Acute lymphoblastic leukaemia ( ALL ) is the most common signifier of malignant neoplastic disease in kids, accounting for 30 % of all the paediatric malignances [ 1 ] . Although the clinical and pathological facets of leukaemia are good studied, small is known about the cistrons that affect the susceptibleness to this disease [ 2 ] . The development of ALL has been proposed to originate through a combination of familial sensitivity and exposure to environmental factors. Since the consequence of these external factors is modulated by cistrons, the latter influences the person ‘s susceptibleness to ALL [ 3 ] .
Folate is an indispensable food for the cellular operation because it provides one-carbon givers that are required for DNA synthesis and methylation [ 4 ] . Methotrexate, an antifolic acid agent, has proven to be an effectual chemotherapeutic drug for the intervention of lymphoid malignances, bespeaking an association between the vitamin Bc metamorphosis and the development of such malignances [ 5 ] . Thymidylate Synthase is a important enzyme in this folate metamorphosis and therefore a good campaigner for analyzing the consequence of polymorphisms in folate metamorphosis cistron on the development of malignances.
Thymidylate Synthase, encoded by Thymidylate Synthase ( TS ) cistron located on chromosome 18p11.32 plays a critical function in keeping a balanced supply of deoxy bases required for DNA synthesis and fix [ 6 ] by catalysing the transition of shit to dTMP. The most common polymorphisms in TS are a alone two-base hit ( 2R ) or ternary ( 3R ) 28-base brace tandem repetition sequence in the 5 ‘ untranslated part ( 5’UTR ) of the TS cistron instantly upstream of the induction site which influences protein look in malignant neoplastic disease cells. The presence of a ternary versus dual 28-bp repetition in the foil part has been associated with an increased Thymidylate Synthase look and the transition of shit to dTMP, thereby diminishing uracil degrees and the consequent erroneous incorporation of U into DNA of quickly spliting haematopoietic root cells, which works protectively against the development of ALL [ 2 ] .
The TS 28-bp repetition polymorphism has been shown to modulate the hazard of ALL in assorted populations but the consequences obtained are controversial and necessitate farther probe to corroborate and clear up the consequences [ 2, 4, 7-8 ] .
The purpose of the present survey was to find the frequence of TSER polymorphism and its possible association with the susceptibleness to ALL in the Kashmiri topics ( India ) . The frequence of TSER genotypes with regard to assorted clinico-pathological features was besides evaluated in the survey.
Materials and Methods
A case-control survey was performed affecting 72 patients with ALL and a control group composed of 82 persons without leukaemia. The ALL samples were from SKIMS, Srinagar collected consecutive between March 2009 and January 2011 from patients at the clip of diagnosing. The diagnosing of ALL was based on FAB or immunophenotypic standards. The chief features of the patients are shown in Table 1. All instances of childhood and grownup ALL were grouped because we did non happen any statistical difference in the incidence of the TSER polymorphism between patients aged 16 old ages or younger ( childhood ALL ) and those older than 16 ( adult ALL ) .
Deoxyribonucleic acid Isolation
Three millilitres of venous blood was drawn into a unfertile tubing incorporating EDTA and stored at -20 & A ; deg ; C until the isolation of genomic DNA. Deoxyribonucleic acid was isolated by a standard phenol-chloroform extraction method [ 9 ] .
PCR Analysis for TS 2R>3R genotyping
The PCR primers, PCR reaction conditions were the same as antecedently described [ 10 ] . PCR was performed in a 25µl entire volume reaction mixture incorporating 100 nanogram of templet DNA and 200 nM each of the forward ( 5′-GTGGCTCCTGCGTTTCCCCC-3 ‘ ) and contrary ( 5’-GGCTCCGAGCCGGCCACAGGCATGGCGCGG-3 ‘ ) primers were used. Polymerase concatenation reaction ( PCR ) cycling parametric quantities were a 5 minute denaturation rhythm at 94 & A ; deg ; C and 35 rhythms of the followers: 94 & A ; deg ; C for 30 s, 63 & A ; deg ; C for 30 s, and 72 & A ; deg ; C for 30 s, and a concluding extension at 72 & A ; deg ; C for 5 min. The amplified merchandises were visualized on a 3 % agarose gel utilizing ethidium bromide.
Deoxyribonucleic acid sequencing analysis was carried out to corroborate the consequences of TS genotyping at the 5 ‘ UTR tandem repetition venue 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 so used for direct DNA sequencing. Deoxyribonucleic acid sequencing was carried out at MACROGEN INC, Korea. To govern out the possibility of sequencing artefacts by PCR, merchandises from at least two different PCRs were sequenced utilizing frontward and rearward primers.
