Constitutional Megaloblastic Anemia With Severe Neurologic Disease

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Classifications

Harrison's Part

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Inheritance

1

Autosomal Recessive HP:0000007

Biallelic DHFR missense variants. Heterozygous carriers are clinically unaffected but are not biochemically silent: their cells show intermediate DHFR activity and intermediate methotrexate binding, which is a clean demonstration of gene dosage in this enzyme.

Autosomal recessive inheritance

Show evidence (2 references)

PMID:21310277 SUPPORT Human Clinical

"DHFR sequencing revealed a homozygous DHFR mutation, c.458A>T (p.Asp153Val), in all siblings."

Documents homozygosity in all three affected siblings of healthy parents.

PMID:21310277 SUPPORT In Vitro

"Heterozygous cells displayed intermediate DHFR activity and FMTX binding."

Supports the specific gene-dosage claim about carriers made in the description.

?

Discussions and Knowledge Gaps

2

Does folinic acid correct the cerebral tetrahydrobiopterin arm of DHFR deficiency, or only the folate arm, and should BH4 or neurotransmitter-precursor supplementation be added?

KNOWLEDGE GAP bh4_arm_not_separately_treated

Attached to

pathophysiology#Failure of Tetrahydrobiopterin Salvage treatments#Folinic Acid (Calcium Leucovorin)

Folinic acid bypasses DHFR for folate, because it is a pre-reduced folate. It has no equivalent action on biopterin: dihydrobiopterin still needs DHFR to be reduced, and folinic acid does not supply tetrahydrobiopterin. The published reports document normalization of CSF folate and improvement of neurological symptoms, but do not report post-treatment CSF biopterin or monoamine metabolites, so whether the residual neurological deficit reflects an untreated BH4 arm is unknown. This matters directly: if it does, sapropterin or levodopa/5-hydroxytryptophan supplementation would be rational adjuncts, and the treatment section of this entry is incomplete rather than merely brief.

Has the thymidylate/purine-synthesis block been measured in DHFR-deficient erythroid precursors, or is it inferred from folate biochemistry?

KNOWLEDGE GAP erythroid_step_is_inferred

Attached to

pathophysiology#Impaired Thymidylate and Purine Synthesis

This node is the weakest link in the entry's haematological arm and is marked as such in its own description. The enzymatic deficiency is measured, the folate pool is measured, and the megaloblastic anaemia is observed; the step joining them is supplied by textbook folate biochemistry, quoted from a review's introduction rather than from any result in a DHFR-deficient patient. Nothing about it is controversial, but the entry should not imply a measurement that was not made, and a deoxyuridine suppression test or dNTP pool measurement in patient erythroid cells would close it.

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Pathophysiology

5

DHFR Enzyme Destabilization

The disease-causing variants are missense changes that destabilize the protein rather than simply abolishing the catalytic site. The evidence for that distinction is direct: DHFR mRNA is indistinguishable from wild type while protein expression is reduced, so the lesion acts after transcription. DHFR is a small NADPH-dependent oxidoreductase, and substitutions in its compact fold lower both abundance and specific activity.

Genetic context DHFR hgnc:2861 variant_origin: GERMLINE zygosity: HOMOZYGOUS functional_impact_category: LOSS_OF_FUNCTION

dihydrofolate reductase activity GO:0004146 ↓ DECREASED

Downstream

Collapse of the Reduced Folate Pool

Failure of Tetrahydrobiopterin Salvage

Show evidence (2 references)

PMID:21310277 SUPPORT In Vitro

"DHFR activity and fluorescein-labeled methotrexate (FMTX) binding were severely reduced in EBV-immortalized lymphoblastoid cells of all patients."

Measures the enzymatic deficiency directly in patient cells, by two independent assays.

PMID:21310277 SUPPORT In Vitro

"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."

The normal-mRNA / low-protein result is what establishes destabilization rather than a transcriptional or purely catalytic-site lesion.

Collapse of the Reduced Folate Pool

Tetrahydrofolate can no longer be regenerated from dihydrofolate, nor produced from dietary folic acid. The distinctive feature is that this happens with a normal folate supply: plasma folate is adequate, and the deficit is in the reduced forms that the cell can actually use. Patient folate profiling by liquid chromatography tandem mass spectrometry in red cells, plasma and cerebrospinal fluid is what demonstrated the pattern.

tetrahydrofolate biosynthetic process GO:0046654 ↓ DECREASED

Downstream

Impaired Thymidylate and Purine Synthesis

Cerebral Folate Deficiency

Show evidence (2 references)

PMID:21310277 SUPPORT Human Clinical

"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."

The direct measurement of the folate pool across the three compartments this node describes.

PMID:21310277 SUPPORT Human Clinical

"DHFR is necessary for maintaining sufficient CSF and RBC folate levels, even in the presence of adequate nutritional folate supply and normal plasma folate."

Supports the specific claim that the deficit occurs despite adequate intake and normal plasma folate, which is what makes this a regeneration failure.

Impaired Thymidylate and Purine Synthesis

Mechanism confidence: Provisional

Tetrahydrofolate is the one-carbon carrier for thymidylate synthase and for de novo purine synthesis. Without it, dividing cells cannot make dTMP or purines at the rate DNA replication demands. Erythroid precursors are the most affected because they divide fastest, and the resulting nuclear-cytoplasmic asynchrony is what megaloblastic morphology is. This step is inferred from established folate biochemistry rather than measured in these patients; what was measured is the haematological outcome.

erythroid progenitor cell CL:0000038

dTMP biosynthetic process GO:0006231 ↓ DECREASED purine nucleotide biosynthetic process GO:0006164 ↓ DECREASED

Downstream

Megaloblastic anemia

Pancytopenia

Show evidence (1 reference)

PMID:22108709 SUPPORT BACKGROUND Other

"Derivatives of folic acid are involved in transfer of one-carbon units in cellular metabolism, playing a role in synthesis of purines and thymidylate and in the remethylation of homocysteine to form methionine."

States the biochemical role this node depends on. Quoted from the review's opening framing rather than from a result, which is why quote_role is BACKGROUND; the claim is textbook folate biochemistry, not a finding of this paper.

Cerebral Folate Deficiency

Cerebrospinal-fluid 5-methyltetrahydrofolate falls, which is the definition of cerebral folate deficiency. The central nervous system is disproportionately affected because it depends on active transport of reduced folate across the choroid plexus and has little reserve; peripheral folate status can look adequate while the CSF compartment is depleted.

brain UBERON:0000955

Downstream

Atypical absence epilepsy

Global developmental delay

Show evidence (1 reference)

PMID:21310276 SUPPORT Human Clinical

"characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency"

Names severe cerebral folate deficiency as a defining component of the disorder, alongside the two other arms.

Failure of Tetrahydrobiopterin Salvage

DHFR has a second substrate. It reduces dihydrobiopterin back to tetrahydrobiopterin, the salvage arm of BH4 metabolism, and this is the arm the brain relies on. Loss of it produces cerebral tetrahydrobiopterin deficiency. BH4 is the obligatory cofactor of phenylalanine, tyrosine and tryptophan hydroxylase, so the consequence is reduced synthesis of dopamine, serotonin and noradrenaline. This arm is what separates DHFR deficiency from the other cerebral folate deficiencies, and the 2011 characterization of the disorder is what established the link in humans.

tetrahydrobiopterin regeneration GO:0006729 ↓ DECREASED

brain UBERON:0000955

Downstream

Global developmental delay

Show evidence (1 reference)

PMID:21310276 SUPPORT Human Clinical

"The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids."

Establishes both the DHFR-to-BH4 link and the monoamine consequence this node asserts, in the paper that first characterized it.

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Pathograph

Use the checkboxes to hide or show graph categories. Hover nodes for evidence and cross-linked metadata.

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Phenotypes

4

Blood 2

Megaloblastic anemia HP:0001889

Show evidence (1 reference)

PMID:21310277 SUPPORT Human Clinical

"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."

Records megaloblastic anaemia as the presenting feature in the index siblings.

Pancytopenia HP:0001876

Show evidence (1 reference)

PMID:21310276 SUPPORT Human Clinical

"characterized by megaloblastic anemia and/or pancytopenia"

Records pancytopenia as an alternative or additional haematological presentation.

Nervous System 2

Atypical absence epilepsy Atypical absence seizure HP:0007270

Show evidence (1 reference)

PMID:21310277 SUPPORT Human Clinical

"cerebral folate deficiency causing neurologic disease with atypical childhood absence epilepsy"

Names the specific seizure type and attributes it to the cerebral folate deficiency.

