Document 3JyY5aRDg7jkwYz3arbM3GL9D

Blood First Edition Paper, prepublished online January 25, 2010; DOI 10.1182/blood-2009-10-249722 Genetic variation in the folate metabolic pathway and risk of childhood leukemia Tracy J Lightfoot1*, W Thomas Johnston1, Dan Painter1, Jill Simpson1, Eve Roman1, Chris F Skibola2, Martyn T Smith2, James M Allan3, G Malcolm Taylor4 on behalf of the United Kingdom Childhood Cancer Study 1 Epidemiology & Genetics Unit, Department of Health Sciences, University of York, York, Y010 5DD 2 Division of Environmental Health Sciences, School of Public Health, University of California, Berkeley, California, USA. 3 Department of Biology, University of York, York, YO10 5DD 4Cancer Immunogenetics Group, School of Cancer and Imaging Sciences, University of Manchester, St Mary's Hospital, Manchester, M13 0JH * Corresponding author Dr Tracy Lightfoot Epidemiology & Genetics Unit Area 3, Seebohm Rowntree Building Department of Health Sciences University of York YO10 5DD United Kingdom Email: tracy.lightfoot@egu.york.ac.uk Tel: +44 (0) 1904 321881 Fax: +44 (0) 1904 321899 1 Copyright 2010 American Society of Hematology Abstract Studies of childhood leukemia and the potential etiological role of genetic variation in folate metabolism have produced conflicting findings and have often been based on small numbers. We investigated the MTHFR MTHFRassociation between polymorphisms in key folate metabolism enzymes ( 677 C>T, 1298 SHMT1 MTR TS TSA>C, 1420 C>T, 2756 A>G, 1494del6 and 28bp repeat) in 939 cases of childhood acute lymphoblastic leukemia (ALL) and 89 cases of acute myeloid leukemia (AML) recruited into the United Kingdom Childhood Cancer Study. We also examined the maternal genotypes of 752 of these cases. Data from 824 non-cancer controls recruited were used for comparison. No evidence of an association MTHFRwith 677 was observed for ALL or AML, either in children or their mothers. However, in children an increased risk of ALL (OR 1.88, 95 % CI 1.16-3.07 p=0.010) and AML (OR 2.74, 95% CI 1.07-7.01 p = MTR0.036) was observed with the 2756 GG genotype; the association most pronounced for cases with MLLthe translocation (OR 4.90, 95% CI 1.30-18.45 p = 0.019). These data suggest that genetic variation in methionine synthase could mediate risk of childhood leukaemia, either via effects on DNA methylation or via effects on fetal growth and development. 2 Introduction Leukaemia accounts for around a third of all malignancies diagnosed in childhood with approximately 57000 cases reported worldwide each year. The major morphological subtypes of leukaemia, acute lymphoblastic (ALL) with a B-cell precursor phenotype and acute myeloid leukaemia (AML), are characterised by gross chromosomal abnormalities 1 2, several of which have been shown to originate in utero 3-6. Although there has been much speculation about the nature of the potential agents that could cause such alterations there is, as yet, no consistent evidence to support a link with either specific exposures or modifiers of exposure7. Folate levels along with genetic regulation of folate metabolism have been the focus of many investigations 8-19, predicated on the notion that they may influence the creation and/or expansion of the pre-leukaemic clone via DNA hypomethylation of key regulatory genes as well as uracil misincorporation into DNA leading to double strand breaks and chromosomal aberrations 20;21. A critical component of the folate metabolic pathway is methylene tetrahydrofolate reductase (MTHFR) which controls the balance between DNA methylation and synthesis via the irreversible conversion of 5,10-methylenetetrahydrofolate (5,10-MeTHF), required for DNA synthesis, to 5-methyl tetrahydrofolate (5-MeTHF), a methyl donor for conversion of homocysteine to S-adenosyl methionine (SAM) (Fig. 1). MTHFRTwo common polymorphisms in (677 C>T and 1298 A>C) which result in decreased catalytic activity 22;23 and subsequent availability of 5,10-MeTHF and SAM, have been extensively studied in MTHFRrelation to childhood leukemia, but findings have been inconsistent. However, is only one of over 30 