real-world
evidence, especially for Asian populations [17]. By utilizing this database, we previously
constructed machine learning approaches and prediction models for amyotrophic lateral
sclerosis and demonstrated that a prediction model based on electronic medical records
(EMRs) could provide a novel approach to estimate the risk for a specific disease [18]. To
enhance the effective selection and utilization of specific DNA methylation biomarkers
from stool DNA screening approaches for the diagnosis of CRC, a traditional method
for DNA methylation biomarker selection that incorporates the associated comorbidity
patterns and disease–gene associations are proposed. This study aimed to analyze the
comorbidity patterns of patients with CRC using a comprehensive EMR database annotated
in Taiwan’s NHIRD and determine disease-specific associated genes from the identified
significant comorbidities. By integrating both primarily identified DNA methylation
biomarkers from a genome-wide differential DNA methylation analysis and the secondary
significant disease–gene associations from historic EMR analysis, we believe that the
cross-validated and selected DNA methylation biomarkers could simultaneously increase
biomarker sensitivity and specificity and address the issues regarding race difference.
Genes 2021, 12, 1539 3 of 14

2. Materials and Methods

2.1. Differential DNA Methylation Analysis for Primary Biomarkers
In this study, DNA methylation profiling datasets (Illumina Human Methylation 450K
Bead Chip Array) were downloaded from TCGA database, and the data of a total of 352
subjects, including 314 specimens with CRC and 38 normal specimens, were used for
primary biomarker analysis (Figure 1). First, both CRC and normal control groups were
integrated and analyzed using the Chip Analysis Methylation Pipeline (ChAMP) [19], a
standard pipeline package including quality control and dataset normalization (BMIQ) for
detecting differentially methylated probes (DMPs). Using parameter settings on β value
differences (Abs(∆Beta) ≥ 0.5) and multiple testing correction by Benjamini and Hochberg
method for decreasing false discovery rate, a set of DMPs could be identified as general
primary biomarker candidates. To identify the possible specific biomarker candidates
for different racial groups, different racial populations were individually analyzed and
cross-race compared. Data of the patients with CRC from several different racial groups
were collected from TCGA. We selected three major racial groups (white, Asian, and black)
for cross comparison, comprising 229 white, 12 Asian, and 62 black. DMP analysis for
each racial group was performed individually using the standard pipeline provided by
the ChAMP package with identical parameter settings. Intersection analysis was applied
to discover a set of universal biomarker candidates that could serve as a broad-spectrum
detection toolkit for CRC. However, an exclusive analysis could be performed to identify
unique DMP biomarkers for specific racial groups.

2.2. Comorbidity Analysis for Secondary Biomarkers

The EMRs used in this study were anonymous and partially selected from Tai-
wan’s NHIRD. The partial dataset comprised the data of one million insured people,
and their comprehensive longitudinal medical records were collected between 2000 and
2013 (IRB:105-0504C). The disease codes followed the International Classification of Dis-
eases, Ninth Revision, Clinical Modification (ICD-9-CM). They contained 17 major chapters
and were further classified into 143 disease groups. For example, the disease group of
560–569 is labelled under the classification “Other diseases of intestines and peritoneum”,
where a three-digit code represents a single disease type. To explore the comorbidities
associated with CRC, the data of positive (with CRC) and negative (without CRC) subjects
were retrieved from Taiwan’s NHIRD. From the one million population NHIRD medical
database, a set of 6293 confirmed positive subjects with age 40–80 years were identified,
and a set of near 5-fold negative subjects (30,653 subjects) without CRC were randomly
matched with them using case-control matching with regard to gender and age attributes.
Data of all selected subjects and their EMRs for 3 years before their first diagnosis of CRC
was obtained from Taiwan’s NHIRD. All retrieved medical records were subjected to data
cleaning and integration to yield simplified personal historic medical records for each
subject. Statistical analysis was then performed on each disease code to verify significance
with regard to CRC under the threshold settings of odds ratio > 2, p-value < 0.05, and
supporting rate (minimum number of diagnosed subjects) > 10%. Once significant co-
morbidities were identified, the disease-associated genes could be directly retrieved from
the DisGeNET [20] annotations. These identified disease-associated genes were consid-
ered secondary biomarkers for the following screening processes. Both identified DNA
methylation positions (primary biomarkers) and comorbidity-associated genes (secondary
biomarkers) were combined to yield a set of biomarker candidates for clinical verification.
Genes 2021, 12, 1539 4 of 14

Primary biomarkers Secondary biomarkers

TCGA datasets
NHIRD EMRs
CRC(314) White(229) Asian(12) Black(62)
Normal(38) CRC(6,293) Normal(30,653)
Age (40~80) Male(3,269) Female(3,024)

Difference analysis
Hypo-methylation TCGACRC(314)Normal(38) Hyper-methylation
(ChAMP)
Comorbidity analysis
Odds ratio > 2 p-values < 0.05
Supporting rate > 10%
Cross racial/ethnic group biomarker analysis for
primary biomarkers (Abs(ΔBeta) ≥ 0.5)

Disease-gene association
Incorporating primary and secondary biomarkers analysis for secondary biomarkers

Selected biomarkers for verification

(ADHFE1/PPP2R5C/SDC2)

Figure 1. Flow chart showing the pipeline for identification of primary biomarkers from DNA methylation analysis using TCGA and secondary biomarkers from the Taiwan’s NHIRD.
Genes 2021, 12, 1539 5 of 14

2.3. Specimen Collection

In total, 30 stool specimens were collected from 13 healthy participants, 4 patients
with adenoma, and 13 patients with CRC in the outpatient and inpatient departments of
the Tri-Service General Hospital, respectively. The colorectal status of all participants (age
range, 40–80 years) enrolled in this study was confirmed by using colonoscopy. The de-
mographic characteristics of healthy participants and patients are listed in Supplementary
Table S1. This study was approved by the Institutional Review Board of the Tri-Service
General Hospital (Protocol No. A202105054), and the study protocol was registered on
ClinicalTrials.gov (ID No. NCT04823793). Approximately 5 g of fresh stool specimen was
collected from each participant and deposited into a storage tube prefilled with 25 mL
preservation buffer (BGI Genomics Co., Shenzhen, China). After collection, the samples
were stored at 4 ◦ C up to 7 days prior to DNA extraction.