The ?2 trial was used for the comparing of the allelomorph and genotype frequence between the instances and controls. The distribution of the genotype frequences in both groups did non divert from the Hardy-Weinberg equilibrium. The Odds ratios ( OR ) were calculated as estimations of comparative hazard for disease and 95 % assurance intervals were calculated for all observed allele frequences. A P & A ; lt ; 0.05 was considered statistically important. The SPSS statistical package bundle ( ver. 11.5, SPSS, Chicago, IL ) was used for the statistical analysis.
72 histopathologically confirmed ALL instances and 82 matched controls belonging to the Kashmir division were analyzed for the TSER polymorphism. The homozygotes for the dual repetition ( 2R/2R ) produced a individual set matching to 215 bp. Heterozygotes ( 2R/3R ) produced two fragments matching to 215bp and 243bp, and homozygotes for the ternary repetition ( 3R/3R ) produced a individual 243bp fragment ( Fig 1 and 2 ) . Sequencing analyses showed that these fragments contained the same sequence except for the last 28-bp repetition of both 2R and 3R genotypes which contains a 2-bp interpolation ( CC, Site ) . A individual G>C ( SNP ) at the 12th base of the 2nd repetition of 3R allelomorph was besides observed in some of the samples sequenced ( Fig. 3 and 4 ) .
The TS three-base hit tandem repetition ( 3R ) allelomorph frequence was found to be 73.75 % in the controls and 67.91 % in instances. The difference in frequence was found to be statistically undistinguished with a P value of 0.2713 ( P & A ; gt ; 0.05 ) . The TS 2R/2R genotype was found to be present in 13.88 % of the instances and 9.75 % of the controls, the 2R/3R discrepancy in 31.94 % of the instances and 31.70 % of controls, and the 3R/3R genotype in 47.22 % of instances and 56.09 % of controls. Deoxyribonucleic acid from 05 instances and 02 controls did non magnify ( Table-2 ) .
It was observed that although the proportion of patients who were homozygous for the TS tandem repetition ( 3R/3R ) was lower in instances than in controls, the difference was non statistically important when utilizing 2R/2R genotype as a mention ( OR= 0.5913 ; 95 % CI, 0.2111-1.657 ; P value= 0.3143 ) . Similarly, it was observed the frequence of the heterozygous genotype ( 2R/3R ) when compared with 2R/2R genotype was non much different between the instances 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 frequences of TSER polymorphism associating to age, gender, brooding, FAB type and immunophenotype of the patients. The TSER genotype was merely statistically significantly associated with the gender of the patients ; age, brooding and FAB type showed no association with the TSER polymorphism.
Acute lymphoblastic leukaemia ( ALL ) is the most common malignant neoplastic disease of childhood with an event free endurance rate of near to 80 % in the developed states [ 11 ] . Folate plays an of import function in DNA synthesis and fix. Folate lack consequences in the big scale misincorporation of U into DNA and chromosome interruptions. This in bend induces chromosome harm, delicate site formation, micronucleus formation and elevated uracil degrees in the Deoxyribonucleic acid of the bone marrow cells [ 12 ] . Taking this information into consideration it seems plausible that functional changes due to polymorphisms in the cistrons involved in folate metamorphosis may be associated with the development of malignant neoplastic diseases. Consistent with this hypothesis it has been shown that polymorphisms in the vitamin Bc related cistrons have been associated with the hazard of malignant neoplastic diseases [ 13 ] . Thymidylate Synthase is a important enzyme in this folate metamorphosis and therefore a good campaigner for analyzing the consequence of polymorphisms in folate metamorphosis cistron on the development of malignances. In the present survey the familial association of 5’UTR tandem repetition polymorphism in Thymidylate Synthase cistron with the development of ALL in Kashmiri population was investigated. The repetition polymorphism in the TS cistron was evaluated in 72 ALL instances and 82 ( age, sex and part matched, non malignant ) controls by PCR analysis of DNA obtained from the blood of the topics, followed by direct sequencing of DNA. We observed that the TS three-base hit tandem repetition ( 3R ) allelomorph frequence was 73.75 % in the controls and 67.91 % in instances. This difference in frequence was found to be statistically undistinguished with a P value of 0.2713 ( P & A ; gt ; 0.05 ) . The TS 2R/2R genotype was found to be present in 13.88 % of the instances and 9.75 % of the controls, the 2R/3R discrepancy in 31.94 % of the instances and 31.70 % of controls, and the 3R/3R genotype in 47.22 % of instances and 56.09 % of controls.
We observed that although the proportion of patients who were homozygous for the TS tandem repetition ( 3R/3R ) was lower in instances than in controls, the difference was non statistically important when utilizing 2R/2R genotype as a mention ( OR= 0.5913 ; 95 % CI, 0.2111-1.657 ; P = 0.3143 ) . Similarly, we observed the frequence of the heterozygous genotype ( 2R/3R ) when compared with 2R/2R genotype was non much different between the instances and controls therefore, statistically undistinguished ( OR=0.7077 ; 95 % CI, 0.2389-2.097 ; P value= 0.5317 ) . Therefore, our survey suggests that there is no association between TS tandem repetition polymorphism and the development of ALL in Kashmiri population.