different enzymes involved in this pathway, and functional polymorphisms in other key enzymes MTR TSsuch as methionine synthase ( ), thymidylate synthase ( ) and serine hydroxymethyltransferase 1 SHMT1( ) have been shown to moderate the risk of haematological malignancies 24-26. These MTRpolymorphisms include 2756 (A>G) which moderates the flux of single carbon moieties for DNA SHMT1methylation processes 27; 1420 (C>T) which reduces circulating folate levels thus shunting 5,10- 3 TSMeTHF towards DNA synthesis 28; a 6bp deletion (1494del6) in the 3' UTR of that influences RNA TSlevels 29; and a polymorphic tandem 28bp repeat sequence within the promoter enhancer region of where the triple repeat increases gene expression levels and reduces DNA damage 30. With a view to providing further insight into the association between childhood leukaemia and folate metabolism we analysed polymorphisms in MTHFR, MTR, SHMT1 and TS in over 1000 cases of acute leukaemia and their mothers recruited as part of the United Kingdom Childhood Cancer study (UKCCS). 4 Methods Study population Cases were children aged 0-14 years diagnosed with leukemia between 1991 - 1996 recruited into the UKCCS 31. Samples taken at the time of diagnosis underwent immunophenotype and cytogenetic MLL TEL-AML1analysis 31;32. Specific chromosomal abnormalities including lesions, translocations and hyperdiploidy were identified by a combination of banded karyotyping, RT-PCR and fluorescence in situ hybridization where appropriate 32. In addition, peripheral blood samples were taken in remission from which DNA was extracted for this and other genetic studies 31;32. In total, DNA was available for 1028 Caucasian cases (55.2% male) of which 939 (91.3%) were ALLs and 89 (8.7%) were AMLs. Of the 939 ALLs, 765 (81.5%) were B-lineage (738 pre-cursor B-cell and 27 pro-B cell) and 87 (9.3%) were T-lineage in origin. The remaining 87 (9.3%) were not entered into clinical treatment trials and details of their immunophenotype were not recorded. With respect to common cytogenetic groups, genotype data TEL-AML1were available for 103 cases with a translocation (52.4% male), 316 cases with hyperdiploidy MLL(56.7% male) and 34 cases with an lesion (35.3% male). Maternal DNA was available for 752 of the leukemia cases, which included 685 ALLs (573 B-lineage and 54 T-lineage) and 58 AMLs. DNA was amplified using DNA polymerase Phi-29, which has been previously validated for use in genetic epidemiology studies 33;34. As part of routine quality control procedures, we also compared pre- and post amplified DNA genotyping results on a random sample set. DNA was obtained from peripheral blood samples taken from 824 non-cancer Caucasian controls (54.1% male) selected from population registers as part of a UK-based case-control study. DNA from this control series has been included in several genetic association studies 25;35. Both the UKCCS and the case-control study from which the controls were obtained were carried out with approval from the UK multi-regional ethics committee and in compliance with the Declaration of Helsinki. 5 Genotyping TMGenotyping was carried out using TaqMan Assays-by-Design supplied by Applied Biosystems (ABI) MTHFR(Applied Biosystems, Foster City, CA) with probes and primer sets for 677C>T (rs1801133), MTHFR 1298A>C (rs1801131), SHMT1 1420C>T (rs1979277), MTR 2756A>G (rs1805087), and TS TS1494del6 (rs16430) polymorphisms and the protocol for the 28-bp repeat identical to those previously published 26;36. Case samples were genotyped for all six polymorphisms, whereas analysis of MTHFR MTHFR MTRmothers samples was restricted to 677C>T, 1298A>C and 2756A>G. TaqMan MTHFRgenotyping assays for were verified by running 96 Coriell samples of known genotypes (http://snp500cancer.nci.nih.gov). All other TaqMan assays were verified by direct sequencing or using standard restriction fragment length polymorphism analysis. For added quality assurance, 5% of control samples were selected at random for repeat analysis, four independent control samples were analyzed on each 96-well plate, thirty duplicate DNAs were