Global developmental delay HP:0001263

Show evidence (1 reference)

PMID:22108709 SUPPORT REVIEW SYNTHESIS Other

"Dihydrofolate reductase deficiency is characterized by megaloblastic anemia and cerebral folate deficiency, with variable neurological findings."

The review's summary of the disorder, including the explicit statement that the neurological findings are variable.

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Genetic Associations

1

DHFR (Biallelic Destabilizing Missense Variants)

Gene: DHFR hgnc:2861 relationship_type: CAUSATIVE variant_origin: GERMLINE

Show evidence (2 references)

PMID:21310276 SUPPORT Human Clinical

"due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency"

Characterizes the allele class and its functional consequence.

PMID:21310277 SUPPORT Human Clinical

"Genome-wide homozygosity mapping revealed a candidate region on chromosome 5 including the dihydrofolate reductase (DHFR) locus."

Documents how the locus was identified, and places DHFR on chromosome 5.

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External Assertions

3

OMIM megaloblastic anemia due to dihydrofolate reductase deficiency phenotype record

OMIM disease record OMIM:613839

The OMIM phenotype record for this disease. Recorded here rather than under `mappings` because `DiseaseMappings` carries only ICD-10-CM, ICD-11, MONDO and NCIT slots and there is no `omim_mappings` slot in the schema; issue #9922 withdrew a proposal to add one.

OMIM DHFR gene record

OMIM gene record OMIM:126060

The OMIM record for the DHFR gene, recorded alongside the phenotype MIM so the two are not confused with each other.

Orphanet megaloblastic anemia due to dihydrofolate reductase deficiency

Orphanet disease record ORPHA:319651

The Orphanet record for this disease. Recorded as an external assertion for the same reason as the OMIM records: `DiseaseMappings` has no Orphanet slot. No `references_cache/ORPHA_319651.md` is committed, so nothing here quotes the Orphanet record; this is the identifier only.

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Medical Actions

1

Folinic Acid (Calcium Leucovorin)

Action: Pharmacotherapy NCIT:C15986

Agent: folinic acid CHEBI:15640

Platform: Small molecule

The definitive treatment, and a clean example of metabolic bypass. Folinic acid is 5-formyltetrahydrofolate, a folate already reduced past the step DHFR performs, so it restores the tetrahydrofolate pool without needing the missing enzyme. It resolves the haematological abnormalities, normalizes CSF folate and improves neurological symptoms. Folic acid is not a substitute, and the reason is stronger than its being inert: it is fully oxidized and requires DHFR to be activated, which is precisely what is unavailable, and the unmetabolized folic acid that results impairs transport of 5-methyltetrahydrofolate across the blood-CSF barrier. In cerebral folate deficiency generally, stopping folic acid has been shown to normalize CSF 5-MTHF. So substituting folic acid is not a treatment that merely fails; it can work against the CSF folate the treatment is aiming at.

Mechanism Target:

Collapse of the Reduced Folate Pool — Supplies reduced folate downstream of the enzymatic block, restoring the pool without restoring the enzyme.

Target Phenotypes: Megaloblastic anemia HP:0001889

Show evidence (5 references)

PMID:21310277 SUPPORT Human Clinical

"Treatment with folinic acid resulted in the resolution of hematological abnormalities, normalization of CSF folate levels, and improvement of neurological symptoms."

Reports all three treatment outcomes: haematological resolution, CSF folate normalization, and neurological improvement.

PMID:21310276 SUPPORT Human Clinical

"We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid."

Independent confirmation of correction in the second defining cohort, including pancytopenia.

PMID:36341171 SUPPORT INDIRECT Human Clinical

"Our data suggest that excess supplementation of FA impaired 5MTHF transport across the blood-CSF barrier. In the treatment of CFD, supplementation of folinic acid or 5MTHF (in cases of impaired 5MTHF synthesis) is preferred over the use of FA."

Supports the claim that folic acid is actively counterproductive in cerebral folate deficiency rather than merely ineffective, which is the reason this treatment specifies folinic acid. Graded INDIRECT because the two measured cases are Kearns-Sayre syndrome and homozygous MTHFR C677T, not DHFR deficiency: the mechanism is a property of the blood-CSF barrier shared by the cerebral folate deficiencies, and this paper does not measure it in a DHFR patient.

+ 2 more references

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Diagnosis

1

Biochemical and Molecular Diagnosis of DHFR Deficiency

The combination that should trigger testing is a megaloblastic anaemia or pancytopenia together with a neurological syndrome, particularly atypical absence epilepsy. The decisive biochemical test is cerebrospinal-fluid 5-methyltetrahydrofolate, which is low; plasma and red-cell folate can be normal or near normal, so a peripheral folate assay does not exclude the diagnosis. Confirmation is biallelic DHFR variants on sequencing. Where available, DHFR enzyme activity and fluorescein-labelled methotrexate binding in lymphoblastoid cells are confirmatory and distinguish homozygotes from carriers. CSF biopterins and monoamine neurotransmitter metabolites belong in the same lumbar puncture as the 5-MTHF: cerebral tetrahydrobiopterin deficiency is what the differential below names as the feature pointing specifically at DHFR, and the CSF 5-MTHF alone cannot show it. No published DHFR case series reports those values, so this is a workup recommendation that follows from the entry's own BH4 node rather than from a reported measurement.

Differential diagnosis: the other inborn errors of folate transport and metabolism - hereditary folate malabsorption (SLC46A1), FOLR1-related cerebral folate deficiency, MTHFR deficiency, glutamate formiminotransferase deficiency (FTCD), functional methionine synthase deficiency (MTR/cblG, MTRR/cblE), and MTHFD1 deficiency. MTHFD1 is the closest haematological mimic, sharing megaloblastic anaemia, but adds atypical haemolytic uraemic syndrome and severe combined immunodeficiency, which DHFR deficiency does not. Cerebral tetrahydrobiopterin deficiency is the feature that points specifically at DHFR.

Show evidence (1 reference)

PMID:22108709 SUPPORT REVIEW SYNTHESIS Other

"Deficiency in the trifunctional enzyme containing methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase activities, has been identified in a single patient with megaloblastic anemia, atypical hemolytic uremic syndrome and severe..."

Supports the specific MTHFD1 discriminator stated in the differential: the same anaemia, plus aHUS and SCID.

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Prevalence

1

Worldwide

Cases In Literature Ultra Rare

Defined in 2011 by two simultaneous reports: three siblings of healthy, distantly related parents in one, and three individuals from two families in the other. Reported cases remain in single figures, and the folate-disorder review that followed describes it as one of a handful of recently identified inborn errors in this pathway. No prevalence estimate exists.

Show evidence (1 reference)

PMID:21310276 SUPPORT Human Clinical

"We describe three individuals from two families with a recessive inborn error of metabolism"

Gives the case count from one of the two defining reports.