It has been shown that the ethnicity of an single plays a function in the assorted distributions of polymorphisms of the cistrons involved in folate metamorphosis [ 14, 15 ] . In the present survey in Kashmiri population, the frequences of TSER 3R/3R, 3R/2R and 2R/2R genotypes were ; 57.3 % , 32.7 % and 10 % severally which are non much different ( P=0.25 ) from those among 252 controls in a CML survey in other Indian population [ Sailaja et al,2010 ) . The prevalence of TS 3R/3R has been reported to be 33.1 % in Caucasic kids with ALL, while in Indonesian samples, its frequence reached 76.1 % in ALL kids [ 15 ] whereas in our population the frequence of 3R/3R in ALL patients was 50.7 % which is somewhat different from that observed in Caucasic kids with ALL ( P=0.049 ) and much different from Indonesian kids with ALL ( P=0.0002 ) . Similarly, in comparing with healthy Chinese, Japanese, Caucasian and African population. The consequence of our survey farther adds to the informations from several other surveies which have shown an inconsistent association between the TSER polymorphism and the hazard of ALL.
In the survey of Skibola, et Al. [ 2 ] , persons with TS 2R3R were shown to exhibit a 2.8-fold decrease and those with TS 3R3R exhibited a 4.0-fold decrease in ALL hazard. In contrast, in a western European pediatric ALL patients, the TS 2R discrepancy was reported to hold a protective consequence [ 4 ] .
Lauten, et Al, ( 2003 ) in a survey, affecting 40 ALL Canadian patients and 40 controls, reported that the frequence of TS genotypes was non significantly different [ 7 ] . Besides, in a big figure of ALL patients from the United Kingdom Childhood Cancer survey, the frequence of TS 28-bp repetition was non significantly different compared to controls [ 16 ] . Similarly, in another survey affecting 73 ALL patients and 128 controls from a western Persian population, it was observed that the frequence of 2R allelomorph was non significantly higher in ALL patients as compared to controls [ 17 ] . However, an interaction between TS 28-bp repetition polymorphism and vitamin Bc consumption has been reported with a reduced hazard of ALL in the 3R3R genotype in combination with high vitamin Bc consumption [ 18 ] .
Petra et Al, ( 2007 ) in Slovenia, reported that TS polymorphisms were non significantly associated with ALL risk [ 8 ] . Similarly, the frequence of TS genotypes in the Indonesian ALL patients and controls did non demo any important difference [ 15 ] .
In the present survey, we have shown that although the TS 5 ‘ UTR tandem repetition polymorphism is present in the Kashmiri population but the frequence of 2R allelomorphs or 3R allelomorph was non significantly different between ALL instances and controls.
The grounds for contrary consequences obtained from several surveies remain equivocal and might be attributed to differences in cultural backgrounds and the choice of the population studied, differences in sample sizes, and gene-environment interactions, such as diet, exposure to chemicals, or nutritionary consumption of vitamin Bc and related vitamins.
In add-on, gene-gene interactions and the presence of an extra G>C SNP within the 2nd repetition of the three-base hit tandem has been shown to act upon the transcriptional activity of the Thymidylate Synthase cistron [ 19 ] . In our survey sequencing consequences of some topics did demo the presence of such a SNP in the 2nd repetition of three-base hit tandem which may act upon the transcriptional activity of the cistron and therefore explicate the ground for contrary consequences obtained. However, it needs farther rating and probe to set up the function of this SNP in the development of ALL.
Furthermore, familial prejudices accompanied with infirmary based case-control surveies may besides be attributed to specious findings or false positive consequences. Due to limited surveies describing the influence of cistron polymorphism involved in folate metamorphosis on ALL, more probes are necessary to happen a clear relationship of these cistrons to susceptibleness to ALL [ 20 ] .
In drumhead, our survey has shown the frequence of TS cistron involved in folate metamorphosis with regard to ALL patients within a homogeneous cultural group. It seems that TS 28bp-repeat polymorphism is non a hazard factor for the susceptibleness to ALL in Kashmiri population. Kashmir is a little vale nowadays at a high height, with largely akin matrimonies ensuing in saving of the familial pool. And owing to historical, cultural, spiritual and lingual differences from remainder of India Kashmiri ‘s show broad familial diversenesss. Therefore, extra analysis with a larger sample size is needed to clear up the existent part of this cistron in the susceptibleness to ALL in different universe populations.
We are thankful 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 ) strategy.
List of Abbreviations
ALL Acute Lymphoblastic Leukemia
CML Chronic Lymphocytic Leukemia
TS Thymidylate Synthase
TSER Thymidylate Synthase Enhancer Region