Association of Methylenetetrahydrofolate reductase (MTHFR 677C>T) polymorphisms with Ovarian Malignancy in women of Northern India

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1 214 Association of Methylenetetrahydrofolate reductase (MTHFR 677C>T) polymorphisms with Ovarian Malignancy in women of Northern India Authors:, Rinki Kumari 1, Anamika Tiwari, Aruna Agrwal, G.P.I. Singh, G P Dubey Short Title: MTHFR gene and ovarian cancer. Abstract: Background- Ovarian cancer is a major gynecological issue and associated with hyperhomocysteinemia is an independent risk factor. Folate and Vitamin B12 & B6 deficiency is the most common cause of hyperhomocysteinemia, thereby provoking a possible association between ovarian cancer and MTHFR C677T polymorphism. The Methylenetetrahydrofolate reductase (MTHFR) gene is a polymorphic gene involved in folate metabolism, DNA biosynthesis, methylation and genomic integrity in actively dividing cells. Objective: The aim of this present study was to investigate an association of MTHFR C677T polymorphism with ovarian cancer and level of folic acid, homocysteine, vitamin B12 & B6 in the ovarian cancer patients and their respective, healthy control. Material and Method: In a case-control study, biochemical analysis was carried out by commercial available ELISA Kit while genetic analysis (PCR-RFLP of peripheral leucocytes) was carried out on all women with ovarian cancer as well as controls. Statistical Analysis: Student t test for quantitative and Chi-square test for nominal variables was used. For estimation of risk, odds ratio and 95% confidence interval were calculated. Result: Frequencies of MTHFR C677C, C677T and T677T were 27.11, and 15% in the ovarian cancer subject and 94.50, 5.49 and 0.00% in the controls, respectively. The results of a Chi square analysis indicated that the MTHFR 677T allele was significantly distributed (p = 0.001). Likewise, the MTHFR T677T genotype showed a 1.9fold increased risk for ovarian cancer. In this case control study, we have evaluated levels of homocysteine but decreased level of folic acid, vitamin B12 and B6 in subject as compare control. Folate and homocysteine levels showed statistically significant between the these two groups; however an increasing trend of homocysteine levels associated with increasing rate of ovarian cancer. We observed the negative correlation between homocysteine and biochemical parameter in the cases. In conclusion, Our data suggest that 1 Rinki Kumari Department of kriya Sharir, Faculty of Ayurveda, Institute of Medical Sciences, B.H.U. Varanasi (U.P) : INDIA

2 215 the MTHFR C677T polymorphism and low folate and vitamin B12 & B6, associated with ovarian cancer and act as risk factor for ovarian cancer. KEY WORDS: Ovarian cancer, Folate, Vitamin B12, Vitamin B6, Homocysteine Introduction Ovarian cancer is considered to be the most common deadly gynecological cancer and is the fourth leading cause of women cancer, worldwide1.like other malignancies, it is also a hereditary disease with multiple genetic events leading to the cancerous phenotype with high mortality. Importantly, the interaction between Gene-Gene and Gene-Environmental involved in the pathogenesis of this cancer and additionally, another factor such as family history, tobacco smoking, infertility and hormone replacement therapy2. Several studies have suggested that deficiency of nutrients, such as folate, vitamins, and microelements, has also been associated with increased risk for ovarian cancer2. Food supplement are affecting gene expression through interaction with genetic polymorphism and modulation of DNA methylation. Deficient nutrient (folate and vitamin B12) are involved in the disturbance of equilibrium between vitamin-dependent and folate metabolism, associated with heart disease, neural tube defects and cancer included endometrium cancer, ovarian cancer by influencing DNA methylation3. Low folate (dietary deficiency of folate or genetic MTHFR deficiency) causes hyperhomocysteinemia, involved in the pathogenesis of several clinical condition such as heart disease, neural tube defects, mental retardation and cancer including ovarian cancer4 and modulate expression of oncogenes, and synthesis of purine and thymidylate, which is vital for DNA repair5. Generally, hyperhomocysteinemia, is reduced activity of MTHFR, an enzyme involved in folate- dependent remethylation of homocysteine to methionine6. A common thermolabile mutation in the MTHFR gene, consisting of a cytosine (C) to thymidine (T) substitution at nucleotide position 677, leads to the exchange of a highly conserved alanine to valine (677C T), resulting in reduced activity of this enzyme and, hence, folate distribution in the blood. The MTHFR 677 TT genotype led to elevated homocysteine levels and DNA hypomethylation in folate-depleted subjects4,7. A number of studies found a positive correlation between hyperhomocysteinemia and low folate and ovarian cancer. Association between ovarian cancer and MTHFR C677T polymorphism has been investigated in various population groups. Since there is no evidence

