Home Print this page Email this page Small font sizeDefault font sizeIncrease font size
Users Online: 274

 

Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Advertise Contacts Login 
     

  Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 57  |  Issue : 4  |  Page : 242-245  

DNA methylation analysis of secreted frizzled-related protein 2 gene for the early detection of colorectal cancer in fecal DNA


Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Web Publication12-Aug-2016

Correspondence Address:
Rasoul Salehi
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan 81744-176
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0300-1652.188357

Rights and Permissions
   Abstract 

Background: The early detection of colorectal cancer (CRC) with high sensitivity screening is essential for the reduction of cancer-specific mortality. Abnormally methylated genes that are responsible for the pathogenesis of cancers can be used as biomarkers for the detection of CRC. The methylation status of the secreted frizzled-related protein 2 (SFRP2) gene was evaluated for their use as a marker in the noninvasive detection of CRC. Materials and Methods: Methylation-specific polymerase chain reaction was performed to analyze the promoter CpG methylation of SFRP2 in the fecal DNA of 25 patients with CRC and 25 individuals exhibiting normal colonoscopy results. Results: Promoter methylation levels of SFRP2 in CRC patients and in healthy controls were 60% and 8%, respectively. Methylation of the SFRP2 promoter in fecal DNA is associated with the presence of colorectal tumors. Conclusion: Hence, the detection of aberrantly methylated DNA in fecal samples may present a promising, noninvasive screening method for CRC.

Keywords: Colorectal cancer, DNA methylation, secreted frizzled-related protein, stool DNA test


How to cite this article:
Babaei H, Mohammadi M, Salehi R. DNA methylation analysis of secreted frizzled-related protein 2 gene for the early detection of colorectal cancer in fecal DNA. Niger Med J 2016;57:242-5

How to cite this URL:
Babaei H, Mohammadi M, Salehi R. DNA methylation analysis of secreted frizzled-related protein 2 gene for the early detection of colorectal cancer in fecal DNA. Niger Med J [serial online] 2016 [cited 2021 May 14];57:242-5. Available from: https://www.nigeriamedj.com/text.asp?2016/57/4/242/188357


   Introduction Top


The incidence of colorectal cancer (CRC) and its mortality rate has increased in the recent years, and it gives rise to difficulties for many health systems in the world. [1] CRC affects close to 150,000 patients in the United States annually and it is the cause of nearly 50,000 deaths. [2] CRC patients have more chance to be treated if it is detected in the early stage of the disease, [3] therefore early detection can reduce mortality and improve survival rates. [4] Although there are many techniques to detect CRC such as the fecal occult blood test (FOBT) and colonoscopy that are more commonly used, [5] they have their limitation to detect, and majority of tumors can remain undetected and it can lead to overtreatment or undertreatment of disease with increasing false-positive and false-negative test results. [6],[7] In addition, FOBT screening decreases only CRC mortality but not the incidence of CRC, and it is not able to detect precursor lesions. [7],[8] Colonoscopy is an invasive method that causes adverse effects including postpolypectomy bleeding and perforation; [8] for this reason, the primary goal is to identify useful screening tools such that it could increase the sensitivity and specificity of screening without invasive actions. [8],[9] Molecular and genetic study can play a key role in the detection of CRC because genetic alterations are the main causes of colorectal neoplasia. [10] Some studies have already tried to show the correlation between various SNP and CRC. [11],[12],[13] One thing is obvious enough that stool-based tests are a noninvasive method, but, in contrast, structural exams are invasive. [8] Scientists are hoping for improving CRC screening by potential benefits of stool-based DNA tests. [14] Interestingly enough, the number of genes silenced by epigenetic mechanisms is more than the number of genetic mutations in CRC that it put forward an important role of epigenetic alterations. [15] There are a great number of genes that will be used as DNA methylation biomarkers ahead that would increase the sensitivity of noninvasive screening tests for CRC. [9],[16] SFRPs genes have a key role in the inhibitory modulators of a tumorigenic pathway (the Wnt signaling pathway), and hence silencing of the SFRP genes is the leading cause of Wnt pathway activation that gives rise to the genesis of the colorectal tumor. [17] It is said that loss of APC gene activity is a prevalent event in sporadic colorectal tumorigenesis that occurs in nearly 80% of cases and because it functions within the Wnt/β-catenin arm of the Wnt signaling pathway, hence it is likely to be expected that changes in this pathway such as silencing of SFRPs genes may be seen in colorectal tumor. In some papers, the secreted frizzled-related protein 2 (SFRP2) gene methylation has been demonstrated as the most sensitive single DNA-based marker in stool for identification of CRC due to the fact that the epigenetic inactivation of SFRP2 gene gives rise to the constitutive Wnt signaling in these putative precursors of CRC. [18],[19],[20]