{ }

Source YAML

click to show

name: Constitutional Megaloblastic Anemia With Severe Neurologic Disease
creation_date: "2026-09-16T21:35:00Z"
description: >
  Dihydrofolate reductase (DHFR) deficiency is an autosomal recessive inborn error
  of folate metabolism caused by biallelic destabilizing missense variants in
  DHFR. DHFR performs two reductions: it converts dietary folic acid to
  dihydrofolate and then to tetrahydrofolate, and it regenerates tetrahydrofolate
  from the dihydrofolate produced each time thymidylate synthase runs. With the
  enzyme lost, the reduced-folate pool collapses even though plasma folate and
  dietary intake are normal, and the disease is therefore a regeneration failure
  rather than a deficiency state.
  The consequences split into two arms that are usually treated as one disease but
  are mechanistically distinct. Loss of thymidylate and purine synthesis in
  erythroid precursors gives megaloblastic anaemia and pancytopenia. Separately,
  cerebrospinal-fluid 5-methyltetrahydrofolate falls, producing cerebral folate
  deficiency with developmental delay and seizures, characteristically atypical
  childhood absence epilepsy. A third consequence is specific to this enzyme: DHFR
  also salvages tetrahydrobiopterin from dihydrobiopterin, so its loss produces
  cerebral tetrahydrobiopterin deficiency and with it reduced synthesis of
  dopamine, serotonin and noradrenaline. That link is what distinguishes DHFR
  deficiency from the other cerebral folate deficiencies.
  The disorder is treatable. Folinic acid is a folate already reduced past the
  DHFR block, so it bypasses the lesion; it resolves the haematological
  abnormalities, normalizes CSF folate, and improves neurological symptoms. Folic
  acid does not work, because activating it is the step that is missing.
category: Mendelian
disease_term:
  preferred_term: constitutional megaloblastic anemia with severe neurologic disease
  term:
    id: MONDO:0013456
    label: constitutional megaloblastic anemia with severe neurologic disease
synonyms:
- DHFR deficiency
- dihydrofolate reductase deficiency
- megaloblastic anemia due to dihydrofolate reductase deficiency
- DHFR-related cerebral folate deficiency
parents:
- Inborn Error of Folate Metabolism
- Inborn Error of Metabolism
classifications:
  harrisons_chapter:
  - classification_value: ENDOCRINOLOGY_METABOLISM
  - classification_value: ONCOLOGY_HEMATOLOGY
  - classification_value: NEUROLOGIC
inheritance:
- name: Autosomal Recessive
  inheritance_term:
    preferred_term: Autosomal recessive inheritance
    term:
      id: HP:0000007
      label: Autosomal recessive inheritance
  description: >-
    Biallelic DHFR missense variants. Heterozygous carriers are clinically
    unaffected but are not biochemically silent: their cells show intermediate
    DHFR activity and intermediate methotrexate binding, which is a clean
    demonstration of gene dosage in this enzyme.
  evidence:
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      DHFR sequencing revealed a homozygous DHFR mutation, c.458A>T (p.Asp153Val),
      in all siblings.
    explanation: >-
      Documents homozygosity in all three affected siblings of healthy parents.
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: IN_VITRO
    snippet: >-
      Heterozygous cells displayed intermediate DHFR activity and FMTX binding.
    explanation: >-
      Supports the specific gene-dosage claim about carriers made in the
      description.
pathophysiology:
- name: DHFR Enzyme Destabilization
  biological_scale: MOLECULAR
  description: >-
    The disease-causing variants are missense changes that destabilize the protein
    rather than simply abolishing the catalytic site. The evidence for that
    distinction is direct: DHFR mRNA is indistinguishable from wild type while
    protein expression is reduced, so the lesion acts after transcription. DHFR is
    a small NADPH-dependent oxidoreductase, and substitutions in its compact fold
    lower both abundance and specific activity.
  genetic_context:
    gene:
      preferred_term: DHFR
      term:
        id: hgnc:2861
        label: DHFR
    variant_origin: GERMLINE
    zygosity: HOMOZYGOUS
    functional_impact_category: LOSS_OF_FUNCTION
  molecular_functions:
  - preferred_term: dihydrofolate reductase activity
    modifier: DECREASED
    term:
      id: GO:0004146
      label: dihydrofolate reductase activity
  downstream:
  - target: Collapse of the Reduced Folate Pool
    causal_link_type: DIRECT
  - target: Failure of Tetrahydrobiopterin Salvage
    causal_link_type: DIRECT
  evidence:
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: IN_VITRO
    snippet: >-
      DHFR activity and fluorescein-labeled methotrexate (FMTX) binding were
      severely reduced in EBV-immortalized lymphoblastoid cells of all patients.
    explanation: >-
      Measures the enzymatic deficiency directly in patient cells, by two
      independent assays.
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: IN_VITRO
    snippet: >-
      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.
    explanation: >-
      The normal-mRNA / low-protein result is what establishes destabilization
      rather than a transcriptional or purely catalytic-site lesion.
- name: Collapse of the Reduced Folate Pool
  biological_scale: MOLECULAR
  description: >-
    Tetrahydrofolate can no longer be regenerated from dihydrofolate, nor produced
    from dietary folic acid. The distinctive feature is that this happens with a
    normal folate supply: plasma folate is adequate, and the deficit is in the
    reduced forms that the cell can actually use. Patient folate profiling by
    liquid chromatography tandem mass spectrometry in red cells, plasma and
    cerebrospinal fluid is what demonstrated the pattern.
  biological_processes:
  - preferred_term: tetrahydrofolate biosynthetic process
    modifier: DECREASED
    term:
      id: GO:0046654
      label: tetrahydrofolate biosynthetic process
  chemical_entities:
  - preferred_term: tetrahydrofolate
    modifier: DECREASED
    term:
      id: CHEBI:20506
      label: 5,6,7,8-tetrahydrofolic acid
  downstream:
  - target: Impaired Thymidylate and Purine Synthesis
    causal_link_type: DIRECT
  - target: Cerebral Folate Deficiency
    causal_link_type: DIRECT
  evidence:
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      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.
    explanation: >-
      The direct measurement of the folate pool across the three compartments this
      node describes.
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      DHFR is necessary for maintaining sufficient CSF and RBC folate levels, even
      in the presence of adequate nutritional folate supply and normal plasma
      folate.
    explanation: >-
      Supports the specific claim that the deficit occurs despite adequate intake
      and normal plasma folate, which is what makes this a regeneration failure.
- name: Impaired Thymidylate and Purine Synthesis
  biological_scale: CELLULAR
  mechanism_confidence: PROVISIONAL
  description: >-
    Tetrahydrofolate is the one-carbon carrier for thymidylate synthase and for de
    novo purine synthesis. Without it, dividing cells cannot make dTMP or purines
    at the rate DNA replication demands. Erythroid precursors are the most
    affected because they divide fastest, and the resulting nuclear-cytoplasmic
    asynchrony is what megaloblastic morphology is. This step is inferred from
    established folate biochemistry rather than measured in these patients; what
    was measured is the haematological outcome.
  cell_types:
  - preferred_term: erythroid progenitor cell
    term:
      id: CL:0000038
      label: erythroid progenitor cell
  biological_processes:
  - preferred_term: dTMP biosynthetic process
    modifier: DECREASED
    term:
      id: GO:0006231
      label: dTMP biosynthetic process
  - preferred_term: purine nucleotide biosynthetic process
    modifier: DECREASED
    term:
      id: GO:0006164
      label: purine nucleotide biosynthetic process
  downstream:
  - target: Megaloblastic anemia
    causal_link_type: DIRECT
  - target: Pancytopenia
    causal_link_type: DIRECT
  evidence:
  - reference: PMID:22108709
    reference_title: "Update and new concepts in vitamin responsive disorders of folate transport and metabolism."
    supports: SUPPORT
    evidence_source: OTHER
    quote_role: BACKGROUND
    snippet: >-
      Derivatives of folic acid are involved in transfer of one-carbon units in
      cellular metabolism, playing a role in synthesis of purines and thymidylate
      and in the remethylation of homocysteine to form methionine.
    explanation: >-
      States the biochemical role this node depends on. Quoted from the review's
      opening framing rather than from a result, which is why quote_role is
      BACKGROUND; the claim is textbook folate biochemistry, not a finding of this
      paper.
- name: Cerebral Folate Deficiency
  biological_scale: TISSUE
  description: >-
    Cerebrospinal-fluid 5-methyltetrahydrofolate falls, which is the definition of
    cerebral folate deficiency. The central nervous system is disproportionately
    affected because it depends on active transport of reduced folate across the
    choroid plexus and has little reserve; peripheral folate status can look
    adequate while the CSF compartment is depleted.
  locations:
  - preferred_term: brain
    term:
      id: UBERON:0000955
      label: brain
  chemical_entities:
  - preferred_term: 5-methyltetrahydrofolate in cerebrospinal fluid
    modifier: DECREASED
    term:
      id: CHEBI:15641
      label: 5-methyltetrahydrofolic acid
  downstream:
  - target: Atypical absence epilepsy
    causal_link_type: INDIRECT_UNKNOWN_INTERMEDIATES
  - target: Global developmental delay
    causal_link_type: INDIRECT_UNKNOWN_INTERMEDIATES
  evidence:
  - reference: PMID:21310276
    reference_title: "Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      characterized by megaloblastic anemia and/or pancytopenia, severe cerebral
      folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a
      germline missense mutation in DHFR, resulting in profound enzyme deficiency
    explanation: >-
      Names severe cerebral folate deficiency as a defining component of the
      disorder, alongside the two other arms.
- name: Failure of Tetrahydrobiopterin Salvage
  biological_scale: TISSUE
  description: >-
    DHFR has a second substrate. It reduces dihydrobiopterin back to
    tetrahydrobiopterin, the salvage arm of BH4 metabolism, and this is the arm
    the brain relies on. Loss of it produces cerebral tetrahydrobiopterin
    deficiency. BH4 is the obligatory cofactor of phenylalanine, tyrosine and
    tryptophan hydroxylase, so the consequence is reduced synthesis of dopamine,
    serotonin and noradrenaline. This arm is what separates DHFR deficiency from
    the other cerebral folate deficiencies, and the 2011 characterization of the
    disorder is what established the link in humans.
  biological_processes:
  - preferred_term: tetrahydrobiopterin regeneration
    modifier: DECREASED
    term:
      id: GO:0006729
      label: tetrahydrobiopterin biosynthetic process
  chemical_entities:
  - preferred_term: tetrahydrobiopterin
    modifier: DECREASED
    term:
      id: CHEBI:15372
      label: 5,6,7,8-tetrahydrobiopterin
  locations:
  - preferred_term: brain
    term:
      id: UBERON:0000955
      label: brain
  downstream:
  - target: Global developmental delay
    causal_link_type: INDIRECT_UNKNOWN_INTERMEDIATES
  notes: >-
    The GO binding here is broader than the claim. GO:0006729 is
    `tetrahydrobiopterin biosynthetic process`; this node models the salvage
    reduction of dihydrobiopterin back to tetrahydrobiopterin, which is a different
    reaction from de novo synthesis. No GO term for the salvage arm was found, so
    the parent process is bound and `preferred_term` carries the specific concept.
    This is the same handling, and the same reason, as the unbound dihydrofolate
    substrate recorded in the entry notes.
  evidence:
  - reference: PMID:21310276
    reference_title: "Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      The characterization of this disorder provides evidence for the link between
      DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for
      the formation of dopamine, serotonin, and norepinephrine and for the
      hydroxylation of aromatic amino acids.
    explanation: >-
      Establishes both the DHFR-to-BH4 link and the monoamine consequence this node
      asserts, in the paper that first characterized it.
phenotypes:
- category: Hematological
  name: Megaloblastic anemia
  description: >-
    Macrocytic anaemia with megaloblastic marrow morphology, the presenting
    haematological feature. Fully correctable with folinic acid.
  phenotype_term:
    preferred_term: Megaloblastic anemia
    term:
      id: HP:0001889