3 216 for Northern Indian women, we designed this study in an effort to investigate a possible correlation between ovarian cancer, its biochemical parameters, and MTHFR C677T polymorphism. Materials and Methods Subject: A case-control study was designed taking 59 young women aged years with ovarian cancer from Department of Obstetrics & Gynecology, Adesh medical Hospital, Bhatinda, Punjab. Controls were randomly selected from the hospital who requested general health examinations in the same hospital during the same period and they had no history of any type of cancer. All Indian female cases newly diagnosed with primary ovarian cancer in the hospital between April 2013 and February 2015 was invited to face-to-face interviews within two months after diagnosis. All cases recruited in this study were histological confirmed. Blood samples were collected from all subjects after an overnight fast into two anticoagulant tubes. One tube stored at 4 C for genomic DNA isolation. Another, tubes were immediately placed on crushed ice, protected from light and centrifuged within 20 minutes at 2000xg for minutes and separate the plasma were stored at -80 C until analysis of homocysteine, folate, and Vitamin B 12& B6. The study was dually approved by ethical committee of the Institute of Medical Sciences and samples were collected after written consent either from the patients or their attendant. Biochemical tests Plasma levels of folic acid, vitamin B12 & B6 and homocysteine were determined using commercially available ELISA Kit (cloud clone crop- folic acid, Abnova -vitamin B12 and homocysteine and for Vitamin B6 Uscn Life Science Inc. Wuhan). Isolation of genomic DNA and genetic analysis Genomic DNA was prepared from peripheral blood by commercially available Bioner DNA isolation Kit. MTHFR (C677T gene was analyzed by polymerase chain reaction (PCR) of genomic DNA by using the following primer pairs: MTHFR C677T (F=5 -TGA AGG AGA AGG TGT CTG CGG GA-3 and R=5 -TGA GAG TGG GGT GCA GGG AGC TT-3 ). We have developed PCR specific strategies using forward and reverse primers in total volume of

4 µl contain ng of DNA, 20 pmole of each primer, 200µM of each dntps mix with Taq buffer (10mM Tris-HCl ph 8.3, 50mM KCl), 3.0mM MgCl2 and 3 unit of Taq polymerase (New England Biolab). Cycling conditions were 4 min at 94ºC for initial denaturation, 58 ºC/1min for annealing followed by 35 cycles and 72 ºC/7min for final extension.rflp analysis was carried out for the polymorphism analysis of MTHFR C677T allele. PCR product (6 µl) were digested at 37ºC for 3hr. in a reaction volume of 25 µl containing 1U of Hinf-I restriction enzyme (New England, Biolabs) and NEB buffer (2.5 µl). The digested product of RFLP was separated on 3% agarose gel stained with EtBr and visualized on Gel Doc system (SR Biosystem). Statistical analyses All the values were calculated as Mean ± SD (standard deviation). Correlation of total homocysteine with other biological parameters was calculated by Person s correlation. Mean values obtained for the different subgroups were compared using one-way ANOVA. Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 16 software. The significance (p<0.05) differences between cases and controls group were evaluated using x2 test. Statistical analysis was further carried out to compare the expected and observed value by using Hardy Weinberg equilibrium to determine the allele frequency (C/T). The odd- ratio was computed at 95% confidence interval to evaluate the risk factor between cases and controls. Result: The case control association study was carried out with 59 ovarian cancer patients and 91 controls. The average ages of cases and controls were ± 4.75 years and ± 4.22 years, respectively (Table 1). There were no significant differences between cases and controls in age and BMI. The anthropometric and biochemical of the present study (cases and controls) were compared in Table 1 and showing the statically significant differences between subject and control. Table-2 shows the Pearson correlation of homocysteine versus folic acid, vitamin B12 and vitamin B6. Significant negative correlations between plasma homocysteine levels and folate levels, vitamin B12 levels and vitamin B6 levels were determined in the study population. The Pearson correlation coefficient between homocysteine and folic acid was in subjects as compared to 0.47 in controls indicating that homocysteine levels