2.4. Stool DNA Extraction and Bisulfite Conversion

Specimen DNA was extracted using the Stool DNA Isolation kit (BGI Genomics
Co., Shenzhen, China). Briefly, 200 µL stool specimen was applied to DNA extraction.
Stool lysis buffer (mainly Triton-X 100) was added, followed by vortexing for 10 min and
centrifugation at 12,000 rpm for 3 min. The supernatant was incubated with Protease K at
70 ◦ C for 20 min. After incubation, stool DNA was precipitated using ethanol and separated
using magnetic beads. Washing was performed three times to remove redundant salts and
proteins. Half of the isolated DNA was modified with bisulfite using the DNA Bisulfite
Conversion kit (BGI Genomics Co., Shenzhen, China) according to the manufacturer’s
instructions.

2.5. Quantitative Methylation Specific PCR (qMSP)

Due to high costs of probe design for qMSP, probes for only a few potential biomarkers
could be designed and verified through clinical experiments. In this study, we selected three
biomarkers, alcohol Dehydrogenase Iron Containing 1 (ADHFE1), syndecan-2 (SDC2), and
protein phosphatase 2 regulatory subunit B, γ (PPP2R5C), from the integrated primary and
secondary biomarker candidate set, mainly based on comprehensive consideration, such
as literature survey, genetic locations of DMPs, primer design, functional analysis, and β
value differences (hypermethylated biomarkers). The methylation levels of ADHFE1, SDC2,
and PPP2R5C were quantitated using DNA Methylation Detection Kit (BGI Genomics
Co., Shenzhen, China) and GAPDH was used as an internal reference gene. Each PCR
reaction contained 9 µL bisulfite-converted DNA in a 20 µL reaction mixture, including
four primer and probe pairs. PCR procedures were performed using LightCycler 480 II
with the following thermal program: 95 ◦ C for 10 min, 10 cycles of 95 ◦ C for 15 s, 65 ◦ C for
30 s, and 72 ◦ C for 30 s, 35 cycles of 95 ◦ C for 15 s, 55 ◦ C for 30 s, and 72 ◦ C for 30 s. Notably,
the annealing temperature in the first 10 cycles was kept at 65 ◦ C and then reduced by
1 ◦ C for each cycle till 56 ◦ C. DNA methylation levels of the target genes were determined
using crossing point (dCp) values obtained by the following formula: Cp value of target
gene—Cp value of GAPDH.

2.6. Fecal Immunochemical Test (FIT)

The FIT was conducted using the OC-SENSOR io analyzer (Eiken Chemical Co., Tokyo,
Japan), with a 100 ng/mL cutoff hemoglobin level.

2.7. Statistical Analysis

Processing procedures of DNA methylation profiles from TCGA database, a multiple
testing correction by Benjamini and Hochberg method was performed to decrease false
discovery rate. The identified significant DMPs with their corresponding adjusted p-values
are shown in the Supplementary Table S2. Statistical analysis and dot plots for methylation
level visualization in Results Section were performed using GraphPad Prism 8 software
(GraphPad Software Inc., San Diego, CA, USA). The area under the receiver operating
Genes 2021, 12, 1539 6 of 14

characteristic curve (AUC-ROC) method was employed to define the cutoff values used to
determine the sensitivity and specificity of ADHFE1, SDC2, and PPP2R5C. The unpaired
parametric t-test was used to determine differences in methylation level between the two
participant groups. The significant results are presented as two-tailed p-values (p < 0.05).

3. Results
3.1. Primary Biomarkers from Differentially Methylated Positions (DMPs)
DNA methylation profiles of 314 patients pathologically diagnosed with CRC were
compared with those of 38 normal subjects for identifying primary DMP biomarkers. Using
parameter settings on β value difference (Abs(∆Beta) ≥ 0.5), 125 DMPs with significant
adjusted p-values could be identified. In addition, three major racial groups, white, Asian,
and black, were further individually analyzed and cross compared. As a result, 79 uni-
versal DMPs could be identified in these three racial groups, and three exclusive DMPs in
white (n = 229), five in black (n = 62), and none in Asian (n = 12) groups were validated.
The identified common and exclusive DMPs and their corresponding methylation probe
IDs, gene names, loci, β value differences, p-values, and adjusted p-values are shown in
Supplementary Table S2.