randomly distributed across the entire plate series and three duplicate plates were included in each genotype analysis. Statistical Analysis Estimates of the odds ratios (OR) for having leukemia were obtained for each polymorphism using univariate logistic regression models (Genmod procedure)37. Genotypes were considered as classes in the regression models so there was no pre-determined expectation of a particular dose-response relationship between the number of variant alleles and the risk of having leukemia. Bivariate gene-gene interactions were assessed by adding multiplicative interaction terms between pairs of genes, one at a time, to a multiple logistic regression model that included all of the genes as covariates. Only individuals with non-missing genotype data for all six polymorphic sites were included in the multiple regression. To be included in a specific analysis, individuals must have had non-missing genotype information for all SNPs included as covariates in the regression model being assessed. Associations in the distributions of 6 gene polymorphisms in pairs of genes were assessed separately for cases and controls using a series of chi-squared tests. 7 Results Genotype distributions for leukemia cases and controls and are shown in Table 1. The control frequencies for MTHFR 677C>T, MTHFR 1298A>C, SHMT1 1420C>T, MTR 2756A>G, TS 1494del6 or TS 28- bp repeat were all in Hardy-Weinberg equilibrium (data not shown) and are similar to those reported in other Caucasian populations 10;11;14-18 24;36. MTHFR SHMT1No statistically significant case-control differences in the distribution of the 677C>T, TS1420C>T, or 28-bp repeat polymorphisms were observed for ALL or AML (Table 1). However, a dose- MTRresponse relationship between the numbers of copies of the 2756 G-allele and increased risk of ALL, specifically that of B-lineage ALL, as well as AML was observed (Table 1). Specifically, heterozygosity (AG) was associated with a 1.24 fold increased risk of ALL (95% CI 1.00-1.53 p=0.05), and homozygosity for the variant allele (GG) with a 1.88 fold increased risk of ALL (95% CI 1.16-3.07 p=0.01) and 2.74 fold increased risk of AML (95% CI 1.07-7.01 p=0.036). Findings were similar for B and T-lineage ALL. In TSaddition, homozygosity for the 1494del6 polymorphism (6bp-/6bp-) was associated with an increased risk of ALL (OR 1.46, 95% CI 1.02-2.08 p = 0.04), B-lineage ALL (OR 1.44, 95% CI 1.00-2.08 p = 0.05) and AML (OR 2.04, 95% CI 1.03-4.03 p = 0.04) (Table 1). There was also limited evidence to suggest that the MTHFR 1298 variant C allele was associated with total ALL (OR 0.79, 95% CI 0.65-0.97) and B-lineage ALL (OR 0.75, 955 CI 0.61-0.93) (Table 1). When data for all polymorphisms were included in a multiple logistic regression model, similar trends were observed to those shown in Table 1 (data not shown). When data were stratified by sex, no differences between boys and girls were observed with respect to MTHFR 677C>T, SHMT1 1420C>T, MTR 2756A>G, TS 1494del6 or TS 28-bp repeat polymorphisms (data MTHFRnot shown). However, there was some evidence that homozygosity for the 1298 A>C polymorphism (CC) was associated with a decreased risk of ALL in girls (OR 0.51, 95% CI 0.30-0.89 p=0.02) and B-lineage ALL (OR 0.48, 95% CI 0.27-0.87 p=0.02), but not in boys (ALL OR 0.96, 95% CI 0.65- 1.43; B-cell ALL OR 0.95, 95% CI 0.62-1.46) (data not shown). 8 Genotype data were also stratified according to the presence of specific chromosomal abnormalities MLL TEL-AML1 MTRincluding and lesions, as well as hyperdiploidy (Table 2). Homozygosity for the 2756 MLLpolymorphism (GG) was strongly associated with positive leukemia (OR 4.90, 95% CI 1.30-18.45 p = MLL0.02). Similar findings were also observed when genotypes from positive leukemias were compared MTR MTRto those for all other leukemias combined ( 2756 AG OR 2.21 95% CI 1.01-4.84; 2756 GG OR MTR2.60, 95% CI 0.71-9.49), and to those from cases with a normal cytogenetic profile ( 2756 AG OR MTR2.16, 95% CI 0.90-5.19; 2756 GG OR 3.94, 95% CI 0.78-19.88). There was also evidence to suggest TSan association, although not