Axial T2 (A-D), midsagittal T1 (E-H), and right parasagittal T1 (I-L) sequences obtained at 3 months (A, E, I), 13 months (B,F, J), 26 months (C,G, K), and 6 years (D, H, L) of age. The initial MRI at 3 months demonstrates mild lateral ventriculomegaly and delayed myelination for age, best appreciated as absence of posterior limb of the internal capsule myelination despite presence of optic radiation myelination (A). Mild cerebellar fissure widening is also appreciated in I. By 13 months, rudimentary white matter myelination has occurred centrally on T1 weighted imaging in the corpus callosum (F) and periventricular white matter (J). However, myelination remains profoundly delayed, and the corpus callosum has not thickened as expected, now falling below the third percentile for age. By 26 months of age, the myelination within the optic radiations on T2 weighted imaging (C) and the central white matter on T1 weighted imaging (G,K) has become more robust. However, there is no appreciable progression in anatomic extent and the overall myelination remains markedly delayed. On the subsequent MRIs, no further myelination has occurred allowing differences in MRI field strength as shown on the MRI performed at 6 years of age (D, H, L). Hypoplasia of the corpus callosum, widening of the cerebellar fissures, and lateral ventriculomegaly persist without change though the foramen Magendie has grown more patulous over time potentially indicating subtle volume loss (E-H). Posterior limb of the internal capsule (A-D): white arrow (solid stem). Corpus callosum (E-H): white arrow (dashed stem). Periventricular white matter (I-L): white arrowheads. Widened cerebellar fissures (I-L): dashed circle. Widened foramen Magendie (H): asterisk.

Graph of CSF 5-methyltetrahydrofolate (5-MTHF) and red blood cell (RBC) folate levels over time, and on various treatments. The dotted horizontal line represents the lower limit of the normal range for CSF 5-MTHF levels (40 nmol/L). RBC folate levels are in ng/mL and CSF 5-MTHF levels are in nmol/L.

Axial T2 (A, D, E, F) and sagittal T1 (B,C) images of Patient 2 at 16 months (A-C), 5 years (D), 6 years (E), and 8 years (F) of age. The initial MRI at 16 months (A-C) demonstrates delayed myelination on T1 weighted imaging (absent subcortical white matter myelination) and T2 weighted imaging (absent posterior limb of the internal capsule myelination). Follow-up imaging through 8 years of age (D-F) demonstrates no significant progress in myelination. Posterior limb of the internal capsule (A, D-F) : white arrow (solid stem). Corpus callosum (B): white arrow (dashed stem). Periventricular white matter (C): white arrowheads. Widened cerebellar fissures (C): dashed circle.

Time dependency of MTHFS enzyme activity as shown by the production of 5,10-methenylTHF as measured by LC-MS/MS. The product was measured in one control line of skin fibroblasts and fibroblasts derived from patient 1 at 37°C for 0, 6, 12, 18, 24 and 30 minutes as described in the Material and Methods section. The final protein concentration in the reaction mixture was 2mg/mL and each data point is the average of duplicate measurements. Immediately after reconstitution of the reaction mixture, blank samples for both disease and the control lines were obtained by boiling the time zero samples for 3 minutes. The time zero blank samples were subtracted from each data point. The above figure is representative of one of 3 experiments. The R square value for the linear regression curve for the control samples is 0.9903.

5-formyl-THF accumulates in fibroblasts of the affected patient. (A) Total ion chromatogram (TIC) of m/z = 472.157 corresponding to the molecular weight of 5 and 10-formyl-THF. 2 peaks are present representing 10-formyl-THF (RT= 7.91 min) and 5-formyl-THF (RT= 8.36 min) species. (B) Normalized abundances of folate species in control (EC and BJ) and patient (1183–01) fibroblasts (mean ± stdev, n=3).

Folate metabolism pathway. Folate metabolites are in bold. Enzymes are in italics. The dashed line represents inhibition of the enzyme serine hydroxymethyltransferase by 5-formyl THF polyglutamate. THF = tetrahydrofolate, DHF = dihydrofolate, MTHFS = 5,10-methenyltetrahydrofolate synthetase, FGAR = formylglycinamide ribotide, GAR=glycinamide ribotide, AICAR = aminoimidazole carboxamide ribotide, FAICAR = formylaminoimidazole carboxamide ribotide, dTMP = deoxythmidine monophosphate, dUMP = deoxyuridine monophosphate.