5 218 increase with the decrease in folic acid in subjects. An inverse correlation was observed between vitamin B12 & B6 and homocysteine levels in both cases and controls. This study was showing the evidence of deviation from the Hardy-Weinberg equilibrium among the case and control groups. Genotypic and allelic frequencies of MTHFR C677T in ovarian cancer and control, shown in Table 2. Although a higher level of heterozygocity (57.63%) was observed in subject than control, statistically significant difference was observed with respect to MTHFR genotypic frequencies. The 677C variant allele frequencies among ovarian cancer and control were, respectively, 0.55 and Also the distribution of the T677T genotype showed, significant difference among control and case (Table 2). Considering the 677CT+677CC genotype combination as a reference, extremely significant association was observed for any of the combinations examined (Table 3). The sample size used in the study had a sufficient statistical power (>90%) to detect a possible association. However, CT genotype was times more in ovarian cancer as compared to controls. No homozygous mutant (TT) genotype was found in the controls but 15% women whose were suffering from ovarian cancer. In cases, the anthropometric and biochemical parameters were compared in relation to the genotypes (CC, CT and TT) (Table 4). Plasma folic acid was more in wild-type genotype (CC) genotype (7.52±0.26 ng/ml) as compared to both genotype (CT & TT) genotype in women with ovarian cancer but not a significant difference between CC and CT whereas it was extremely significant with TT (CC/TT). Similarly, homocysteine was more in mutant homozygous (TT) genotype (11.13±1.6μmol/L) as compared to normal (CC) genotype (10.72±1.75). Difference between wild homozygous (CC) and heterozygous were exit but did not reach significant while significant differences occur only between CC and TT. Table 1: Demographic, anthropometric and biochemical parameters of the cases and controls. Parameter Ovarian cancer Control P value

6 219 N=59 N=91 Age (years) 27.08± ±4.22 NS BMI (kg/m2 ) 26.97± ±1.49 NS Family history of Ovarian 39(66.10) 10 (10.98) ---- cancer, n (%) Folic acid (ng/ml) 7.18± ±0.76 <0.0001* Homocysteine (μmol/l) 11.01± ± * Vitamin B12 (pg/ml) ± ±77.92 <0.0001* Vitamin B6(nmol/L) 38.59± ±1.36 <0.0001* All the values are expressed as Mean±SD. Significant values are expressed as *P<0.05, **P<0.01 and ***P<0.001 compared with Normal (one Way ANOVA followed by Turkey s test) N= number; Unpaired t test.*sd - standard deviation; BMI - body mass index. Reference value for homocysteine-5-15 micromoles /L; Vitamin B nmol/l;vitamin B pg/ml and 3-17 ng/ml for folic acid were considered for comparison of test results. Table-2: Correlation between total homocysteine and biological parameters. Case (n=59) Control (n=91) Biological parameters r value P value r value P value Folate (mcg/l) Vitamin B12 (pg/ml) *.015 Vitamin B6 (pg/ml) Table-3 :Genotype distribution and allele frequency of MTHFR C677T polymorphism in ovarian cancer and Controls) along with Odds ratio and 95% CI. Genotype Ovarian Control Chi square OR 95% CI p-value cancer N=59 N=91 CC 16(27.11) 86(94.50) ( ) <0.001 CT 34(57.62) 5(5.49) ( ) <0.001 TT 9(15) 0(0) Inf(2.93-inf) <0.001 C (66) 0.55 (179) ( ) <0.001

7 220 T (52)0.44 (5) ( ) <0.001 CT+CC vers TT 59(39%) 9(61%) ( ) <0.005 TT+CT vers CC 59(41%) 86(59%) ( ) <0.001 CT vers CC 50( 35%) 91(65%) ( ) <0.001 Correlation coefficient value-r value. Table 4: Anthropometric and biochemical parameter of ovarian cancer according to the genotypes CC, CT and TT of MTHFR gene C677T polymorphism Parameters. Parameter CC CT TT (N=16) (N=34) (N=9) Age (years) 25.22± ± ±4.17 BMI (kg/m2 ) 26.71± ± ±0.26 Folic acid (ng/ml) 7.52± ± ±0.95 Homocysteine (μmol/l) 10.72± ± ±1.6 Vitamin B12 (pg/ml) ± ± ± Vitamin B6 (pg/ml) 40.99± ± ±4.92 Discussion: Epidemiological studies suggest this gynecological cancer (ovarian cancer), one of the most frequent malignancies and is a major public health issue in both developing and developed country8,9. In this present study, we have undertaken a case-control study to investigate the role of the anthropometric and biochemical parameter along with MTHFR (C677T) gene polymorphisms and their susceptibility in ovarian cancer. In the present study age and Body mass index (BMI) were normal the ovarian cancerous subject as like healthy female and was not a risk factor for ovarian cancer. Numerous studies found the deficiency of food supplements or folate along with macronutrients such as vitamin B12 & B6 associated with the development of several clinical condition and cancer 1,10,11. A large number of studies supports the key role of folate in the human health whereas low folate associated with increased risk of neural tube defect including other clinical condition and several cancer as like colon cancer breast, cervical cancer and ovarian cancer etc1,5,911,13. Our results of present studies showing the same finding associations between biochemical measures of low folate & low Vitamin B12- B6 status and risk of ovarian cancer when compared a control.