statistically significant at the conventional 5% level, with the 28bp repeat TSpolymorphism, 3R/3R (OR 3.53, 95% CI 0.98-12.71). Furthermore, homozygosity for the 6bp deletion polymorphism (6bp-/6bp-) was related to hyperdiploidy (OR 1.69, 95% CI 1.07-2.68 p =0.02). No significant associations were observed between TEL-AML1 positive leukemia and the polymorphisms MTHFRstudied with the exception of 1298 where the presence of the C-allele appeared to be related to a decreased risk of TEL-AML1 positive leukemia (OR 0.52, 95% CI 0.33-0.81 p = 0.01). When we investigated associations between the six polymorphisms we observed, as expected, MTHFR MTHFR TS TSassociations between 677 and 1298, and between 6bp deletion and 28bp repeat MTR TSpolymorphisms. In addition we also detected an interaction between the 2756 and 6bp deletion MTRpolymorphisms among ALL cases (p=0.05), such that heterozygotes for the 2756 polymorphism were more likely to have at least one copy of the allele with the 6bpdeletion present. When we examined the effects of gene-gene interactions on leukemia risk, we observed some evidence of an interaction between MTHFR 1298 and SHMT1 1420 polymorphisms and between MTHFR 1298 and TS 28bp repeat polymorphisms for ALL (p=0.09 and p=0.11, respectively) and also B-lineage ALL (p=0.16 and p=0.07, respectively). There were too few individuals in the other subsets of cases to support this analysis. 9 The relationship between maternal genotype and risk of childhood leukaemia was also explored. No MTHFR MTHFR MTRassociation between 677, 1298, or 2756 polymorphisms and any leukemia subtype was observed in either univariate (Table 3) or multiple regression models (data not shown). Furthermore, no differences were seen when data were stratified by sex of the child or cytogenetic subtype (data not shown). When maternal and child genotype data were included in a single regression model, results were generally similar to when the child's genotype alone was considered, although for MTRsome polymorphisms the findings were more striking. For example, homozygosity for 2756 (GG) in MLLboth mother and child was more strongly associated with positive leukemia when compared to other leukemias (OR 8.78, 95% CI 1.92-40.13 p=0.005) and to leukemias with a normal cytogenetic profile (OR 18.75, 95% CI 1.60-220.00 p=0.02), than when the child's genotype alone was considered (all MTR MTRleukemias combined: 2756 GG OR 2.60, 95% CI 0.71-9.49; normal cytogenetic profile 2756 GG OR 3.94, 95% CI 0.78-19.88). 10 Discussion MTRWe have demonstrated that the 2756 A>G genetic polymorphism is associated with increased risk MLLof both childhood ALL and AML, and that this risk is further increased in the subset of cases with an chromosomal abnormality. However, in contrast to others9-11;13;18;38, our data do not support the MTHFRhypothesis that the 677 C>T polymorphism modifies the risk of ALL or AML in the UK. SHMT TSFurthermore, no associations were observed for either 1420 C>T, or 28bp polymorphisms. In addition, our findings provide no evidence for a role for maternal genetic variation in folate metabolism in the etiology of childhood leukemia. MTR encodes a vitamin B12- dependant enzyme which catalyses the remethylation of homocysteine to methionine, the precursor to S-adenosylmethionine (SAM) the universal methyl group donor 27. The activity of MTR is dependent on vitamin B12 being available, as well as methionine synthase reductase (MTRR), which maintains the methionine synthase-bound B12 in its fully reduced active state as methylcob(III)alamin 39. The MTR reaction also releases tetrahydrofolate which is re-methylated to 5,10-MeTHF MTRfor nucleotide synthesis. It has been suggested that the 2756 polymorphism may alter enzyme activity 27 and that the G-variant could enhance the flux of one-carbon moieties available for DNA methylation processes 40. This may provide a possible mechanism by which this polymorphism could mediate risk since hypermethylation is important in acute leukemia41. In addition, in a prospective MTRcohort study investigating pregnancy complications presence of the fetal 2756 G-allele was associated with uteroplacental insufficiency 42 suggesting it plays a part in normal fetal development, which combined with the knowledge that childhood ALL and AML originate in-utero provides further support for its role in disease aetiology. The only other study to investigate the association between MTR 2756 and childhood leukaemia is that by Gast et al 43, and whilst no evidence was found to support MTR MTRRa role for , a protective effect with polymorphisms in was observed. 