INTRODUCTION:

Colorectal cancer is the third most common cancer worldwide^1,2, with projections suggesting a 60% increase in new cases to reach 2.2 million new cases and 1.1 million deaths by the year 2030³, with a survival rate of approximately 60-64%⁴

Data from the World Health Organization indicates that the situation in Syria mirrors the worldwide trends, with colorectal cancer being the third most frequently diagnosed cancer in both men and women.⁵

A lot of risk factors could be involved in colorectal cancersuch as: age, level of education (higher levels of education correlated with lower incidence of colorectal cancer)⁶,obesity, low physical activity, red meat consumption, high salt diets, smoking⁷,and alcohol consumption⁸.

In addition to the risk factors mentioned above Colorectal carcinogenesis is a complicated and multi-step process including changes in many oncogenes and tumor suppressor genes.⁹

A lot of recent studies determined the role of folic acid metabolism in colorectal cancer¹⁰. Folate plays a crucialrole in various cellular processes such as: DNA synthesis, purine and pyrimidine metabolism and DNA methylation. A deficiency in folate leads to ahuge incorporation of uracil into DNA and causes DNA breaks, which might lead to carcinogenesis ¹¹

The MTHFR gene, also known as 5,10-methylenetetrahydrofolate reductase gene is located on the short arm of chromosome 1 (1p36.3) ¹², the gene encodes a 74.6 kDa protein (656 amino acids)¹³.

MTHFR is a key enzyme in folate metabolism, it mediates the transformation of 5,10-methylene-tetrahydrofolate (THF) to 5-methyl tetrahydrofolate (THF) which provides methyl groups for the formation of S-Adenosylmethionine which is vital for DNA methylation reactions, additionally 5,10-methyleneTHF the substrate of the MTHFR enzyme is essential for thymidine synthesis through thymidylate synthase (TS) and purine synthesis via the conversion to 10-formyl-THF, ultimately contributing to DNA synthesis¹⁴,

One of the most common polymorphisms of MTHFR is A1298C (rs1801131).

The A1298C polymorphismis in exon 7, leads to a glutamate to alanine replacement at codon 429, this polymorphism lies in the C-terminal end of the enzyme and potentially result in a decrease of a 40% in enzyme activity of the variant genotype ¹⁴.

MTHFR polymorphisms is being a point of interest in many health conditions¹⁵, including cardiovascular diseases (MTHFR deficiency can result inhigh levels of homocysteine in the blood contributing to the development of arterial and venous thrombosis as well as atherosclerosis)¹⁶¹⁷¹⁸. Moreover MTHFR polymorphisms have been also linked to conditions such as schizophrenia ¹⁹, Alzheimer ̓s²⁰ and cancer, particularlycolorectal cancer, since colon cells are subjected to continuous regeneration, and need high rates of DNA synthesis. Furthermore, DNA repair is crucial in the colonic mucosa and DNA repair mechanisms can be influenced by methylation status and by nucleotides availability ²¹.

Few studies have addressed the association between this polymorphism and colorectal cancer. Some studies suggested that this polymorphism is associated with lower susceptibility to colorectal cancer ^{22, 23}, but other studies demonstrated that this polymorphism has no effect on developing colorectal cancer ¹³

The objective of this research is to investigate the frequency of the A1298C gene variant among colorectal cancer patients and healthy individuals, we also aimed to determine the potential relation between the mentioned polymorphism and developing colorectal cancer in patients from Syriaas cancer is a significant public health globally ^{24, 25}, being one of the five main causes of death in all societies ^{26, 27}.

It is important to report the impact of genetic factors such as A1298C variant on colorectal cancer in different populations including those in Syria.

MATERIALS AND METHODS:

Patients and samples:

The research was conducted on a group of 50 patients with colorectal cancer, consisting of 32 with colon cancer and 18 with rectal cancer from the oncology department at Tishreen University Hospital in Latakia, Syria. The average age of the patients was 57.54±11.08 years old. Additionally, a control group of 20 healthy individuals with an average age of 47.65±5.90 years old was included in the study. Peripheral blood samples were collected on EDTA tubes, followed by DNA isolation for further analysis.

DNA extraction:

DNA was extracted from whole blood by the salting out method using theAmplisens® extraction kit according to the attached instructions and keptat -20 C˚ until usage.

