Cancer Epidemiology 36 (2012) e227–e231

Methylenetetrahydrofolate reductase C677T genetic polymorphisms andrisk of leukaemia among the North Indian population

Syed Rizwan Hussain a, Hena Naqvi a, Syed Tasleem Raza a,*, Faisal Ahmed a, Sunil G. Babu c,Ashutosh Kumar d, Zeashan Haider Zaidi b, Farzana Mahdi a

a Department of Biotechnology, Era’s Lucknow Medical College and Hospital, Lucknow 226003, Indiab Department of Statistics, Era’s Lucknow Medical College and Hospital, Lucknow 226003, Indiac Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow 226025, Indiad Department of Pathology, CSM Medical University, Lucknow 226003, India

A R T I C L E I N F O

Article history:

Received 6 November 2011

Received in revised form 11 February 2012

Accepted 15 February 2012

Available online 20 March 2012

Keywords:

MTHFR

Genetic polymorphism

AML

CML

CLL

ALL

Folic acid

A B S T R A C T

Background: Leukaemia is a heterogeneous disease in which haematopoietic progenitor cells acquire

genetic lesions that lead to a block in differentiation, increased self-renewal, and unregulated

proliferation. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR), involved in folate

metabolism, plays a crucial role in cells because folate availability is important for DNA integrity. The

aim of this case–control study was to evaluate the association of the C677T MTHFR gene polymorphism

with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia

(CML) and chronic lymphocytic leukaemia (CLL). Materials and methods: A total of 275 leukaemia cases –

including AML (n = 112), ALL (n = 81), CML (n = 43), CLL (n = 39) – and 251 age/sex-matched healthy

control individuals participated in this study. MTHFR C677T polymorphisms in the cases and controls

were evaluated by polymerase chain reaction and restriction fragment length polymorphism (PCR-

RFLP). Results: The average MTHFR 677CC, 677CT, 677TT genotype frequencies of total leukaemia cases

were 68.73%, 19.64%, and 11.64% in cases, and 71.71%, 24.30%, and 3.98% in healthy controls,

respectively. The average frequency of the MTHFR 677T allele was 21.45% among the cases compared to

16.13% among the controls. Conclusions: In the present case–control study we have observed a higher

frequency of the MTHFR 677TT genotype in cases of leukaemia (AML, ALL, CML and CLL) as compared

with controls; this might be due to ethnic and geographic variation. As per our findings, although the

frequency of the MTHFR 677T allele is moderately high in AML, ALL and CLL, no statistically significant

association was found; on the other hand statistically significant association was found in the context of

CML cases.

� 2012 Elsevier Ltd. All rights reserved.

Contents lists available at SciVerse ScienceDirect

Cancer EpidemiologyThe International Journal of Cancer Epidemiology, Detection, and Prevention

jou r nal h o mep age: w ww.c an cer ep idem io log y.n et

1. Background

Leukaemia is a heterogeneous disease in which haematopoieticprogenitor cells acquire genetic lesions that lead to a block indifferentiation, increased self-renewal and unregulated prolifera-tion. Leukaemia usually arises as a result of DNA translocations,inversions or deletions in genes that regulate blood-cell develop-ment or homeostasis. Folate deficiency has been associated withuracil misincorporation into DNA which results in double-strandbreaks during uracil excision repair, thus increasing the risk of

* Corresponding author. Tel.: +91 522 2408122/2408123; fax: +91 5222407824.

E-mail addresses: rizwan59elmc@gmail.com (S.R. Hussain),

naqvi.hena@gmail.com (H. Naqvi), tasleem24@gmail.com (S.T. Raza),

faizbms@gmail.com (F. Ahmed), sunil_gos@yahoo.com (S.G. Babu),

ashutosh.kumar@hotmail.com (A. Kumar), zeashanzaidi@gmail.com (Z.H. Zaidi),

farzana.mahdi@gmail.com (F. Mahdi).