11 MTHFR 677 C>T and 1298 A>C polymorphisms have been the focus of many investigations of genetic variation in the folate metabolic pathway. However, results are conflicting, with some studies reporting MTHFRprotective effects for 677 TT 9-11;13;18;38 and 1298 CC 9-11, whilst others, including our own, have yielded little or no evidence of effect at least for 677 12;15;17;44;45. There are several possible reasons for these inconsistencies, one of which relates to the small case population of most previous studies. Here, however we present data on almost 1000 childhood ALLs, which is the largest single study to date. In MTHFRaddition, it is likely that the complexity of the folate metabolic pathway may be important as is only one of over 30 enzymes involved in the pathway. An alternative explanation, suggested in relation to colorectal cancer 46, relates to differences in circulating folate levels between populations. In the mid to late 1990s fortification of foods with folic acid became mandatory in several countries, including the USA, but not the UK, resulting in marked increases in folate intake. At the same time, recommendations were made for folate supplementation during pregnancy. Interestingly, when data from a Canadian study of 270 cases of ALL were stratified by year of birth to take account of these recommendations (pre and post 1996), protective effects of the MTHFR MTHFR677 T-allele and 1298 C- allele were only observed in children born prior to 1996 11. Analogous findings have been observed for colorectal cancer where associations between polymorphisms in genes involved in the folate pathway and colorectal cancer risk appear to be modified by folate levels 47;48. MTHFR genotypes with lower enzyme activity (677 TT and 1298 CC) favour increased availability of the non-methylated form of folate (5,10-MeTHF) for DNA synthesis and decreased levels of 5-MeTHF for MTHFRDNA methylation, i.e. decreased activity alters the normal intracellular distribution of folate substrates in favour of precursors for nucleotide synthesis. Thus, if adequate levels of folate are MTHFRavailable, even if activity is low, there is sufficient conversion of 5-MeTHF for DNA methylation whilst still shunting 5,10-MeTHF towards the synthesis of dUMP to dTMP and therefore preventing uracil 12 incorporation and chromosomal damage. This suggests that differences in folate availability may MTHFRinfluence functional effects of polymorphisms, which could possibly account for different findings between studies. In the absence of folate intake data, it is not possible to investigate this further within the UKCCS. Folate plays an important role in embryogenesis and early fetal development via its effects on DNA methylation and synthesis49. As such, the well documented in utero origin of ALL has led to hypotheses that folate intake may be important in disease etiology. However, unlike for Down syndrome and neural tube defects, few studies of leukaemia have investigated maternal genotype and folate intake 8;16;19, focusing instead on the role of the child's genotype 9-13;15;17;18;38;44;45. Our findings for mothers are, however, consistent with those reported by the only other studies to have investigated this topic, albeit on smaller populations and fewer polymorphisms 11;16 . This is the largest study to date to investigate the association between genetic variation in the folate metabolic pathway and the risk of childhood leukemia. The pathway is, however, complex and our analyses were restricted to several key enzymes and excluded other possible candidates including methionine synthase reductase as well as reduced folate carrier, which has previously been linked to risk MTHFRof childhood ALL18. In conclusion, whilst our data do not support a role for 677 C>T, they suggest MLLthat methionine synthase may be important for both ALL and AML, especially in cases with translocations. 