3.2. Secondary Biomarkers from Comorbidity Analysis

From the one million NHIRD dataset, the association between each comorbidity code
and CRC was statistically evaluated. When the statistical parameters were defined as odds
ratio > 2, p-value < 0.05, and supporting rate > 10%, there were only four disease groups
associated with CRC that could be identified. “Other Diseases of Intestines and Peritoneum
(560–569)”, “Diseases of Veins and Lymphatics, And Other Diseases of Circulatory System
(451–459)”, “Other Diseases of Digestive System (570–579)”, and “Diseases of Esophagus,
Stomach, And Duodenum (530–539)”. For each identified disease group, the corresponding
disease-associated genes could be retrieved from the DisGeNet database. Moreover, 1142,
430, 2693, and 1469 annotated genes could be found associated with the four identified
disease groups. In addition, the 10,437 annotated disease–gene associations are known for
CRC (ICD9: 153 and 154). All CRC associated genes were considered to be the secondary
biomarker candidates and served as a screening filter for advanced selection. The secondary
biomarkers identified from the comorbidities are listed in Supplementary Table S3. After
performing intersection analysis using the DMP primary biomarkers, a total of 51 candidate
genes, including 48 genes that were found to be hypermethylated in CRC tissues, were
identified. In addition, we compared the results of 125 primary DMPs obtained by single
use TCGA methylation profiles and the results of 51 DMPs by integrating comorbidity
constraints based on average sensitivity, specificity, and accuracy measurements. For the
initially selected 125 primary biomarkers, the average sensitivity, specificity, and accuracy
achieved 68.15%, 99.95%, and 71.58%, respectively, (by setting the β-difference cutoff value
larger than 0.5) from the collected TCGA specimens, while performance of the constrained
51 biomarkers could be slightly increased to 70.36%, 99.89% and 73.55%, respectively,
under the same cutoff settings. The detailed sensitivity, specificity, and accuracy for each
primary biomarker from TCGA dataset are also provided in Supplementary Table S3,
individually. Incorporating comorbidity analytics and associated disease-gene constraints
provided an effective approach to decrease the number of suitable biomarkers from 125 to
51 DMP candidates. Nevertheless, the development of a usable probe and primer for DNA
methylation assessment is time-consuming and laborious. To verify a multi-gene panel
that can cover people of all ethnicities for the purpose of CRC screening, we summarized
several commercial methylation kits on the market, shown in Table 1. In comparison with
these commercial kits, Colotect, as a stool-based DNA methylation test, with three selected
genes (ADHFE1, SDC2, and PPP2R5C) from the 48 candidate genes, was designed for
further clinical verification.
Genes 2021, 12, 1539 7 of 14

Table 1. Commercial methylation kits for colorectal cancer detection.

Sensitivity/Specificity
Manufacture Product Name Target Genes Specimen Type
for CRC 1
Exact Sciences Cologuard NDRG4, BMP3 Stool DNA 92%/90%
Epigenomics Epi proColon SEPT9 Plasma DNA 68%/89%
New Horizon Health
ColoClear NDRG4, BMP3 Stool DNA 96%/87%
Limited
ADHFE1, SDC2,
BGI Genomics Colotect Stool DNA 90%/89%
PPP2R5C
Ammunition Life
IColocomf SDC2, TFPI2 Stool DNA 93%/95%
Technology
Creative Biosciences Colosafe SDC2 Stool DNA 84%/98%
BioChain Institute mSEPT9 SEPT9 Plasma DNA 77%/86%
RealBio Technology COLOWELL SDC2, SFRP2 Stool DNA 84%/94%
1 Sensitivity and specificity values were provided by manufacturer instructions or published articles.

3.3. Verification of Stool DNA Methylation in Individual Specimen

In this study, we estimated the methylation levels of ADHFE1, SDC2, and PPP2R5C in
stool DNA from normal participants (n = 13), patients with adenoma (n = 4), and patients
with CRC (n = 13). The methylation levels (dCp) of these three genes are presented as
dot plots in Figure 2. Lower dCp values represent higher methylation. These three genes
were significantly hypermethylated in stool DNA (p < 0.001) collected from patients with
CRC (Figure 2A). However, only SDC2 revealed high methylation in adenoma patient’s
stool DNA (p < 0.001) (Figure 2A). ROC curves and the associated AUCs of the diagnostic
prediction model using dCp values in the CRC and normal cohorts were performed using
GraphPad Prism Version 8.0 (Figure 2B). The AUCs of ADHFE1, SDC2, and PPP2R5C were
0.8935, 0.8402 and 0.8817, respectively. The Youden index = sensitivity + (specificity-1)
represents the difference between the diagnostic performance of the test and the best
possible performance. The optimal cutoff based on each gene was set at the cutoff that gave
the highest Youden index. The sensitivity and specificity of ADHFE1 were 84.6% and 100%,
respectively, when the cutoff value of dCp was defined as 5.02 (Youden index = 84.62)
The sensitivity and specificity of SDC2 were 69.2% and 92.3%, respectively, when the
cutoff value of dCp was defined as 7.50 (Youden index = 61.54). The sensitivity and
specificity of PPP2R5C were 69.2% and 100%, respectively, when the cutoff value of dCp
was defined as 9.33 (Youden index = 69.23) (Table 2). The details of the cutoff value
settings and corresponding sensitivity and specificity for the three genes are shown in the
Supplementary Table S4, respectively. Furthermore, we have compared “OR” combination
and linear combination of the three gene dCp values using R package (version 3.3.2). The
results showed no difference between OR operation and a linear combination from the three
genes. The weighted coefficients obtained by using a logistic regression model of the three
genes were formulated as 4.5545 − 0.1397 × (dCP of ADHFE1) − 0.1200 × (dCp of SDC2)
− 0.1909 × (dCp of PPP2R5C). The details of the comparison are shown in Supplementary
Table S5. Notably, 75% (3/4) of adenoma specimens revealed hypermethylation of SDC2,
but only one specimen revealed hypermethylation of ADHFE1 (Table 3). These observations
indicate that the methylation status of ADHFE1 showed a good performance for the
detection of CRC, whereas that of SDC2 showed a good performance for the detection of
both adenoma and CRC. Furthermore, FITs yielded accurate results for late-stage CRC
detection but less accurate results for early-stage cancer and precancer detection. Moreover,
the combination of methylation markers and the FIT revealed the best sensitivity for both
adenoma and CRC screening at all stages (Table 4).
Genes 2021, 12, 1539 8 of 14

Figure 2. Stool DNA methylation levels of the three candidate genes. (A) The dot plots represent
crossing point (dCp) values for methylation status of ADHFE1, SDC2, and PPP2R5C. The average
methylation levels are displayed as horizontal bars in the middle of the scattered dots. (ns: not
significant; ***: p < 0.001). (B) Area under the receiver operating characteristic curve (AUC-ROC) for
the DNA methylation status of ADHFE1, SDC2, and PPP2R5C in CRC stool DNA. The cutoff values
of dCp of ADHFE1, SDC2, and PPP2R5C are 5.02, 7.50 and 9.33, respectively.

