Folates, including folic acid, in particular have been administered to DS patients based on a preliminary study conducted some time ago and not meeting current protocol standards 
. Assessments of the efficacy and safety of this treatment were required. Our intention-to-treat analysis showed no positive effect of leucovorin. However, the per-protocol analysis of a restricted group examined by the same psychologist at the beginning and end of trial revealed a positive effect of leucovorin. In the per-protocol analysis, the DA of DS children was significantly higher on leucovorin treatment than on placebo (53.1% vs. 44.4%, p
0.031) (). The per-protocol analysis also identified several cofactors, including thyroid treatment. DS patients receiving leucovorin and thyroxin treatment had a DA of 59.7%, whereas those receiving thyroxin only had a DA of 40.3% (p
The choice to use folinic acid/leucovorin rather than folic acid is debatable. Leucovorin may be a good alternative to folic acid because it is transported by the RFC (SLC19A1 gene) and efficiently transformed into 5,10-CH2
THF and 5-CH3
THF, which may be reduced by three methyl transferases—DNMT3L, PRMT2, N6AMT1—and by CBS activity (). Several studies have reported this molecule to have a higher bioavailability than folic acid 
. No side effect of this drug was observed, despite its use at more than 10 times the RDA. Studies on folic acid have also reported an absence of adverse events 
. As some positive effects were detected in our patients, we believe this dose of LV to be appropriate and safe for folate supplementation.
We also hypothesized that age at the start of folate supplementation might be important, as young neurons may be more sensitive to treatment, resulting in more efficient rescue from folate deficiency. We therefore enrolled very young patients in this study (3 months to 30 months). Our study population was representative of the general DS population in terms of birth weight, length, occipito-frontal circumference and malformation frequency 
. However, the age range studied may have been too wide as major neurological acquisitions occur early in life and are highly variable. This may have resulted in heterogeneity of the population, potentially accounting, at least in part, for the negative result of the intention-to-treat analysis.
Several clinical trials had reported negative results regarding the effect of vitamin supplementation including folates 
. The results of these studies, although carefully monitored, may be challenged. Indeed, clinical trials on large DS populations are not easy to perform, as centres specialising in DS treatment do not have large patient cohorts. This is a major drawback of all studies. By contrast, the most recent study 
included 156 patients with Down's syndrome and a mix of 5 antioxidants or folinic acid was given in a blinded manner. Folinic acid was not found to be active on cognitive functions. However, we believe the daily dose of folinic acid (0.1 mg vs. 1.0±0.3 mg/kg in our study) was too low to be truly active. The negative results of our intent-to-treat analysis may be due to our lack of expertise in managing large trials. Psychometric analysis is a critical factor in such trials. When we first wrote the protocol, we thought it would be possible for the same psychometric examiner to carry out all three evaluations (V1, V2 and V3). However, as some of our patients came from far away and had limited flexibility in terms of appointments, it was sometimes difficult for the same psychologist to carry out assessments at the beginning and end of the study (12 months later). As even well trained professionals may vary in terms of the running of tests, we believe that analyses in which the same psychologist carried out at least two of the tests (per protocol analysis) are likely to be more accurate, although this violates the intention-to-treat analysis protocol. These pitfalls should be carefully avoided in subsequent studies, and the use of internationally validated scales would be required for international trials. However such scales are not always available, may be complicated to use, may result in young DS patients systematically obtaining scores towards the bottom of the scale or may not be linear over long periods. The Brunet-Lezine scale has several advantages: it is well established, easy to use and reproducible. However, we will probably use international scales, such as the Griffith's scale in future studies. Nevertheless, the development of reliable scales remains a key issue.
The relationship between leucovorin and thyroxin treatment was not expected, although thyroid status is known to be important for the mental development of patients with trisomy 21. Several hypotheses may be proposed to explain the possible synergistic effect of LV and thyroid treatment. The first hypothesis is that the antioxidant effect of folinic acid may have been amplified by the oxidative stress induced by thyroxin treatment. Indeed, thyroid hormone has been shown to increase mitochondria metabolism and thus production of reactive oxygen species (particularly in patients with hypothyroidism) and to induce an antioxidant imbalance 
. Furthermore, thyroid hormone has been shown to down regulate the expression of a gene involved in the catabolism of free oxygen radicals, superoxide dismutase-1 
. All together, these facts may explain why thyroxin may have stimulated the cognitive development of the children and how folinic acid may have limited the thyroxin drawbacks linked to oxidative stress.
The second hypothesis is that homocysteine, which is involved in both the folate pathway and thyroid status, may play a key role in the relationship between LV and thyroid treatment. Homocysteine is remethylated or leaves the cycle by being further transformed by CBS (cataplerosis) (). Thyroid hormones probably induce the expression of certain genes, but these results must be interpreted with caution, given the complexity of the different metabolic pathways. Plasma total homocysteine (tHcy) concentration is inversely correlated with thyroid hormone levels 
and returns to normal levels when euthyroidism is restored 
. Hypothyroid patients also have low plasma folate concentrations, and folate supplementation in addition to thyroid treatment is recommended 
. A relationship between thyroid status and folates has also been shown for methyl tetrahydrofolate reductase (MTHFR) activation in humans and rats 
. Some studies have reported that IQ is related to tHcy concentrations in DS patients, and to polymorphisms of two enzymes of the remethylation pathway—MTHFR (C677T) and a transcobalamin (TCN C776G) 
. Unfortunately, tHcy levels and MTHFR, TCN2 and MTHFD1 polymorphisms were not investigated in our patients. Future studies should consider these factors, to improve our understanding of therapeutic mechanisms. However, these findings suggest that homocysteine may play a negative role in the neurons of DS patients alone or via its metabolites 
The improvement of cognitive functions associated with LV treatment may increase the autonomy of the DS population. If maintained over time, it could increase quality of life and may have economic implications. A follow-up study is planned for these patients at the age of seven years, to check whether the LV-induced enhancement of development is maintained over time. The interaction between folates and thyroxin also requires investigation. Further studies should also focus on the cofactors identified in this trial. Despite technical difficulties, this trial strongly suggests that the psychomotor development of DS patients may be improved by treatments acting on metabolic pathways. These encouraging results should lead to a search for specific drugs targeting the metabolic disturbances induced by the overexpression of chromosome 21 genes.