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Acute lymphoblastic leukemia (ALL) is the most common form of cancer in children, accounting for 30% of all the pediatric malignancies . Although the clinical and pathological aspects of leukemia are well studied, little is known about the genes that affect the susceptibility to this disease . The development of ALL has been proposed to arise through a combination of genetic predisposition and exposure to environmental factors. Since the effect of these external factors is modulated by genes, the latter influences the individual's susceptibility to ALL .
Folate is an essential nutrient for the cellular functioning because it provides one-carbon donors that are required for DNA synthesis and methylation . Methotrexate, an antifolic acid agent, has proven to be an effective chemotherapeutic drug for the treatment of lymphoid malignancies, indicating an association between the folate metabolism and the development of such malignancies . Thymidylate Synthase is a crucial enzyme in this folate metabolism and hence a good candidate for studying the effect of polymorphisms in folate metabolism gene on the development of malignancies.
Thymidylate Synthase, encoded by Thymidylate Synthase (TS) gene located on chromosome 18p11.32 plays a vital role in maintaining a balanced supply of deoxy nucleotides required for DNA synthesis and repair  by catalyzing the conversion of dUMP to dTMP. The most common polymorphisms in TS are a unique double (2R) or triple (3R) 28-base pair tandem repeat sequence in the 5' untranslated region (5'UTR) of the TS gene immediately upstream of the initiation site which influences protein expression in cancer cells. The presence of a triple versus double 28-bp repeat in the enhancer region has been associated with an increased Thymidylate Synthase expression and the conversion of dUMP to dTMP, thereby decreasing uracil levels and the consequent erroneous incorporation of uracil into DNA of rapidly dividing hematopoietic stem cells, which works protectively against the development of ALL .
The TS 28-bp repeat polymorphism has been shown to modulate the risk of ALL in various populations but the results obtained are controversial and require further investigation to confirm and clarify the results [2, 4, 7-8].
The aim of the present study was to determine the frequency of TSER polymorphism and its possible association with the susceptibility to ALL in the Kashmiri subjects (India). The frequency of TSER genotypes with respect to various clinico-pathological characteristics was also evaluated in the study.
Materials and Methods
A case-control study was performed involving 72 patients with ALL and a control group composed of 82 individuals without leukemia. The ALL samples were from SKIMS, Srinagar collected sequentially between March 2009 and January 2011 from patients at the time of diagnosis. The diagnosis of ALL was based on FAB or immunophenotypic criteria. The main characteristics of the patients are shown in Table 1. All cases of childhood and adult ALL were grouped because we did not find any statistical difference in the incidence of the TSER polymorphism between patients aged 16 years or younger (childhood ALL) and those older than 16 (adult ALL).
Three milliliters of venous blood was drawn into a sterile tube containing EDTA and stored at -20°C until the isolation of genomic DNA. DNA was isolated by a standard phenol-chloroform extraction method .
PCR Analysis for TS 2R→3R genotyping
The PCR primers, PCR reaction conditions were the same as previously described . PCR was performed in a 25µl total volume reaction mixture containing 100 ng of template DNA and 200 nM each of the forward (5'-GTGGCTCCTGCGTTTCCCCC-3') and reverse (5'-GGCTCCGAGCCGGCCACAGGCATGGCGCGG-3') primers were used. Polymerase chain reaction (PCR) cycling parameters were a 5 minute denaturation cycle at 94°C and 35 cycles of the following: 94°C for 30 s, 63°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. The amplified products were visualized on a 3% agarose gel using ethidium bromide.
DNA sequencing analysis was carried out to confirm the results of TS genotyping at the 5' UTR tandem repeat locus in a subset of 12 representative samples. PCR amplicons were recovered from agarose gel, followed by purification with DNA recovery Kit (Zymo Research, USA) and then used for direct DNA sequencing. DNA sequencing was carried out at MACROGEN INC, Korea. To rule out the possibility of sequencing artifacts by PCR, products from at least two different PCRs were sequenced using forward and reverse primers.
The χ2 test was used for the comparison of the allele and genotype frequency between the cases and controls. The distribution of the genotype frequencies in both groups did not deviate from the Hardy-Weinberg equilibrium. The Odds ratios (OR) were calculated as estimates of relative risk for disease and 95% confidence intervals were calculated for all observed allele frequencies. A P < 0.05 was considered statistically significant. The SPSS statistical software package (ver. 11.5, SPSS, Chicago, IL) was used for the statistical analysis.
