Dihydrofolate reductase deficiency
Dihydrofolate reductase deficiency is an ultra-rare condition with less than 20 reported cases within the literature, as of 2025. It is an inherited disorder of folate metabolism caused by biallelic pathogenic mutations in the DHFR gene. Functionally, loss or significant reduction of DHFR activity impairs biological processes such as the conversion of dihydrofolate (DHF) to tetrahydrofolate (THF), the conversion of folic acid to dihydrofolate, and the recycling of tetrahydrobiopterin from dihydrobiopterin. These disrupt processes important for normal DNA replication and cell division, as well as neurotransmitter synthesis.
Presented symptoms vary greatly, but include megaloblastic anemia, cerebral folate deficiency, significantly reduced or absence of cerebrospinal-fluid folate and neurological symptoms of varying type and severity such as seizures, developmental delay, and microcephaly.The disorder follows an autosomal recessive mode of inheritance pattern. Diagnosis is typically conducted through combinations of genetic testing, biochemical assessment of specific metabolites in the body, and measurement of DHFR enzyme presence and activity. Treatment with folinic acid has been shown to improve some patient symptoms. The first confirmed cases of the condition were reported in 2011.
Dihydrofolate reductase protein
Dihydrofolate reductase is a conserved enzyme found in all organisms that consists of 187 amino acids and exhibits an α/β-fold structure. It catalyzes the reaction of dihydrofolate to tetrahydrofolate through stereospecific hydride transfer, as well as the folic acid to DHF reaction at a lower rate.THF and its derivatives are important in de novo synthesis of purines, thymidines, and various amino acids, which are important in the creation of DNA, cell proliferation and cell growth. It has also been shown to have a role in the recycling of tetrahydrobiopterin from dihydrobiopterin, which is a key cofactor for enzymes that synthesize important neurotransmitters including serotonin, melatonin, dopamine, norepinephrine, epinephrine, and nitric oxide.Tetrahydrobiopterin is also used as a cofactor in the metabolism of phenylalanine.
While the protein is expressed in most tissues with the exception of connective tissue, soft tissue and the eye, its RNA is expressed in all tissues in varying amounts.
The enzyme’s core is formed by an eight-stranded β-sheet surrounded by four α-helices on each side. Its active site, located near the N-terminus, forms an opening that binds both the cofactor NADPH and the substrate folate or dihydrofolate, facilitating the enzyme’s catalytic activity. Within this active site, three protein domains called the Met20, F-G, and G-H loops act as a flexible lid that encloses the cofactor NADPH and the substrate, stabilizing their interaction during catalysis of the reaction. Gene variants that affect this mechanism are common reasons for loss of function.
Another structural class of DHFR exists in bacterial chromosomes that is evolutionarily unrelated to the mammalian class. This class of DHFR is has much greater variation in mechanism due to evolutionary pressures from antibiotics compared to its mammalian counterpart, leading to many "types" being classified under this class.
