Colorectal cancer and polymorphism of methylenetetrahydrofolate reductase (C677T) in Morocco

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ORIGINAL ARTICLE /ARTICLE ORIGINAL

Colorectal cancer and polymorphism of methylenetetrahydrofolate reductase ( C677T ) in Morocco

Cancer colorectal et polymorphisme du méthylènetétrahydrofolate réductase (C677T) au Maroc

B. Diakite · A. Benmoussa · K. Hamzi · H. Jouhadi · S. Nadifi

Revised: 23 November 2011, Accepted: 14 June 2012

AbstractIntroduction: Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism. It is involved in the synthesis, repair, and methylation of deoxyribonucleic acid (DNA). The most frequently studied MTHFRgene polymorphism is C677T, which is involved in the pathogenesis of many diseases including cancer. This case-control study was undertaken to analyze the association ofMTHFR C677Tpolymorphism and the risk of colorectal cancer (CRC).

Methods: We determined the genotypes and alleles of MTHFR gene in 36 patients with histological confirmed CRC (19 women and 17 men) and in 36 normal controls

matched for sex without a history of cancer. DNA was iso- lated from peripheral blood samples, and genotypes were determined by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP).

Results: In light of our results, the association of combined CT and TT genotypes and T allele was associated with an increased risk of CRC odds ratio 6.88 [95% confidence interval (CI): 2.30–20.49, P = 0.0005), 5.91 (95% CI:

2.14–16.34,P= 0.0006), and 2.96 (95% CI: 1.4–6.25,P= 0.0045), respectively.TThomozygous was not a protective factor in our study with an odds ratio of 2.36 (95% CI:

0.63–17.65, P = 0.16). Relative risk, individuals carrying at least oneTallele have a 2-fold greater risk of developing CRC.

Conclusion: The MTHFR C677T gene variant was a risk factor for CRC in our population under study.

Keywords Colorectal cancer ·MTHFR· Polymorphism · Association

RésuméPrésentation: La méthylènetétrahydrofolate réduc- tase (MTHFR) est une enzyme clé dans le métabolisme foli- que. Elle est impliquée dans la synthèse, la réparation et la méthylation de l’ADN. Le polymorphisme du gèneMTHFR le plus souvent étudié est leC677T, qui est impliqué dans la pathogenèse de nombreuses maladies, dont le cancer. Cette étude, portant sur un groupe cas-témoins apparié, a été menée pour analyser l’association du polymorphisme MTHFR C677Tet du risque de cancer colorectal.

Méthodes: Nous avons déterminé les génotypes et les allèles du gèneMTHFRchez 36 patients présentant un cancer colorectal confirmé histologiquement (19 femmes et 17 hommes) et chez 36 témoins normaux appariés selon le sexe sans anté- cédents de cancer. L’ADN a été isolé à partir de sang périph- ériques et les génotypes ont été déterminés par PCR-RFLP.

Résultats : Nos résultats montrent que la combinaison de génotypes CTetTTet d’allèles T est associée à un risque accru de CRC de 6,88 (avec un intervalle de confiance [IC]

B. Diakite · A. Benmoussa · K. Hamzi · S. Nadifi (*) Department of Genetic and Molecular Pathology Laboratory (LGPM), Faculty of Medicine and Pharmacy14,

Tarek-ibn-Ziad, QH, BP 9154 Casablanca, Morocco e-mail : nadifisel@yahoo.fr

H. Jouhadi

Oncology Department, Ibn Rochd Hospital, Casablanca, Morocco

LIST OF ABBREVIATIONS 5-MTHF: 5-methyltetrahydrofolate

5,10-MTHF: 5,10-methylene tetrahydrofolate reductase ADK: adenocarcinoma

al: collaborator

DNA: deoxyribonucleic acid

dTMP: deoxythymidylate monophosphate dUMP: deoxyuridylate monophosphate CI: confidence interval