Table 2. The performance of ADHFE1, SDC2, and PPP2R5C for detection of CRC.

Gene Name
ADHFE1 SDC2 PPP2R5C ADHFE1 + SDC2 + PPP2R5C
Cutoff value 1 5.02 7.50 9.33 Any one positive
Sensitivity 84.6% 69.2% 69.2% 84.6%
(Positive number/case) 2 (11/13) (9/13) (9/13) (11/13)
Specificity 100% 92.3% 100% 92.3%
(Positive number/control) 3 (13/13) (12/13) (13/13) (12/13)
1 Cutoff values of methylation level are determined by AUC-ROC calculation. 2 The numbers in parentheses
represent the CRC cases identified by hypermethylated gene/total CRC cases. 3 The numbers in parentheses
represent that the control subject number was able to identify as normal/total control subjects.
Genes 2021, 12, 1539 9 of 14

Table 3. Quantitative methylation specific PCR results and FIT detection status for individual participants.

No. Status Size/Stage FIT mADHFE1 mSDC2 mPPP2R5C

Participant-001 Normal
Participant-002 Normal
Participant-003 Normal
Participant-004 Normal
Participant-005 Normal
Participant-006 Normal
Participant-007 Normal
Participant-008 Normal
Participant-009 Normal
Participant-010 Normal
Participant-011 Normal
Participant-012 Normal •
Participant-013 Normal
Participant-014 Adenoma 0.4 cm •
Participant-015 Adenoma 0.2 cm •
Participant-016 Adenoma 0.4 cm • •
Participant-017 Adenoma 0.5 cm
Participant-018 CRC IIIC • • • •
Participant-019 CRC IV • • • •
Participant-020 CRC IIIB • • • •
Participant-021 CRC IIIA • • •
Participant-022 CRC IIIB •
Participant-023 CRC IV •
Participant-024 CRC IIIB • • •
Participant-025 CRC IIA • • • •
Participant-026 CRC IV • • •
Participant-027 CRC I • • • •
Participant-028 CRC I • •
Participant-029 CRC IIIB • • • •
Participant-030 CRC IIA • • • •

Table 4. The combined performance of methylation markers and FIT for detection of CRC and
adenoma.

Methylation Markers
Methylation
(ADHFE1 + SDC2 + FIT
Markers + FIT
PPP2R5C)
Sensitivity 75.0% 0% 75.0%
(Positive number/adenoma cases) (3/4) (0/4) (3/4)
Sensitivity 84.6% 92.3% 100%
(Positive number/CRC cases) (11/13) (12/13) (13/13)

4. Discussion
DNA methylation is an important molecular mechanism associated with human
tumorigenesis. In particular, abnormal DNA methylation patterns are related to the
diagnosis and prognosis of many types of cancers. This study aimed to identify potential
DNA methylation-based biomarkers for CRC. We identified a total of 51 methylation-
driven genes through a comprehensive analysis of TCGA and EMRs annotated in Taiwan’s
NHIRD. We present a system to visually analyze the comorbidities associated with CRC
using multidimensional categorical EMR data, of 6293 patients with CRC and 30,653
normal subjects. Our primary aim was to identify primary biomarkers from TCGA and
secondary biomarkers from EMRs annotated in Taiwan’s NHIRD, which might share a
common mechanism for further biomarker exploration and selection. “Other Diseases
of Intestines and Peritoneum (560–569)”, “Diseases of Veins and Lymphatics, And Other
Genes 2021, 12, 1539 10 of 14

Diseases of Circulatory System (451–459)”, “Other Diseases of Digestive System (570–579)”,

and “Diseases of Esophagus, Stomach, And Duodenum (530–539)” were found to be
commonly associated with CRC (ICD9: 153 and 154). These identified comorbidity disease–
gene associations were considered as secondary biomarkers for the subsequent clinical
verification process.
The present study showed that a stool DNA methylation test could facilitate early
detection of CRC and adenoma. Compared to the detection rates of the FIT, the stool DNA
methylation test could detect 75.0% (3/4) of adenoma (≥0.2 cm) cases and 84.6% (11/13) of
CRC cases. These observations suggested that the DNA methylation test showed better
performance for predicting advanced adenoma than that of the FIT. This difference may be
attributed to the DNA methylation markers for the early detection of CRC.
Notably, most information obtained from TCGA was based on the Caucasian pop-
ulation (>75.0%). Robust evidence is available regarding the relationship between CRC
and SEPT9, a well-known hypermethylated gene in CRC, in the Caucasian population.
Nevertheless, the diagnostic sensitivity of blood-based mSEPT9 in CRC detection varied
among published reports [21]. Lee et al. [22] found a very low sensitivity of mSEPT9
(36.6%) among the Korean population. Therefore, we present an integrative approach that
combines TCGA with NHIRD-based information to identify biomarker panels for CRC.
The CRC incidence rate in many Asian countries has rapidly increased in recent
decades [23,24]. Because DNA methylation alternations differ based on patients’ race [25],
the race difference is considered an indispensable factor for DNA methylation biomarker
selection. To our knowledge, this is the first study to integrate the genome-wide differential
DNA methylation analysis and disease–gene associations retrieved from Taiwan’s NHIRD
for the identification of DNA methylation biomarkers for CRC in Asian populations. Three
genes, ADHFE1, SDC2, and PPP2R5C, were identified and found to be hypermethylated
in Taiwanese patients with CRC using qMSP analysis (Figures 1 and 2). A comparison
of our results showed that overlap in both ADHFE1 and SDC2 genes was reported to be
highly methylated in Asian populations with CRC, including Chinese and South Korean
populations [26–28]. Therefore, our proposed DNA methylation biomarker identification
approach is beneficial to improve the biomarker feasibility for CRC detection in Asian
populations.
Thus far, the FIT and colonoscopy are widely used to screen CRC. However, the
sensitivity of FITs for early CRC detection needs to be improved [29]. While colonoscopy
is regarded as a valuable tool for CRC detection, its use is limited due to the high cost,
invasiveness, and possible complications [30]. Stool DNA methylation level detection has
the potential to overcome the abovementioned limitations, and, thus, it has currently be-
come an alternative approach for CRC screening [30]. While the efficacy of a single marker
is often limited, a multi-marker signature can have greater diagnostic value. Our study
demonstrated that the assessment of methylation levels of ADHFE1, SDC2 and PPP2R5C in
stool has a better detection rate for CRC screening. The sensitivities of methylated ADHFE1,
SDC2 and PPP2R5C, and the combination of these three genes for CRC detection were
84.6%, 69.2%, 69.2% and 84.6%, respectively, and their specificities were 100%, 92.3%, 100%
and 92.3%, respectively (Table 2). Methylated SDC2 is known as a valuable biomarker
for CRC detection. The sensitivity and specificity by methylated SDC2 for CRC detection
in three commercial methylation kits, IColocomf (Ammunition Life Technology Co., Ltd.,
Wuhan, China), Colosafe (Creative Biosciences Co., Ltd., Guangdong, China), and COLOW-
ELL (RealBio Technology Co., Ltd., Shanghai, China) were 77% and 98%, 84% and 98%, and
71% and 94%, respectively [28,31,32]. Although methylated SDC2 had a lower sensitivity
for CRC screening in the present study than that reported previously, the sensitivity and
specificity of the three gene combination for CRC detection were comparable to those
results previously (Table 2). These observations support that the assessment of methylation
levels of multiple genes might improve the detection rate of CRC [33]. Compared with
SDC2 and PPP2R5C, ADHFE1 showed a better ranking as well as the highest sensitivity for
CRC detection, indicating that the ranking computed via our biomarker selection approach
Genes 2021, 12, 1539 11 of 14