The aim of our research is to study the methylation status of SFRP2 gene in stool samples from patients with CRC and normal cases, making use of methylation-specific polymerase chain reaction (MSP), as an effective way to screen and detect in the early stages of CRC.


   Materials and methods Top


Fifty stool samples were collected from 25 CRC patients and 25 healthy volunteers, as control group without any history of familial cancer, according to their own colonoscopy. The experimental design was approved by the Ethics Committee of Isfahan University of Medical Sciences, and informed consent was obtained from each patient and healthy individual. All stool samples were stored in − 80°C after labeling them to prevent any enzymatic degradation of DNA.

DNA extraction was done by the use of QIAamp DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. The quality of the DNA extract was examined by spectrophotometry and gel electrophoresis. Then, all the DNA extracted were transferred to −20°C.

DNA treatment by EpiTect Bisulfit was used to convert all unmethylated cytosines to uracil while leaving methyl cytosines unaltered (EpiTect Bisulfite Kit, Qiagen) and eluted in 50 μL of elution buffer. We used myogenic differentiation gene as positive control due to the fact that this gene does not have any CpG island so that any cytosine will be converted to uracil after treatment by bisulfite. In addition, methylated DNA without any treatment by bisulfite was used as a negative control.

MSP was performed with specific primers for either methylated or unmethylated DNA, as previously described. [Table 1] shows the MSP primers. Briefly, a 2 μg DNA sample was used in each amplification reaction. In addition, 17.87 μl O2HDD, 2.5 μl polymerase chain reaction (PCR) buffer 10X, 2-μl dNTP, 0.25 μl forward and 0.25 μl reverse primer, and 0.125 μl TakaRa Taq HS were used in MSP reaction. The MSP procedures for SFRP2 gene were performed as follows: 95°C 10 min, 95°C 45 s 45 cycles, 50°C 30 s 45 cycles (annealing temperature for unmethylated primer pairs), 62°C 30 s 45 cycles (annealing temperature for methylated primer pairs), 72°C for 30 s, and 72°C 5 min for final extension. We used negative and positive controls as described above.
Table 1: Secreted frizzled-related protein gene 1 primers sequences, annealing temperature, and product size for methylation-specific polymerase chain reaction assays

Click here to view


Statistical analysis

Statistical analyses were carried out by the aid of a digital computer, using the Statistical Package for Social Science (version 13.0; SPSS Inc., Chicago, IL, USA) program. Pearson's Chi-squared test was used to assess the association between the methylation status of the SFRP2 promoter in the DNA from all stool samples, as well as to assess the association between methylated SFRP2 promoter (positive or negative), tumor location (colon vs. rectum), patient group (control vs. CRC), and demographic variables, such as age and gender. P > 0.05 was considered to be significant.


   Results Top


In this study, the status of the SFRP2 gene methylation was assessed by MSP reaction in patients and control groups [Figure 1].
Figure 1: Methylation status of secreted frizzled-related protein 2 gene in stool samples

Click here to view


[Table 1] summarizes the characteristics of the twenty patients in the study (65% males, 35% females); the mean ± standard deviation age was 58 years.

The methylation levels of the SFRP2 gene were assessed in the patient and control groups that give rise to the following findings: 12 from 20 patients were methylated and in control group, unlike the patients, 2 from 20 individuals were methylated. For SFRP2 gene, sensitivity and specificity were 60% and 92%, respectively. Methylation status (positive vs. negative) of SFRP2 gene between CRC and control groups was significantly different (P = 0.006).