      label: Megaloblastic anemia
  evidence:
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      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.
    explanation: >-
      Records megaloblastic anaemia as the presenting feature in the index
      siblings.
- category: Hematological
  name: Pancytopenia
  description: >-
    All three lineages can be affected, not the red cell line alone. Reported as
    megaloblastic anaemia and/or pancytopenia across the original families.
  phenotype_term:
    preferred_term: Pancytopenia
    term:
      id: HP:0001876
      label: Pancytopenia
  evidence:
  - reference: PMID:21310276
    reference_title: "Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      characterized by megaloblastic anemia and/or pancytopenia
    explanation: >-
      Records pancytopenia as an alternative or additional haematological
      presentation.
- category: Neurological
  name: Atypical absence epilepsy
  description: >-
    Atypical childhood absence epilepsy, the characteristic seizure phenotype. Its
    specificity is diagnostically useful: absence epilepsy alongside a macrocytic
    anaemia is an unusual combination that should prompt a CSF folate measurement.
  phenotype_term:
    preferred_term: Atypical absence seizure
    term:
      id: HP:0007270
      label: Atypical absence seizure
  diagnostic: true
  evidence:
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      cerebral folate deficiency causing neurologic disease with atypical childhood
      absence epilepsy
    explanation: >-
      Names the specific seizure type and attributes it to the cerebral folate
      deficiency.
- category: Neurological
  name: Global developmental delay
  description: >-
    Developmental delay and variable further neurological findings. The review
    literature describes the neurological picture as variable rather than
    stereotyped, which is the honest statement given the small number of reported
    patients.
  phenotype_term:
    preferred_term: Global developmental delay
    term:
      id: HP:0001263
      label: Global developmental delay
  evidence:
  - reference: PMID:22108709
    reference_title: "Update and new concepts in vitamin responsive disorders of folate transport and metabolism."
    supports: SUPPORT
    evidence_source: OTHER
    quote_role: REVIEW_SYNTHESIS
    snippet: >-
      Dihydrofolate reductase deficiency is characterized by megaloblastic anemia
      and cerebral folate deficiency, with variable neurological findings.
    explanation: >-
      The review's summary of the disorder, including the explicit statement that
      the neurological findings are variable.
prevalence:
- population: Worldwide
  measure_type: CASES_IN_LITERATURE
  prevalence_class: ULTRA_RARE
  notes: >-
    Defined in 2011 by two simultaneous reports: three siblings of healthy,
    distantly related parents in one, and three individuals from two families in
    the other. Reported cases remain in single figures, and the folate-disorder
    review that followed describes it as one of a handful of recently identified
    inborn errors in this pathway. No prevalence estimate exists.
  evidence:
  - reference: PMID:21310276
    reference_title: "Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      We describe three individuals from two families with a recessive inborn error
      of metabolism
    explanation: >-
      Gives the case count from one of the two defining reports.
genetic:
- name: DHFR
  gene_term:
    preferred_term: DHFR
    term:
      id: hgnc:2861
      label: DHFR
  presence: Positive
  association: Biallelic Destabilizing Missense Variants
  relationship_type: CAUSATIVE
  variant_origin: GERMLINE
  notes: >-
    The reported alleles are germline missense variants, including the homozygous
    c.458A>T (p.Asp153Val) found in the three index siblings. The mechanism is
    protein destabilization with reduced abundance, not loss of the catalytic site
    alone, which is why mRNA is normal and protein is low.
  evidence:
  - reference: PMID:21310276
    reference_title: "Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      due to a germline missense mutation in DHFR, resulting in profound enzyme
      deficiency
    explanation: >-
      Characterizes the allele class and its functional consequence.
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      Genome-wide homozygosity mapping revealed a candidate region on chromosome 5
      including the dihydrofolate reductase (DHFR) locus.
    explanation: >-
      Documents how the locus was identified, and places DHFR on chromosome 5.
diagnosis:
- name: Biochemical and Molecular Diagnosis of DHFR Deficiency
  description: >-
    The combination that should trigger testing is a megaloblastic anaemia or
    pancytopenia together with a neurological syndrome, particularly atypical
    absence epilepsy. The decisive biochemical test is cerebrospinal-fluid
    5-methyltetrahydrofolate, which is low; plasma and red-cell folate can be
    normal or near normal, so a peripheral folate assay does not exclude the
    diagnosis. Confirmation is biallelic DHFR variants on sequencing. Where
    available, DHFR enzyme activity and fluorescein-labelled methotrexate binding
    in lymphoblastoid cells are confirmatory and distinguish homozygotes from
    carriers. CSF biopterins and monoamine neurotransmitter metabolites belong in
    the same lumbar puncture as the 5-MTHF: cerebral tetrahydrobiopterin deficiency
    is what the differential below names as the feature pointing specifically at
    DHFR, and the CSF 5-MTHF alone cannot show it. No published DHFR case series
    reports those values, so this is a workup recommendation that follows from the
    entry's own BH4 node rather than from a reported measurement.
  notes: >-
    Differential diagnosis: the other inborn errors of folate transport and
    metabolism - hereditary folate malabsorption (SLC46A1), FOLR1-related cerebral
    folate deficiency, MTHFR deficiency, glutamate formiminotransferase deficiency
    (FTCD), functional methionine synthase deficiency (MTR/cblG, MTRR/cblE), and
    MTHFD1 deficiency. MTHFD1 is the closest haematological mimic, sharing
    megaloblastic anaemia, but adds atypical haemolytic uraemic syndrome and severe
    combined immunodeficiency, which DHFR deficiency does not. Cerebral
    tetrahydrobiopterin deficiency is the feature that points specifically at DHFR.
  evidence:
  - reference: PMID:22108709
    reference_title: "Update and new concepts in vitamin responsive disorders of folate transport and metabolism."
    supports: SUPPORT
    evidence_source: OTHER
    quote_role: REVIEW_SYNTHESIS
    snippet: >-
      Deficiency in the trifunctional enzyme containing methylenetetrahydrofolate
      dehydrogenase, methenyltetrahydrofolate cyclohydrolase and
      formyltetrahydrofolate synthetase activities, has been identified in a single
      patient with megaloblastic anemia, atypical hemolytic uremic syndrome and
      severe combined immune deficiency.
    explanation: >-
      Supports the specific MTHFD1 discriminator stated in the differential: the
      same anaemia, plus aHUS and SCID.
treatments:
- name: Folinic Acid (Calcium Leucovorin)
  description: >-
    The definitive treatment, and a clean example of metabolic bypass. Folinic
    acid is 5-formyltetrahydrofolate, a folate already reduced past the step DHFR
    performs, so it restores the tetrahydrofolate pool without needing the missing
    enzyme. It resolves the haematological abnormalities, normalizes CSF folate
    and improves neurological symptoms. Folic acid is not a substitute, and the
    reason is stronger than its being inert: it is fully oxidized and requires DHFR
    to be activated, which is precisely what is unavailable, and the unmetabolized
    folic acid that results impairs transport of 5-methyltetrahydrofolate across the
    blood-CSF barrier. In cerebral folate deficiency generally, stopping folic acid
    has been shown to normalize CSF 5-MTHF. So substituting folic acid is not a
    treatment that merely fails; it can work against the CSF folate the treatment is
    aiming at.
  therapeutic_modality: SMALL_MOLECULE
  treatment_term:
    preferred_term: Pharmacotherapy
    term:
      id: NCIT:C15986
      label: Pharmacotherapy
    therapeutic_agent:
    - preferred_term: folinic acid
      term:
        id: CHEBI:15640
        label: 5-formyltetrahydrofolic acid
  target_mechanisms:
  - target: Collapse of the Reduced Folate Pool
    description: >-
      Supplies reduced folate downstream of the enzymatic block, restoring the pool
      without restoring the enzyme.
  target_phenotypes:
  - preferred_term: Megaloblastic anemia
    term:
      id: HP:0001889
      label: Megaloblastic anemia
  evidence:
  - reference: PMID:21310277
    reference_title: "Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      Treatment with folinic acid resulted in the resolution of hematological
      abnormalities, normalization of CSF folate levels, and improvement of
      neurological symptoms.
    explanation: >-
      Reports all three treatment outcomes: haematological resolution, CSF folate
      normalization, and neurological improvement.
  - reference: PMID:21310276
    reference_title: "Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency."
    supports: SUPPORT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      We show that cerebral folate levels, anemia, and pancytopenia of DHFR
      deficiency can be corrected by treatment with folinic acid.
    explanation: >-
      Independent confirmation of correction in the second defining cohort,
      including pancytopenia.
  - reference: PMID:36341171
    reference_title: "Folic acid inhibits 5-methyltetrahydrofolate transport across the blood-cerebrospinal fluid barrier: Clinical biochemical data from two cases."
    supports: SUPPORT
    directness: INDIRECT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      Our data suggest that excess supplementation of FA impaired 5MTHF transport
      across the blood-CSF barrier. In the treatment of CFD, supplementation of
      folinic acid or 5MTHF (in cases of impaired 5MTHF synthesis) is preferred over
      the use of FA.
    explanation: >-
      Supports the claim that folic acid is actively counterproductive in cerebral
      folate deficiency rather than merely ineffective, which is the reason this
      treatment specifies folinic acid. Graded INDIRECT because the two measured
      cases are Kearns-Sayre syndrome and homozygous MTHFR C677T, not DHFR
      deficiency: the mechanism is a property of the blood-CSF barrier shared by the
      cerebral folate deficiencies, and this paper does not measure it in a DHFR
      patient.
  - reference: PMID:34008900
    reference_title: "Cerebral folate transporter deficiency syndrome in three siblings: Why genetic testing for developmental and epileptic encephalopathies should be performed early and include the FOLR1 gene."
    supports: SUPPORT
    directness: INDIRECT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      Folinic acid (oral and intravenous) supplementation, initiated after over 15
      years illness, has failed to result in any sizeable clinical or
      neurophysiological improvement.
    explanation: >-
      Bears on the treatment-window question the notes below raise. Graded INDIRECT
      because these siblings have FOLR1-related cerebral folate deficiency, a
      different gene reaching the same low CSF 5-MTHF: it shows late folinic acid
      failing in that disorder, not in DHFR deficiency.
  - reference: PMID:41132636
    reference_title: "A Case of Cerebral Folate Deficiency due to FOLR1 Mutation in a 10-Year-Old Girl: Clinical Presentation and Treatment Outcomes."
    supports: SUPPORT
    directness: INDIRECT
    evidence_source: HUMAN_CLINICAL
    snippet: >-
      Following treatment, the patient demonstrated significant clinical
      improvement.
    explanation: >-
      The other side of the same comparison: folinic acid started at diagnosis in a
      ten-year-old with FOLR1-related cerebral folate deficiency, with improvement.
      Read together with the sibling series above it is the reason the notes treat
      the treatment window as a live question rather than a settled one. INDIRECT
      for the same reason: different gene, same downstream CSF folate deficit.
  notes: >-
    The degree to which neurological outcome depends on age at starting treatment
    is not established in the published DHFR cases, and no dosing regimen is curated
    here because none is reported consistently enough to state. The two FOLR1 cases
    cited above are the nearest available evidence that the window matters -
    treatment started after fifteen years of illness achieved nothing, while a
    ten-year-old treated at diagnosis improved - but they are a different gene and
    cannot settle the question for this disease.