may provide a good recommendation for biomarker utilization in CRC screening. However,
the performance of methylated ADHFE1 for adenoma detection was poorer than that of
methylated SDC2 (Tables 3 and 4), suggesting that further refinement of our biomarker
selection approach for precancer detection is necessary.
The putative biological relevance of ADHFE1, SDC2, and PPP2R5C in carcinogen-
esis may provide additional support to our methodology. ADHFE1 is involved in the
metabolism of 4-hydroxybutyrate in mammalian tissues [34]. Previous studies have re-
ported the hypermethylation of ADHFE1 in CRC and adenoma tissues. Hypermethy-
lated ADHFE1 might induce CRC occurrence via the stimulation of tumor cell prolifera-
tion [35,36]. SDC2 is a transmembrane heparan sulfate proteoglycan, serving as a regulator
in the mitogen-activated protein kinase (MAPK) pathway [37]. Detection of SDC2 methy-
lation status in blood and stool specimens has been widely adopted in the assessment of
CRC and adenoma [38,39]. Protein phosphatase 2A (PP2A) is composed of a structural
subunit (A), a regulatory subunit (B), and a catalytic subunit, which is a serine/threonine
phosphatase. As a tumor suppressor, PP2A is involved in the regulation of Wnt signaling
and MAPK pathways [40]. PPP2R5C belongs to the B subunit family of PP2A, which
acts as a negative regulator of cell cycle transition [41]. The methylation of PPP2R5C is
considered a biomarker for CRC and breast cancer detection [42,43]. Notably, these three
genes are involved in the MAPK pathway and affect cell proliferation, which may impact
the progression of CRC.
DNA methylation tests can be performed using blood and stools. Both approaches
are expected to improve the screening compliance for CRC in the general population.
Although the blood-based DNA methylation test, which has recently been approved by
the U.S. Food and Drug Administration (FDA), is believed to have a better adherence,
the examination is only suggested for people who refuse any other screen modalities due
to its relatively low sensitivity and specificity and uncertain testing interval [44]. Unlike
the blood-based DNA methylation test, stool-based DNA methylation test is one of the
regularly recommended CRC screening tests in the latest National Comprehensive Cancer
Network (NCCN) guidelines [44]. Current stool-based tests include the FIT and the stool
DNA test. The stool DNA test combines multiple molecular biomarkers with the FIT. A
methylation panel comprising ADHFE1, SDC2, and PPP2R5C correctly identified CRC
with a sensitivity of 84.6% and specificity of 92.4% in stool samples, and it had a higher
sensitivity than FIT alone in detecting advanced precancerous lesions. However, we still
found two (2/13) false-negative CRC cases, which yielded positive findings in FIT. To
enhance the sensitivity of the stool-based test, a combined FIT–fecal multitarget DNA test
is suggested.
The present study has several limitations. First, the small sample size decreases the
statistical power of our findings. Second, although we have collected specimens from
one hospital, publication bias may still exist. Third, we have searched only for articles
written in English, while many articles written in other languages were ignored. The
candidate biomarkers identified from TCGA and Taiwan’s NHIRD selection model need
to be validated using larger sample sizes to confirm their accuracy and clinical utility.
Fourth, because the sample size in this study was limited, we were unable to incorporate
multiple factors, such as sex, age, and tumor localization, into our biomarker selection
approach. Therefore, future studies need to analyze the relationship between other such
factors and biomarker selection. Fifth, according to our biomarker selection approach,
ADHFE1, SDC2, and PPP2R5C are supposed to display hypermethylation in the CRC
tissues from white, Asian, and black populations; however, 5% of ADHFE1, 10% of SDC2,
and 10% of PPP2R5C methylation levels in CRC tissues from the white population did not
coincide with these findings. To ensure the feasibility of the methylation level biomarker
in CRC tissues from Asian populations, the methylation level analysis of three genes in
CRC tissue was performed. ADHFE1, SDC2, and PPP2R5C were hypermethylated in all
(18/18) CRC tissues samples compared with adjacent normal tissues (p < 0.05) in an Asian
population (2021, unpublished data).
Genes 2021, 12, 1539 12 of 14

