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1 University of Zurich Zurich Open Repository and Archive Winterthurerstr. 190 CH-8057 Zurich Year: 2009 Association of genetic variants of methionine metabolism with methotrexate-induced CNS white matter changes in patients with primary CNS lymphoma Linnebank, M; Moskau, S; Jurgens, A; Simon, M; Semmler, A; Orlopp, K; Glasmacher, A; Bargart, C; Vogt-Schaden, M; Urbach, H; Schmidt-Wolf, I G; Pels, H; Schlegel, U Linnebank, M; Moskau, S; Jurgens, A; Simon, M; Semmler, A; Orlopp, K; Glasmacher, A; Bargart, C; Vogt-Schaden, M; Urbach, H; Schmidt-Wolf, I G; Pels, H; Schlegel, U (2009). Association of genetic variants of methionine metabolism with methotrexate-induced CNS white matter changes in patients with primary CNS lymphoma. Neuro Oncology, 11(1):2-8. Postprint available at: Posted at the Zurich Open Repository and Archive, University of Zurich. Originally published at: Neuro Oncology 2009, 11(1):2-8.

2 Association of genetic variants of methionine metabolism with methotrexate-induced CNS white matter changes in patients with primary CNS lymphoma Abstract Methotrexate (MTX) is an important anticancer drug and the most efficient chemotherapy component in primary central nervous system lymphoma (PCNSL). A typical side effect of intravenous high-dose MTX is the occurrence of confluent CNS white matter changes (WMC). Since MTX directly interferes with methionine metabolism, we analyzed the impact of genetic variants of methionine metabolism on the occurrence of WMC as a model of MTX toxicity. In a retrospective analysis of 68 PCNSL patients treated with MTX-based polychemotherapy with (n=42) or without (n=26) intraventricular treatment, ten genetic variants influencing methionine metabolism were analyzed. Pearson's Chi-square test and multinominal regression analysis were used to define the relevance of these genetic variants for the occurrence of WMC. In this patient sample, the occurrence of WMC was significantly predicted by the TT genotype of methylenetetrahydrofolate reductase c.677c>t (chi(2)=8.67; p=0.013; df=2), the AA genotype of methylenetetrahydrofolate reductase c.1298a>c (chi(2)=13.5; p=0.001; df=2) and the GG genotype of transcobalamin 2 c.776c>g (chi(2)=19.73; p<0.001) in addition to male gender (chi(2)=11.95; p=0.001). These data strengthen the hypothesis that MTX effects are influenced by methionine metabolism which may offer new strategies to improve MTX-based therapies.

3 Association of genetic variants of methionine metabolism with MTX-induced CNS white matter changes in patients with primary central nervous system lymphoma Linnebank M 1,2, Moskau S 1, Jurgens A 3, Simon M 4, Semmler A 1, Orlopp K 5, Glasmacher A 5, Bargart C 6, Vogt-Schaden M 7, Urbach H 8, Schmidt-Wolf IGH 5, Pels H 3, Schlegel U 3 1 University Hospital Bonn, Dept. Neurology, Bonn, Germany 2 University Hospital Zurich, Dept. Neurology, Zurich, Switzerland 3 University Hospital Bochum, Knappschaftskrankenhaus, Dept. Neurology, Bochum, Germany 4 University Hospital Bonn, Dept. Neurosurgery, Bonn, Germany 5 University Hospital Bonn, Dept. Internal Medicine, Bonn, Germany 6 University Hospital Cologne, Dept. Radiology, Cologne, Germany 7 University Hospital Heidelberg, Dept. Neurology, Heidelberg, Germany 8 University Hospital Bonn, Dept. Radiology, Bonn, Germany Correspondence: Dr. Michael Linnebank, University Hospital Zurich, Dept. Neurology, Frauenklinikstrasse 26, CH-8091 Zurich, Switzerland. Phone: Fax: Michael.Linnebank@usz.ch Grant: This study was supported by the "Deutsche Krebshilfe", grant-no (M. Linnebank and U. Schlegel). 1