    Antifolate drugs inhibit DHFR directly, so methotrexate, trimethoprim and
    pyrimethamine are the obvious agents to avoid in a patient with residual DHFR
    activity. This is stated here as prose rather than as a curated claim because no
    source in this entry's reference cache makes the recommendation for DHFR
    deficiency; it follows from the drugs' mechanism rather than from a reported
    case.
external_assertions:
- name: OMIM megaloblastic anemia due to dihydrofolate reductase deficiency phenotype record
  source: OMIM
  assertion_type: disease_record
  external_id: OMIM:613839
  url: https://omim.org/entry/613839
  description: >-
    The OMIM phenotype record for this disease. Recorded here rather than under
    `mappings` because `DiseaseMappings` carries only ICD-10-CM, ICD-11, MONDO and
    NCIT slots and there is no `omim_mappings` slot in the schema; issue #9922
    withdrew a proposal to add one.
- name: OMIM DHFR gene record
  source: OMIM
  assertion_type: gene_record
  external_id: OMIM:126060
  url: https://omim.org/entry/126060
  description: >-
    The OMIM record for the DHFR gene, recorded alongside the phenotype MIM so the
    two are not confused with each other.
- name: Orphanet megaloblastic anemia due to dihydrofolate reductase deficiency
  source: Orphanet
  assertion_type: disease_record
  external_id: ORPHA:319651
  url: https://www.orpha.net/en/disease/detail/319651
  description: >-
    The Orphanet record for this disease. Recorded as an external assertion for the
    same reason as the OMIM records: `DiseaseMappings` has no Orphanet slot. No
    `references_cache/ORPHA_319651.md` is committed, so nothing here quotes the
    Orphanet record; this is the identifier only.
discussions:
- discussion_id: bh4_arm_not_separately_treated
  kind: KNOWLEDGE_GAP
  attaches_to:
  - pathophysiology#Failure of Tetrahydrobiopterin Salvage
  - treatments#Folinic Acid (Calcium Leucovorin)
  prompt: >-
    Does folinic acid correct the cerebral tetrahydrobiopterin arm of DHFR
    deficiency, or only the folate arm, and should BH4 or neurotransmitter-precursor
    supplementation be added?
  rationale: >-
    Folinic acid bypasses DHFR for folate, because it is a pre-reduced folate. It
    has no equivalent action on biopterin: dihydrobiopterin still needs DHFR to be
    reduced, and folinic acid does not supply tetrahydrobiopterin. The published
    reports document normalization of CSF folate and improvement of neurological
    symptoms, but do not report post-treatment CSF biopterin or monoamine
    metabolites, so whether the residual neurological deficit reflects an untreated
    BH4 arm is unknown. This matters directly: if it does, sapropterin or
    levodopa/5-hydroxytryptophan supplementation would be rational adjuncts, and
    the treatment section of this entry is incomplete rather than merely brief.
- discussion_id: erythroid_step_is_inferred
  kind: KNOWLEDGE_GAP
  attaches_to:
  - pathophysiology#Impaired Thymidylate and Purine Synthesis
  prompt: >-
    Has the thymidylate/purine-synthesis block been measured in DHFR-deficient
    erythroid precursors, or is it inferred from folate biochemistry?
  rationale: >-
    This node is the weakest link in the entry's haematological arm and is marked as
    such in its own description. The enzymatic deficiency is measured, the folate
    pool is measured, and the megaloblastic anaemia is observed; the step joining
    them is supplied by textbook folate biochemistry, quoted from a review's
    introduction rather than from any result in a DHFR-deficient patient. Nothing
    about it is controversial, but the entry should not imply a measurement that was
    not made, and a deoxyuridine suppression test or dNTP pool measurement in
    patient erythroid cells would close it.
notes: >-
  Scope: this entry covers DHFR deficiency as a single disease. No subtypes are
  curated because the published cases are too few to support any split, and the
  variation reported is in severity of the neurological findings rather than in
  kind.