13 Acknowledgments The United Kingdom Childhood Cancer Study (UKCCS) is sponsored and administered by the Leukaemia Research Fund (LRF). This study was conducted by twelve teams of investigators (ten clinical and epidemiological, and two biological). The work was coordinated by a Management Committee chaired by Sir Richard Doll. It was supported by the UK Children's Cancer Study Group of paediatric oncologists and by the National Radiological Protection Board. Financial support was provided by Cancer Research UK (previously Imperial Cancer Research Fund and Cancer Research Campaign), the Leukaemia Research Fund and the Medical Research Council through grants to their units; by the Leukaemia Research Fund, the Department of Health, the Electricity Association, the Irish Electricity Supply Board (ESB), the National Grid Company plc and Westlakes Research (Trading) Ltd through grants for the general expenses of the study; by the Kay Kendall Leukaemia Fund for the associated laboratory studies. The investigation in Scotland was funded by The Scottish Office, Scottish Power plc, Scottish Hydro-Electric plc, and Scottish Nuclear Ltd. We thank the members of the UK Childhood Cancer Study Group for their support and staff of local hospitals, general practitioners, general practice staff and UKCCS interviewers and technicians. We especially thank the families of the children included in the study, without whose participation, this investigation would not have been possible. Data and sample collection for the lymphoma case control study, as well as DNA amplification of the UKCCS samples were supported by Leukaemia Research. Genotyping of all UKCCS samples was funded by Cancer Research UK and Leukaemia Research. Genotyping for the lymphoma case control was funded through NIH grant CA104862 from the U.S. National Cancer Institute and by the National Foundation for Cancer Research. We thank all consultants, hospital staff, general practitioners, and interviewees who participated in the study. Our thanks also go to all the staff involved in sample collection and processing. 14 Author contributions TJL designed and secured funding for the current investigation, contributed to UKCSS genotyping and was responsible for conception of the article, interpretation of data, and producing the first draft and revisions. WTJ devised and carried out statistical analyses. DP carried out laboratory analyses of UKCCS samples and JS was responsible for data co-ordination and management. ER is responsible for UKCCS data integrity, is involved in all aspects of UKCCS design and conduct, and oversaw data acquisition and interpretation. CFS designed the original assays and carried out laboratory analysis of control samples. MTS was involved in design and conduct of the analysis of the control samples. JMA contributed to the current study design, UKCCS genotyping and pre/post WGA quality control. GMT is sample custodian for the UKCCS, secured funding for the WGA and contributed to the current study design. All authors reviewed and revised the manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests 15 Reference List (1) Rowley JD. The critical role of chromosome translocations in human leukemias. Annu Rev Genet 1998;32:495-519. (2) Pui CH. 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Toxicol Appl Pharmacol 2004;198 (2):75- 85. 