1877-7821/$ – see front matter � 2012 Elsevier Ltd. All rights reserved.

doi:10.1016/j.canep.2012.02.008

chromosomal aberrations [1]. Documented causes of leukaemia –such as ionizing radiation, benzene and cancer chemotherapy –are responsible for only a small proportion of the total cases [2].The quantitative and qualitative changes in folate metabolism areenhancing risk factors for leukaemia [3]. The 5,10-methylenete-trahydrofolate reductase (MTHFR) gene is located on the shortarm of chromosome 1 at locus 1p36.3. The cDNA sequence of thisgene is 2.2 kb long, composed of 11 exons (103–432 bp), and themajor product of the MTHFR locus in humans is a 77-kDa protein[4]. The MTHFR directs 5,10-methylenetetrahydrofolate (5,10-methylene-THF) towards methionine synthesis at the expense ofDNA synthesis. A lower MTHFR activity leads to increased plasmalevels of homocysteine and decreased 5-methyl-THF formation.This contributes to the pool of methylene-THF, methyl groupdonor for the conversion of dUMP to dTMP by thymidylatesynthase. Therefore, it is thought that the lower activity of MTHFRmight favour optimal DNA synthesis by reducing the uracil

S.R. Hussain et al. / Cancer Epidemiology 36 (2012) e227–e231e228

misincorporation rate, a potential cause of double-strand breaksduring excision repair processes [5,6]. Defects or polymorphism inthe genes of the folate-dependent enzymes and deficiency ofmicronutrients may influence cancer susceptibility [5,7–9].Investigations on mutations in the MTHFR gene have focusedon the catalytic domain and the two polymorphisms C677T andA1298C, which slightly change enzymatic activity [10]. C677Tpolymorphism in exon 4, which causes alanine to be substitutedby valine at codon 222, is the most frequently studied polymor-phism in this gene. This allele is commonly termed thermolabilebecause its activity decreases above 37 8C [11]. Individuals withthe homozygous MTHFR 677TT genotype have 30% enzymeactivity compared with those having the wild-type allele, whilethe enzyme activity is 60% in individuals with the heterozygousMTHFR 677CT allele [4]. The reduced MTHFR activities were foundto be correlated with a decreased risk of leukaemia, lymphomaand colorectal carcinoma. Although clinical, pathological andimmunological phenotypic features of the disease are welldocumented, little is known about leukaemogenesis [12]. TheMTHFR gene C677T presents a higher risk of cardiovasculardisease and birth defects, particularly neural tube defects,coronary artery disease [4], cerebrovascular disease, venousthrombotic disease [13], squamous-cell carcinoma [14], breastcancer [15], colorectal cancer [16,17], Turner’s syndrome [18],endometrial cancer [13], schizophrenia [19], and hypertension[20]. The C677T polymorphism in the MTHFR gene and the risk ofdifferent kinds of leukaemia chiefly accentuate a lower risk ofacute lymphoid leukaemia (ALL) in people possessing the 677TTvariant [21]. However, conclusions about this correlation withother acute and chronic forms of leukaemia are controversial[3,22–25]. Currently, there are no data concerning the linkbetween the MTHFR polymorphism and susceptibility to leukae-mia in the North Indian population. Thus, we designed a case–control study to evaluate the MTHFR C677T polymorphism incases of ALL, acute myeloid leukaemia (AML), chronic myeloidleukaemia (CML) and chronic lymphocytic leukaemia (CLL)among the North Indian population.

2. Materials and methods

2.1. Study samples

The population we studied consisted of 275 cases of leukaemiaand 251 healthy controls. Ethical approval was obtained from theinstitutional ethical committee of Era’s Lucknow Medical andHospital, Lucknow, Uttar Pradesh, India. This study was carried outbetween March 2006 and December 2010.

Fig. 1. Polyacrylamide gel electrophoresis analysis of MTHFR C677T polymorphism. Lane

genotype (98, 175, 23 bp), Lane 1 & 12 100 bp Ladder.