Polymorphism Identification:

The genomic type of each samplewasdetermined using gbHemoA1298C kit by GeneriBiotech®.

This kit operates based on the allelic discrimination principle. To conduct the test, we followed kit instructions: Initially, the kit tubes were defrosted and centrifuged. Subsequently,16µl of the Assay qPCR (detection assay) was transferred into micro tubes along with 4µl of the sample (after allowing the samples to reach room temperature). Additionally, 16µl of the Assay qPCR was added to 4µl of each standard (wild type, heterozygote, mutant) as a positive control, and to deionized water as a negative control (no template). Next, we configured the real-time PCR as per kit's manual and determined the genetic type according to the kit's instruction

Statistical analysis:

The data were subjected to analysis utilizing the Statistical Package for Social Science (SPSS) version 20 and were presented as percentages, logistic regression was utilized to compute the odds ratio (OR).

Statistical significance was attributed to results with a p-value less than or equal to 0.05 in accordance with standard academic conventions.

RESULTS:

A1298C prevalence:

We studied the prevalence of each genotype in patients (colon and rectal cancer combined then each one separately) and in healthy individuals.

The results, presented in Table 1, show that a majority of patients with colon and rectal cancer combined (62%) exhibited the AA (wild type) genotype, followed by 26% with the AC (heterozygote) genotype, and 12% with the CC (mutant) genotype. The frequency of the normal allele A was found to be 75%, while the mutant allele C was 25%.

Upon examining the data for colon cancer patients alone (Table 2), it was observed that 59.4% had the AA genotype, 34.4% had the AC genotype, and 6.2% had the CC genotype. The normal allele A frequency was 76.5%, and the mutant allele C frequency was 23.5%.

For patients with rectal cancer alone (Table 3), 66.7% of the patients had AA genotype, 11.1% had AC and 22.2% had CC. the frequency of the normal allele A was 72.2%and for the mutant allele C was 27.8%.

Lastly, we determined the prevalence of each genotype in the healthy individuals (table 4) and 40% had AA genotype, 30% had AC and 30% had CC. the frequency of the normal allele A was 55%and for the mutant allele was 45%.

Table 1: Prevalence of A1298C in colorectal cancer patients

Number	A1298C polymorphism (N=50, 100%)			Allele frequency
Number	AA	AC	CC	A	C
N	31	13	6	0.75	0.25
%	62	26	12	0.75	0.25

Table 2: Prevalence of A1298C in colon cancer patients

Number	A1298C polymorphism (N=32, 100%)			Allele frequency
Number	AA	AC	CC	A	C
N	19	11	2	0.765	0.235
%	59.4	34.4	6.2	0.765	0.235

Table 3: Prevalence of A1298C in rectal cancer patients

Number	A1298C polymorphism (N=18, 100%)			Allele frequency
Number	AA	AC	CC	A	C
N	12	2	4	0.722	0.278
%	66.7	11.1	22.2	0.722	0.278

Table 4: Prevalence of A1298C in healthy individuals

Number	A1298C polymorphism (N=20, 100%)			Allele frequency
Number	AA	AC	CC	A	C
N	8	6	6	0.55	0.45
%	40	30	30	0.55	0.45

Association of A 1298 C polymorphism with colorectal cancerrisk:

After determining the prevalence of each genotype in patients and healthy individuals, we studied the association of genotype with risk of colorectal cancer.

The risk of developing cancer when an individual's genotype changes from normal to other types (heterozygous mutant or homozygous mutant) was studied as a single independent variable using the Binary Logistic Regression test (table 5).The analysis revealedthat there is no significant association between the A1298C polymorphism and the risk of colorectal cancer (P value>0.05)

Further analysis was conducted to explore the association of each genotype with the risk of colon cancer and rectal cancer separately. The study indicated that the CC genotype served as a protective factor against colon cancer (OR=0.140, CI=95%, P value =0.033). However,there was no observed association between the A1298C polymorphism and the risk of rectal cancer (P value>0.05)

Table 5: Association of A1298C polymorphism with risk of colorectal cancer

Genotype	Odds Ratio	p-value	95% Confidence Interval
Genotype	Odds Ratio	p-value	Low Limit	High Limit
AA	Reference	-	-	-
AC	0.559	0.358	0.162	1.934
CC	0.258	0.053	0.065	1.018