4 Abstract Methotrexate (MTX) is an important anticancer drug and the most efficient chemotherapy component in primary central nervous system lymphoma (PCNSL). A typical side-effect of intravenous high-dose MTX is the occurrence of confluent CNS white matter changes (WMC). As MTX directly interferes with methionine metabolism, we aimed to analyze the impact of genetic variants of methionine metabolism on the occurrence of WMC as a model of MTX toxicity. In a retrospective analysis of 68 PCNSL patients treated with MTX-based polychemotherapy with (n=42) or without (n=26) intraventricular treatment, eleven genetic variants influencing methionine metabolism were analyzed. Pearson s Chi-square test and multinominal regression analysis were used to define the relevance of these genetic variants for the occurrence of WMC. In this patient sample, the occurrence of WMC was significantly predicted by genotypes of methylenetetrahydrofolate reductase c.677c>t (χ 2 =8.67; p=0.013), methylenetetrahydrofolate reductase c.1298a>c (χ 2 =13.5; p=0.001) and transcobalamin 2 c.776c>g (χ 2 =19.73; p<0.001) in addition to male gender (χ 2 =11.95; p=0.001). These data strengthen the hypothesis that MTX effects are influenced by methionine metabolism which may offer new strategies to improve MTX-based therapies. 2

5 Introduction The anticancer and anti-inflammatory drug methotrexate (MTX) is a competitive inhibitor of several enzymes involved in folate and methionine metabolism such as dihydrofolate reductase (DHFR), aminoimidazole carboxamide synthase, glycinamide ribonucleotide transformylase and thymidylate synthethase (TS). 1,2 Manifestations of MTX-toxicity include nausea, mucositis, myelosuppression, and renal, liver and pulmonary dysfunction. Neurotoxicity of MTX may present with acute, subacute and chronic encephalopathy as well as with clinically asymptomatic subacute confluent white matter changes (WMC), which are diagnosed on T2 weighted MRI sequences. 3 Response to MTX-based chemotherapy and manifestation of toxicity show a high inter-individual variability. It is therefore important to identify factors influencing MTX effects in order to improve treatment outcome. Uptake of folates into the cells occurs through organic bi-directional anion carriers like the reduced folate carrier 1 (RFC1) as well as through the unidirectionally folate receptors alpha and beta, that mediate folate uptake into cells via endocytosis. These receptors as well as several transporters that unidirectionally export folates from cells, are also involved in uptake and export of MTX. 4 The intracellular DHFR-inhibiting effect of MTX leads to a depletion of 5,10-CH 2 - FH 4 and thereby to both an impairment of nucleic acid synthesis and of homocysteine remethylation via 5,10-methylenetetrahydrofolate reductase (MTHFR) and 5- methyltetrahydrofolate-homocysteine S-methyltransferase (MTR). As this is the only pathway of homocysteine remethylation in the CNS, application of MTX can lead to a marked increase of neurotoxic homocysteine within the CNS and to a lack of S-adenosylmethionine (SAM), which is necessary for myelination within the CNS, neuronal membrane stability and neurotransmission. 2,5-8. 3

6 MTX is the most efficient drug in the treatment of primary central nervous system lymphomas PCNSL, 9 which account for 3% of all primary brain tumors in the United States (Central Brain Tumor Registry of the United States, 2004). Prognosis of PCNSL, which are highly aggressive tumors, is poor with a median survival of approximately two months in untreated patients. Brain irradiation, chemotherapy and combination of these result in a substantially improved survival. 10,11 The Bonn protocol to treat PCNSL is a high-dose MTX (cycles 1, 2, 4 and 5) based systemic polychemotherapy regimen including alkylating agents, vinca-alkaloids and dexamethasone combined with intraventricular MTX, prednisolone, and ARA-C, which has resulted in a five-year-survival fraction of 75% in patients <60 years. 12 In a recent study we observed that the presence of at least one of the genotypes MTHFR c.677tt, MTR c.2756ag/gg or transcobalamin 2 (Tc2, transport protein of the MTR cofactor vitamin B12) c.776gg was associated with the occurrence of WMC in a sample of 42 MTXtreated PCNSL patients, but we failed to identify a significant independent association of any of these genotypes with WMC. 13 In the present study we extended the patient sample to 68 patients and analyzed a larger set of in total eight polymorphisms of methionine metabolism. Here, we identified three functional polymorphisms that independently predicted the occurrence of WMC. Patients and methods The study population consisted of 68 consecutive PCNSL patients (32 male, 36 female) of Caucasian origin, who had been treated in two prospective chemotherapy trials: Until December 2002 treatment included intraventricular drug administration (MTX, prednisolone and Ara-C) ( original Bonn protocol, n=42). 12. Afterwards, patients were treated without intraventricular therapy ( modified Bonn protocol, n=26; unpublished). Median age at diagnosis was 59 years (range 28 to 77). 4