72 histopathologically confirmed ALL cases and 82 matched controls belonging to the Kashmir division were analyzed for the TSER polymorphism. The homozygotes for the double repeat (2R/2R) produced a single band corresponding to 215 bp. Heterozygotes (2R/3R) produced two fragments corresponding to 215bp and 243bp, and homozygotes for the triple repeat (3R/3R) produced a single 243bp fragment (Fig 1 and 2). Sequencing analyses showed that these fragments contained the same sequence except for the last 28-bp repeat of both 2R and 3R genotypes which contains a 2-bp insertion (CC, Site). A single G→C (SNP) at the 12th nucleotide of the second repeat of 3R allele was also observed in some of the samples sequenced (Fig. 3 and 4).
The TS triple tandem repeat (3R) allele frequency was found to be 73.75 % in the controls and 67.91% in cases. The difference in frequency was found to be statistically insignificant with a P value of 0.2713(P> 0.05). The TS 2R/2R genotype was found to be present in 13.88% of the cases and 9.75% of the controls, the 2R/3R variant in 31.94% of the cases and 31.70% of controls, and the 3R/3R genotype in 47.22% of cases and 56.09% of controls. DNA from 05 cases and 02 controls did not amplify (Table-2).
It was observed that although the proportion of patients who were homozygous for the TS tandem repeat(3R/3R) was lower in cases than in controls, the difference was not statistically significant when using 2R/2R genotype as a reference (OR= 0.5913; 95% CI, 0.2111-1.657; P value= 0.3143). Similarly, it was observed the frequency of the heterozygous genotype (2R/3R) when compared with 2R/2R genotype was not much different between the cases and controls (OR=0.7077; 95% CI, 0.2389-2.097; P value= 0.5317).
TS genotypes and clinicopathological variables
Table -3 shows the genotype frequencies of TSER polymorphism relating to age, gender, dwelling, FAB type and immunophenotype of the patients. The TSER genotype was only statistically significantly associated with the gender of the patients; age, dwelling and FAB type showed no association with the TSER polymorphism.
Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood with an event free survival rate of near to 80% in the developed countries . Folate plays an important role in DNA synthesis and repair. Folate deficiency results in the large scale misincorporation of uracil into DNA and chromosome breaks. This in turn induces chromosome damage, fragile site formation, micronucleus formation and elevated uracil levels in the DNA of the bone marrow cells . Taking this information into consideration it seems plausible that functional alterations due to polymorphisms in the genes involved in folate metabolism may be associated with the development of cancers. Consistent with this hypothesis it has been shown that polymorphisms in the folate related genes have been associated with the risk of cancers . Thymidylate Synthase is a crucial enzyme in this folate metabolism and hence a good candidate for studying the effect of polymorphisms in folate metabolism gene on the development of malignancies. In the present study the genetic association of 5'UTR tandem repeat polymorphism in Thymidylate Synthase gene with the development of ALL in Kashmiri population was investigated. The repeat polymorphism in the TS gene was evaluated in 72 ALL cases and 82 (age, sex and region matched, non malignant) controls by PCR analysis of DNA obtained from the blood of the subjects, followed by direct sequencing of DNA. We observed that the TS triple tandem repeat (3R) allele frequency was 73.75 % in the controls and 67.91% in cases. This difference in frequency was found to be statistically insignificant with a P value of 0.2713(P> 0.05). The TS 2R/2R genotype was found to be present in 13.88% of the cases and 9.75% of the controls, the 2R/3R variant in 31.94% of the cases and 31.70% of controls, and the 3R/3R genotype in 47.22% of cases and 56.09% of controls.
We observed that although the proportion of patients who were homozygous for the TS tandem repeat(3R/3R) was lower in cases than in controls, the difference was not statistically significant when using 2R/2R genotype as a reference (OR= 0.5913; 95% CI, 0.2111-1.657; P = 0.3143). Similarly, we observed the frequency of the heterozygous genotype (2R/3R) when compared with 2R/2R genotype was not much different between the cases and controls hence, statistically insignificant (OR=0.7077; 95% CI, 0.2389-2.097; P value= 0.5317). Thus, our study suggests that there is no association between TS tandem repeat polymorphism and the development of ALL in Kashmiri population.