Signs and symptoms
Verified cases of individuals with DHFR deficiency have included cases with both megaloblastic anemia and neurological manifestations. One report described considerable variation in clinical manifestations, noting that some patients had marked neurological symptoms with hematological abnormalities, whereas others had mild findings or no clear clinical signs.Hematologic features
Megaloblastic anemia has been reported in several individuals with DHFR deficiency. Macrocytosis without anemia and megaloblastic anemia have been reported in affected siblings. Pancytopenia has been reported in some individuals with DHFR deficiency. Thrombocytopenia has also been reported in affected individuals. Leukopenia has been described in at least one affected individual. Peripheral blood smear examination in one affected individual showed both macrocytes, microcytes, in addition to hyper segmented neutrophils. Low folate levels in red blood cells has been reported in some individuals with DHFR deficiency. Normal serum folate concentrations have been described in multiple affected individuals. Decreased or undetectable cerebrospinal fluid 5-methyltetrahydrofolate levels have been reported in affected individuals. Increased serum lactate dehydrogenase levels have been documented in some individuals with DHFR deficiency.Bone marrow features
Bone marrow investigations have revealed megaloblastic erythropoiesis in several individuals with DHFR deficiency. In one affected individual, bone marrow aspiration showed megaloblastic erythropoiesis with early and late megaloblasts, giant metamyelocytes, excess siderocytes, and reduced megakaryocytes. In another affected individual, bone marrow biopsy demonstrated severely impaired erythroid outgrowth with a myeloid differentiation defect, the presence of multiple megaloblasts, an almost complete absence of late-stage red blood cell precursors, and giant myeloid bands. In a further case, a bone marrow smear showed decreased erythropoiesis with megaloblasts and abnormal myeloid cell production with hypersegmented neutrophils.Neurological features
Seizures and epilepsy have been reported in several individuals with DHFR deficiency.Neurodevelopmental impairments have been described in several affected individuals, including moderately severe developmental disorders with epilepsy, severe developmental delay, and later-emerging neurodevelopmental delay unresponsive to treatment. Learning difficulties have been reported alongside atypical absence epilepsy in childhood in at least one patient. Developmental delay with central hypotonia and poor head control has been described in one affected child. Microcephaly has been reported in several individuals with DHFR deficiency, and secondary microcephaly has been documented in one affected individual. Neuroimaging in one affected individual has shown cerebellar vermis hypoplasia, cerebellar and cerebral atrophy, a thin corpus callosum, delayed myelination, increased cerebrospinal fluid volume, and ventricular dilatation. Additional neuroimaging findings reported in separate individuals include underdeveloped or thin corpus callosum, brain atrophy, and presence of calcium deposits within brain tissue; cortical laminar necrosis, hemorrhagic leukomalacia, vermis inferior hypoplasia, and widespread supratentorial and infratentorial tissue loss. Post-mortem neuropathologic examination has revealed a small brain with ventricular dilatation, white-matter atrophy, and calcifications in the basal ganglia and subcortical white matter in one affected individual.Infectious and immunologic features
Respiratory infections have been reported in several individuals with DHFR deficiency, including frequent lower respiratory tract infections, Pneumocystis jirovecii infection, and bronchopneumonia. The infant with Pneumocystis jirovecii infection had leukocytosis despite normal absolute lymphocyte counts, CD4/CD8 ratio, naive/memory lymphocyte proportions, and lymphocyte proliferation. The affected child with bronchopneumonia had decreased IgA and IgM concentrations. Death due to Klebsiella aerogenes pneumonia has been reported in one infant with DHFR deficiency. In another case, death occurred following severe pneumonia and respiratory failure in early infancy. Herpes stomatitis has also been reported in one affected infant.Gastrointestinal and constitutional features
Hepatomegaly has been reported in several infants with DHFR deficiency. Other symptoms described in affected patients include: pallor, reduced oral intake with icterus, failure to thrive, coughing, and vomiting, fever, loose stools, pale complexion, and reduced urine output. Feeding via gastrostomy has been reported in one affected child. One infant presented after a choking episode with hypothermia and profound anemia and died shortly after arrival, with post-mortem examination showing hepatosplenomegaly and enlarged lymph nodes.Cardiopulmonary features
Respiratory failure and dyspnea have been reported in infants with DHFR deficiency affected by Pneumocystis jirovecii infection. Respiratory failure has also been described in one affected infant with severe pneumonia. Pulmonary hypertension and repeating respiratory insufficiency leading to death from pulmonary complications at four months of age has been reported in one affected infant. Cyanosis with a decrease in heart rate preceding death has been reported in one affected infant. Congenital heart disease with an ostium secundum atrial septal defect has been reported in one affected infant.Ophthalmologic and sensory features
Frequent episodes of impaired vision with blinking and winking, partially associated with impaired consciousness and repetitive eyeball movements during sleep, have been reported in one affected patient. Post-mortem examination in one affected infant has revealed gliosis in the periventricular white matter with possible optic atrophy.Genetics
DHFR deficiency is consistent with a autosomal recessive inheritance model. The gene that codes for the functional variant has 6 exons, and 5 introns. The gene is located on the long arm, or q arm of chromosome 5, in the q14.1 region, between nucleotide 79613515 and nucleotide 82033415 on the Human Genome Assembly, however, processed pseudogenes lacking introns have been found on separate chromosomes. Specifically, pseudogene-1 was mapped to chromosome 18, pseudogene-2 was mapped to chromosome 6, pseudogene-3 was mapped to chromosome 2, and pseudogene-4 was mapped to chromosome 3. DHFR RNA is expressed in all tissues in varying amounts, however functional DHFR protein is not expressed in connective tissue, soft tissue and the eye. The disease causing variant differs across affected cases on different exons. Cases of affected patients showed both compound heterozygote and homozygous genotypes for a single disease-causing variant.The compound heterozygote variants were not found in public population databases or local control samples that were used in the study, which they deemed "hallmarks of a rare variant." These variants were given "likely pathogenic variants" classification by the American College of Medical Genetics and Genomics guidelines. The other variants were classified as "pathogenic."