7 Of the 68 patients, 55 completed therapy (six cycles of polychemotherapy), and all 68 underwent at least two cycles. MRI was carried out before initiation of therapy. The second MRI, which was used to define occurrence of WMC, was performed after two cycles of chemotherapy to assess treatment response as part of the trial protocol. 12 None of the patients underwent brain irradiation prior to MRI assessment. WMC were defined as new, confluent and symmetrical hyperintense lesions of the supratentorial white matter detectable on FLAIR and T2-weighted images. 3 Genotyping was performed by amplification of genomic DNA applying polymerase chain reaction (PCR) and subsequent restriction enzyme digestion or by allele specific PCR, followed by agarosegel-electrophoresis. For analysis of the ATIC polymorphism c.347c>g. 14, we used the PCR primers 5 -AGCTGTAAACCACATGAGTGG and 5 -AGGCAGAGGTTGCAGTCAGC with an annealing temperature of 61. The PCR product of 471bp was digested with the restriction enzyme HpyCH4III (New England Biolabs, Frankfurt, Germany) resulting in fragments of 26bp, 96bp, 349bp for the G-allele, and 21bp, 26bp, 96bp, 328bp for the C-allele. The other PCR and restriction analysis conditions were essentially as described Genetic analyses and correlations with WMC were done in a retrospective approach. Biochemical parameters like plasma levels of folate, vitamin B6 or vitamin B12, were not available. Diagnosis of WMC was independently made by the chairman of the subdivision of Neuro- Oncology (US) and by the chairman of the subdivision of Neuro-Radiology (HU). For all patients, both made the same diagnosis (newly developed WMC after the 2 nd cycle of chemotherapy, yes or no). Diagnosis of WMC was made blinded for the results from the genetic analyses and vice versa. Mix-up of asymptomatic WMXC with severe leukoencephalopathy in MRI-based diagnoses was unlikely, because leukoencephalopathies generally present different in MRI and develop later than the respective MRI was done. 23 5

8 Nominal regression analysis (α=0.05) was used to test the independent predictive value of the covariables polymorphisms, age, gender, Karnofsky score before therapy, and application of intraventricular therapy (yes or no) on the incidence of WMC (definition given above) as endpoint. For the tested genotypes the Hardy-Weinberg equilibrium was calculated for all PCNSL patients together (with or without WMC) with a Chi 2 goodness-of-fit-test (alpha=0.05). Informed written consent was obtained from all patients or their legal guardians. This study was approved by the local ethics committees. Results Of the 68 patients, one patient died after the 2nd cycle before MRI could be carried out, and suitable MRI studies from 2 further patients were not available for other reasons. Thus, a total of 65 PCNSL patients were analyzed for the occurrence of WMC (Table 1). The Hardy-Weinberg equilibrium of the observed genotype frequencies was fulfilled for each genetic variant. The C- allele of the rare polymorphism CBS c.833t>c (I278T) was not detected in this study s sample, and this polymorphism was excluded from further analyses. Overall, 20 (31%) of the 65 patients showed clinically asymptomatic WMC, and 45 did not. In all 20 cases, WMC was detectable in the MRI after the 2 nd cycle of therapy; no-one of the 55 patients who completed the six cycles of therapy developed WMC later than after the 2 nd cycle. In no patient, WMC occurred thereafter. Multivariate nominal regression analysis revealed that the occurrence of WMC was predicted by male gender (χ 2 =11.95; p=0.001; OR=2.54, CI 95% = ). In addition, three genetic variants independently predicted the occurrence of WMC in our patient sample: MTHFR c677c>t (χ 2 =8.67; p=0.013; OR conferred by the TT genotype: 6.0, CI 95% = ), MTHFR c.1298a>c (χ 2 =13.5; p=0.001; OR conferred by the 6