5. Conclusions
In summary, through analysis of 450K methylation data from TCGA and EMR data
collected from Taiwan’s NHIRD, we identified a set of biomarker genes for CRC detection
in this study. The potential disease relevance of the three selected genes was verified using
the stool DNA-based methylation test. Notably, in our proof-of-concept study, we found
that the multitarget stool DNA methylation test with a FIT could precisely detect CRC and
adenoma in the early stages. Furthermore, we revealed a panel of noninvasive methylomic
biomarkers for CRC. Among 48 candidate genes, there were 27 candidate genes (AEBP1,
AGRN, AMPH, CHST2, COL25A1, CPLX1, FAM110B, GFRA1, GLRB, GRASP, GSG1L, IRF4,
IRX5, LMO1, MPPED2, PPP2R5C, PREX2, PTPRN2, RALYL, SND1, SPOCK1, THBD, TLL1,
TLX1, USP44, VIPR2, and VSX1) with less known roles in the management of colorectal
cancer. These novel findings also enable us to search for similar predictive methylation
markers in the future. These epigenetic-based biomarkers to detect early-stage colon cancer
require further investigation.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/genes12101539/s1, Table S1: Demographic characteristics of testing subjects, Table S2: 152
Primary DMPs, Table S3: 51 Candidate DMPs, Table S4: Cutoff values of selected biomarkers,
Table S5: OR combination vs linear combination of selected biomarkers.
Author Contributions: Conceptualization, Y.-C.C. (Yi-Chiao Cheng), T.-W.P. and P.-H.W.; methodol-
ogy, Y.-J.C. and T.-W.P.; software, C.-H.Y., J.-L.H. and Y.-H.T.; validation, Y.-J.C. and H.-H.H.; formal
analysis, Y.-J.C. and H.-H.H.; investigation, Y.-C.C. (Yu-Ching Chou) and T.-W.P.; resources, S.-W.J.,
C.-C.W. and P.-K.C.; data curation, Y.-J.C. and H.-H.H.; writing—original draft preparation, Y.-C.C.
(Yi-Chiao Cheng), Y.-J.C. and T.-W.P.; writing—review and editing, T.-W.P.; visualization, Y.-J.C.;
supervision, T.-W.P.; project administration, Y.-C.C. (Yu-Ching Chou) and T.-W.P.; funding acquisition,
Y.-C.C. (Yu-Ching Chou) and T.-W.P. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was partially supported by the grant from NTUT-TMU Research Center
Project 110TN013.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Institutional Review Board of Chang Gung Memorial
Hospital (Protocol No. 105-0504C) and Tri-Service General Hospital (Protocol No. A202105054).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study. Written informed consent has been obtained from the patients to publish this paper.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Hung-Cheng Lai and Polly Shu-Chuan Lin (Guzip Biomarkers Corp.)
for technical assistance. We thank BGI Genomics Co., Ltd. for the gift of the DNA Methylation
Detection Kit for Human SDC2, ADHFE1 and PPP2R5C Genes.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.

References
1. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN
Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249. [CrossRef]
2. Cancer Registry Annual Report, 2018 Taiwan. Available online: https://www.hpa.gov.tw/EngPages/Detail.aspx?nodeid=1061
&pid=6071 (accessed on 6 September 2021).
3. Brenner, H.; Kloor, M.; Pox, C.P. Colorectal cancer. Lancet 2014, 383, 1490–1502. [CrossRef]
4. Balchen, V.; Simon, K. Colorectal cancer development and advances in screening. Clin. Interv. Aging 2016, 11, 967–976. [CrossRef]
5. Navarro, M.; Nicolas, A.; Ferrandez, A.; Lanas, A. Colorectal cancer population screening programs worldwide in 2016: An
update. World J. Gastroenterol. 2017, 23, 3632–3642. [CrossRef] [PubMed]
6. Morikawa, T.; Kato, J.; Yamaji, Y.; Wada, R.; Mitsushima, T.; Shiratori, Y. A comparison of the immunochemical fecal occult blood
test and total colonoscopy in the asymptomatic population. Gastroenterology 2005, 129, 422–428. [CrossRef]
Genes 2021, 12, 1539 13 of 14