Table 6: Association of A1298C polymorphism with risk of colon cancer

Genotype	Odds Ratio	p-value	95% Confidence Interval
Genotype	Odds Ratio	p-value	Low Limit	High Limit
AA	Reference	-	-	-
AC	0.772	0.695	0.212	2.813
CC	0.14	0.033	0.023	0.85

Table 7: Association of A1298C polymorphism with risk of rectal cancer

Genotype	Odds Ratio	p-value	95% Confidence Interval
Genotype	Odds Ratio	p-value	Low Limit	High Limit
AA	Reference	-	-	-
AC	0.222	0.108	0.036	1.39
CC	0.444	0.305	0.094	2.093

DISCUSSION:

Colorectal cancer has become a growing concern in recent years, with the most recent statistics highlighting its increasing prevalence. As we have previously discussed, colorectal cancer is a complex disease with multiple contributing factors. Numerous recent studies have pointed to the potential role of folate in the development of colorectal cancer, suggesting a possible inverse relationship between folate levels and the risk of developing cancer.²⁸

Folate metabolism is closely involved in processes crucial to cellular proliferation, DNA synthesis, purine and pyrimidine metabolism and DNA methylation. It is possible that dysregulated folate metabolism might cause abnormal cellular proliferation and predispose to carcinogenesis¹⁰

MTHFR gene encodes a very important enzyme that mediates and regulates folatemetabolism, any malfunction or mutations can lead to impairment in cell function and increase the risk of developing various types of cancer, including colorectal cancer.

In this study we aimed to investigate the prevalence of A1298C polymorphism and association with risk of colorectal cancer.

After examining the data from the entire group of patients, we found out that A1298C polymorphism is not associated with colorectal cancer (table 5) (P> 0.05),These findings align with previous studies, including one conducted byFernández,et alin Madrid, Spain, which indicated that there is no significant association between A1298C polymorphism with overall risk of colorectal cancer ¹⁴ and a study by Ramin lak,et alin Tehran, Iran that also indicated no significant association between A1298C polymorphism and colorectal cancer risk¹³.

After examining the association with colorectal cancer in general we wanted to detect if there is a difference in association between the mentioned polymorphism with colon cancer risk and rectal cancer risk each separately.

This is due to the fact that there is a range of incompatible and indecisive data concerning the impact of this polymorphism on both types of cancer. Furthermore, there have been very few studies that have specifically examined how this polymorphism affects the risk of colon cancer and rectal cancer separately, which could be different since colon and rectal cancer are different in pathogenesis, molecular carcinogenesis, treatment strategies ²⁹.

We found out that actually there is a difference between the two cancers, where the A>C polymorphism was associated with reduced risk with colon cancer (table 6) (OR=0.1440, Pvalue= 0.033), but not associated with risk of rectal cancer (OR=0.444, Pvalue= 0.305).Our findings about colon cancer matched a studyonAfrican Americans and whites by keku,et al³⁰, and a study in India by wang, et al³¹. However, our finding about rectal cancer didn’t match possibly due to the difference in sample sizes.Interestinglyour results are in line with a study in Japan by Matsuo, et al, which also found no association between rectal cancer and the A>C polymorphism.

We could possibly explain our results regarding colon cancer that, A1298C polymorphism is in the C-terminal of MTHFR enzyme and causes an Alanine to Glutamate substitution near to the binding site for the MTHFR inhibitor S-adenosyl-methionine (SAM), may probably affect feedback inhibition. Furthermore, the balance of DNA synthesis and DNA methylation may affect the regulation of gene expression impacting cancer risk ³¹. Further assessment of our findings on rectal cancer is required on a larger sample size for validation.

We conclude that A1298C polymorphism is associated with lower risk of colon cancer and we did not find any association between the mentioned polymorphism and rectal cancer.We recommend conducting this study on a larger group taking in consideration gene-gene interaction, and gene-environment interaction.

We also recommend conducting separate studies on colon cancer and rectal cancer to gain a deeper understanding of how MTHFR polymorphisms are related to each specific type of cancer.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Oncology and Laboratory Departments in Tishreen University Hospital, Lattakia, Syria for their kind support during the research.

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Received on 08.04.2024 Modified on 08.06.2024

Research J. Pharm. and Tech 2024; 17(10):4694-4698.

DOI: 10.52711/0974-360X.2024.00723