9 AA genotype: 6.00, CI 95% = ) and Tc2 c.776c>g (χ 2 =19.7; p<0.001; OR conferred by the GG genotype: 2.15, CI 95% = ). The DHFR c del19bp variant showed a significant prediction of WMC formation in nominal regression analysis (χ 2 =6.63; p=0.036), however, this result was due to an over-representation of heterozygous cases only, which is not plausible. Thus, this result must be disregarded if not confirmed in other studies. The polymorphism ATIC c.346c>g was associated with WMC for trend (χ 2 =4.97; p=0.083; OR conferred by the GG genotype: 3.5, CI 95% = ) Discussion The antifolate MTX is an important anticancer drug. The understanding of mechanisms involved in MTX toxicity is important to improve MTX therapies. WMC represent signs of mild MTXinduced neurotoxicity, which in its extreme variant may present as necrotising encephalopathy after intraventricular MTX application 24 or as a dementing disease after combination of systemic MTX with whole brain radiotherapy. 25 Using this model we aimed to analyze whether genetic variants of methionine metabolism modify the susceptibility to MTX toxicity. We cannot decide to what extend the results of our study may be transferred to different treatment protocols. Male gender was significantly associated with WMC. The methionine metabolism is influenced by endogenous steroids, 26 which may provide an explanation for gender-specific differences of the WMC rates. In contrast to our data, Hoekstra and co-workers reported that MTX withdrawal as putative sign of side-effects was more frequent in female than in male patients treated with low-dose oral MTX ( 15mg/week). 27 Speculatively, the impact of gender on MTX toxicity may differ between therapy regimens (long-term low-dose versus high-dose) and organs, however, gender-specific MTX effects and side-effects have not been clearly evaluated, so far. 7

10 We have previously reported preliminary data suggesting that a combined genotype of polymorphisms of methionine and folate metabolism may be associated with the occurrence of WMC in MTX-treated patients who carry at least one of the genotypes MTHFR c.677tt, MTR c.2756ag/gg or Tc2 c.776gg, but that study did not identify any significant independent association of one of these genotypes with WMC. 13 In the present study we attempted to identify clinical and genetic risk factors for the formation of WMC in a larger series of PCNSL patients treated with high-dose MTX. In multivariate analysis, the Tc2 c.776c>g and the two MTHFR missense polymorphisms c.677c>t and c.1298a>c showed a significant association with the occurrence of WMC, and the missense polymorphism ATIC c.346c>g showed an association for trend. The MTHFR polymorphism c.677c>t (p.a222v) leads to a reduced MTHFR activity disposing a lower remethylation rate of homocysteine to methionine and, thus, to lower SAM concentrations and increased homocysteine levels (figure 1). 16 The missense polymorphism Tc2 c.776c>g (p.p259r) is known to alter the affinity of Tc2 to cobalamin also leading to a reduced remethylation of homocysteine to methionine. 15,28,29 Thus, the T allele of MTHFR c.677c>t and the G-allele of Tc2 c.776c>g may both plausibly pronounce the homocysteine-increasing and the SAM-decreasing effect of MTX leading to a higher risk of MTX-induced WMC as observed in our study. Anecdotally, we have previously reported serial data on CSF analyses of four PCNSL patients treated with high-dose MTX showing that those two of four patients with the GG genotype of Tc2 c.776c>g had the lowest CSF levels of SAM. One of these Tc2 c.776gg carriers was the only one of those four patients who developed WMC. 30 In that study we also reported that during MTX treatment, homocysteine CSF levels increased up to 17-fold in patients treated with intraventricular MTX. As we found that even slight elevations of homocysteine levels lead to neuronal dysfunction in cultured cells as well as in primary 8

