Methylenetetrahydrofolate Reductase Gene C677T and A1298C Polymorphic Sequence Variations Influences the Susceptibility to Chronic Myeloid Leukemia in Kashmiri Population

Background: Methylenetetrahydrofolate reductase (MTHFR) gene is a crucial regulator of folate metabolism and its two prominent polymorphic variants C677T and A1298C lead to decreased MTHFR enzyme activity. Aim of the Study: We planned this case–control study based on numerous studies supporting the association of MTHFR polymorphisms (C677T and A1298C) with CML risk in different ethnic populations. Therefore, the influence of these polymorphisms on CML susceptibility was investigated among Kashmiri population (North India). Materials and Methods: Polymerase chain reaction/restriction fragment length polymorphism (RFLP) technique was employed for genotyping MTHFR C677T and A1298C SNP's in 125 CML patients as against 150 age and gender matched healthy controls. Results: A significant difference was observed in frequency of 677CT genotype between cases and controls [46.4 vs. 27.3% (p = 0.0005)]. Similarly combined 677CT+TT genotype showed significant difference between cases and controls [50.4 vs. 28.6% (p = 0.0002)]. Both MTHFR 677CT and 677CT+TT genotypes imposed greater than 2-fold risk of developing CML (OR = 2.4, 95%CI: 1.46–4.05; OR = 2.5, 95%CI: 1.53–4.16). In case of A1298C SNP, the frequency of 1298AC genotype was higher in controls (64.0%) as compared to CML cases (48.8%) (p = 0.04) and imparted a significant protective role from CML predisposition. Furthermore, haplotype analysis revealed only “677CT/1298AA” haplotype significantly increased the risk of CML predisposition [(p = 0.008) (OR = 3.2, 95% CI: 1.3–7.4)]. Conclusion: We conclude that both MTHFR C677T and A1298C polymorphisms may be important genetic modifiers and seem to have a plausible role to confer risk of CML in Kashmiri population, where C677T SNP strongly increases the risk of CML while as A1298C SNP has a protective effect.

Background: Methylenetetrahydrofolate reductase (MTHFR) gene is a crucial regulator of folate metabolism and its two prominent polymorphic variants C677T and A1298C lead to decreased MTHFR enzyme activity.
Aim of the Study: We planned this case-control study based on numerous studies supporting the association of MTHFR polymorphisms (C677T and A1298C) with CML risk in different ethnic populations. Therefore, the influence of these polymorphisms on CML susceptibility was investigated among Kashmiri population (North India).
Materials and Methods: Polymerase chain reaction/restriction fragment length polymorphism (RFLP) technique was employed for genotyping MTHFR C677T and A1298C SNP's in 125 CML patients as against 150 age and gender matched healthy controls.

INTRODUCTION
Chronic Myeloid Leukemia (CML) whose incidence and prevalence has dramatically increased in recent past is the most common myeloid malignancy. CML is a disorder of the hematopoietic stem cell and leads to the buildup of myeloid precursors in bone marrow, peripheral blood and body tissues (1). CML results from t(9;22) that leads to the generation of hybrid fusion gene BCR-ABL1 which generates genomic instability by disrupting the balance between proliferation and apoptosis of cells and drives the leukemic changes in CML (2,3). Although the t(9;22) is the primary cause for the development of CML, the factors that lead to the induction of Philadelphia chromosome are still unknown. Despite welldocumented clinical features and pathology of the disease, very little is known about the individual's susceptibility to CML (3)(4)(5).
Hereditary or familial associations with the development of CML have not been reported yet.
Association studies have provided evidence which suggests single nucleotide polymorphisms in key genes might increase the risk of CML (6,7). Therefore, identification of CML susceptibility genes will elucidate disease pathogenesis and provide insights about the risk assessment of the disease. Epidemiological studies indicate an effective association between low folate intake and increased cancer risk. The notion that pathogenesis of CML is linked to the folate metabolism has also been hypothesized (8). Among the diverse group of enzymes that affect the metabolism of folic acid, the most promising role has been attributed to 5,10-methylenetetrahydrofolate reductase (MTHFR).
MTHFR gene in humans is found to be positioned on chromosome one comprising of 11 protein-coding sequences (9). The fate of folate metabolism is regulated by MTHFR catalyzed one-step irreversible reduction of 5, 10methylenetetrahydrofolate to 5-methylTHF. The latter is found to be a typical form of folate in serum. 5-methylTHF is the major carbon donor for the conversion of homocysteine to methionine, which is later on used for DNA synthesis (10). Weak activity of MTHFR may dampen the 5-methyltetrahydrofolate pathway leading to 5-methyltetrahydrofolate build up that causes reduction in the conversion of dUMP to dTMP. Once dUMP to dTMP methylation is deficient, Uracil can misincorporate into DNA, consequentially resulting in DNA double-stranded breaks that leads to numerous anomalies including hematological malignancies (11,12).
The decreased functional activity of MTHFR has been attributed to two single gene variants (rs1801133) and (rs1801131). For (rs1801133), the SNP arises due to a missense mutation at nucleotide position 222 in exon 4 of MTHFR gene causing C > T substitution that results in the replacement of alanine with valine. The mutation leads to increased plasma homocysteine via production of MTHFR with lesser enzyme activity at 37 • C (13). It has been shown that the carriers of homozygous genotype (MTHFR 677 TT) possess only 30 percent MTHFR enzyme activity in comparison to the wild genotype, whereas 60% enzyme activity is found in individuals with heterozygous (MTHFR 677 CT) genotype (10).
In exon 7, A1298C polymorphism at codon 429 results from the substitution of glutamine with alanine, which results in diminished enzyme activity up to 33% (14). Numerous studies evaluating the possible link between these two MTHFR SNPs in CML have been reported but the findings of these correlation studies are controversial (4,15).
Since such a possibility has never been investigated in our ethnically conserved population, the current case-control study was attempted to investigate the prominent MTHFR C677T and A1298C SNPs as a measure of risk for CML development in ethnic Kashmiri population.