   Discussion Top


It is reasonable to screen CRC in individuals aged >50 years for reduction in the incidence and mortality of CRC. There is no doubt that genetic diagnosis may provide a noninvasive highly sensitive screening test. To find a proper screening test, it is necessary to consider some aspects of tests such as (a) sensitivity and specificity, (b) safety, and (c) acceptability, which often determine compliance, (d) cost, (e) efficacy (the extent to which medical interventions improve health under ideal circumstances), and (f) effectiveness, which are important because they indicate the accuracy of detecting and removing precancerous lesions. [21] There are several limitations for routine screening methods including colonoscopy and FOBT. Colonoscopy is an invasive method that patients may be not willing to do it and although FOBT is a noninvasive method, the sensitivity of FOBT is approximately 15-35% that could not detect those tumors without bleeding. Although colonoscopy is more sensitive, it is an invasive method that may cause perforation and bleeding and there are more disadvantages such as high costs, difficulty in preparation for the patients, and the need for sedation. [22] Epigenetic diagnosis is noninvasive and highly sensitive in contrast to colonoscopy and FOBT screening. Highly sensitive, specific, and easily analyzable markers are required for noninvasive stool-based CRC screening. Several studies have been done in detecting DNA mutations in the feces of CRC patients. Lu et al. [23] analyzed stool samples from 56 patients. This study assayed methylation status of SFRP2, GATA4/5, NDRG4, and VIM genes. Sensitivity and specificity were 96.4% and 65%, respectively. There are limitations to use multitarget panels due to the high cost and a difficult collection process. It is estimated by a meta-analysis that there is an overall sensitivity of 62% and a specificity of 80% for colorectal neoplasia by using methylated genes in the feces of CRC patients. Wnt signaling pathway acts as an oncogenic way that aberrant activation of the Wnt pathway may give rise to the variety of human cancers, especially in CRC. SFRP2 gene, an important member of the SFRP family, functions as a negative regulator of the oncogenic Wnt pathway through competing with frizzled membrane-bound receptors that can use it as an appropriate marker in CRCs screening. [9] Mόller et al. [24] reported that SFRP2 hypermethylation exhibits a sensitivity of 77-90% with regard to identifying patients with CRC. Huang et al. [25] reported that methylation of SFRP2 occurs in 94.2% of the patients with CRC, with occurrences of 52.4, 37.5, and 16.7% in adenomas, hyperplastic polyps, and ulcerative colitis, respectively. In the recent study, it has been demonstrated the status of methylation of SFRP2 gene in stool DNA of the patients with CRC that could be used as a noninvasive and high sensitive method to choose for screening test in future. [26] Here, we demonstrate that SFRP2 is significantly hypermethylated and downregulated in CRCs when compared with nontumor samples. For SFRP2 gene, sensitivity and specificity were 60% and 92%, respectively, and methylation status (positive vs. negative) of SFRP2 gene between CRC and control groups was significantly different (P = 0.006). In summary, the detection of tumor-derived DNA alterations in the stool is a fascinating new approach with a considerable potential for the noninvasive detection of CRC. Our results demonstrate that the hypermethylation of SFRP2 in fecal samples shows promise for the accurate detection of CRC.


   Conclusion Top


Incidence of CRC is increasing and effective early detection of colon cancer would be beneficial to reduce mortality and costs. DNA methylation, one of the molecular mechanisms in carcinogenesis of colon cancer can be used as biomarker to achieve our aim to early diagnosis of CRC. DNA methylation markers paved the way for developing noninvasive diagnostic assays. The methylation status of SFRP2 gene has a high potential to use as non-invasive method to detect CRC patients in the early stages of tumor. We conclude that the methylation pattern of SFRP2 gene in stool DNA may offer a good alternative in the early noninvasive detection of CRC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Holdenrieder S, Dharuman Y, Standop J, Trimpop N, Herzog M, Hettwer K, et al. Novel serum nucleosomics biomarkers for the detection of colorectal cancer. Anticancer Res 2014;34:2357-62.  Back to cited text no. 1
    
2.
Yang D, Hillman SL, Harris AM, Sinicrope PS, Devens ME, Ahlquist DA. Patient perceptions of stool DNA testing for pan-digestive cancer screening: A survey questionnaire. World J Gastroenterol 2014;20:4972-9.  Back to cited text no. 2
    
3.
Tsang AH, Cheng KH, Wong AS, Ng SS, Ma BB, Chan CM, et al. Current and future molecular diagnostics in colorectal cancer and colorectal adenoma. World J Gastroenterol 2014;20:3847-57.  Back to cited text no. 3
    