11 neurons, 13 the increase of neurotoxic homocysteine during MTX treatment is a possible mechanism of MTX neurotoxicity in addition to SAM-depletion. Polymorphisms of methionine metabolism may be relevant modifiers. Concerning the second genetic variant with significant influence on the occurrence of WMC in multivariate analysis, MTHFR c.1298a>c (p.e429a), we have shown that this polymorphism influences MTHFR activity in human fibroblasts (about 80% activity in CC fibroblasts as opposed to AA fibroblasts). 31 However, the A-allele, which was associated with higher MTHFR activity in our as well as in other studies, 32,31 was observed in association with WMC. This is in contrast to our hypothesis on elevated homocysteine and reduced SAM levels as reasons for MTX neurotoxicity. However, the MTHFR c.1298a>c and c.677c>t variants are in linkage disequilibrium, i.e. c.677t allele is linked to c.1298a 33. Thus, an over-representation of c.677t alleles accounts for an over-representation of c.1298a alleles. The limited number of patients with different haplotypes in this series precludes the identification of a MTHFR c.677c>t;c.1298a>c haplotype predisposing to the development of WMC. Thus, we cannot decide whether the linkage disequilibrium with MTHFR c.677c>t or a different mechanism may explain the observed association of the wildtype A-allele of MTHFR c.1298a>c with WMC. The enzyme ATIC is involved in folate metabolism catalyzing the two final steps of purine synthesis. Previous studies did not observe any biological effect of the ATIC polymorphism c.346c>g (p.t116s), 14 which showed an association with WMC for trend only. Thus, especially the effect of the ATIC polymorphism on the occurrence of WMC has to be retested in an independent patient sample. In summary, within the limitations of any association study, the present study supports the 9

12 hypothesis that methionine metabolism modifies MTX (neuro)toxicity. The manipulation of methionine metabolism, e.g. by supplementation of SAM (figure 1), may be a promising strategy to improve MTX tolerance. 10

13 References 1. Mudd SH, Levy HL, Kraus JP. Disorders of transsulfuration. Volume The Metabolic and Molecular Bases of Inherited Disease. New York: Mc Graw-Hill: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler K, Vogelstein B. Editors; p Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem. 1990;1: Fliessbach K, Helmstaedter C, Urbach H, et al. Neuropsychological outcome after chemotherapy for primary CNS lymphoma: a prospective study. Neurology. 2005;64: Matherly LH, Goldman DI. Membrane transport of folates. Vitam Horm. 2003;66: Quinn CT, Griener JC, Bottiglieri T, Arning E, Winick NJ. Effects of intraventricular methotrexate on folate, adenosine, and homocysteine metabolism in cerebrospinal fluid. J Pediatr Hematol Oncol. 2004;26: Quinn CT, Griener JC, Bottiglieri T, Hyland K, Farrow A, Kamen BA. Elevation of homocysteine and excitatory amino acid neurotransmitters in the CSF of children who receive methotrexate for the treatment of cancer. JClinOncol. 1997;15: Surtees R, Leonard J, Austin S. Association of demyelination with deficiency of cerebrospinal-fluid S-adenosylmethionine in inborn errors of methyl-transfer pathway. Lancet. 1991;338: Surtees R, Clelland J, Hann I. Demyelination and single-carbon transfer pathway metabolites during the treatment of acute lymphoblastic leukemia: CSF studies. JClinOncol. 1998;16:

14 9. Batchelor T, Loeffler JS. Primary CNS lymphoma. J Clin Oncol. 2006;24: Plotkin SR, Batchelor TT. Advances in the therapy of primary central nervous system lymphoma. Clin Lymphoma. 2001;1: ; discussion Ferreri AJ, Abrey LE, Blay JY, et al. Summary statement on primary central nervous system lymphomas from the Eighth International Conference on Malignant Lymphoma, Lugano, Switzerland, June 12 to 15, J Clin Oncol. 2003;21: Pels H, Schmidt-Wolf IG, Glasmacher A, et al. Primary central nervous system lymphoma: results of a pilot and phase II study of systemic and intraventricular chemotherapy with deferred radiotherapy. J Clin Oncol. 2003;21: Linnebank M, Pels H, Kleczar N, et al. MTX-induced white matter changes are associated with polymorphisms of methionine metabolism. Neurology. 2005;64: Gellekink H, Blom HJ, den Heijer M. Associations of common polymorphisms in the thymidylate synthase, reduced folate carrier and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase genes with folate and homocysteine levels and venous thrombosis risk. Clin Chem Lab Med. 2007;45: Afman LA, Lievers KJ, van der Put NM, Trijbels FJ, Blom HJ. Single nucleotide polymorphisms in the transcobalamin gene: relationship with transcobalamin concentrations and risk for neural tube defects. Eur J Hum Genet. 2002;10: Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. NatGenet. 1995;10:

