Dihydrofolate Reductase Deficiency Is Caused by a Homozygous DHFR Mutation and Leads to Congenital Megaloblastic Anemia and Cerebral Folate Deficiency

Abstract 1006

Folate deficiency can be associated with megaloblastic anemia, neurological and mental disorders, cardiovascular disease, embryonic defects, in particular neural tube defects, and, possibly, malignancies. The importance of an intact intracellular folate metabolite cycle is illustrated by the severity of symptoms in rare inborn disorders of folate metabolism.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) and at a lower rate of folic acid (FA) to DHF. DHFR plays a key role in maintaining intracellular folate homeostasis. A distinct genetic defect associated with DHFR deficiency has not been described.

We examined three children of healthy distantly related parents presenting with megaloblastic anemia and neurological symptoms of varying severity. Total serum folate and cobalamin, homocysteine, urinary excretion of methylmalonic acid, orotic acid, formiminoglutamic acid, and cobalamin binding capacity were normal. Red blood cell (RBC) folate was below the normal range in all patients. Cerebrospinal fluid (CSF) analysis revealed very low 5-methyl-tetrahydrofolate (5MTHF) in one and the absence of detectable CSF 5MTHF in two siblings. Treatment with folinic acid (5-formyl-tetrahydrofolate; 5FTHF) increased RBC folate in all patients associated with a normalization of RBC indices and bone marrow morphology and led to CSF 5MTHF normalization.

According to a recessive inheritance model, we conducted a screen for homozygous chromosomal regions using genome-wide analysis of single nucleotide polymorphisms. This analysis revealed a short overlap on the long arm of chromosome 2 and a second common region on the long arm of chromosome 5. This region included the DHFR gene locus. Genomic sequence analysis of DHFR exons and neighboring nucleotides revealed a homozygous mutation DHFR c.458A>T in exon 5 (RefSeq NM_000791) leading to the amino acid change DHFR p.Asp153Val (p.D153V) in all siblings.

Assuming DHFR deficiency we analyzed folate metabolites in RBCs and plasma by liquid chromatography tandem mass spectrometry (LC-MS/MS). The folate profile in RBCs and plasma was compatible with DHFR deficiency. To examine DHFR function we studied the formation of THF out of DHF in EBV-immortalized lymphoblastoid cells from patients, their heterozygous mother and healthy control individuals. DHFR activity was severely reduced in all patient cells (12, 88, 51 pmol THF per hour and mg protein, respectively) as compared to controls (median 3741, range 1441–6828 pmol/h_*mg) whereas cells of the heterozygous mother exhibited DHFR activity close to the lower limit of the control range (1156 pmol/h_*mg). A fluorescent Methotrexate (MTX) binding assay showed a markedly reduced MTX binding capacity in EBV-immortalized lymphoblastoid cells of all three patients. Cells of the heterozygous mother presented an intermediate binding capacity. To assess whether the reduced DHFR activity was caused by an altered mRNA and protein expression due to the DHFR p.Asp153Val mutation we performed RT-PCR and western blot analyses. RT-PCR revealed a single ∼450bp cDNA fragment without significant quantitative differences between DHFR wildtype, DHFR p.Asp153Val heterozygous, or homozygous cells. In contrast, DHFR protein expression was reduced in EBV-immortalized lymphoblastoid cells carrying the DHFR mutation as compared to wildtype cells. Semiquantitative assessment revealed a reduction of the amount of intact DHFR protein in heterozygous lymphoblastoid cells to about 70 percent, in homozygous cells from one patient to 50 percent and from the other patients to 20–30 percent as compared to wildtype cells.

In conclusion, the homozygous mutation DHFR p.Asp153Val causes DHFR deficiency and leads to a complex hematological and neurological disease which can successfully be treated with 5FTHF.