MATERIALS AND METHODS
The present case-control study included 125 CML patients from Kashmir who visited the outpatient clinic or inpatient wards of departments of Medical Oncology and Clinical Hematology, Sher-i-Kashmir Institute of Medical Sciences (SKIMS), J&K (North India).
All patients were assessed clinically and diagnosed by standard methods like blood picture, bone marrow analysis, and detection of Philadelphia chromosome by peripheral blood/bone marrow Cytogenetics. The diagnosis was confirmed by molecular detection of BCR-ABL1 hybrid fusion gene by reverse transcriptase polymerase chain reaction (RT-PCR) and real time polymerase chain reaction (Rq-PCR). Patient details were collected by personal interaction with the patients and laboratory parameters like total leukocyte count (TLC), differential leukocyte count (DLC), and platelet counts were recorded from the routine CBC/bone marrow reports. We further evaluated risk scores like Sokal and EUTOS (European Treatment and Outcome Study) for the CML patients. Sokal and EUTOS scores were calculated from the clinical parameters recorded at diagnosis before start of treatment. Sokal score was determined as per the age, spleen size, platelet count, peripheral blood blasts, and patients were risk stratified as low-risk (Sokal score <0.8), intermediate-risk (Sokal score 0.8-1.2) and highrisk (>1.2) (16). EUTOS score was calculated on the spleen size and percentage of basophils in the peripheral blood. Patients were categorized as low-risk (score <87) and high-risk (score ≥87) (17). We determined the Sokal and EUTOS scores using online formulas on the BloodRef network site (http://www. bloodref.com). The control group comprised of age, sex, and ethnicity matched 150 healthy blood donors with no history of any malignancy and living in the same geographical area as the patients. This study was conducted only after taking informed consent from all participants and approval from the local SKIMS Ethics committee.

DNA Extraction
Genomic DNA from the peripheral blood/bone marrow leukocytes of CML cases and peripheral blood of control subjects was extracted using QIAamp DNA Blood Mini Kit (Cat No. 51104), Qiagen (Germany) and its concentration and purity was analyzed by Bio spectrophotometer (Eppendorf AG; Serial No: 6137EQ102539; Germany).

Statistics
The significance of the present study was evaluated by Chisquare or Fisher's exact test. Frequency and percentage were used to express qualitative data. Descriptive analyses included frequencies of genotypes and alleles. Odds ratio (OR) as an estimate of relative risk and the 95% confidence interval (CI) was calculated using SPSS version 12 data analysis software. For all analysis, P-values at the level of 0.05 were taken as statistically significant.  (Tables 1, 2).

RESULTS
In the current study, as per the Sokal score, 28 (22.4%) patients were stratified as low risk, 68 (54.4%) as intermediate risk and 29 (32.2%) as high risk group patients. According to the EUTOS score, 89 (71.2%) patients were categorized as low risk and 36 (28.8%) as high risk patients. Genotypic distribution of the two MTHFR SNPs did not relate either with the Sokal risk groups (p = 0.16 and p = 0.79, respectively) or EUTOS score (p = 0.34 and p = 0.42, respectively) (Tables 1, 2).