15 17. Harmon DL, Shields DC, Woodside JV, et al. Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations. GenetEpidemiol. 1999;17: Johnson WG, Stenroos ES, Spychala JR, Chatkupt S, Ming SX, Buyske S. New 19 bp deletion polymorphism in intron-1 of dihydrofolate reductase (DHFR): a risk factor for spina bifida acting in mothers during pregnancy? AmJMedGenetA. 2004;124: Linnebank M, Homberger A, Junker R, Nowak-Goettl U, Harms E, Koch HG. High prevalence of the I278T mutation of the human cystathionine beta-synthase detected by a novel screening application. ThrombHaemost. 2001;85: van der Put NM, Gabreels F, Stevens EM, et al. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? AmJHumGenet. 1998;62: Winkelmayer WC, Eberle C, Sunder-Plassmann G, Fodinger M. Effects of the glutamate carboxypeptidase II (GCP2 1561C>T) and reduced folate carrier (RFC1 80G>A) allelic variants on folate and total homocysteine levels in kidney transplant patients. Kidney Int. 2003;63: Dervieux T, Furst D, Lein DO, et al. Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis. Arthritis Rheum. 2004;50: Lai R, Abrey LE, Rosenblum MK, DeAngelis LM. Treatment-induced leukoencephalopathy in primary CNS lymphoma: a clinical and autopsy study. 13

16 Neurology. 2004;62: Shapiro WR, Chernik NL, Posner JB. Necrotizing encephalopathy following intraventricular instillation of methotrexate. Arch Neurol. 1973;28: Abrey LE, DeAngelis LM, Yahalom J. Long-term survival in primary CNS lymphoma. J Clin Oncol. 1998;16: Wong DL, Zager EL, Ciaranello RD. Effects of hypophysectomy and dexamethasone administration on central and peripheral S-adenosylmethionine levels. J Neurosci. 1982;2: Hoekstra M, van Ede AE, Haagsma CJ, et al. Factors associated with toxicity, final dose, and efficacy of methotrexate in patients with rheumatoid arthritis. Ann Rheum Dis. 2003;62: Lievers KJ, Afman LA, Kluijtmans LA, et al. Polymorphisms in the transcobalamin gene: association with plasma homocysteine in healthy individuals and vascular disease patients. Clin Chem. 2002;48: von Castel-Dunwoody KM, Kauwell GP, Shelnutt KP, et al. Transcobalamin 776C->G polymorphism negatively affects vitamin B-12 metabolism. Am J Clin Nutr. 2005;81: Becker A, Vezmar S, Linnebank M, et al. Marked elevation in homocysteine and homocysteine sulfinic acid in the cerebrospinal fluid of lymphoma patients receiving intensive treatment with methotrexate. Int J Clin Pharmacol Ther. 2007;45: Linnebank M, Linnebank A, Jeub M, et al. Lack of genetic dispositions to 14

17 hyperhomocysteinemia in Alzheimer disease. AmJMedGenetA. 2004;131: van der Put NM, van Straaten HW, Trijbels FJ, Blom HJ. Folate, homocysteine and neural tube defects: an overview. Exp Biol Med (Maywood). 2001;226: Linnebank M, Homberger A, Nowak-Gottl U, Marquardt T, Harms E, Koch HG. Linkage disequilibrium of the common mutations 677C > T and 1298A > C of the human methylenetetrahydrofolate reductase gene as proven by the novel polymorphisms 129C > T, 1068C > T. EurJPediatr. 2000;159:

18 Table 1. Factors predicting the incidence of white matter changes (WMC) therapy ivt + ivt regression WMC + (n=20) χ 2 =0.652; p=0.420 WMC (n=45) Age mean sd WMC + (n=20) χ 2 =2.32; p=0.128 WMC (n=45) Karnofsky mean sd WMC + (n=20) χ 2 =0.011; p=0.917 WMC (n=45) gender female male WMC + (n=20) χ 2 =11.95; p=0.001 WMC (n=45) ATIC CC CG GG c.346c>g WMC + (n=20) χ 2 =4.97; p=0.083 WMC (n=45) CBS del/del ins/del ins/ins c.844_845ins68 16