2.2. DNA isolation

Specimens were collected from 275 routinely processedunstained bone-marrow slides and blood diagnosed as leukaemiafrom the Department of Pathology at Era’s Lucknow MedicalCollege and other hospitals and pathology centres available inLucknow. Genomic DNA extraction was done using a commerciallyavailable DNA extraction kit (Medox, India) and the DNA wasstored at �20 8C.

2.3. Genotyping of the MTHFR gene C677T polymorphism

The MTHFR C677T polymorphism was analysed by thepolymerase chain reaction (PCR) followed by restriction fragmentlength polymorphism (RFLP). Genomic DNA was amplified (MJMini Thermocycler, Bio-Rad, UK) using the following PCRconditions: 94 8C for 4 min, 34 cycles at 94 8C for 30 s, 60.7 8Cfor 45 s, 72 8C for 45 s, and finally 72 8C for 12 min. The primersused for amplification of the MTHFR C677T gene polymorphismwere as follows: forward primer 50-TGA AGG AGA AGG TGT CTGCGG GA-30 and reverse primer 50-AGG ACG GTG CGG TGA GAG TG-30 [21]. Amplification was performed with 25 mL PCR reactionmixture containing 100 ng template DNA, 10 pmol of each primerand 2� PCR master mixes (Fermentas, Germany). Amplificationsuccess of samples was monitored by 2% agarose gel electropho-resis. Hereafter the PCR products were subjected to digestion byHinf1 enzyme (NEB, UK) to screen for the C677T polymorphism.The enzymatic mixture contained 0.6 mL restriction enzyme, 2 mL10� buffer, 10 mL PCR products and 7 mL distilled water; themixture was incubated overnight at 37 8C for digestion. Thedigested product was electrophoresed on 10% non-denaturatingpolyacrylamide gel electrophoresis (PAGE) at 110 V for 2 h. In thecase of C677T polymorphism, an undigested 198 bp band showedwild-type CC genotype, while two bands of 175 and 23 bpconfirmed mutant TT genotype and three bands of 198, 175 and23 bp were detected in the heterozygous CT genotype [21] (Fig. 1).

2.4. Statistical analysis

The significance of this study was evaluated by Chi-square orFisher’s exact test. Odds ratio (OR) was calculated as an estimate ofrelative risk of having disease according to the relative frequency ofdifferent genotypes among the cases as well as the controls(adjusting for age and sex). ORs are given with 95% confidenceinterval (CI). P-value was considered significant at <0.05. Allstatistical tests were performed using SPSS (Statistical Package forthe Social Sciences) version 12 software.

6, 8–11 CC genotype (198 bp), Lane 4 TT genotype (175, 23 bp), Lane 2, 3, 5 & 7 CT

Ta

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.

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(71

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CT

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S.R. Hussain et al. / Cancer Epidemiology 36 (2012) e227–e231 e229

3. Results

In our study we covered 275 leukaemia cases, including 142males and 133 females, their ages ranging from 2 to 68 years.Calculated mean age of cases was 39.46 years, SD � 19.47. Themean age of 251 healthy controls was 41.53 years and SD � 11.72.Cases were further categorized as AML (n = 112), ALL (n = 81), CML(n = 43) and CLL (n = 39). All the cases and controls were successfullygenotyped by PCR-RFLP. The average MTHFR 677CC, 677CT and677TT genotype frequencies in total leukaemia cases were 68.73%,19.64, 11.64%, and 71.71%, 24.30% and 3.98% in healthy controls,respectively.

In 112 AML cases, MTHFR 677T allele frequency was 21.42%,while the genotype frequencies of 677CC, 677CT and 677TT were66.96%, 23.21% and 9.82%, suggesting no statistically significantdifference as compared with controls (OR = 2.6, 95% CI: 1.0–6.4;OR = 3.0, 95% and CI: 1.4–6.3).