CRC: colorectal cancer

LGPM: genetics and molecular pathology laboratory Ml: milliliter

MS: methionine synthase

MTHFR: methylene tetrahydrofolate reductase OR: odd ratio

PCR: polymerase chain reaction

RFLP: restriction fragment length polymorphism RNA: ribonucleic acid

RR: relative risk

SAM: S-adenosylmethionine TNM: tumor, node, metastasis DOI 10.1007/s12558-012-0233-x

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de 95 % : 2,30–20,49, p = 0,0005), de 5,91 (IC 95 % : 2,14–16,34, p= 0,0006) et de 2,96 (IC 95 % : 1,4–6,25, p= 0,0045). Les homozygotes TT n’ont pas été un facteur de protection dans notre étude, avec un taux de 2,36 (IC 95 % : 0,63–17,65,p= 0,16). Concernant le risque rela- tif, une personne porteuse d’au moins un allèle T présente un risque deux fois plus élevé de développer un CRC.

Conclusion: La variante génétiqueMTHFR C677Tconsti- tue un facteur de risque de cancer colorectal dans la population que nous avons étudiée.

Mots clésCancer colorectal ·MTHFR· Polymorphisme · Association

Introduction

Colorectal cancer (CRC) is a major health problem in the world. It is the third cancer after breast and lung cancers [1]. In Morocco, it ranks first among other digestive cancers (38.1% of the colon adenocarcinoma and 38.8% of the rectum adenocarcinoma) [2]. There is a geographic variation in the incidence of CRC in the world, especially Africa and Asia, where the incidence of CRC is up to 20 times less than in Northern Europe or in the United States [3]. This difference could be explained by the mode of living, type of diet, or genetic predisposition.

The colorectal mucosa is in constant renewal. Deoxyribo- nucleic acid (DNA) synthesis is more active in these tissues and, therefore, there is more chance for error. Moreover, more folates are needed that are essential not only for synthesis but also for the phenomenon of methylation DNA [4,5]. Cell folate status depends not only on their food intake

but also on genetic determinants of two polymorphisms enzymes of folate cycle: methylene tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) [6].MTHFRis a key enzyme in folate metabolism (Fig. 1). It catalyzes the irreversible reduction of 5,10-methylene tetrahydrofolate reductase (5,10-MTHF) to 5-methyltetrahydrofolate (5-MTHF) [7] that serves as a substrate for the remethylation of homocysteine to methionine to give S-adenosylmethionine (SAM) [8], which is the main donor of methyl radicals in human, and is therefore essential for the methylation reactions [9]. In addition, 5,10-MTHF that is the substrate of MTHFR is used for methylation of deoxyuridylate monophosphate (dUMP) into deoxythymidylate monophosphate (dTMP), and thus participates in the pyrimidine synthesis (DNA). Moreover, the folate cycle contributes to the synthesis of purine DNA and ribonucleic acid (RNA) [10]. A lack of the enzymeMTHFRis responsible for an imbalance of methylation reactions leading to hypomethylation of genomic DNA and aberrant promoter hypermethylation of some genes [11,12].

A common variant of theMTHFRenzyme was identified in 1988 [13]: the MTHFR 677C → T (A222V) polymorphism, which is the thermolabile form of the enzyme MTHFR [14]. This polymorphism is the most common genetic cause of hyperhomocysteinemia and has been involved in several multifactorial disorders, such as cardiovascular disease and cancer [15–18]. It has been shown that inadequate intake of folate coupled with a methionine deficiency and/or chronic alcoholism may increase the risk of CRC [6]. This risk is even greater when the subject has the TTgenotype in theMTHFRgene [19].CTheterozygous has approximately 60% of normal enzyme activity, while TT homozygous has only 30% of this activity. So with this

Fig. 1 MTHFRmetabolic pathway (atlasgeneticsoncology.org)

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form of enzyme, there is an accumulation of 5,10-MTHF orienting to more DNA synthesis [20]. However, several studies have demonstrated that with adequate folate intake, the677TTgenotype has protective effect associated with a decreased risk of acute lymphocytic leukemia and CRC [21–23]. The mechanism proposed to explain this phenomenon is the redistribution of folates, facilitating the synthesis of thymidine and reducing the risk of DNA damage. How- ever, other studies showed no association [4,11,24]; hence, the association ofTTgenotype and the CRC remains contro- versial [25]. In Morocco, the lack of studies on the association of677C T MTHFRgene polymorphism and CRC has paved a way for us to perform this work to contribute to a better assessment of this risk.