18 MTHFR MTHFR MTR SHMT1 TS TSTable 1 677C>T, 1298A>C, 2756A>G, 1420C>T, 1494del6 and 28-bp repeat genotype frequencies, odds ratios (OR) and 95% confidence intervals (CI) in acute leukemia cases and controls Controls Acute lymphoblastic leukemia (ALL) Acute Myeloid Leukemia (AML) Total ALL B-lineage T-lineage n n OR (95% CI) n TOTAL* MTHFR (677 C>T) CC CT TT CC v CT/TT MTHFR (1298A>C) AA AC CC AA v AC/CC SHMT1 (1420C>T) CC CT TT CC v CT/TT TS 28bp repeat# 2R/2R 2R/3R 3R/3R 2R2R v others TS (1494 del6) 6bp+/6bp+ 6bp+/6bp6bp-/6bp6bp+/6bp+ v6bp+/6bp-/6bp-/6bp- MTR 2756 (A>G) AA 824 359 (47.2) 317 (41.7) 84 (11.1) 401 (52.8) 350 (46.1) 332 (43.7) 77 (10.2) 409 (53.9) 351 (46.1) 318 (41.8) 92 (12.1) 410 (53.9) 181 (24.0) 368 (48.8) 205 (27.2) 575 (76.0) 373 (49.0) 331 (43.4) 58 (7.6) 389 (51.0) 510 (67.2) 939 374 (46.4) 341 (42.4) 90 (11.2) 431 (53.6) 408 (51.9) 305 (38.8) 73 (9.3) 378 (48.1) 401 (49.0) 320 (39.1) 97 (11.9) 417 (51.0) 193 (25.4) 344 (45.3) 222 (29.3) 570 (74.6) 423 (49.5) 336 (39.3) 96 (11.2) 432 (50.5) 531 (61.0) 1.00 1.03 (0.84-1.27) 1.03 (0.74-1.43) 1.03 (0.85-1.26) 1.00 a 0.79 (0.64- 0.97) 0.81 (0.57-1.15) c 0.79 (0.65-0.97) 1.00 0.88 (0.71-1.09) 0.92 (0.67-1.27) 0.89 (0.73-1.09) 1.00 0.88 (0.68-1.13) 1.02 (0.77-1.34) 0.93 (0.74-1.17) 1.00 0.90 (0.73-1.10) e 1.46 (1.02-2.08) 0.98 (0.81-1.19) 1.00 765 302 (46.0) 275 (41.8) 80 (12.2) 355 (54.0) 343 (53.2) 243 (37.7) 59 (9.1) 302 (46.8) 330 (49.0) 259 (38.5) 84 (12.5) 343 (51.0) 165 (26.6) 274 (44.2) 181 (29.2) 457 (73.4) 353 (50.4) 268 (38.3) 79 (11.3) 347 (49.6) 431 (60.6) OR (95% CI) 1.00 1.03 (0.83-1.29) 1.13 (0.80-1.59) 1.05 (0.85-1.30) 1.00 b 0.75 (0.60-0.93) 0.78 (0.54-1.13) d 0.75 (0.61-0.93) 1.00 0.87 (0.69-1.08) 0.97 (0.70-1.35) 0.89 (0.72-1.10) 1.00 0.82 (0.63-1.06) 0.97 (0.72-1.3) 0.87 (0.68-1.11) 1.00 0.86 (0.69-1.06) f 1.44 (1.00-2.08) 0.94 (0.77-1.16) 1.00 n 87 OR (95% CI) n 89 37 (50.7) 29 (39.7) 7 (9.6) 36 (49.3) 1.00 0.89 (0.53-1.48) 0.81 (0.35-1.88) 0.87 (0.54-1.41) 47 (59.5) 21 (26.6) 11 (13.9) 32 (40.5) 33 (48.5) 32 (47.1) 3 (4.4) 35 (51.5) 1.00 1.02 (0.61-1.70) 0.41 (0.12-1.38) 0.91 (0.55-1.49) 41 (52.6) 26 (33.3) 11 (14.1) 37 (47.4) 35 (47.9) 33 (45.2) 5 (6.9) 38 (52.1) 1.00 1.04 (0.63-1.71) 0.55 (0.21-1.43) 0.93 (0.57-1.50) 41 (52.6) 28 (35.9) 9 (11.5) 37 (47.4) 13 (18.1) 33 (45.8) 26 (36.1) 59 (81.9) 1.00 1.25 (0.64-2.43) 1.77 (0.88-3.54) 1.43 (0.77-2.66) 15 (18.5) 40 (49.4) 26 (32.1) 66 (81.5) 36 (46.8) 32 (41.6) 9 (11.7) 41 (53.2) 1.00 1.00 (0.61-1.65) 1.61 (0.74-3.51) 1.09 (0.68-1.75) 41 (48.8) 30 (35.7) 13 (15.5) 43 (51.2) 48 (61.6) 1.00 43 (55.8) 19 OR (95% CI) 1.00 0.51 (0.30-0.87) 1.00 (0.50-2.01) 0.61 (0.38-0.98) 1.00 0.67 (0.40-1.12) 1.22 (0.60-2.48) 0.77 (0.48-1.23) 1.00 0.75 (0.46-1.25) 0.84 (0.39-1.79) 0.77 (0.48-1.23) 1.00 1.31 (0.71-2.44) 1.53 (0.79-2.98) 1.38 (0.77-2.49) 1.00 0.82 (0.5-1.35) g 2.04 (1.03-4.03) 1.02 (0.64-1.58) 1.00 AG GG AA v AG/GG 223 (29.4) 26 (3.4) 249 (32.8) 288 (33.1) 51 (5.9) 339 (39.0) h 1.24 (1.00-1.53) j 1.88 (1.16-3.07) m 1.31 (1.07-1.60) 240 (33.8) 40 (5.6) 280 (39.4) i 1.27 (1.02-1.59) k 1.82 (1.09-3.03) n 1.33 (1.07-1.65) 26 (33.3) 4 (5.1) 30 (38.4) 1.24 (0.75-2.05) 1.63 (0.55-4.88) 1.28 (0.79-2.07) 28 (36.4) 6 (7.8) 34 (44.2) 1.49 (0.90-2.46) 2.74 (1.07-7.01)l 1.62 (1.01-2.60)o * Totals include individuals for whom a result was not available, and varied between SNPs # includes two cases with 1R/1R; one case with 2R/4R, one case with 3R/4R; two controls with 3R/4R genotypes a b c defghi j k l mn o p = 0.03, p = 0.01, p = 0.02, p = 0.01, p = 0.03, p = 0.05, p = 0.04, p = 0.05, p = 0.03, p = 0.01, p = 0.02, p = 0.04, p= 0.01, p = 0.01 , p = 0.05 20 MTHFR MTHFR MTRTable 2 Number (%) of cases and controls, odds ratios (OR) and 95% confidence intervals (CI) by leukemia subgroup for SHMT1 TS TS2756A>G, 1420C>T, 1494del6 and 28-bp repeat 677C>T, 1298A>C, TOTAL* MTHFR (677 C>T) CC CT TT CC v CT/TT MTHFR (1298A>C) AA AC CC AA v AC/CC SHMT1 (1420C>T) CC CT TT CC v CT/TT TS 28bp repeat# 2R/2R 2R/3R 3R/3R 2R/2R v all others TS (1494 del6) 6bp+/6bp+ 6bp+/6bp6bp-/6bp6bp+/6bp+ v6bp+/6bp-/6bp-/6bp- MTR 2756 (A>G) AA AG Control s n (%) 824 359 (47.2) 317 (41.7) 84 (11.1) 401 (52.8) 350 (46.1) 332 (43.7) 77 (10.2) 409 (53.9) 351 (46.1) 318 (41.8) 92 (12.1) 410 (53.9) 181 (23.9) 368 (48.7) 205 (27.1) 575 (76.1) 373 (49.0) 331 (43.4) 58 (7.6) 389 (51.0) 510 (67.2) 223 (29.4) n (%) 34 MLL OR (95% CI) 13 (44.8) 12 (41.4) 4 (13.8) 16 (55.2) 1.00 1.05 (0.47-2.32) 1.32 (0.42-4.14) 1.10 (0.52-2.32) 16 (55.2) 8 (27.6) 5 (17.2) 13 (44.8) 1.00 0.53 (0.22-1.25) 1.42 (0.51-1.40) 0.70 (0.33-1.47) 15 (48.4) 13 (41.9) 3 (9.7) 16 (51.6) 1.00 0.96 (0.45-2.04) 0.76 (0.22-2.69) 0.91 (0.45-1.87) 3 (10.3) 14 (48.3) 12 (41.4) 26 (89.7) 1.00 2.30 (0.65-8.09) 3.53 (0.98-12.71) 2.73 (0.82-9.12) 18 (54.5) 10 (30.3) 5 (15.2) 15 (45.5) 1.00 0.63 (0.28-1.38) 1.79 (0.64-5.00) 0.80 (0.40-1.61) 12 (41.4) 14 (48.3) 1.00 d 2.67 (1.21-5.86) TEL-AML1 n (%) OR (95% CI) 103 38 (42.2) 36 (40.0) 16 (17.8) 52 (57.8) 1.00 1.07 (0.66-1.73) 1.80 (0.96-3.38) 1.23 (0.79-1.91) 56 (62.2) 28 (31.1) 6 (6.7) 34 (37.8) 1.00 a 0.53 (0.33-0.85) 0.49 (0.20-1.17) b 0.52 (0.33-0.81) 35 (38.5) 44 (48.3) 12 (13.2) 56 (61.5) 1.00 1.39 (0.87-2.22) 1.31 (0.65-2.62) 1.37 (0.88-2.14) 20 (24.7) 40 (49.4) 21 (25.9) 61 (75.3) 1.00 0.98 (0.56-1.73) 0.93 (0.49-1.77) 0.96 (0.56-1.63) 55 (56.7) 34 (35.1) 8 (8.2) 42 (43.3) 1.00 0.70 (0.44-1.10) 0.94 (0.42-2.06) 0.73 (0.48-1.12) 58 (59.8) 34 (35.1) 1.00 1.34 (0.85-2.11) Hyperdiploidy n (%) OR (95% CI) 316 124 (45.9) 117 (43.3) 29 (10.8) 146 (54.1) 1.00 1.07 (0.80-1.43) 1.00 (0.63-1.60) 1.05 (0.80-1.39) 137 (50.9) 109 (40.5) 23 (8.6) 132 (49.1) 1.00 0.84 (0.63-1.12) 0.76 (0.46-1.27) 0.82 (0.62-1.09) 148 (52.7) 98 (34.9) 35 (12.4) 133 (47.3) 1.00 0.73 (0.54-0.98) 0.90 (0.58-1.39) 0.77 (0.58-1.01) 70 (26.4) 116 (43.8) 79 (29.8) 195 (73.6) 1.00 0.82 (0.58-1.15) 1.00 (0.68-1.46) 0.88 (0.64-1.21) 137 (47.7) 114 (39.7) 36 (12.6) 150 (52.3) 1.00 0.94 (0.70-1.25) c 1.69 (1.07-2.68) 1.05 (0.80-1.38) 181 (60.5) 101 (33.8) 1.00 1.28 (0.96-1.71) 21 GG AA v AG/GG 26 (3.4) 249 (32.8) 3 (10.3) 17 (58.6) e 4.90 (1.30-18.45) f 2.90 (1.36-6.17) 5 (5.1) 39 (40.1) 1.69 (0.63-4.57) 1.37 (0.88-2.14) 17 (5.7) 118 (39.1) 1.84 (0.98-3.47) g 1.34 (1.01-1.76) * Total includes cases for whom a result was not available, and varied between SNPs # includes three controls with 3R/4R genotype ab c d ef g p=0. 01, p = 0.001, p = 0.02, p = 0.01, p=0.02, p=0.006, p = 0.04 22 Table 3 The distribution of MTHFR 677C>T, MTHFR 1298A>C and MTR 2756A>G polymorphisms among mothers of children with acute leukemia. Controls n Total ALL n OR (95% CI) Case mothers Acute lymphoblastic leukemia (ALL) B-cell ALL T-cell ALL n OR (95% CI) n OR (95% CI) Acute Myeloid Leukemia (AML) n OR (95% CI) TOTAL* MTHFR (677 C>T) CC CT TT CC v CT/TT MTHFR (1298A>C) AA AC CC AA v AC/CC MTR 2756 (A>G) AA AG GG AA v AG/GG 378 685 573 54 58 157 (44.7) 159 (45.2) 35 (10.0) 194 (55.2) 315 (47.1) 277 (41.4) 77 (11.5) 354 (52.9) 1.00 0.87 (0.66-1.14) 1.10 (0.70-1.71) 0.91 (0.70-1.18) 261 (46.7) 229 (41.0) 69 (12.3) 298 (53.3) 1.00 0.87 (0.65-1.15) 1.19 (0.75-1.86) 0.92 (0.71-1.21) 26 (49.1) 21 (39.6) 6 (11.3) 27 (50.9) 1.00 0.80 (0.43-1.48) 1.04 (0.40-2.70) 0.84 (0.47-1.50) 24 (45.3) 24 (45.3) 5 (9.4) 29 (54.6) 157 (44.9) 151 (43.1) 42 (12.0) 193 (55.1) 334 (50.1) 254 (38.1) 78 (11.8) 332 (49.9) 1.00 0.79 (0.60-1.04) 0.87 (0.57-1.33) 0.81 (0.62-1.05) 285 (51.2) 205 (36.9) 66 (11.9) 231 (48.8) 1.00 0.75 (0.56-1.00) 0.87 (0.56-1.34) 0.77 (0.59-1.01) 27 (50.9) 24 (45.3) 2 (3.8) 26 (49.1) 1.00 0.92 (0.51-1.67) 0.28 (0.06-1.21) 0.78 (0.44-1.40) 24 (46.2) 18 (34.6) 10 (19.2) 28 (53.8) 239 (68.3) 97 (27.7) 14 (4.0) 111 (31.7) 429 (65.7) 201 (31.2) 33 (5.1) 234 (36.3) 1.00 1.15 (0.86-2.50) 1.31 (0.69-2.50) 1.17 (0.89-1.55) 358 (64.4) 168 (30.2) 30 (5.4) 198 (35.6) 1.00 1.16 (0.86-1.56) 1.43 (0.74-2.75) 1.19 (0.90-1.58) 35 (66.0) 16 (30.2) 2 (3.8) 18 (34.0) 1.00 1.13 (0.60-2.13) 0.98 (0.21-4.48) 1.11 (0.60-2.04) 31 (57.4) 21 (38.9) 2 (3.7) 23 (42.6) 1.00 0.99 (0.54-1.81) 0.93 (0.33-2.62) 0.98 (0.55-1.75) 1.00 0.78 (0.41-1.49) 1.56 (0.69-3.51) 0.95 (0.53-1.70) 1.00 1.67 (0.91-3.05) 1.10 (0.24-5.08) 1.60 (0.89-2.87) * Totals include individuals for whom a result was not available, and varied between SNPs OR odds ratio CI confidence interval 23 Figure 1 Metabolic folate pathway Metabolites: 5-methylTHF, 5-methyltetrahydrofolate; 10-formylTHF, 10-formyltetrahydrofolate; SAM- adenosylmethionine; SAH, S-adenosylhomocysteine; DHF, dihydrofolate; THF, tetrahydrofolate; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate Enzymes: MTR, methionine synthase; SHMT, serine hydroxymethyltransferase; MTHFR, 5,10-methylenetetrahydrofolate reductase; TS, thymidylate synthase 24