This study investigated the association between six folate pathway polymorphisms and neurocognitive deficits in childhood ALL survivors, with the premise that these polymorphisms would modulate neurocognitive function in the setting of intermittent folate depletion due to MTX. In a previous study of 48 childhood ALL survivors, Krull et al reported a 7.4-fold increased risk of attention deficit disorder, as measured by parental questionnaire, in survivors with the MTHFR
1298AC/CC genotype [29
]. Results of the current study, which includes 24 additional survivors and new independent measures that evaluate child performance directly rather than through a proxy rater, corroborate this earlier finding. Specifically, patients with the MTHFR
1298AC/CC genotypes had lower mean scores for direct performance measures of shifting attention and processing speed, namely TMTB. Additionally, the current study provides a more comprehensive investigation of the role of folate pathway polymorphisms in the development of neurocognitive deficits after ALL therapy. We genotyped a total of six folate pathway polymorphisms in the current study, and our findings suggest that MS
2756A>G and TSER
polymorphisms may also contribute to the interindividual variation in neurocognitive function seen in ALL survivors. The combined effect of multiple polymorphisms on the folate pathway, as measured by the GRS, may be more important than one single polymorphism.
The MTHFR, MS
, and TS
genes have key functions in the regulation of folate and homocysteine. The MTHFR enzyme catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-MTHF [31
], and the MTHFR
1298CC genotype results in 60% of the usual MTHFR enzyme activity [32
]. MS catalyzes the remethylation of homocysteine to methionine with methylcobalamin as a cofactor [31
], and MS deficiency results in elevated plasma homocysteine levels [33
]. The MS
2756A>G polymorphism leads to an amino acid substitution, and the variant G allele has been associated with lower plasma homocysteine levels [33
], possibly conferring a protective effect. Finally, TS catalyzes the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate [31
]. The promoter enhancer region of the TS
gene may contain two (2R) or three (3R) 28-bp tandem repeat sequences that function as transcriptional enhancer elements. The TSER
3R/3R genotype yields higher gene expression levels in vitro
and higher enzyme activity in vivo
than the 2R/2R genotype [35
]. The 3R/3R genotype is associated with reduced folate and higher homocysteine levels, particularly in individuals with low dietary folate intake [36
While the MTHFR
2756A>G, and TSER
polymorphisms do not directly lead to neurodegenerative disease in normal individuals, they may become critical in the setting of intermittently exaggerated folate depletion caused by MTX therapy for childhood ALL. Alternatively, it is possible that these polymorphisms are not directly associated with neurocognitive dysfunction but are surrogate markers for tightly linked polymorphisms that are in fact responsible for the associations seen in our study. MTHFR
1298A>C and MS
2756A>G were associated with impairment on the DIVERGT battery, suggesting an impact on global intellectual function. MTHFR
2756A>G, and TSER
polymorphisms also appeared to be related specifically to deficits in attention and processing speed, as indicated by TMTA and TMTB. In a study of ALL patients that included patients with cranial irradiation, Krajinovic et al found that polymorphisms in MTHFR
2756A>G, and MTRR
66A>G were not related to change in IQ scores over the first four years after diagnosis of ALL. These patients were diagnosed in a similar treatment era as patients on our study but had differences in CNS-directed therapy and did not have long-term neurocognitive evaluations that allow comparison to our study [37
The combined effect of multiple folate pathway polymorphisms may best predict which children are at risk for treatment-related neurocognitive deficits. Survivors with GRS ≥ 6 performed consistently worse on measures of attention and processing speed and were more likely to demonstrate clinical impairment in these domains. An individual with several at-risk genotypes may have striking variation in folate or homocysteine levels that leads to increased risk for neurocognitive deficits after MTX therapy. As the effects of additional folate pathway polymorphisms are evaluated in a larger sample size, the GRS may be refined to provide a more accurate prediction of which children are at risk for treatment-related toxicity.