Material and methods Patients and samples

This is a case-control study carried out on 72 subjects.

Thirty-six patients with colorectal adenocarcinoma were admitted at the oncology department of Ibn Rochd Univer- sity Hospital Centre of Casablanca. Clinic pathological data have been collected from their medical records. For each patient, a family investigation was conducted and a blood sample was performed. Another set of 36 controls was from healthy general population with no history of CRC.

Informed consent was signed by each patient and control.

DNA extraction

Genomic DNA was extracted from 5 ml of blood samples by the standard salt method. The quality and quantity of extracted DNA was checked on 1% agarose gel using a spectrophotometer.

Genotyping of MTHFR (C677T)

The genotypes for MTHFR (C677T) gene polymorphism were detected by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) as described by Frosst et al. [16].

Statistical analysis

Statistical analysis was performed using the statistical pro- gram SPSS 11.0 (SPSS Inc., Chicago, IL, USA) and Med- Calc 11.5.1.0 user-friendly software. Test of Hardy–Wein- berg equilibrium was carried out separately for the (C677T)MTHFRpolymorphism in cases and controls. The clinical and histological features and the relation to different genotypes were made by chi-squared (χ²) test. APvalue of

less than 0.05 was considered significant. Associations between genotypes and CRC risk were estimated by calcu- lating odds ratio (OR) with confidence intervals (CI) of 95%.

The risk of developing cancer was calculated from the relative risk (RR).

Results

We studied 36 patients with CRC and 36 cases of age- and sex-matched controls recruited from the oncology department over a year. The statistical study showed that both sample groups (patients and case-control) are in Hardy– Weinberg equilibrium for the distribution ofMTHFRpoly- morphism (χ²= 2.48,P= 0.12;χ²= 1. 88,P= 0.17).

The correlation betweenMTHFRpolymorphism (Fig. 2), clinical and histological features (Fig. 3), showed no

Fig. 2 3% agarose gel showing MTHFR after digestion (HinfI enzyme), M: Weight Ladder, 1: Homozygous CC, 2: Heterozygous CT, and 3: Homozygous TT

Fig. 3 Photo of a histological section of a colic adenocarcinoma after staining with hematoxylin/eosin

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significant difference between patient group and case- control relative to age, sex, family history, location, and histology of the tumor (Table 1).

The genotypes frequencies found in patients and controls were 27.8%CC, 61.1%CT, 11.1%TTand 69.4%CC, 22.2%

CT, 8.3%TT, respectively (Table 2). The allele frequencies ofCandTalleles were 58.3% and 41.7% for patients against 80.6% and 19.4% for controls (Fig. 4).

Our results show that theTallele andCTheterozygous are significantly associated with a higher risk of CRC with OR of 2.96 (95% CI: 1.4–6.26, P = 0.0045) and 6.88 (2.31–20.49,P= 0.0005), respectively (Table 2). The combined association ofCTandTTgenotypes in cases compared to controls was statistically significant with OR 5.91 (95%

CI: 2.14–16.34,P = 0.0006). In light of our findings, TT homozygous was not a protective factor of CRC with an OR of 3.33. This shows thatMTHFR C677Tpolymorphism

may increase the risk of CRC in the Moroccan population (Fig. 4).

In this study, we also investigated the RR between cases versus controls. The RR of having the disease was two times higher in individuals carrying at least oneTallele (TTorCT) thanCCcarriers (Table 3).