19 WMC + (n=20) χ 2 =0.857; p=0.355 WMC (n=45) DHFR del/del ins/del ins/ins c del19bp WMC + (n=20) χ 2 =6.63; p=0.036 WMC (n=45) MTHFR c.677c>t CC CT TT WMC + (n=20) χ 2 =8.67; p=0.013 WMC (n=45) MTHFR c.1298a>c AA AC CC WMC + (n=20) χ 2 =13.5; p=0.001 WMC (n=45) MTR c.2756a>g AA AG GG WMC + (n=20) χ 2 =0.702; p=0.402 WMC (n=45) RFC1 c.80g>a WMC + (n=20) χ 2 =3.35; p=0.187 WMC (n=45)

20 TC2 c.776c>g CC CG GG WMC + (n=20) χ 2 =19.73; p<0.001 WMC (n=45) TSER 28bp repeat 2/2 2/3 3/3 WMC + (n=20) χ 2 =3.193; p=0.203 WMC (n=45) Nominal regression with all parameters as listed as covariables. Karnofsky: before therapy. sd: standard deviation. ivt: intraventricular therapy. WMC: white matter changes. For MTR, the AG and the GG genotype were pooled for multiple nominal regression analysis. Genetic analysis included the genetic variants 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) c.346c>g (p.t116s; rs ), cystathionine beta-synthase (CBS) c.833t>c (missense mutation p.i278t; not detected in our study and not included in Table 1), CBS c.844_855ins68 (splice alteration affecting the transcript level; GenBank S ), dihydrofolate reductase (DHFR) c del19bp (intronic deletion supposedly affecting the transcript level; GenBank NM_ ), methylenetetrahydrofolate reductase (MTHFR) c.677c>t (missense mutation p.a222v; rs ) and MTHFR c.1298a>c (missense mutation p.e429a; rs ), methionine-homocysteine S-methyltransferase (MTR) c.2756a>g (missense mutation p.d919g; rs ), reduced folate carrier 1 (RFC1) c.80g>a (missense mutation p.r27h; rs ), transcobalamin 2 (Tc2) c.776c>g (missense mutation p.p259r; rs ), and the 28bp repeat of the thymidylate synthase (TYMS) enhancer region (TSER; 2 or 3 repeats; GenBank NM_001071). 18

21 Figure legend Figure 1 Human methionine metabolism Activated methionine (S-adenosylmethionine, SAM) is the methyl group donor for numerous reactions. The degradation product of SAM is S-adenosylhomocysteine (SAH), which is hydrolysed to homocysteine by SAH-hydrolase. Homocysteine can be transsulfurated to cystathionine by the vitamin B6-dependent cystathionine beta-synthase (CBS), and subsequently, via the vitamin B6-dependent cystathionine gamma-lyase, to cysteine. Cysteine is essential for glutathione synthesis. Alternatively, homocysteine can be remethylated to methionine by 5- methyltetrahydrofolate-homocysteine S-methyltransferase (MTR, also called methionine synthase). MTR requires methylcobalamin (a derivate of vitamin B12) and 5- methyltetrahydrofolate (5-CH 3 -FH 4 ) as cofactors. Transcobalamin 2 (Tc2) is the major transporter protein for cobalamin, 5-CH 3 -FH 4 is synthesized from 5,10- methylenetetrahydrofolate (5,10-CH 2 -FH 4 ) by 5,10-methylenetetrahydrofolate reductase (MTHFR), and 5,10-CH 2 -FH 4 is synthesized by dihydrofolate reductase (DHFR) in two subsequent steps from the folate pool. 5,10-CH 2 -FH 4 is also needed for nucleic acid synthesis, in which thymidylate synthase (TYMS) and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) are involved. Thus, MTHFR activity regulates whether a one-carbon unit of tetrahydrofolate is utilized for methionine synthesis or for nucleic acid synthesis, and thus for DNA synthesis, stability and repair. 5,10-CH 2 -FH 4 is regenerated from 5-CH 3 -FH 4 by MTR during methionine synthesis. 19

22 20