Examples of these variants include homozygous c.61G>A; p.Gly21Arg, homozygous c.458A>T; p.Asp153Val, homozygous c.238C>T; p. Leu80Phe, homozygous c.335 T > G; p.Met112Arg and compound heterozygous c.77C > T; p.Gly18Val and c.53G > T; p.Pro26Leu.
Pathophysiology
DHFR catalyzes the dihydrofolate to tetrahydrofolate reaction, as well as the folic acid to dihydrofolate reaction. Proper folate metabolism by this enzyme is key for the de novo synthesis of purines, thymidines, and various amino acids, which are important in DNA synthesis, cell proliferation and cell growth.DHFR deficiency would also impact recycling of tetrahydrobiopterin from dihydrobiopterin, impacting neurotransmitter synthesis and phenylalanine metabolism. One study linked the lack of cerebral tetrahydrobiopterin to neurodevelopmental symptoms that arise.Differences in disease causing variants causes unique protein level pathophysiology in different cases:
| Variant | Location | Protein Level Impact |
| Homozygous c.238C>T;p.Leu80Phe | Exon 3 | The addition of a phenylalanine alters the position of a critical lysine 55 amino acid residue, creating steric hindrance and stopping cofactor binding for the altered protein. DHFR activity was 100 times lower than wild-type, non-altered DHFR. |
| Compound Heterozygous c.77C>T;p.Gly18Val and c.53G>T;p.Pro26Leu | Both located in the active-site region at the N-terminus. | Gly18Val: This variant altered the torsion angle of the proteins, which negatively affects the thermodynamic stability of the proteins. This led to lower protein expression and activity. Furthermore, Valine is larger than glycine, changing the shape of the substrate binding pocket for NADPH. |
| Compound Heterozygous c.77C>T;p.Gly18Val and c.53G>T;p.Pro26Leu | Both located in the active-site region at the N-terminus. | Pro26Leu: This variant altered the torsion angle of the proteins, which negatively affects the thermodynamic stability of the proteins. This led to lower protein expression and activity. The variant also disrupted the folding of the Met20 loop protein domain. |
| Homozygous c.61G>A;p.Gly21Arg | active site region at the N terminus | The Gly21, situated in the substrate binding pocket for NADPH, is replaced with a highly polar, larger arginine. This significantly affects the ability of NADPH to bind within the active site, reducing catalytic efficiency and potentially protein stability as well. A complete absence of DHFR activity was noted. |
| Homozygous c.458A>T;p.Asp153Val | Exon 5 | Asp153 normally stabilizes the "F-G" loop via a hydrogen bond. The mutation disrupts the fold/dynamics of the "F-G" and "Met20" loops, leading to reduced catalytic efficiency and protein instability. DHFR activity in lymphoblastoid cells of all three patients was severely reduced to less than 10% of control levels, and protein expression was reduced to 20%-50% compared to control. Fibroblasts showed 70%-80% of control protein expression across the three patients. mRNA expression was not significantly different between wildtype and control subjects. |
| Homozygous c.335T>G;p.Met112Arg | Not described | Not described |
Diagnosis
Genetic analysis
Genetic diagnosis of DHFR deficiency has been established by identifying mutations in the DHFR gene in affected individuals. Whole-exome sequencing has been employed in several reported families to identify homozygous or compound-heterozygous mutations DHFR variants. In children from first-cousin parents, autozygosity mapping on SNP arrays identified a homozygous region at containing DHFR, and sequencing of this region revealed a homozygous variant. In one case study, children from distantly related patents were subject to Genome-wide homozygosity mapping followed by DHFR sequencing detected a novel homozygous variant in all affected siblings. The authors of a 2025 DHFR deficiency case study suggested that their findings indicate a need for early molecular diagnosis of newborns, given that they presented unexplained megaloblastic anemia, delayed development, or epilepsy.Biochemical analysis