Discussion

MTHFRplays an important role in regulating folate metabolism. MTHFR C677T variant has been associated with an increased risk of several diseases such as cardiovascular disease, neural tube defects [29], complications of pregnancy, neuropsychiatric disorders and cancers [18, 26]. The results obtained by different teams, however, remain contradictory [25]. We conducted this study to better assess the interaction

Table 1 Frequencies ofMTHFR C677Tpolymorphism in patients with CRC according to clinical and histological parameters

Number MTHFR C677TPolymorphism Chi-Squared

CC(%) CT(%) TT(%) Pvalue Test

Age 36 1.0 0,0

≤40 years 18 5 (27.8) 11 (61.1) 2 (11.1)

> 40 years 18 5 (27.8) 11 (61.1) 2 (11.1)

Gender 36 0.12 2.48

Female 19 8 (42.1) 9 (47.1) 2 (10.5)

Male 17 2 (11.8) 13 (76.5) 2 (11.8)

Family antecedent 36 0.62 4.42

CRC 12 3 (25.0) 7 (58.3) 2 (16.7)

Other cancers 8 3 (37.5) 4 (50.0) 1 (12.5)

CRC + Other cancers 5 - 5 (100.0) -

No family history 11 4 (36.4) 6 (54.5) 1 (9.1)

Localization of cancer 36 0.17 9.02

Right colon 9 4 (44.4) 4 (44.4) 1 (11.1)

Left colon 7 3 (42.9) 4(57.1) -

Rectum 7 - 7(100.0) -

Left colon-rectum 13 3 (23.1) 7 (53.8) 3(23.1)

Histology component 36 0.40 0.71

ADK simple 21 5 (23.8) 13 (61.9) 3 (14.3)

ADK coloid mucinous 15 5 (33.3) 9 (60.0) 1 (6.7)

Differenciation 36 0.82 1.54

Well 11 2 (18.2) 8 (72.7) 1 (9.1)

Moderate 13 4 (61.5) 8 (61.5) 1 (7.7)

Poor 12 4 (33.3) 6 (50.0) 2 (16.7)

TNM 36 0.62 2.62

Stage II 7 3 (42.9) 4 (57.1) -

Stage III 25 6 (24.0) 15 (60.0) 4 (16.0)

Stage IV 4 1 (25.0) 3 (75.0) -

ADK: adenocarcinoma; CRC: colorectal cancer; MTHFR genotype: wild-type homozygous (CC); heterozygous (CT); and mutant homozygous (TT).

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betweenMTHFR C677Tgene and CRC in Moroccan population. Regarding the correlation betweenMTHFRpolymor- phism and clinical features, we found that this polymorphism would have no impact on clinical events and histological tumor and may not be predictive factor of these parameters. This finding is consistent with what has been described by Carmen S.P. Lima et al. [27] and Toffoli

et al. [28], but not with the results published by Park et al.

[29] and Shannon et al. [11], who reported thatTTgenotype, respectively, could play a role in the prognosis of CRC with a statistically significant difference (P= 0.003) depending on the age and the presence of metastatic lymph nodes.

These contradictory results could be explained partly by the small size of our sample.

Table 2 The frequencies of theMTHFR C677Tgene polymorphism in colorectal adenocarcinoma patients and controls MTHFR

genotype

Cases (n = 36)

Controls (n= 36)

OR (95% CI)

P value

CC 10 (27.8) 25 (69.4) 1.0 Reference –

CT 22 (61.1) 8 (22.2) 6.88 (2.30–20.49) *** 0.0005

TT 4 (11.1) 3 (8.3) 3.33 (0.63–17.65) 0.16

CT-TT 26 (72.2) 11 (30.6) 5.91 (2.14–16.34) *** 0,0006

Callele 42 (58.3) 58 (80.6) 1.0 Reference –

Tallele 30 (41.7) 14 (19.4) 2.96 (1.4–6.25) ** 0.0045

MTHFRgenotype: wild-type homozygous (CC); heterozygous (CT); and mutant homozygous (TT); Odds ratio (OR); Confidence interval (CI).