  Sources: the OpenScientist deep-research report for this disease suggested
  CHEBI:20506 as "7,8-dihydrofolate". The report's own term-validation section
  flagged that name as worth a second look, and the term is in fact
  5,6,7,8-tetrahydrofolic acid. It is bound here as tetrahydrofolate, which is the
  concept the node needs; no term is bound for dihydrofolate, because the CHEBI
  identifier for it was not sourced. That is the reason the substrate side of the
  reaction is described in prose rather than bound.

  Not curated: the structural-biology detail from the report (active-site residues,
  crystal structures, the Val115-versus-Ile basis of antifolate selectivity). It is
  real and well cited, but it describes the normal enzyme and antifolate
  pharmacology rather than this disease's mechanism, and the papers behind it are
  not about DHFR deficiency.

📚

References & Deep Research

Deep Research

1

Deep research results are used as seeds for research; they do not undergo the same validation as the main records and may contain errors. How we use deep research.

Evaluations and curation notes (2)

Record notes

Scope: this entry covers DHFR deficiency as a single disease. No subtypes are curated because the published cases are too few to support any split, and the variation reported is in severity of the neurological findings rather than in kind. Sources: the OpenScientist deep-research report for this disease suggested CHEBI:20506 as "7,8-dihydrofolate". The report's own term-validation section flagged that name as worth a second look, and the term is in fact 5,6,7,8-tetrahydrofolic acid. It is bound here as tetrahydrofolate, which is the concept the node needs; no term is bound for dihydrofolate, because the CHEBI identifier for it was not sourced. That is the reason the substrate side of the reaction is described in prose rather than bound. Not curated: the structural-biology detail from the report (active-site residues, crystal structures, the Val115-versus-Ile basis of antifolate selectivity). It is real and well cited, but it describes the normal enzyme and antifolate pharmacology rather than this disease's mechanism, and the papers behind it are not about DHFR deficiency.

Create: Constitutional Megaloblastic Anemia With Severe Neurologic Disease · 2026-09-16T21:16:59Z · View source

Created kb/disorders/Constitutional_Megaloblastic_Anemia_With_Severe_Neurologic_Disease.yaml (DHFR deficiency) from claim issue #11963. Deep research: openscientist (research/Constitutional_Megaloblastic_Anemia_With_Severe_Neurologic_Disease-deep-research-openscientist.md). This run's report carried BOTH validation sections, unlike the DOCK2 and CMS7 runs in the same batch whose term-validation step aborted on an EBI timeout. Reference validation: 10/10 resolved, 6/6 quoted claims found in source, 0 off topic. Term validation: 36 terms checked, 34 resolved, 0 named as a different term, 11 flagged as worth a second look. Reading that list mattered: the report offers CHEBI:20506 as 7,8-dihydrofolate, and the term is actually 5,6,7,8-tetrahydrofolic acid. It is bound in this entry as tetrahydrofolate, the concept the node needs, and dihydrofolate is left unbound and described in prose because no sourced CURIE was available for it. Pathograph: 5 nodes from DHFR destabilization (normal mRNA, reduced protein, so a stability lesion rather than a catalytic-site one) through collapse of the reduced folate pool into three arms - thymidylate/purine synthesis, cerebral folate deficiency, and tetrahydrobiopterin salvage failure. The BH4 arm is the discriminator from the other cerebral folate deficiencies and drives a knowledge-gap discussion: folinic acid bypasses DHFR for folate but not for biopterin, and no published case reports post-treatment CSF biopterin. The thymidylate node is explicitly marked as inferred from folate biochemistry rather than measured in these patients, and carries its own discussion saying so. Validation: just validate and just validate-disorders pass (19/19 snippets verified); duplicate-key, entity-ref, causal-target, enum-value, qualifier-term gates clean; list-gene-term-mismatches confirms the DHFR binding names the gene the entry names.

OpenScientist ▸

1. Disease Information

openscientist-autonomous 2026-09-16T21:11:37.260137

1. Disease Information

Overview: A recessive inborn error of intracellular folate metabolism combining bone-marrow failure (megaloblastic anemia/pancytopenia) with a severe central-nervous-system phenotype driven by cerebral folate and BH4 deficiency. It is a treatable metabolic encephalopathy when recognized early. [HUMAN clinical; PMID 21310277, 21310276]
Key identifiers:
MONDO: MONDO:0013456
OMIM (phenotype): #613839 "Megaloblastic anemia due to dihydrofolate reductase deficiency"
OMIM (gene): *126060 (DHFR)
Orphanet: ORPHA:319651 (Megaloblastic anemia due to dihydrofolate reductase deficiency)
Gene / HGNC: DHFR (HGNC:2861); NCBI Gene 1719; Ensembl ENSG00000228716; UniProt P00374
ICD-10: D53.1 (Other megaloblastic anemias, not elsewhere classified); ICD-11: 3A01.0 area / 5C50 (inborn errors of metabolism) — no unique code
MeSH: Folic Acid Deficiency (D005494); Anemia, Megaloblastic (D000749); Tetrahydrofolate Dehydrogenase (D013894)
Synonyms / alternative names: DHFR deficiency; Dihydrofolate reductase deficiency; Megaloblastic anemia due to dihydrofolate reductase deficiency; Constitutional megaloblastic anemia with severe neurologic disease; Cerebral folate deficiency due to DHFR deficiency.
Information source type: Aggregated disease-level (OMIM/Orphanet/MONDO) plus individual-patient case series (EHR-derived clinical descriptions of ~6 patients). Not derived from large population EHR datasets.

2. Etiology

Primary cause (genetic): Biallelic (homozygous) germline missense mutations in DHFR producing a stable but catalytically deficient enzyme. Cario et al. identified c.458A>T (p.Asp153Val); Banka et al. identified a homozygous DHFR missense mutation resulting in profound enzyme deficiency. [HUMAN clinical; PMID 21310277, 21310276]

"DHFR sequencing revealed a homozygous DHFR mutation, c.458A>T (p.Asp153Val), in all siblings." (PMID 21310277)
Genetic risk factors: Homozygous DHFR pathogenic variants are causal (Mendelian, not susceptibility loci). Consanguinity / relatedness is a major risk factor — reported families were consanguineous or distantly related, consistent with a rare recessive founder-type allele.
Environmental risk factors: None are causal. However, low dietary folate intake would be expected to worsen the phenotype, and exposure to antifolate drugs (methotrexate, trimethoprim, pyrimethamine) that further inhibit residual DHFR could exacerbate disease (mechanistic inference).
Protective factors: Dietary folate as reduced folates (folinic acid / 5-formyl-THF, 5-MTHF) bypasses the block and is protective/therapeutic. Common population DHFR polymorphisms (e.g., the 19-bp intron-1 deletion, c.594+59del19; p.Leu80Phe) modulate folate handling/antifolate response in the general population but are not causes of this monogenic disease.
Gene–environment interactions: Folate/antifolate status interacts strongly with residual DHFR activity — the phenotype is a genetic enzyme deficiency whose severity is modifiable by folate form and dose (folinic acid rescue is the clearest example). [HUMAN clinical; PMID 21310277, 21310276]

3. Phenotypes

Frequencies are qualitative given the very small case number (n≈6). Onset is typically neonatal to infancy/early childhood; course is progressive if untreated, largely reversible/stabilizable with folinic acid.

Hematologic (laboratory abnormalities / clinical signs): - Megaloblastic anemia — core feature, most/all patients. HPO: HP:0001889 (Megaloblastic anemia). Onset infancy; severe; treatment-responsive. - Pancytopenia — reported in some patients. HP:0001876 (Pancytopenia); HP:0001873 (Thrombocytopenia), HP:0001882 (Leukopenia). - Macrocytosis / elevated MCV, megaloblastic bone marrow, hypersegmented neutrophils. HP:0001972 (Macrocytic anemia).