Mean scores for TMTA, TMTB, and DIG in our study population were similar to population norms. However, the lack of baseline neurocognitive testing makes it impossible to evaluate whether declines occurred in these domains over time for individual patients. ALL survivors overall scored lower on tests of fine motor speed than expected for age-adjusted population norms. Similar findings have been reported in other studies and have been postulated to be related to vincristine chemotherapy [38
]. Decreased verbal fluency performance has also been previously reported in childhood ALL survivors and do not appear to be related to demographic or treatment variables [39
One of the strengths of our study was that the study population was drawn from long-term survivors, with median time off-therapy of 4.4 years, ensuring that ample time had elapsed for determination of neurocognitive dysfunction. Additionally, the potential for selection bias is decreased with the use of a validated neurocognitive screening battery, which can be administered in a time-efficient manner during the survivor's annual clinic visit, thereby promoting testing for all ALL survivors.
This study had several important limitations. The study was adequately powered to detect a statistically significant difference on neurocognitive tests of one standard deviation or greater. However, small differences between genotype groups may not have been detected, and we are unable to investigate potential gene-gene or gene-environment interactions. Validation of these results is clearly needed in a larger study. Additionally, the leukemia protocols used to treat participants in this study were heterogeneous and involved some differences in chemotherapy regimens for CNS prophylaxis. These differences between regimens are partially accounted for by inclusion of the important covariates of cumulative IV MTX dose and number of intrathecal MTX doses. We were unable to analyze the dosing schedule of MTX or leucovorin rescue in the current pilot study. Other limitations included a lack of information in this retrospective study on parental education, socioeconomic status, or dietary folate intake, all of which would be important to include in a future study.
Previous studies have shown conflicting results regarding the association between MTX dose intensity and long-term neurocognitive impairment. In a study of 79 survivors of high-risk ALL, neurocognitive performance did not vary between children treated with high-dose MTX and children treated with very high-dose MTX [5
]. However, in another study of 36 survivors, those who had received intensified treatment that included high-dose MTX were more likely to have attention deficits than survivors who had received intermediate-dose IV MTX on standard regimens [40
]. In our study, we did not observe a relationship between cumulative IV MTX dose or number of intrathecal MTX doses and performance on any of the neurocognitive tests. The study was not designed to investigate this association, so sample size may have been inadequate to detect small differences in test scores among treatment groups. It is also possible, however, that MTX “exposure” from an administered dose, as well as the downstream effects on the folate pathway and neurodevelopment, are moderated by genetic polymorphisms involving folate pathway enzymes. Possible interaction between genotype and MTX exposure will be important to investigate in a larger future study.
Early MTX-related neurotoxicity may predict long-term neurocognitive impairment, but many patients without a history of early MTX-related neurotoxicity also demonstrated long-term neurocognitive deficits. Although this study was not designed to detect an association between the rare outcome of early MTX-related neurotoxicity and folate pathway polymorphisms, further studies may investigate this relationship. Further investigation is also needed to evaluate the association between folate pathway polymorphisms and neurocognitive function in healthy populations. The MTHFR
677C>T polymorphism, frequently in the setting of hyperhomocysteinemia, has been associated with vascular dementia, cognitive decline with aging, depression, and schizophrenia in non-cancer populations [41
]. In a recent study of healthy adolescents, the MTHFR 677TT genotype was associated with a mild reduction in cognitive performance when compared to those with the MTHFR 677CC/CT genotypes [44
]. Further evaluation of the other folate pathway polymorphisms and neurocognitive outcomes in the general population may help clarify the impact of MTX therapy and possible gene-environment interactions.
In summary, we present additional evidence that folate pathway polymorphisms may modulate the development of neurocognitive deficits in childhood ALL survivors. Future directions include validation of these findings in a larger sample of research subjects and investigation of variation in other folate pathway genes, including cystathione beta synthase, reduced folate carrier 1, and dihydrofolate reductase. Additional pathways that may be important to investigate include other drug metabolism pathways, oxidative stress pathways, and glucocorticoid receptor signaling pathways. Results from these studies may allow us to identify ALL patients at highest risk for neurocognitive impairment and therefore develop interventions to prevent this important late effect of therapy.