It has been shown that the ethnicity of an individual plays a role in the various distributions of polymorphisms of the genes involved in folate metabolism [14, 15]. In the present study in Kashmiri population, the frequencies of TSER 3R/3R, 3R/2R and 2R/2R genotypes were; 57.3%, 32.7% and 10% respectively which are not much different (P=0.25) from those among 252 controls in a CML study in other Indian population[Sailaja et al,2010). The prevalence of TS 3R/3R has been reported to be 33.1% in Caucasian children with ALL, while in Indonesian samples, its frequency reached 76.1% in ALL children  whereas in our population the frequency of 3R/3R in ALL patients was 50.7% which is slightly different from that observed in Caucasian children with ALL(P=0.049) and much different from Indonesian children with ALL(P=0.0002). Similarly, in comparison with healthy Chinese, Japanese, Caucasian and African population. The result of our study further adds to the data from several other studies which have shown an inconsistent association between the TSER polymorphism and the risk of ALL.
In the study of Skibola, et al. , individuals with TS 2R3R were shown to exhibit a 2.8-fold reduction and those with TS 3R3R exhibited a 4.0-fold reduction in ALL risk. In contrast, in a western European pediatric ALL patients, the TS 2R variant was reported to have a protective effect .
Lauten, et al, (2003) in a study, involving 40 ALL Canadian patients and 40 controls, reported that the frequency of TS genotypes was not significantly different . Also, in a large number of ALL patients from the United Kingdom Childhood Cancer study, the frequency of TS 28-bp repeat was not significantly different compared to controls . Similarly, in another study involving 73 ALL patients and 128 controls from a western Iranian population, it was observed that the frequency of 2R allele was not significantly higher in ALL patients as compared to controls . However, an interaction between TS 28-bp repeat polymorphism and folate intake has been reported with a decreased risk of ALL in the 3R3R genotype in combination with high folate intake .
Petra et al, (2007) in Slovenia, reported that TS polymorphisms were not significantly associated with ALL risk . Similarly, the frequency of TS genotypes in the Indonesian ALL patients and controls did not show any significant difference .
In the present study, we have shown that although the TS 5' UTR tandem repeat polymorphism is present in the Kashmiri population but the frequency of 2R allele or 3R allele was not significantly different between ALL cases and controls.
The reasons for contrary results obtained from several studies remain ambiguous and might be attributed to differences in ethnic backgrounds and the selection of the population studied, differences in sample sizes, and gene-environment interactions, such as diet, exposure to chemicals, or nutritional intake of folate and related vitamins.
In addition, gene-gene interactions and the presence of an additional G→C SNP within the second repeat of the triple tandem has been shown to influence the transcriptional activity of the Thymidylate Synthase gene . In our study sequencing results of some subjects did show the presence of such a SNP in the second repeat of triple tandem which may influence the transcriptional activity of the gene and hence explain the reason for contrary results obtained. However, it needs further evaluation and investigation to establish the role of this SNP in the development of ALL.
Furthermore, inherited biases accompanied with hospital based case-control studies may also be attributed to spurious findings or false positive results. Due to limited studies reporting the influence of gene polymorphism involved in folate metabolism on ALL, more investigations are necessary to find a clear relationship of these genes to susceptibility to ALL .
In summary, our study has shown the frequency of TS gene involved in folate metabolism with respect to ALL patients within a homogenous ethnic group. It seems that TS 28bp-repeat polymorphism is not a risk factor for the susceptibility to ALL in Kashmiri population. Kashmir is a small valley present at a high altitude, with mostly consanguineous marriages resulting in preservation of the genetic pool. And owing to historical, cultural, religious and linguistic differences from rest of India Kashmiri's show wide genetic diversities. Thus, additional analysis with a larger sample size is needed to clarify the real contribution of this gene in the susceptibility to ALL in different world populations.
We are grateful to Syed Sameer , Muzamil Makhdoomi and Beenish Iqbal for helpful suggestions.
This work was supported by grants from the Centre for Scientific and Industrial
Research (CSIR), India under the Junior Research Fellowship (JRF) scheme.
List of Abbreviations
ALL Acute Lymphoblastic Leukemia
CML Chronic Lymphocytic Leukemia
TS Thymidylate Synthase
TSER Thymidylate Synthase Enhancer Region