Detection of SPG20 gene promoter-methylated DNA, as a novel epigenetic biomarker, in plasma for colorectal cancer diagnosis using the MethyLight method

Abstract

Abstract. Aberrant promoter methylation of genes is a common epigenetic alteration in colorectal cancer (CRC). In the present study, spastic paraplegia 20 (SPG20) promoter-methylated DNA, as a potential diagnostic biomarker, was investigated in plasma and tumor tissue samples from patients with CRC. To the best of our knowledge, the quantification of SPG20 promoter-methylated DNA in plasma samples remains unreported. SPG20 promoter methylation was investigated in 32 paired tumor and healthy adjacent tissues, 37 plasma samples from patients with CRC, and in 37 plasma samples from a healthy control group, using the MethyLight method. The percentage of methylated reference (PMR) values was determined for each sample, and the sensitivity and specificity of this unique biomarker were evaluated. PMR values were significantly higher in plasma samples from patients with CRC compared with in those from the control group (P<0.05). Plasma specimens from patients and healthy controls exhibited median PMR values of 7.7 (95% CI, 4.15-15.28) and 0.59 (95% CI, 0.14-1.12), respectively. Notably, the median PMR values were identified as 42.39 (95% CI, 27.69-72.26) and 3.61 (95% CI, 1.07-5.29) in tumor and adjacent healthy tissues, respectively. Using receiver-operating characteristics curve analysis, the area under curve (AUC) was demonstrated to be 0.984 for plasma samples, exhibiting a sensitivity of 81.1% and a specificity of 96.9%. Furthermore, the AUC was 0.996 for tissue samples, revealing a sensitivity of 93.8% and specificity of 99.96%.Results from the present study indicate that the identification of SPG20 promoter-methylated DNA in plasma is a potential diagnostic biomarker for the detection of CRC. Furthermore, the results demonstrate a satisfactory sensitivity and specificity, indicating the importance of SPG20 methylation as a novel noninvasive biomarker. IntroductionColorectal cancer (CRC) is one of the most common malignancies worldwide, accounting for an estimated 1.3 million new cases and >500,000 mortalities/year (1). CRC is the fourth leading cause of cancer-associated mortality worldwide, with a rapidly increasing incidence rate in the worldwide (2,3). In the Asian population, the annual prevalence rate of CRC has steadily increased over the past two decades (4). The Ministry of Health and Medical Education in Iran states that cancer is the third most common cause of mortality in Iran (5). Currently, the gold standard method for CRC detection is internal imaging of the colon with colonoscopy followed by a biopsy examination; however, it remains an invasive procedure with possible serious complications and limitations (6). Cancer is a multistep process resulting from a gradual accumulation of genetic and epigenetic changes to the genome (7). The methylation of 5'-C-phosphate-G-3' (CpG) islands has been suggested to be associated with gene silencing, serving an important role in cancer development (8,9). The aberrant promoter methylation of genes is a primary occurrence in the process of CRC carcinogenesis, whic...

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“…The SPG20 gene encodes a protein called Spartin that has been found to be involved in intracellular epidermal growth factor receptor trafficking; SPG20 promoter has been found to be hypermethylated in colorectal cancer, resulting in gene silencing and cytokinesis arrest [ 40 ]. SPG20 promoter hypermethylation was also validated as a novel noninvasive biomarker [ 41 ]. MCM2 is one of six highly conserved proteins recruited to form the MCM protein complex, a cell ubiquitous hexamer that works as molecular motor performing DNA duplex unwinding and fork progression during DNA replication [ 42 ].…”

DNA methylation markers detected in blood, stool, urine, and tissue in colorectal cancer: a systematic review of paired samples

“…In the present systematic review, we identified two studies investigating this biomarker in more than one type of material, although none of these studies included adenomas. Rezvani et al found methylated SPG20 in 94% of CRC tissues and correspondingly in 81% of the plasma samples from these patients [ 41 ]. The specificity in tissue samples was 99% and in plasma samples 97% [ 41 ].…”

“…Rezvani et al found methylated SPG20 in 94% of CRC tissues and correspondingly in 81% of the plasma samples from these patients [ 41 ]. The specificity in tissue samples was 99% and in plasma samples 97% [ 41 ]. Zhang et al found methylated SPG20 in 85% of CRC tissues and correspondingly in 80% of stool samples from these patients [ 42 ].…”

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“…The other two approaches for MSP quantification included two control PCR products: ALU–C4 ( ALU ) as a DNA input normalizer and commercial methylated bisulfite-converted human DNA (Qiagen) as a fully methylated control [32,33]. Equations used for these two approaches were:PMR= M/U / ALU for sample M/U / ALU for methylated control and PMR= M / ALU for sample M/ ALU for methylated control where in all three approaches for the quantification of MSP: PMR > 1 indicates a strong MGMT promoter methylation (hypermethylated), PMR = 0 (no M primer MSP product detectable) indicates an unmethylated MGMT promoter and PMR < 1 indicates weak MGMT promoter methylation.…”