Out of 81 ALL cases, the frequency of the MTHFR 677T allele wasfound to be 27.16% while the genotype frequencies of 677CC,677CT and 677TT were 72.84%, 16.05% and 11.11%, whichestablishes no statistically significant difference as compared withthe controls (OR = 2.7, 95% CI: 1.0–7.0; OR = 3.0, 95% CI: 1.4–6.3).

MTHFR 677T allele frequency was 34.88% in 43 CML cases. Thegenotype frequencies of 677CC, 677CT and 677TT were 65.12%,18.60% and 16.28%, which indicates a statistically considerabledifference compared with the controls (OR = 4.5, 95% CI: 1.5–12.7;OR = 3.0, 95% CI: 1.4–6.3).

In 39 CLL cases the MTHFR 677T allele frequency was observedas 21.79%. The genotype frequencies of 677CC, 677CT and 677TTwere 69.23%, 17.95% and 12.82%, which represents no statisticallysignificant difference compared with the controls (OR = 3.3, 95% CI:1.0–10.4; OR = 3.0, 95% CI: 1.4–6.3).

The frequency of MTHFR C677T polymorphism and statisticalanalysis of the cases and controls are shown in Table 1.

4. Discussion

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a keyenzyme in folate metabolism, and changes in its activity resultingfrom polymorphisms in the MTHFR gene could modify suscepti-bility to cancer. The MTHFR 677T polymorphism shows significantethnic and geographical variations. During one of the studies of7000 newborns from 16 regions worldwide, the 677T allele variedfrom 26.6% to 46% in Italy, 25.7% in the Middle East, and 44.2% innorthern China [26]. In addition, a study in Lebanon reported thefrequency of the MTHFR 677TT homozygous genotype to be 7.66%and 13.08% in Muslim Arabs and Christians, respectively [27].

A detailed review of MTHFR 677T allele and 677TT genotypefrequency is included in Table 2. Analysis of control groupsconfirms a relatively low frequency of MTHFR 677TT polymor-phism among the Iranian population when compared to otherAsian populations, particularly the eastern countries [11,15,28].There are few studies about the role of MTHFR polymorphism indeveloping chronic leukaemia, especially CML. In the studyperformed by Moon and colleagues, MTHFR 677T allele did notinfluence the risk of CML in Korean populations [23]. Individually,there was no evidence that MTHFR 677T polymorphism affects therisk of CLL or, restricting the analysis to cases, provided noevidence of an association between MTHFR polymorphism and therisk of CLL [29]. Among the results contrasting with theaforementioned studies, Hur and colleagues observed that norelationship was found between MTHFR C677T variants (CT andTT) and CML, AML and ALL development [24]. However, Deligezerand colleagues observed a similar pattern in the distribution ofMTHFR C677T polymorphism in myeloid and lymphoid leukaemia,in such a way that the frequency of MTHFR 677TT genotype in all

Table 2Comparison of the frequencies of MTHFR C677T polymorphisms among leukaemia cases and controls in different ethnic groups.

Populations Study No. of total

cases/control

Leukaemia Control Reference

CC CT TT CC CT TT

United Kingdom

CLL 832/886 361 (43.38%) 381 (45.79%) 90 (10.81%) 383 (43.22%) 397 (44.80%) 106 (11.96%) [29]

North and Southwest England

ALL 71/114 35 (50.7%) 29 (42.0%) 5 (7.2%) 61 (53.5%) 39 (34.2%) 14 (12.3%)

AML 237/377 134 (60.1%) 66 (29.6%) 23 (10.3%) 196 (53.1%) 130 (35.2%) 43 (11.7%) [21]

Italian

ALL 174/110 65 (37.4%) 71 (40.8%) 38 (21.8%) 35 (31.8%) 55 (50%) 20 (18.2%) [31]

Japanese

ALL 97/243 44 (45.36%) 39 (40.20%) 14 (14.43%) 81 (33.33%) 126 (51.85%) 31 (12.75%) [13]