4.
Xu XH, Wu XB, Wu SB, Liu HB, Chen R, Li Y. Identification of miRNAs differentially expressed in clinical stages of human colorectal carcinoma-an investigation in Guangzhou, China. PLoS One 2014;9:e94060.  Back to cited text no. 4
    
5.
Wang X, Kuang YY, Hu XT. Advances in epigenetic biomarker research in colorectal cancer. World J Gastroenterol 2014;20:4276-87.  Back to cited text no. 5
    
6.
Vernon SW. Participation in colorectal cancer screening: A review. J Natl Cancer Inst 1997;89:1406-22.  Back to cited text no. 6
    
7.
Bretthauer M, Kalager M. Principles, effectiveness and caveats in screening for cancer. Br J Surg 2013;100:55-65.  Back to cited text no. 7
    
8.
de Wijkerslooth TR, Bossuyt PM, Dekker E. Strategies in screening for colon carcinoma. Neth J Med 2011;69:112-9.  Back to cited text no. 8
    
9.
Salehi R, Mohammadi M, Emami MH, Salehi AR. Methylation pattern of SFRP1 promoter in stool sample is a potential marker for early detection of colorectal cancer. Adv Biomed Res 2012;1:87.  Back to cited text no. 9
[PUBMED]  Medknow Journal  
10.
Goel A, Boland CR. Epigenetics of colorectal cancer. Gastroenterology 2012;143:1442-60.e1.  Back to cited text no. 10
    
11.
Daraei A, Salehi R, Mohamadhashem F. DNA-methyltransferase 3B 39179 G>T polymorphism and risk of sporadic colorectal cancer in a subset of Iranian population. J Res Med Sci 2011;16:807-13.  Back to cited text no. 11
[PUBMED]  Medknow Journal  
12.
Daraei A, Salehi R, Mohamadhashem F. PTGS2 (COX2) -765G>C gene polymorphism and risk of sporadic colorectal cancer in Iranian population. Mol Biol Rep 2012;39:5219-24.  Back to cited text no. 12
    
13.
Daraei A, Salehi R, Salehi M, Emami MH, Janghorbani M, Mohamadhashem F, et al. Effect of rs6983267 polymorphism in the 8q24 region and rs4444903 polymorphism in EGF gene on the risk of sporadic colorectal cancer in Iranian population. Med Oncol 2012;29:1044-9.  Back to cited text no. 13
    
14.
Bosch LJ, Mongera S, Terhaar Sive Droste JS, Oort FA, van Turenhout ST, Penning MT, et al. Analytical sensitivity and stability of DNA methylation testing in stool samples for colorectal cancer detection. Cell Oncol (Dordr) 2012;35:309-15.  Back to cited text no. 14
    
15.
Sakai E, Nakajima A, Kaneda A. Accumulation of aberrant DNA methylation during colorectal cancer development. World J Gastroenterol 2014;20:978-87.  Back to cited text no. 15
    
16.
Mitchell SM, Ross JP, Drew HR, Ho T, Brown GS, Saunders NF, et al. A panel of genes methylated with high frequency in colorectal cancer. BMC Cancer 2014;14:54.  Back to cited text no. 16
    
17.
Qi J, Zhu YQ, Luo J, Tao WH. Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor. World J Gastroenterol 2006;12:7113-7.  Back to cited text no. 17
    
18.
Caldwell GM, Jones CE, Ashley AM, Wei W, Hejmadi RK, Morton DG, et al. Wnt signalling in adenomas of familial adenomatous polyposis patients. Br J Cancer 2010;103:910-7.  Back to cited text no. 18
    
19.
Pretlow TP, Pretlow TG. Mutant KRAS in aberrant crypt foci (ACF): Initiation of colorectal cancer? Biochim Biophys Acta 2005;1756:83-96.  Back to cited text no. 19
    
20.
Oberwalder M, Zitt M, Wöntner C, Fiegl H, Goebel G, Zitt M, et al. SFRP2 methylation in fecal DNA - A marker for colorectal polyps. Int J Colorectal Dis 2008;23:15-9.  Back to cited text no. 20
    
21.
Brezina R, Schramek S, Kazár J. Selection of chlortetracycline-resistant strain of Coxiella burnetii. Acta Virol 1975;19:496.  Back to cited text no. 21
    