DISCUSSION
CML, which arises due to genetic damages to pluripotent hematopoietic stem cells, has peculiar clinical and biological features (19). Although information about the factors which predispose individuals to CML is scanty (3), but its risk may be increased due to quantitative and qualitative changes in folate metabolism caused by these genetic damages (20).  Folate acts as a transporter of carbon moiety which is used for converting homocysteine to methionine and also for the production of purine and pyrimidine (21). MTHFR is one of the main regulators of folate metabolism and polymorphic variations like C677T and A1298C in this gene leads to variant alleles which results in decreased MTHFR activity in vitro in comparison to the wild-type allele and thereby increases CML susceptibility (22,23).
Keeping in view the conserved gene pool of our ethnic population due to the common practice of interrelated marriages, the pattern of polymorphic variations always remains an important issue to evaluate, for identification of individuals with increased disease susceptibility. Therefore, this case-control study is the first from our region that aims to observe whether sequence polymorphic variations at C677T and A1298C in MTHFR gene predisposes individuals to CML in our population (Kashmiri, North India). Studies in varying ethnic groups regarding the possible association between MTHFR polymorphic sequence variations and susceptibility to CML have been carried out but the reported results are conflicting ( Table 5).
In this study, the frequencies of MTHFR C677T variant genotypes CT and TT in CML patients were 46.4 and 4.0%, respectively, which was higher when compared to controls. The frequency of MTHFR C677T genotypes found was almost similar to that reported by other authors from different ethnic regions (3,24,25). The variation in genotype frequencies can result from geographic and ethnic differences. However, higher frequencies of TT genotypes were reported in Iran by Vahid et al. and in India by Hussain et al., but the number of cases in these studies was comparatively less (5,26).
We observed a higher 677T allele frequency (27.2%) in our CML cases which depict that 677T allele significantly influences the risk of CML in Kashmiri population (p = 0.0004). In likewise studies by Hussain et al. and Banescu et al., the authors also reported a significant impact of the 677T allele on risk of CML in patients from North Indian and Romania (26,27). However, a study conducted by Moon et al. reported no significant influence of MTHFR 677T allele on CML risk in Korean population (28). These observations depict a strong association between MTHFR C677T SNP and predisposition to CML and it could be considered as a risk factor for CML development in our population. However, there are certain studies where no significant association of the same SNP was reported with the risk of CML development (3,28,29).
With respect to MTHFR A1298C polymorphism, the frequency of AC and CC genotypes in cases was 48.8 and 8.0%, respectively, which was close to that reported by Lordelo et al. and Khorshied et al. (3,24). Interestingly we reported higher frequencies of the heterozygous genotype AC in controls than in CML cases (64 vs. 48.8%). A similar scenario was reported by Vahid et al. and Ismail et al. (5,25). Furthermore, we reported a significant 1.7-fold decrease in CML risk in cases with 1298AC genotypes when compared with 1298 AA genotype (OR = 0.57, 95% CI: 034-0.95), (p = 0.04) (   (32,33). This variability in different ethnic populations implies that the functional impact of these SNP's on CML susceptibility is a population dependent phenomenon.
In the present study, we found 677CT/1298AA haplotype associated with more than 3-fold increased risk of developing CML as its frequency was significantly higher in cases than controls (P = 0.008). However, Banescu et al. reported 677CT/1298AC compound heterozygote as a risk factor for developing CML in Romanian population (27).
Various clinicopathological characteristics including, disease phase, Sokal, and EUTOS score in patients with CML were stratified but did not show any correlation with MTHFR 677 C > T and 1298 A > C.
In summary, the main findings of the study emphasize the importance of MTHFR C677T variant genotypes for conferring strong risk to CML. On the other hand, MTHFR A1298C heterozygous genotype selectively is shown to exert a protective impact for CML cases as is reported in several studies. Although the present report had a limitation of relatively small study population, but the findings therein revealed that the incidences of CML are more dependent on folate status. We conclude that both MTHFR C677T and A1298C SNPs may be important genetic modifier and seem to have a plausible role to confer risk of CML in Kashmiri (North Indian) population. The apparent inconsistencies between our results and those of previous studies, in different populations, may be due to the diverse number of patients studied, different genotype frequencies, different ethnicities, and the absence of information on dietary folate intake.
Additional studies, with a larger sample size in various geographical areas are augmented to explore MTHFR C677T and A1298C SNPs in CML pathogenesis. Further the dual role of the two SNPs in CML seems to have a promise for functional analysis to analyze the leukemogenesis of chronic myeloid leukemia.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
This study was carried out in accordance with the recommendations of the Code of Ethics of the World Medical Association. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Sher-i-Kashmir Institute of Medical Sciences Ethics committee.

AUTHOR CONTRIBUTIONS
SB conceptualized, designed and supervised the study, did lab work, and also drafted the manuscript. ZS supervised the study, provided the consumables and logistical support, and submitted the manuscript. KJ performed the lab work. AP did proofreading and corrected the manuscript. JR, SG, and GM provided patient samples. RB performed lab work and drafted the manuscript. SA did compilation and statistical work.