The diagnostic evaluation of DHFR deficiency often involves the biochemical analysis of folate concentrations and metabolite profiles within blood and cerebrospinal fluid. In one report, folate metabolite profiles in red blood cells, plasma, and cerebrospinal fluid were analyzed by liquid chromatography-tandem mass spectrometry as part of the diagnostic evaluation. In another study, red blood cell folate components were measured by liquid chromatography-tandem mass spectrometry to evaluate the distribution of different folate forms during diagnostic assessment. Measurement of cerebrospinal-fluid folate metabolites, including 5-methyltetrahydrofolate, has been performed as part of the diagnostic assessment in several individuals. In some patients, cerebrospinal-fluid neurotransmitter metabolites and tetrahydrobiopterin-related compounds were also measured to assess associated monoamine and BH4 abnormalities.Enzymatic activity assays
DHFR enzymatic activity has been assessed in Epstein-Barr virus-immortalized lymphoblastoid cell lines derived from affected individuals to determine if protein activity is impaired. In one study, DHFR activity in lymphoblastoid cells was quantified by measuring the formation of tetrahydrofolate from dihydrofolate using liquid chromatography-tandem mass spectrometry, and comparison of patient-derived and control cells in this assay was used to confirm reduced DHFR activity. Another report measured DHFR activity using fluorescein-labeled methotrexate binding in lymphoblastoid cells from affected siblings to assess substrate binding and DHFR function, as methotrexate can bind to the active site of DHFR.Expression analysis
Reverse-transcription polymerase chain reaction of DHFR mRNA has been performed in lymphoblastoid cells from affected individuals and controls to assess the expression levels of DHFR mutant transcripts compared to control transcripts. Immunoblot analysis of fibroblasts and Epstein-Barr virus-immortalized lymphoblastoid cells has been used to assess DHFR protein expression in affected individuals and heterozygous parents.Functional characterization of DHFR variants
Functional evaluation of DHFR variants has been performed by transfecting cultured cells with expression constructs carrying wild type or mutant DHFR. Protein levels in these cells have been examined by western blots and enzyme linked immunosorbent assay. Furthermore, a labeled ligand binding assay has also been used to assess DHFR binding in transfected cells.Treatment and management
Folinic acid therapy
Folinic acid supplementation has been reported in several individuals with DHFR deficiency. Folinic acid was initiated in some individuals after cerebrospinal-fluid 5-methyltetrahydrofolate concentrations were found to be low or undetectable. In one report, anemia and pancytopenia improved during folinic acid supplementation whereas severe developmental delay and other neurological features persisted.In multiple individuals, cerebrospinal-fluid 5-methyltetrahydrofolate concentrations increased or normalized following folinic acid supplementation. In one affected infant, oral folinic acid at a dose of 30 mg daily was followed by improvement in anemia, seizure control, and cerebrospinal-fluid 5-methyltetrahydrofolate levels, while profound developmental delay with central hypotonia and poor head control persisted. In another infant, folinic acid at 3 mg/kg per day was reported to improve neurological symptoms before death from severe pneumonia and respiratory failure. In three affected siblings, folinic acid at 1 mg/kg per day was reported to normalize cerebrospinal-fluid 5-methyltetrahydrofolate in two siblings and to increase red blood cell folate concentrations with normalization of mean corpuscular volume and bone-marrow morphology in all three.