Fig. 4 Genotype and allele frequency of MTHFR polymorphism in cases and control

Table 3 Relative risk of cases compared to controls MTHFR

genotype

Cases number (%)

Controls number (%)

RR (95% CI)

P value

CC 10 (27.8) 25 (69.4) 1 Reference –

CT 22 (61.1) 8 (22.2) 2.84 (1.49–5.42)** 0.0016

TT 4 (11.1) 3 (8.3) 2.67 (0.69–10.31) 0.16

CT + TT 26 (72.2) 11 (30.6) 2.36 (1.39–4.03)** 0.002

Callele 42 (58.33) 58 (80.56) 1 Reference –

Tallele 30 (41.67) 14 (19.44) 2.14 (1.24–3.69)** 0.006

RR: relative risk; wild-type homozygous, heterozygous (CT); and mutant homozygous (TT).

** Significant.

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The study of RR in patients with at least one T allele showed they have twice the risk of developing CRC (Table 3). This would imply a dominant effect of this polymorphism in our population.

These results are similar to those observed recently by Naghibalhossaini et al. [30] who reported in the Iranian population an increased risk of developing CRC (incidence OR = 2.4 and a 95% CI = 1.08–4.04) for genotype CT and combined CT and TT association (OR = 2.4, 95%

CI = 1.6–3.6). The same result was found in Dutch population by Heijmans et al. [19], whereTTgenotype has an RR of 3.65 (1.07–12.5) to develop the CRC in men.

Suggesting that functional activity ofMTHFRin a tumor could play an important role in the survival and progression of CRC,Tallele was statistically significant in our study and the individuals with at least one 677Tallele are likely to develop CRC. A meta-analysis conducted in 2007 on 10,131 patients and witnesses 15,362 by Yan Huang et al.

showed thatTallele may have a protective role against CRC [25]. These conflicting results could be explained by various factors including the mode of living. Indeed, it is known that folate metabolism depends not only on genetic determinants but also on dietary folate .It is now accepted that inadequate intake of folate increases the risk of developing precancerous lesions (adenomas) and thus the risk of CRC. Several polymorphisms are involved at theMTHFRgene or methionine synthase. It was demonstrated that theC677T MTHFRpoly- morphism has a small influence on the level of plasma homocysteine in Caucasian populations and northern Eur- ope. By cons its impact is largest in Asia and Africa where poor nutrition is promoting the imbalance in folate metabolism and the risk of cancer. This risk of developing cancer could be explained through global hypomethylation of DNA and genomic instability by deficiency of methyl radicals and the accumulation of uracil with defects in DNA repair result- ing in strand breaks DNA [6].

Conclusion

We confirmed thatTallele,CTheterozygote, and association betweenCTandTTgenotypes ofMTHFRgene are associated with an increased risk of CRC development and therefore could be a risk factor for genetic susceptibility to CRC in the Moroccan population. Future case-control studies must be conducted with a higher sample taking into account the determination of folate and vitamin B12 essay to eluci- date the mechanism of this polymorphism in colorectal carcinogenesis.

AcknowledgmentThis study is supported by Genetics and Molecular Pathology Laboratory, Faculty of Medicine and Pharmacy, University Hassan II, Casablanca-Morroco. We

thank personnel of oncology department, university hospital Center, Ibn Rochd of Casablanca for their collaboration in collecting clinical and histological data on patients.

Authors’contribution

DB participated in the design of this study, drafted the manuscript, carried out the molecular genetic studies, and participated in the interpretation of data and performed the statistical analysis. BA carried out the DNA extraction, took the samples of patients, and helped to draft the manuscript. HK participated in the sequence alignment and helped to draft the manuscript. JH carried out the patient recruitment and the histology studies. NS conceived of the study, designed and coordinated the experiments, and drafted the manuscript. All authors read and approved the final manuscript.

Conflict of interest:The authors do not have any conflict of interest.

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