Egyptian

ALL 88/311 39 (44.3%) 42 (47.7%) 7 (8%) 156 (50.2%) 135 (43.4%) 20 (6.4%) [33]

Iranian

AML 106/97 58 (54.71%) 39 (36.79%) 9 (8.49%) 56 (57.73%) 37 (38.14%) 4 (4.12%) [30]

CML 38/97 24 (63.15%) 11 (28.94%) 3 (7.89%)

North Indian

AML 112/251 75 (66.96%) 26 (23.12%) 11 (9.82%) 180 (71.71%) 61 (24.30%) 10 (3.98%) Our study

ALL 81/251 59 (72.84%) 13 (16.05%) 9 (11.11%)

CML 43/251 28 (65.12%) 8 (18.60%) 7 (16.28%)

CLL 39/251 27 (69.23%) 7 (17.95%) 5 (12.82%)

AML, acute myeloid leukaemia, ALL, acute lymphoblastic leukaemia; CML, chronic myeloid leukaemia; CLL, chronic lymphocytic leukaemia.

S.R. Hussain et al. / Cancer Epidemiology 36 (2012) e227–e231e230

three malignancies of CML, AML and ALL was lower than those ofthe controls, although the differences were not statisticallysignificant [25]. Vahid et al. reported the frequency of MTHFR677T 23.19% and 26.88% in controls and AML cases respectively,while the frequencies of MTHFR 677TT genotype were 4.12% and8.49% in the controls and cases, respectively, in the Iranianpopulation [30]. In our study, we have detected a low MTHFR677TT genotype frequency (3.98%) in the controls compared tocases, and the highest MTHFR 677TT genotype frequency (16.28%)was detected in CML cases, while in AML, CLL and ALL thefrequencies of the MTHFR 677TT genotype were found to be 9.82%,12.82% and 11.11% respectively. The frequencies of the MTHFR677T allele in AML, ALL, CML, CLL and controls were found to be21.42%, 27.16%, 34.88%, 21.79% and 16.13%, respectively.

The result of MTHFR 677TT genotype polymorphism in ourstudy in AML cases (9.82%; OR = 2.64, 95% CI: 1.07–6.4) iscomparable to the MTHFR 677TT genotype polymorphism of8.49% in Iranian AML cases [30], and MTHFR 677TT genotypepolymorphism in ALL cases (11.11%; OR = 2.74, 95% CI: 1.06–7.08)is comparable to the 14.43% MTHFR 677TT genotype polymor-phism in ALL cases in the Japanese population [13]. Apparently thefrequency of MTHFR 677TT genotype polymorphism in CML cases(16.28%; OR = 4.5, 95% CI: 1.58–12.7) was significantly highcompared to the earlier study of MTHFR 677TT genotypepolymorphism cases of CML (7.89%) in the Iranian population[30]. The MTHFR 677TT genotype polymorphism in cases of CLLwas 12.82% (OR = 3.33, 95% CI: 1.05–10.4) which is comparable toMTHFR 677TT genotype polymorphism in cases of CLL of 11.96% inthe United Kingdom population [29].

In the present case–control study, we have observed higherfrequencies of MTHFR 677TT genotype in cases of leukaemia (AML,ALL, CML and CLL) as compared with controls; this might be due toethnic and geographic variation. As per our findings, although thefrequency of the MTHFR 677T allele is moderately high in AML, ALLand CLL, no statistically significant association was found; on theother hand a statistically significant association was found in thecontext of CML cases. The involvement of a high MTHFR 677T allelefrequency (34.88%) observed for CML cases compared to AML, ALL,and CLL is a specification for prospective investigations, as thegenetic pattern may affect and consequently lead to CML in theNorth Indian population. Future studies involving a larger groupmight be required to validate this study.

Conflict of interest

The authors have no conflict of interests in this article.

Acknowledgements

We are grateful that the study was supported by an intramuralgrant from the Era’s Lucknow Medical College and Hospital,Lucknow, Uttar Pradesh, India.

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