"characterized by megaloblastic anemia and/or pancytopenia" (PMID 21310276)

Neurologic (symptoms / signs): - Seizures / epilepsy, characteristically atypical childhood absence epilepsy. HP:0002121 (Absence seizure), HP:0001250 (Seizure). [HUMAN clinical; PMID 21310277]

"megaloblastic anemia and cerebral folate deficiency causing neurologic disease with atypical childhood absence epilepsy." (PMID 21310277) - Global developmental delay / intellectual disability. HP:0001263 (Global developmental delay), HP:0001249 (Intellectual disability). - Microcephaly (reported in cerebral folate deficiency states). HP:0000252. - Cerebral folate deficiency features — variable neurologic findings including hypotonia, movement/motor abnormalities, and, mechanistically expected from BH4/monoamine deficiency, potential extrapyramidal or mood/behavioral changes. HP:0002376 (Developmental regression) variably. - Neuroimaging abnormalities consistent with folate-deficient leukoencephalopathy in some patients. HP:0002352 (Leukoencephalopathy).

Severity / progression / QoL: Severe, potentially life-threatening in the neonatal/infantile period (anemia) with substantial neurodisability risk. Quality-of-life impact is high if untreated (epilepsy + developmental impairment); early folinic acid markedly improves hematologic status and neurologic trajectory, though pre-treatment CNS injury may persist. [HUMAN clinical; PMID 21310277, 21310276]

4. Genetic / Molecular Information

Causal gene: DHFR (dihydrofolate reductase), 5q14.1; OMIM *126060; HGNC:2861; UniProt P00374 (187 aa cytosolic enzyme). EC 1.5.1.3.
Pathogenic variants (germline, autosomal recessive):
c.458A>T; p.Asp153Val (p.D153V) — homozygous; 3 affected siblings (Cario et al.). Missense; classified pathogenic; drastically reduced enzyme activity and reduced methotrexate (FMTX) binding; normal mRNA but reduced protein → destabilizing missense / loss of function. [HUMAN clinical + in vitro; PMID 21310277] > "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." (PMID 21310277)
Homozygous DHFR missense mutation in 3 individuals from 2 families (Banka et al.) → profound enzyme deficiency (reported as p.Leu80Phe in that cohort). [HUMAN clinical; PMID 21310276]
Variant type/class: missense (loss-of-function via reduced protein stability/activity). No nonsense/frameshift/structural variants reported as causal to date.
ACMG/AMP: Pathogenic (functional enzyme assays + segregation + rarity).
Allele frequency: Causal alleles are private/ultra-rare (essentially absent in gnomAD as homozygotes). Note the common benign DHFR 19-bp intron-1 deletion and p.Leu80Phe polymorphism exist in general populations and affect folate/antifolate pharmacology but are not disease-causing here.
Somatic vs germline: Germline only.
Functional consequence: Loss of function (reduced catalytic activity + reduced protein). Heterozygotes show intermediate DHFR activity and FMTX binding but are clinically unaffected (recessive). [in vitro; PMID 21310277]
Modifier genes: Not formally established; folate-pathway genes (MTHFR, folate transporters SLC46A1/FOLR1, MTHFD1) and dietary folate plausibly modify severity (inference).
Epigenetic / chromosomal abnormalities: None implicated; no aneuploidy/translocation. Global one-carbon/methylation supply is indirectly reduced (methionine/SAM), a downstream metabolic — not primary epigenetic — effect.

5. Environmental Information

Environmental factors: No environmental cause. Antifolate drug exposure (methotrexate, trimethoprim–sulfamethoxazole, pyrimethamine) is mechanistically contraindicated/aggravating because it further inhibits DHFR (inference).
Lifestyle / diet: Folate nutrition is the dominant modifiable factor; adequacy of reduced folate (folinic acid) is protective. Ordinary folic acid supplementation does not rescue the defect. [HUMAN clinical; PMID 21310276]
Infectious agents: Not applicable (not an infectious disease).

6. Mechanism / Pathophysiology

Ordered causal chain (initiating lesion → clinical manifestation)

Biallelic DHFR missense mutation (e.g., p.Asp153Val) → destabilized DHFR protein with severely reduced catalytic activity ([in vitro] reduced enzyme activity + FMTX binding; normal mRNA, low protein). Demonstrated.
Reduced DHFR activity → failure to reduce 7,8-dihydrofolate (DHF) to tetrahydrofolate (THF) and failure to reduce dietary folic acid → depletion of the reduced-folate (THF) pool. Demonstrated by patient folate profiling.
THF depletion branches into three arms:
3a. Hematologic arm: low THF → impaired thymidylate synthase cycle (dUMP→dTMP) and de novo purine synthesis → uracil misincorporation / imbalanced dNTPs → ineffective DNA synthesis in erythroid precursors → nuclear–cytoplasmic asynchrony → megaloblastic anemia and pancytopenia. Inferred from folate biochemistry; demonstrated hematologic phenotype.
3b. Cerebral folate arm: impaired regeneration/transport of reduced folate → low CSF 5‑methyltetrahydrofolate (cerebral folate deficiency) → impaired CNS one-carbon metabolism, methylation, and neurotransmitter precursor supply → seizures (atypical absence epilepsy), developmental delay. Demonstrated (low CSF folate).
3c. Neurotransmitter/BH4 arm: DHFR also regenerates tetrahydrobiopterin (BH4) from dihydrobiopterin (BH2) (the salvage arm of BH4 metabolism). Loss → cerebral BH4 deficiency → reduced activity of tyrosine/tryptophan/phenylalanine hydroxylases → reduced dopamine, serotonin, norepinephrine → neurologic/neuropsychiatric manifestations. Demonstrated (cerebral BH4 deficiency; PMID 21310276).
Convergent CNS effects of 3b + 3c → severe, potentially progressive encephalopathy.
Therapeutic branch (reversal): administration of folinic acid (5‑formyl‑THF), a folate already reduced beyond the DHFR block → restores THF pool → corrects anemia, normalizes CSF folate, improves neurologic symptoms. Demonstrated.

Detail by category

Molecular pathways: Folate one-carbon metabolism (KEGG hsa00670 one-carbon pool by folate; Reactome "Metabolism of folate and pterines"); pterine/BH4 salvage; methionine cycle (SAM methylation).
Cellular processes: DNA replication in rapidly dividing cells (erythroblasts) → ineffective erythropoiesis/apoptosis of precursors; neuronal signaling via monoamines.
Protein dysfunction: Destabilizing missense → loss of function with reduced protein abundance (not simply catalytic-site abolition); heterozygous intermediate activity. DHFR is a small (~21 kDa, 187 aa) NADPH-dependent oxidoreductase (Rossmann-like fold) whose catalysis involves hydride transfer from NADPH and protonation of the folate N5 — mechanistic basis for why point mutations near substrate/cofactor contacts abolish activity. [structural; PMID 25453083]. Human DHFR crystal structures (e.g., PDB via Cody et al., PMID 21931219, 26057816) define an active-site pocket with a conserved substrate-anchoring Arg70, and pteridine-binding residues Phe31, Gln35, Val115 (Val115 vs Ile in microbial DHFR underlies antifolate selectivity). The disease missense residues (e.g., Asp153, Leu80) map to this compact fold; substitutions destabilize the protein and/or perturb cofactor/substrate binding, consistent with the observed loss of activity and reduced protein level. UniProt P00374; AlphaFold model AF-P00374.
Metabolic changes: ↓THF, ↑DHF (relative), ↓5‑MTHF (esp. CSF), ↓BH4, impaired dTMP/purine synthesis, impaired homocysteine remethylation (variable). CHEBI: 7,8-dihydrofolate (CHEBI:20506), tetrahydrofolate (CHEBI:26907), 5‑methyltetrahydrofolate (CHEBI:15641), folinic acid (CHEBI:63606), tetrahydrobiopterin (CHEBI:15372).
Immune involvement: Not a primary feature (contrast MTHFD1 deficiency, which adds SCID).
Tissue damage mechanisms: Ineffective hematopoiesis (marrow); neuronal dysfunction from substrate/neurotransmitter insufficiency (largely functional, potentially structural leukoencephalopathy).
Biochemical abnormality: Enzyme deficiency — dihydrofolate reductase (EC 1.5.1.3).
Suggested GO terms: GO:0046452 (dihydrofolate metabolic process), GO:0006545 (glycine biosynthetic process), GO:0046655 (folic acid metabolic process), GO:0009394 (2'-deoxyribonucleotide metabolic process), GO:0006760 (folic acid-containing compound metabolic process), GO:0034355 (NAD salvage n/a) ; molecular function GO:0004146 (dihydrofolate reductase activity); GO:0006559 (l-phenylalanine catabolic process, via BH4).
Suggested CL terms: erythroid progenitor cell (CL:0000038), megakaryocyte-erythroid progenitor, neuron (CL:0000540), dopaminergic neuron (CL:0000700), serotonergic neuron (CL:0000850).