7. Whitlock, E.P.; Lin, J.S.; Liles, E.; Beil, T.L.; Fu, R. Screening for Colorectal Cancer: A Targeted, Updated Systematic Review for the
U.S. Preventive Services Task Force. Ann. Intern. Med. 2008, 149, 638–658. [CrossRef] [PubMed]
8. De Wijkerslooth, T.R.; Stoop, E.M.; Bossuyt, P.M.; Meijer, A.G.; van Ballegooijen, M.; van Roon, A.H.C.; Stegeman, I.; Kraaijen-
hagen, A.R.; Fockens, P.; van Leerdam, E.M.; et al. Immunochemical Fecal Occult Blood Testing Is Equally Sensitive for Proximal
and Distal Advanced Neoplasia. Am. J. Gastroenterol. 2012, 107, 1570–1578. [CrossRef] [PubMed]
9. Imperiale, T.; Ransohoff, D.F.; Itzkowitz, S.H.; Levin, T.R.; Lavin, P.; Lidgard, G.P.; Ahlquist, D.A.; Berger, B.M. Multitarget Stool
DNA Testing for Colorectal-Cancer Screening. N. Engl. J. Med. 2014, 370, 1287–1297. [CrossRef]
10. Lee, J.K.; Liles, E.G.; Bent, S.; Levin, T.R.; Corley, D.A. Accuracy of fecal immunochemical tests for colorectal cancer: Systematic
review and meta-analysis. Ann. Intern. Med. 2014, 160, 171. [CrossRef]
11. Brenner, H.; Niedermaier, T.; Chen, H. Strong subsite-specific variation in detecting advanced adenomas by fecal immunochemical
testing for hemoglobin. Int. J. Cancer 2017, 140, 2015–2022. [CrossRef]
12. Kim, C.W.; Kim, H.; Kim, H.R.; Kye, B.-H.; Min, B.S.; Oh, T.J.; An, S.; Lee, S.-H. Colorectal cancer screening using a stool
DNA-based SDC2 methylation test: A multicenter, prospective trial. BMC Gastroenterol. 2021, 21, 173. [CrossRef]
13. Ebner, D.W.; Kisiel, J.B. Stool-Based Tests for Colorectal Cancer Screening: Performance Benchmarks Lead to High Expected
Efficacy. Curr. Gastroenterol. Rep. 2020, 22, 1–9. [CrossRef]
14. Park, S.-K.; Baek, H.L.; Yu, J.; Kim, J.Y.; Yang, H.-J.; Jung, Y.S.; Choi, K.Y.; Kim, H.; Kim, H.O.; Jeong, K.U.; et al. Is methylation
analysis of SFRP2, TFPI2, NDRG4, and BMP3 promoters suitable for colorectal cancer screening in the Korean population? Intest.
Res. 2017, 15, 495–501. [CrossRef]
15. Cooper, G.S.; Markowitz, S.D.; Chen, Z.; Tuck, M.; Willis, J.E.; Berger, B.M.; Brenner, D.E.; Li, L. Performance of multitarget stool
DNA testing in African American patients. Cancer 2018, 124, 3876–3880. [CrossRef]
16. Hsieh, C.-Y.; Su, C.-C.; Shao, S.-C.; Sung, S.-F.; Lin, S.-J.; Kao Yang, Y.-H.; Lai, E.C.-C. Taiwan’s National Health Insurance Research
Database: Past and future. Clin. Epidemiol. 2019, 11, 349–358. [CrossRef]
17. Huang, C.-H.; Yip, B.-S.; Taniar, D.; Hwang, C.-S.; Pai, T.-W. Comorbidity Pattern Analysis for Predicting Amyotrophic Lateral
Sclerosis. Appl. Sci. 2021, 11, 1289. [CrossRef]
18. Morris, T.J.; Butcher, L.M.; Feber, A.; Teschendorff, A.E.; Chakravarthy, A.R.; Wojdacz, T.K.; Beck, S. ChAMP: 450k Chip Analysis
Methylation Pipeline. Bioinformatics 2014, 30, 428–430. [CrossRef] [PubMed]
19. Piñero, J.; Queralt-Rosinach, N.; Bravo, À.; Deu-Pons, J.; Bauer-Mehren, A.; Baron, M.; Sanz, F.; Furlong, L.I. DisGeNET: A
discovery platform for the dynamical exploration of human diseases and their genes. Database 2015, 2015, bav028. [CrossRef]
[PubMed]
20. Xie, L.; Jiang, X.; Li, Q.; Sun, Z.; Quan, W.; Duan, Y.; Li, D.; Chen, T. Diagnostic Value of Methylated Septin9 for Colorectal Cancer
Detection. Front. Oncol. 2018, 8, 247. [CrossRef]
21. Lee, H.S.; Hwang, S.M.; Kim, T.S.; Kim, D.-W.; Park, D.J.; Kang, S.-B.; Kim, H.-H.; Park, K.U. Circulating Methylated Septin 9
Nucleic Acid in the Plasma of Patients with Gastrointestinal Cancer in the Stomach and Colon. Transl. Oncol. 2013, 6, 290–296.
[CrossRef]
22. Sung, J.J.; Lau, J.Y.; Goh, K.L.; Leung, W.K. Asia Pacific Working Group on Colorectal Cancer. Increasing incidence of colorectal
cancer in Asia: Implications for screening. Lancet Oncol. 2005, 6, 871–876. [CrossRef]
23. Soon, M.-S.; Soon, A.; Lin, T.-Y.; Lin, O.S. Distribution of colon neoplasia in Chinese patients: Implications for endoscopic
screening strategies. Eur. J. Gastroenterol. Hepatol. 2008, 20, 642–647. [CrossRef]
24. Kwabi-Addo, B. Epigenetic Biomarkers and Racial Differences in Cancer. In Epigenetic Mechanisms in Cancer; Saldanha, S., Ed.;
Academic Press: Boston, MA, USA, 2018; Chapter 10; pp. 243–273.
25. Han, Y.D.; Oh, T.J.; Chung, T.-H.; Jang, H.W.; Kim, Y.N.; An, S.; Kim, N.K. Early detection of colorectal cancer based on presence
of methylated syndecan-2 (SDC2) in stool DNA. Clin. Epigenet. 2019, 11, 51. [CrossRef]
26. Hu, Y.-H.; Ma, S.; Zhang, X.-N.; Zhang, Z.-Y.; Zhu, H.-F.; Ji, Y.-H.; Li, J.; Qian, X.-L.; Wang, Y.-X. Hypermethylation of ADHFE1
Promotes The Proliferation of Colorectal Cancer Cell Via Modulating Cell Cycle Progression. OncoTargets Ther. 2019, 12, 8105–8115.
[CrossRef]
27. Zhao, G.; Liu, X.; Liu, Y.; Ma, Y.; Yang, J.; Li, H.; Xiong, S.; Fei, S.; Zheng, M.; Zhao, X. Methylated SFRP2 and SDC2 in stool
specimens for Colorectal Cancer early detection: A cost-effective strategy for Chinese population. J. Cancer 2021, 12, 2665–2672.
[CrossRef]
28. Niedermaier, T.; Balavarca, Y.; Brenner, H. Stage-Specific Sensitivity of Fecal Immunochemical Tests for Detecting Colorectal
Cancer: Systematic Review and Meta-Analysis. Am. J. Gastroenterol. 2020, 115, 56–69. [CrossRef] [PubMed]
29. Issa, A.I.; Noureddine, M. Colorectal cancer screening: An updated review of the available options. World J. Gastroenterol. 2017,
23, 5086–5096. [CrossRef] [PubMed]
30. Wang, J.; Liu, S.; Wang, H.; Zheng, L.; Zhou, C.; Li, G.; Huang, R.; Wang, H.; Li, C.; Fan, X.; et al. Robust performance of a novel
stool DNA test of methylated SDC2 for colorectal cancer detection: A multicenter clinical study. Clin. Epigenet. 2020, 12, 1–12.
[CrossRef]
31. Zhang, W.; Yang, C.; Wang, S.; Xiang, Z.; Dou, R.; Lin, Z.; Zheng, J.; Xiong, B. SDC2 and TFPI2 Methylation in Stool Samples as an
Integrated Biomarker for Early Detection of Colorectal Cancer. Cancer Manag. Res. 2021, 13, 3601–3617. [CrossRef] [PubMed]
Genes 2021, 12, 1539 14 of 14