8 221 Folate deficiency induces hyperhomocysteinemia that induces genetic instability because low folate decrease DNA methylation, which is a nearly universal feature of early tumor genesis. Although, insufficient DNA methylation may promote carcinogenesis by the suppression of proto-oncogene s and were associated with a higher misincorporationn of uracil into DNA as well as with increased incidence of micronucleus in peripheral lymphocytes and it was also positively correlated to homocysteine levels in the blood. However, ovarian cancer have been developed through a large number of the activation of different oncogenes7. MTHFR, an enzymes that play a crucial role in the folate metabolic pathway due to involvement in both DNA synthesis and DNA methylation. Interestingly, in the recent year we have observed the genetic susceptibility in various types of tumors leads to a growing attention to the MTHFR C677T gene polymorphisms associated risk in tumurogenesis10. As we know that association of MTHFR and cancer susceptibility which arises through two pathway- folate metabolism ( DNA methylation processes that are dependent on S-adenosyl methionine (SAM)), and thymidylate synthesis, contributes to DNA replication and cell division. There were large number of evidence have supports the reduced activity of MTHFR deecrease the methylation of homocysteine to methionine and in turn the level of SAM, resulting in DNA hypomethylation. However, low level of MTHFR substrate, 5,10- methylene THF, required for thymidylate synthesis could lead to uracil mis incorporation into DNA, diminished DNA repair, and increased frequency of chromosomal breaks and damage and these are derived the malignant cell and involved in rapid cell division.12 MTHFR have two common low-function polymorphic variants one is T variant at nucleotide 677 (677 C T) and C variant at nucleotide 1298 (1298 A C). MTHFR 677 CT genotype, exchange highly conserved alanine to valine (677C T, alanine valine), resulting in reduced activity of this enzyme whereas MTHFR 677 TT genotype led to elevated homocysteine levels and DNA hypomethylation in folate-depleted subjects. However, unbalanced diet makes different variant. Although the regulation of homocysteine metabolism is complex and is dependent on multiple vitamin (act as cofactor),however, MTHFR mutations also affect folate metabolism, resulting in increased homocysteine The results of our study show a novel relation between MTHFR C677T polymorphism and ovarian cancer. The finding of this study assumes importance. The finding of increased risk of MTHFR C677T polymorphism in ovarian cancer together with the finding that heterozygous genotype (CT) is associated with decrease the enzymatic activity and TT with

9 222 hyperhomocysteinemia signify that this can be a cause of women with ovarian cancer. Simultaneous, estimation of homocysteine and folate levels would have given better insight into the relation between dietary MTHFR deficiency, MTHFR C677T polymorphism, hyperhomocysteinemia, and ovarian cancer. Our finding have supported by above mentioned studies. Conclusion In conclusion, the present case-control study observes statistically significant levels of folic acid among cases and controls and also observed that functional polymorphisms MTHFR- C677T, is a key folate metabolism enzyme were associated with ovarian cancer. Mutation in the MTHFR affects the level of homocysteine and DNA damaging and a trend was noticed in the association between increasing plasma total homocysteine levels with ovarian cancer, although this was statistically significant. For the one- carbon metabolism pathway, there are co-factor need for completion of process and Vitamin B12 & B6 act as co-factor for MTHFR enzyme. The results of the present study need to be confirmed with a larger sample size. As an adjunct to the preliminary findings reported here, we propose to increase the sample size and to study the prevalence of the 677 C T MTHFR gene. Acknowledgments We would like to thank the Adesh University, Punjab for their assistance and support to this study. The authors wish to express their gratitude to Miss Nauratan Kour and patients and the population who come under our study and all staff member of our laboratory. Conflict of interests The authors declare that they have no conflict interests. References: 1. Corona G, Fabris M, Viel A, Zarrelli A, Donada C, Boiocchi M. Original Paper Homocysteine Accumulmaation in Human Ovarian Carcinoma AsciticKystic Fluids Possibly Caused by Metabolic Alteration of the Methionine Cycle in Ovarian Carcinoma Cells. 1997;33(8):

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