In one previously asymptomatic sibling who received folinic acid irregularly, focal epilepsy was reported three years later as the first neurological impairment. In one additional sibling, folinic acid therapy was followed by a period of transient independence from anticonvulsive medications without additional neurological symptoms, and later irregular folinic acid supplementation was associated with recurrence of epileptic symptoms.
Clinical deterioration occurred in some infants despite improvements in hematologic or biochemical measures during folinic acid supplementation. One report described an affected infant with no detectable dihydrofolate-reductase activity who died in early infancy despite folinic acid supplementation and noted that earlier cases with residual activity had shown reported improvement. The same reported suggested that residual enzyme activity might relate to those responses while concluding that the overall effect of folinic acid supplementation remains uncertain.
Folic acid, vitamin B12, and hydroxocobalamin
In one infant, folic acid was started for megaloblastic anemia, and the anemia resolved with initially satisfactory neurodevelopmental progress before folic acid discontinuation was followed by status epilepticus. One patient with macrocytosis without anemia and atypical absence epilepsy was given folic acid at 5 mg per day, but their neurological symptoms persisted. Hydroxocobalamin was started in an affected infant after a working diagnosis of transcobalamin II deficiency, and the hematological profile remained unresponsive to this treatment. In another infant, neither vitamin B12 nor folic acid improved clinical or laboratory abnormalities.Adjunctive therapies and acute management
In one infant with DHFR deficiency and Pneumocystis jirovecii infection, treatment included high-dose co-trimoxazole, prednisolone, extracorporeal membrane oxygenation, and intravenous folinic acid supplementation. The infant showed hematological recovery and extracorporeal membrane oxygenation support was discontinued, but later experienced a clinical deterioration and died despite receiving folinic acid therapy. The authors noted that high-dose co-trimoxazole treatment for Pneumocystis jirovecii infection may have affected the clinical course, because trimethoprim inhibits the dihydrofolate reductase enzyme, and they stated that a negative contribution of this treatment could not be excluded.Anticonvulsant medications have been used to manage seizures in several individuals. In one infant, phenobarbital treatment was reported to terminate seizures initially, but seizures recurred the following day and prompted further evaluation and introduction of folinic acid therapy. One infant had generalized and focal seizures that were unresponsive to phenytoin, benzodiazepines, and pyridoxine before partial seizure control was achieved with phenobarbitone and levetiracetam.
Individuals without DHFR-specific treatment
Some individuals with DHFR deficiency have been described to not receive disease-specific treatment at the time of reporting. One patient with anemia and treatment-resistant seizures of undefined cause died in infancy before DHFR deficiency was recognized and before folinic acid supplementation was introduced in the family.One report indicated that identifying and initiating treatment for the disorder early in development would result in a better prognosis, and for families with a history of DHFR deficiency, genetic counselling and prenatal screening were suggested.
History
In 1967, there was a report of a patient with possible DHFR deficiency, but normal DHFR activity was found in the patient’s fibroblasts later. In 1976, another two cases of Dihydrofolate Reductase Deficiency were described, but it was shown that one patient had methionine synthase reductase deficiency and the other had transcobalamin II deficiency. Before 2011, no molecularly confirmed cases of dihydrofolate reductase deficiency had been reported.In 2011, three affected individuals of European descent were reported with two different pathogenic DHFR variants, and three affected siblings of British Pakistani origin were described in a separate study. In 2017, one affected German individual was identified. In 2022, three additional affected individuals from a Dutch pedigree were reported with homozygous DHFR variants. In 2025, two affected siblings of Chinese ancestry were described with compound-heterozygous DHFR variants.