Mutational Analysis of the MTHFR Gene in Breast Cancer Patients of Pakistani Population

Breast cancer begins in the any part of breast, caused by abnormal cells growth and division. MTHFR association with cancer anomalies also gets a very recent attention. MTHFR gene provides instruction for making an enzyme known as methylenetetrahydrofolate reductase. Methylenetetrahydrofolate reductase (MTHFR) is playing a central role in folate and homocysteine metabolism that catalyzes the conversion of 5-10 methylenetetrahydrofolate to 5-methylenetetrahydrofolate which is circulatory folate which later utilized into homocystein remethylation by methionine (Rosenblatt, 1995). MTHFR gene is located on short arm (p) of the chromosome 1 (1p36.3) and has


Introduction
Breast cancer begins in the any part of breast, caused by abnormal cells growth and division.MTHFR association with cancer anomalies also gets a very recent attention.MTHFR gene provides instruction for making an enzyme known as methylenetetrahydrofolate reductase.Methylenetetrahydrofolate reductase (MTHFR) is playing a central role in folate and homocysteine metabolism that catalyzes the conversion of 5-10 methylenetetrahydrofolate to 5-methylenetetrahydrofolate which is circulatory folate which later utilized into homocystein remethylation by methionine (Rosenblatt, 1995).MTHFR gene is located on short arm (p) of the chromosome 1 (1p36.3)and has (FSH) (Gaughan et al., 2000).MTHFR gene has two promoters and isoform (70 kDa and 77 kDa) (Tran et al., 2002).Human MTHFR gene is composed of eleven (11) exons that encode a protein of 656 amino acids.Analysis of promoter regions of MTHFR gene revealed that it does not have a TATA box that contain CpG islands, which have

Mutational Analysis of the MTHFR Gene in Breast Cancer Patients of Pakistani Population
Muhammad Akram, FA Malik, Mahmood Akhtar Kayani* MTHFR gene is involved in diseases was firstly inborn error of folate metabolism.In 1988, a MTHFR assays of lymphocyte extract in cardiovascular disease patients (Kang et al., 1988).Different epidemiological studies showed that folate low level in the development of the cancer in several organs as cervix, colorectal, lungs, brain, pancreas and breast (Kim, 1999).The variation in MTHFR gene C677T and A1298C have been associated with reduced activity of MTHFR enzyme that increased the availability of folate for thymidilate and purine synthesis.These variants have relation with invasive breast cancer, which was investigated by multiethnic cohort study of man and postmenopausal women of Japan (Goyette et al., 1998).The role of MTHFR C677T gene polymorphism in breast cancer risk is very controversial.Allele T seems to be involved in cancer risk (Henríquez et al., 2010).While the study of treated patients with adjuant therapy show the C/C variant of MTHFR C677T polymorphism suffer a four times higher risk of multifocal lesions in the tumor (Henríquez et al., 2010).
About 9 to 12% of the general population in Taiwan population is TT homozygous effected lacking the enzyme responsible for metabolizing folic acid.44% are CT affected which cannot fully metabolized folic acid to its active form (Molloy, 1997).MTHFR gene has two most nucleotide addition which result in change of amino acids C677T ala222val and A1298C to Glu429Ala (Goyette et al., 1998;Kim, 2000).
In this study, MTHFR germline and sporadic mutations of breast cancer patients are investigated.So far no single study in relation to MTHFR germline mutations in breast cancer in Pakistan population has been documented yet.We are primarily interested to explore local ethnic sporadically affected breast cancer patients for any polymorphism/mutation.As germline involvements may further impart as an associated risk for this disease.

Materials and Methods
110 samples of breast cancer patients along with 110 normal individuals without any occurred disease (taken with no earlier family history of any type of cancer were involved in this study.Postmenopausal stage and unilateral or multifocal breast cancer affected patients were included. from Combined Military Hospital (CMH, Rawalpindi), Nuclear and Oncology Radiation Therapy Institute (NORI, Islamabad) and Allied Hospital (Faisalabad) in Pakistan.Informed consents of these patients with a prior and oncologists were taken prior to sampling.Blood samples (5ml) were drawn from these diagnosed patients and controls.Samples were transported to laboratory for further processing under proper storage conditions.
DNA extraction was carried out from blood samples by using organic method as mentioned in the previous protocol with minor modifications (Helms, 1990).Electrophoresis of these DNA samples was carried out on 1% agarose gel for 45min at 120V.Gel was later immersed in 0.5µg/ml ethedium bromide solution for DNA staining MTHFR gene is amplified by different markers that may design by using Primer 3 software.Primers designed in such a way that intron and exon junctions were also screened in this study.The coding exons and using polymerase chain reaction (PCR) 20 µl volume, consist of 0.3 mM deoxyribonucleotide (dNTPS), 1X PCR buffer (10 mM), 2 mM Mgcl2, 0.5 µM each primers (forward and reverse), 1.5 U Taq Polymerase and 50ng of the genomic DNA.The thermal cycling consisting of PCR product was electrophoretically separated by using 2% agarose gel with ethidium bromide stained.Bands of PCR were visualized by UV transilluminator and gel doc.
Single stranded conformational polymorphism (SSCP) was done following the previously mentioned protocol (Orita et al., 1989) and Restriction fragment length using SSCP for any mobility shift and alterations and product of desired samples was purified using DNA Ontario).