22.
Itzkowitz SH, Jandorf L, Brand R, Rabeneck L, Schroy PC 3 rd , Sontag S, et al. Improved fecal DNA test for colorectal cancer screening. Clin Gastroenterol Hepatol 2007;5:111-7.  Back to cited text no. 22
    
23.
Lu H, Huang S, Zhang X, Wang D, Zhang X, Yuan X, et al. DNA methylation analysis of SFRP2, GATA4/5, NDRG4 and VIM for the detection of colorectal cancer in fecal DNA. Oncol Lett 2014;8:1751-6.  Back to cited text no. 23
    
24.
Müller HM, Oberwalder M, Fiegl H, Morandell M, Goebel G, Zitt M, et al. Methylation changes in faecal DNA: A marker for colorectal cancer screening? Lancet 2004;363:1283-5.  Back to cited text no. 24
    
25.
Huang Z, Li L, Wang J. Hypermethylation of SFRP2 as a potential marker for stool-based detection of colorectal cancer and precancerous lesions. Dig Dis Sci 2007;52:2287-91.  Back to cited text no. 25
    
26.
Zhang H, Zhu YQ, Wu YQ, Zhang P, Qi J. Detection of promoter hypermethylation of Wnt antagonist genes in fecal samples for diagnosis of early colorectal cancer. World J Gastroenterol 2014;20:6329-35.  Back to cited text no. 26
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1]


This article has been cited by
1 A systematic review and meta-analysis of the DNA methylation in colorectal Cancer among Iranian population
Soraya Bilvayeh,Zohreh Rahimi,Kheirollah Yari,Shayan Mostafaei
Gene Reports. 2021; : 101080
[Pubmed] | [DOI]
2 Methylation as a critical epigenetic process during tumor progressions among Iranian population: an overview
Iman Akhlaghipour,Amir Reza Bina,Mohammad Reza Abbaszadegan,Meysam Moghbeli
Genes and Environment. 2021; 43(1)
[Pubmed] | [DOI]
3 Performance evaluation of stool DNA methylation tests in colorectal cancer screening: a systematic review and meta-analysis
Mahir Gachabayov,Edward Lebovics,Aram Rojas,Daniel M Felsenreich,Rifat Latifi,Roberto Bergamaschi
Colorectal Disease. 2021;
[Pubmed] | [DOI]
4 A systematic review and quantitative assessment of methylation biomarkers in fecal DNA and colorectal cancer and its precursor, colorectal adenoma
Rongbin Liu,Xuan Su,Yakang Long,Dalei Zhou,Xiao Zhang,Zulu Ye,Jiangjun Ma,Tao Tang,Fang Wang,Caiyun He
Mutation Research/Reviews in Mutation Research. 2019; 779: 45
[Pubmed] | [DOI]
5 Association between SFRP promoter hypermethylation and different types of cancer: A systematic review and meta-analysis
Jun Yu,Yang Xie,Mengying Li,Fenfang Zhou,Zhenyang Zhong,Yuting Liu,Feng Wang,Jian Qi
Oncology Letters. 2019;
[Pubmed] | [DOI]
6 Hypermethylated Promoters of Secreted Frizzled-Related Protein Genes are Associated with Colorectal Cancer
Haochang Hu,Tiangong Wang,Ranran Pan,Yong Yang,Bin Li,Cong Zhou,Jun Zhao,Yi Huang,Shiwei Duan
Pathology & Oncology Research. 2018;
[Pubmed] | [DOI]
7 Comparison of SFRP2 promoter methylation in stool sample and cfDNA regarding patients with colorectal cancer
Mohsen Mohammadi,Seyed-Mojtaba Jafari,Mohammad-Ali Mohaghegh,Ali Ghasemi,Niloofar Momenzadeh
Comparative Clinical Pathology. 2017;
[Pubmed] | [DOI]
8 TET2 and MEG3 promoter methylation is associated with acute myeloid leukemia in a Hainan population
Hongxia Yao,Mengling Duan,Lie Lin,Congming Wu,Xiangjun Fu,Hua Wang,Li Guo,Wenting Chen,Li Huang,Dan Liu,Ruo Rao,Shuwen Wang,Yipeng Ding
Oncotarget. 2017; 8(11): 18337
[Pubmed] | [DOI]



 

Top
  
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and me...
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed2433    
    Printed58    
    Emailed0    
    PDF Downloaded32    
    Comments [Add]    
    Cited by others 8    

Recommend this journal