Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease

Abstract

The importance of intracellular folate metabolism is illustrated by the severity of symptoms and complications caused by inborn disorders of folate metabolism or by folate deficiency. We examined three children of healthy, distantly related parents presenting with megaloblastic anemia and cerebral folate deficiency causing neurologic disease with atypical childhood absence epilepsy. Genome-wide homozygosity mapping revealed a candidate region on chromosome 5 including the dihydrofolate reductase (DHFR) locus. DHFR sequencing revealed a homozygous DHFR mutation, c.458A>T (p.Asp153Val), in all siblings. The patients' folate profile in red blood cells (RBC), plasma, and cerebrospinal fluid (CSF), analyzed by liquid chromatography tandem mass spectrometry, was compatible with DHFR deficiency. DHFR activity and fluorescein-labeled methotrexate (FMTX) binding were severely reduced in EBV-immortalized lymphoblastoid cells of all patients. Heterozygous cells displayed intermediate DHFR activity and FMTX binding. RT-PCR of DHFR mRNA revealed no differences between wild-type and DHFR mutation-carrying cells, whereas protein expression was reduced in cells with the DHFR mutation. Treatment with folinic acid resulted in the resolution of hematological abnormalities, normalization of CSF folate levels, and improvement of neurological symptoms. In conclusion, the homozygous DHFR mutation p.Asp153Val causes DHFR deficiency and leads to a complex hematological and neurological disease that can be successfully treated with folinic acid. DHFR is necessary for maintaining sufficient CSF and RBC folate levels, even in the presence of adequate nutritional folate supply and normal plasma folate.

Figure 1

Identification of the DHFR c.458A>T Mutation in a Family and Tertiary Structure of Human DHFR (A) Pedigree of the family. Genetic analysis was performed in the affected siblings and their parents. (B) Sequence analysis of DHFR exon 5 detected a homozygous base change, c.458A>T, in all siblings and a heterozygous mutation in both parents. Amplifications were performed with the HotStar-Taq DNA Polymerase Kit (QIAGEN) with 35 cycles in a GeneAmp PCR System 9700 (Applied Biosystems). Sequencing reactions were performed with BigDye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems) and analyzed on an ABI 3100 DNA Genetic Analyzer (Applied Biosystems). (C) Crystal structure of human DHFR (yellow ribbon model with highlighted secondary structure, Protein Data Bank entry 2W3M) bound to NADPH (color-coded stick model with green carbons, white phosphors, red oxygens, and blue nitrogens) and DHF (ruby stick model). The mutation site Asp153Val is highlighted as a magenta stick model. Asp153 stabilizes one side of the “F-G” loop, indicating that the mutation alters DHFR activity by influencing the fold and dynamics of the catalytically important “F-G” and “Met20” loops. The figure was prepared with the program PyMOL (Schrödinger).

Figure 2

Functional Characteristics of the DHFR Mutant (A) DHFR activity in lymphoblastoid cells of control individuals (DHFR wild-type), the patients' mother (heterozygous for DHFR p.Asp153Val = p.D153V), and the three siblings (homozygous for DHFR p.D153V) as measured by the formation of THF out of DHF per hour and mg protein. A DHFR Kit (Sigma) was used for the assay (for details, see text). (B) Fluorescence-activated cell sorting (FACS) analysis of lymphoblastoid cells after incubation with 10 μM fluorescein-labeled methotrexate (FMTX; Invitrogen). Cells were additionally stained with 7-Aminoactinomycin D (7-AAD; 2,5 μg/ml) to sort vital cells for FMTX-FACS analysis. (C) DHFR RT-PCR after isolation of mRNA from lymphoblastoid cells of the heterozygous mother (lanes 2 and 9), the three homozygous siblings (lanes 3–5 and 10–12; patients 1–3), and a control individual (DHFR wild-type) (lanes 6 and 13) revealed a single ∼450 bp cDNA fragment (agarose gel, 1.5%). cDNA was synthesized with the use of random hexamers and Superscript II RNase HT (Invitrogen) at 42°C for 50 min. DHFR PCR was performed in 35 cycles at 55°C (for primer sequences, see Table S1). (D) DHFR protein expression in DHFR p.D153V heterozygous (lane 1, mother) and homozygous (lane 2, patient 2; lane 3, patient 3; lane 4, patient 1) lymphoblastoid cells (upper panel) and fibroblasts as compared to control cells (stepwise dilution of total blotted protein: lanes 5–9). In the lower panel, lane 4 is waste because fibroblasts from patient 1 were not available. After separation on a 15% SDS gel, proteins were blotted on an Immobilon-P membrane (Millipore). Primary antibodies (DHFR [E-18], MAPK1 [D-2], Santa Cruz Biotechnology) were diluted to a concentration of 1:200 and 1:10,000; secondary antibodies (anti-goat IgG-HRP, Santa Cruz Biotechnology; goat anti-mouse IgG [H⁺L]-HRP, BioRad), 1:2000 and 1:10,000, respectively. Blots were developed with the use of the Super Script West Pico Chemoluminescent Substrate Kit (Thermo Scientific).