Results
The genotype frequencies of breast cancer patients and controls are summarized in    , 2001;Shrubsole et al., 2001, Feigelson et al., 2003).
of uracil into DNA that causes chromosomal breaks and DNA repairs disrupted has also been reported in literature deficiency may cause uracil misincorporation in the human DNA that induces chromosomal breakage and is involved in cancer progression (Blount et al., 1997& Ames, 1999).Three variants regarding MTHFR gene as C677T, G1298C and G1793A MTHFR polymorphisms extensively reported in relation to different populations.SNP (C677T) codes an alanine amino acid into valine as substitution in the N-terminal catalytic domain the second SNP (A1298C) that codes an alanine amino acid to glutamine addition in the C-terminal of regulatory domain.
The MTHFR gene at the G1793A polymorphism results an arginine to glutamine change at codon 594 (Rady, 2002) not been reported.These two SNPs are correlated with the enzymes thermolability and reduced invitro enzymatic activity (Frosst, 1995;Weisberg, 1998).
In the present study, association of MTHFR gene polymorphism in sporadic breast cancer patients of local population was evaluated.Both coding as well as non-coding regions were included in this screening to identify the penetrance of any splice site variations as well.Nine exons were selected to be screened for mutational analysis i.e. exons 1 up to 9. The polymorphism (C677T and A1298C) or mutation was found in this study as compared with normal samples.Similar results have been found in earlier studies as well in Korean women al., 2004;Perry et al., 2004;Justenhoven et al., 2005).our population is also in accordance to the previously gene polymorphism (677C>T) has been observed while screening 110 breast cancer diagnosed patients along with 110 normal samples.No association among reduced plasma folate level and elevated homocysteine levels were found in relation to the MTHFR polymorphism.
Moreover the polymorphism studies showing the said polymorphism have all been carried out in caucasian population.Only few studies from Asian countries have been published and that has emphasized on the lack of polymorphism involved in breast cancer patients for these variants (Ford and Bowmwn, 1999;Perry et al., 2004).Moreover family history seems to be important for any association of polymorphism/mutations with increased risk of breast cancer.Patients in study showing positive association, for polymorphism for C677T and A1298C SNPs with increased risk of breast cancer had strong family history (Kotsopoulos et al., 2008), whereas all patients recruited in this study had no family history of earlier report conducted in different populations.As in Jewish population, bilateral breast cancer or combined breast and ovarian cancer causes were higher in individual having 677T allele on MTHFR gene (Gershoni et al., 2000).Diet factors and other genetic polymorphism of MTHFR can influence results.That finding gives useful information to comprehend the molecular basis of breast cancer development, malignancy, progression and outcome (Henríquez et al., 2010).to variation in sample size, population involved and folate dietary intake variation.But it is needed to be further explored considering the following possible explanatory reasons for the negative results found in this study.
In conclusions, Pakistani population although may offer potential to explore the contribution to consanguinity for hereditary form of the cancer and it might not be the case for sporadic cancer.It is possible that MTHFR gene polymorphism is present in low frequency as compared with that found in many other studies in of polymorphism for MTHFR gene (C677, A1298C) has been observed in breast cancer patients, the genotypic data of 677TT 677CC, 677CT, and 677TT genotypes were 0.2230, 0.0060, and 0.0770, respectively.While for the 1298AA, 1298AC, and 1298CC genotypes were 0.5545, 0.0092, and 0.0029, respectively.We also found These results require a further through analysis to be Since altered regulation of several gene may also be attributed not only to mutation in coding regions but also to variation in expressional levels of transcriptional factors, promoter methylation and loss or gain of binding sites even upstream regulatory regions of the genes.Hence we strongly recommended a further screening of MTHFR levels in the body with respect to different stages of tumors in a large size cohort study.

Figure 1 .Figure 2 .
Figure 1.Electropherogram of the Ethidium Bromide Stained 1% Agarose Gel Showing Extracted DNA of Sample.The P1 to P6 refer to patients and N refers to standard DNA

Figure
Figure 3. Electropherogram of the Ethidium Bromide Product of Exon 7 Diseased Samples.The P 1-P5 refers to the patients involved in present study

Table 2 .
Polymerase chain reaction product of exons 4 and 7 of MTHFR are shown in samples.The variation of band mobility in patients was