32. Liu, R.; Su, X.; Long, Y.; Zhou, D.; Zhang, X.; Ye, Z.; Ma, J.; Tang, T.; Wang, F.; He, C. A systematic review and quantitative
assessment of methylation biomarkers in fecal DNA and colorectal cancer and its precursor, colorectal adenoma. Mutat. Res. Rev.
Mutat. Res. 2019, 779, 45–57. [CrossRef] [PubMed]
33. Kardon, T.; Noël, G.; Vertommen, D.; Van Schaftingen, E. Identification of the gene encoding hydroxyacid-oxoacid transhydroge-
nase, an enzyme that metabolizes 4-hydroxybutyrate. FEBS Lett. 2006, 580, 2347–2350. [CrossRef] [PubMed]
34. Moon, J.W.; Lee, S.K.; Lee, Y.W.; Lee, J.O.; Kim, N.; Lee, H.J.; Seo, J.S.; Kim, J.; Kim, H.S.; Park, S.-H. Alcohol induces cell
proliferation via hypermethylation of ADHFE1 in colorectal cancer cells. BMC Cancer 2014, 14, 377. [CrossRef] [PubMed]
35. Fan, J.; Li, J.; Guo, S.; Tao, C.; Zhang, H.; Wang, W.; Zhang, Y.; Zhang, D.; Ding, S.; Zeng, C. Genome-wide DNA methylation
profiles of low- and high-grade adenoma reveals potential biomarkers for early detection of colorectal carcinoma. Clin. Epigenet.
2020, 12, 1–13. [CrossRef] [PubMed]
36. Hua, R.; Yu, J.; Yan, X.; Ni, Q.; Zhi, X.; Li, X.; Jiang, B.; Zhu, J. Syndecan-2 in colorectal cancer plays oncogenic role via
epithelial-mesenchymal transition and MAPK pathway. Biomed. Pharmacother. 2020, 121, 109630. [CrossRef] [PubMed]
37. Oh, T.; Kim, N.; Moon, Y.; Kim, M.S.; Hoehn, B.D.; Park, C.H.; Kim, T.S.; Kim, N.K.; Chung, H.C.; An, S. Genome-Wide
Identification and Validation of a Novel Methylation Biomarker, SDC2, for Blood-Based Detection of Colorectal Cancer. J. Mol.
Diagn. 2013, 15, 498–507. [CrossRef]
38. Niu, F.; Wen, J.; Fu, X.; Li, C.; Zhao, R.; Wu, S.; Yu, H.; Liu, X.; Wang, X.; Zou, H. Stool DNA Test of Methylated Syndecan-2 for the
Early Detection of Colorectal Neoplasia. Cancer Epidemiol. Biomark. Prev. 2017, 26, 1411–1419. [CrossRef]
39. Eichhorn, P.J.; Creyghton, M.P.; Bernards, R. Protein phosphatase 2A regulatory subunits and cancer. Biochim. Biophys. Acta BBA
Bioenergy 2009, 1795, 1–15. [CrossRef]
40. Lee, T.Y.; Lai, T.Y.; Lin, S.C.; Wu, C.W.; Ni, I.F.; Yang, Y.S.; Hung, L.Y.; Law, B.K.; Chiang, C.W. The B56gamma3 regulatory subunit
of protein phosphatase 2A (PP2A) regulates S phase-specific nuclear accumulation of PP2A and the G1 to S transition. J. Biol.
Chem. 2010, 285, 21567–21580. [CrossRef]
41. Xie, H.; Mahoney, D.W.; Foote, P.H.; Burger, K.N.; Doering, K.A.; Taylor, W.R.; Then, S.S.; Cao, X.; McGlinch, M.; Berger, C.K.;
et al. Novel Methylated DNA Markers in the Surveillance of Colorectal Cancer Recurrence. Clin. Cancer Res. 2021, 27, 141–149.
[CrossRef]
42. Joo, J.E.; Fab, K.C.; Dowty, J.G.; Milne, R.L.; Wong, E.M.; Dugue, P.-A.; English, D.; Hopper, J.L.; Goldgar, D.E.; Giles, G.G.; et al.
Heritable DNA methylation marks associated with susceptibility to breast cancer. Nat. Commun. 2018, 9, 1–12. [CrossRef]
43. Potter, N.T.; Hurban, P.; White, M.N.; Whitlock, K.D.; Lofton-Day, C.E.; Tetzner, R.; Koenig, T.; Quigley, N.B.; Weiss, G. Validation
of a Real-Time PCR–Based Qualitative Assay for the Detection of Methylated SEPT9 DNA in Human Plasma. Clin. Chem. 2014,
60, 1183–1191. [CrossRef] [PubMed]
44. NCCN Guidelines Version 2.2021—Colorectal Cancer Screening. Available online: https://www.nccn.org/login?ReturnURL=
https://www.nccn.org/professionals/physician_gls/pdf/colorectal_screening.pdf (accessed on 6 September 2021).