Dihydrofolate reductase deficiency (DHFR 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 (5-methyltetrahydrofolate) 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 (tetrahydrobiopterin, folate) 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 (DHFR) is a conserved enzyme found in all organisms that consists of 187 amino acids and exhibits an α/β-fold structure (protein secondary structure). It catalyzes the reaction of dihydrofolate (DHF) to tetrahydrofolate (THF) through stereospecific hydride transfer (transfer of a H- hydride ion(s) that results in the creation of a single specific stereoisomer), 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 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 (anemia with immature abnormally large red blood cells) and neurological manifestations. One report described considerable variation in clinical manifestations, noting that some patients had marked neurological symptoms with hematological (blood-related) 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 (enlarged red blood cells with a normal cell count) and megaloblastic anemia have been reported in affected siblings. Pancytopenia (low count in all blood cell types) has been reported in some individuals with DHFR deficiency. Thrombocytopenia (low platelet count) has also been reported in affected individuals. Leukopenia (low white blood cell count) has been described in at least one affected individual. Peripheral blood smear examination (microscopic analysis of circulating blood cells) in one affected individual showed both macrocytes (enlarged red blood cells), microcytes (abnormally small red blood cells), in addition to hyper segmented neutrophils(white blood cells with excessively segmented nuclei). Low folate levels in red blood cells has been reported in some individuals with DHFR deficiency. Normal serum folate (folate in blood) concentrations have been described in multiple affected individuals. Decreased or undetectable cerebrospinal fluid 5-methyltetrahydrofolate (a tetrahydrofolate derivative) levels have been reported in affected individuals. Increased serum lactate dehydrogenase (an enzyme involved in energy metabolism) levels have been documented in some individuals with DHFR deficiency.
Bone marrow features Bone marrow investigations have revealed megaloblastic erythropoiesis (production of enlarged red blood cells) in several individuals with DHFR deficiency. In one affected individual, bone marrow aspiration (a procedure that collects bone marrow for examination) showed megaloblastic erythropoiesis with early (immature) and late (more developed) megaloblasts (enlarged and immature red blood cell precursors), giant metamyelocytes (immature white blood cells), excess siderocytes (red blood cells containing iron granules), and reduced megakaryocytes (large bone marrow cells that produce platelets). In another affected individual, bone marrow biopsy demonstrated severely impaired erythroid outgrowth (expansion of red blood cell precursors) with a myeloid differentiation (maturation of bone-marrow-derived myeloid cells) 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 (microscopic examination of a sample of bone marrow) showed decreased erythropoiesis (red blood cell production) 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 (epilepsy with slow, irregular episodes of impaired awareness and staring) in childhood in at least one patient. Developmental delay with central hypotonia (low muscle tone from central nervous system dysfunction) and poor head control has been described in one affected child. Microcephaly (abnormally small head size) has been reported in several individuals with DHFR deficiency, and secondary microcephaly (small head size developing after birth) has been documented in one affected individual. Neuroimaging in one affected individual has shown cerebellar vermis hypoplasia (underdeveloped central region of the cerebellum), cerebellar and cerebral atrophy (loss of tissue), a thin corpus callosum, delayed myelination, increased cerebrospinal fluid volume, and ventricular dilatation (enlarged fluid-filled cavities where cerebrospinal fluid circulates). 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 (neuronal death in specific layers of the cerebral cortex), hemorrhagic leukomalacia (white-matter injury with bleeding), vermis inferior hypoplasia (underdeveloped lower cerebellar midline structure), and widespread supratentorial and infratentorial tissue loss (tissue loss above and below the tentorium, the membrane dividing upper and lower brain regions). Post-mortem neuropathologic examination has revealed a small brain with ventricular dilatation, white-matter atrophy, and calcifications in the basal ganglia and subcortical (regions under the cerebral cortex) 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 (high white blood cell count) despite normal absolute lymphocyte counts (the number of lymphocytes detected in blood), CD4/CD8 ratio (the proportion of helper T cells to cytotoxic T cells), naive/memory lymphocyte proportions (the distribution of newly formed and memory lymphocytes), and lymphocyte proliferation. The affected child with bronchopneumonia had decreased IgA (antibody found mainly in mucous membranes and secretions) and IgM (antibody produced early in the immune response) 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 (enlarged liver) has been reported in several infants with DHFR deficiency. Other symptoms described in affected patients include: pallor (unusual paleness of the skin), reduced oral intake with icterus (yellow discoloration of the skin and eyes), failure to thrive, coughing, and vomiting, fever, loose stools, pale complexion, and reduced urine output. Feeding via gastrostomy (a procedure that forms an opening into the stomach) has been reported in one affected child. One infant presented after a choking episode with hypothermia (low body temperature) and profound anemia and died shortly after arrival, with post-mortem examination showing hepatosplenomegaly (enlarged liver and spleen) and enlarged lymph nodes.
Cardiopulmonary features Respiratory failure and dyspnea (difficulty breathing) 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 (elevated blood pressure in the pulmonary circulation) and repeating respiratory insufficiency leading to death from pulmonary complications at four months of age has been reported in one affected infant. Cyanosis (blue coloration of the skin due to low oxygen levels) with a decrease in heart rate preceding death has been reported in one affected infant. Congenital heart disease (a heart condition present at birth) with an ostium secundum atrial septal defect (a defect in the central portion of the wall dividing the atria of the heart) 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 (reduced awareness or alertness) and repetitive eyeball movements during sleep, have been reported in one affected patient. Post-mortem examination in one affected infant has revealed gliosis (buildup of glial cells following nervous system damage) in the periventricular white matter (white matter located around the brain's ventricles) with possible optic atrophy.
Genetics