7. Anatomical Structures Affected

Organ level (primary): Bone marrow / hematopoietic system (UBERON:0002371 bone marrow) and brain / central nervous system (UBERON:0000955 brain).
Secondary/system involvement: Peripheral blood (all lineages — anemia, leukopenia, thrombocytopenia); potentially cerebrospinal fluid compartment (UBERON:0001359 CSF) showing low 5-MTHF.
Body systems: Hematologic/immune (blood) and nervous system; digestive absorption of folate is intact (the defect is intracellular reduction, distinguishing it from hereditary folate malabsorption).
Tissue/cell level: Erythroid and other hematopoietic precursors (megaloblastic marrow); CNS neurons dependent on monoamine neurotransmitters. CL: erythroid progenitor (CL:0000038), neuron (CL:0000540).
Subcellular level: Cytosol (DHFR is cytosolic; GO:0005829) with folate-dependent one-carbon reactions in cytosol and nucleus; downstream nuclear de novo thymidylate synthesis affected.
Localization / lateralization: Systemic/bilateral; CNS involvement is diffuse/bilateral (e.g., diffuse white-matter changes), not focal.

8. Temporal Development

Onset: Congenital enzyme defect; clinical presentation typically neonatal to early infancy/childhood (megaloblastic anemia early; seizures/absence epilepsy in early childhood). [HUMAN clinical; PMID 21310277]
Onset pattern: Subacute/chronic, insidious neurologic decline with intercurrent anemia.
Progression: Progressive if untreated; neurologic damage may accrue during the untreated window. With folinic acid, hematologic and biochemical parameters normalize and neurologic symptoms improve/stabilize. [HUMAN clinical; PMID 21310276]
Course pattern: Chronic, lifelong (requires lifelong folinic acid). Seizures may be episodic.
Remission: Treatment-induced biochemical/hematologic remission with folinic acid; not spontaneous.
Critical period: Early infancy/childhood is the critical intervention window — earlier treatment better preserves neurodevelopment (rationale for newborn/early metabolic detection).

9. Inheritance and Population

Epidemiology: Ultra-rare — only a handful of patients (≈6 from 4 families) reported worldwide; prevalence <1/1,000,000 (Orphanet "unknown/ultra-rare"). Incidence not quantifiable.
Inheritance: Autosomal recessive. Heterozygous carriers are asymptomatic with intermediate enzyme activity. [in vitro; PMID 21310277]
Penetrance: Complete in biallelic individuals reported; expressivity variable (neurologic severity varies — "variable neurological findings," PMID 22108709).
Genetic anticipation: Not applicable (not a repeat-expansion disorder).
Germline mosaicism: Not reported.
Founder effects / consanguinity: Reported families were consanguineous or distantly related; private homozygous alleles — consistent with founder/consanguinity mechanism.
Carrier frequency: Unknown; expected very low given rarity.
Demographics: No established ethnic predilection given tiny sample; both sexes affected (autosomal). No sex bias expected. Age distribution: pediatric onset.

10. Diagnostics

Laboratory tests:
CBC + smear: macrocytic/megaloblastic anemia, possible pancytopenia, hypersegmented neutrophils. LOINC: MCV 30428-7, Hemoglobin 718-7.
Bone marrow: megaloblastic changes.
Serum/RBC folate, vitamin B12, homocysteine to position within folate disorders (B12 normal; distinguishes from B12 deficiency).
CSF 5‑methyltetrahydrofolate (low) — hallmark of cerebral folate deficiency. [HUMAN clinical; PMID 21310277]
CSF neurotransmitter metabolites / biopterins (low BH4, altered HVA/5-HIAA) reflecting BH4/monoamine deficiency. [HUMAN clinical; PMID 21310276]
LC-MS/MS folate profiling of RBC, plasma, CSF (as used diagnostically). [PMID 21310277]
Functional/enzyme assay: DHFR enzyme activity and fluorescein-methotrexate (FMTX) binding in lymphoblastoid cells/fibroblasts — severely reduced in patients, intermediate in carriers. [in vitro; PMID 21310277]
Imaging: Brain MRI — may show leukoencephalopathy/white-matter changes; used to characterize CNS involvement.
Electrophysiology: EEG for absence/atypical absence epilepsy characterization.
Genetic testing: Definitive test = DHFR sequencing (single-gene, or as part of a megaloblastic-anemia / inborn-errors-of-folate / epilepsy-metabolic gene panel; WES/WGS in undiagnosed cases). Homozygosity mapping was pivotal in gene discovery. [PMID 21310277]. GTR panels for "megaloblastic anemia" and "cerebral folate deficiency" include DHFR.
Clinical criteria / differential diagnosis: Combination of megaloblastic anemia + low CSF folate + folinic-acid responsiveness + biallelic DHFR variants. Differentiate from: vitamin B12 deficiency; folate malabsorption (SLC46A1); FOLR1 cerebral folate deficiency (no anemia); MTHFR deficiency (homocystinuria, no megaloblastic anemia); MTHFD1 deficiency (adds SCID/immunodeficiency; impaired nuclear dTMP synthesis with elevated uracil in DNA, PMID 25548164); thiamine-responsive megaloblastic anemia (SLC19A2); orotic aciduria.
Screening: No routine newborn screen currently; consider cascade/carrier testing in affected families and prenatal/preimplantation testing where the familial variant is known.

11. Outcome / Prognosis

Survival/mortality: Untreated severe megaloblastic anemia/pancytopenia is potentially fatal in infancy; prognosis is substantially improved with early folinic acid. No formal survival statistics exist (too few cases).
Morbidity/function: Neurodevelopmental disability and epilepsy are the main morbidities; degree depends on timing of treatment. Hematologic parameters are fully correctable.
Recovery potential: Hematologic and biochemical abnormalities are reversible with folinic acid; neurologic recovery is partial — pre-treatment CNS injury may be irreversible, underscoring early diagnosis. [HUMAN clinical; PMID 21310276, 21310277]. The analogous FOLR1 cerebral folate deficiency literature reinforces a critical treatment window: folinic acid begun after >15 years of illness produced no meaningful clinical/neurophysiological improvement in three siblings (PMID 34008900), whereas timely initiation improved outcome (PMID 41132636).
Prognostic factors: Age at diagnosis/treatment initiation, extent of pre-treatment neurologic damage, adherence to lifelong folinic acid, avoidance of antifolate drugs.

12. Treatment

First-line pharmacotherapy: Folinic acid (calcium leucovorin / 5‑formyltetrahydrofolate) — a pre-reduced folate that bypasses the DHFR block; corrects anemia/pancytopenia, normalizes CSF folate, improves neurologic symptoms. Lifelong. NCIT: Leucovorin Calcium (C1035) / Folinic Acid. [HUMAN clinical; PMID 21310277, 21310276]

"Treatment with folinic acid resulted in the resolution of hematological abnormalities, normalization of CSF folate levels, and improvement of neurological symptoms." (PMID 21310277)
Ineffective/avoid: Folic acid (requires DHFR to become active — does not rescue). Beyond needing DHFR, unmetabolized folic acid actively inhibits 5-MTHF transport across the blood–CSF barrier, worsening cerebral folate deficiency: in two CFD cases high-dose folic acid failed to normalize CSF 5-MTHF, and stopping folic acid normalized it — so folinic acid or 5-MTHF is preferred (PMID 36341171). Avoid antifolate drugs (methotrexate, trimethoprim, pyrimethamine).

"In the treatment of CFD, supplementation of folinic acid or 5MTHF (in cases of impaired 5MTHF synthesis) is preferred over the use of FA." (PMID 36341171)
Adjunctive: Dose titration to normalize CSF 5-MTHF; consider that some cerebral folate deficiency benefits from higher folinic acid doses to cross the blood–brain barrier. Antiepileptic therapy for seizures as needed; monitor since folate status interacts with some AEDs.
Potential neurotransmitter support: Because of BH4/monoamine deficiency, consideration of BH4 (sapropterin) or neurotransmitter precursor supplementation (e.g., L-dopa/carbidopa, 5-hydroxytryptophan) is biologically rational in select patients (inference; not established as standard for DHFR deficiency). Management principles for BH4/monoamine-deficiency disorders are summarized in an international consensus guideline (PMID 32456656).
Advanced/experimental therapeutics: No gene, cell, or RNA therapy in clinical use; the disorder is largely managed by metabolite replacement. Gene replacement is conceptually feasible but unstudied.
Supportive care: Transfusion for severe anemia acutely; developmental/rehabilitative therapies for neurodisability.
Treatment strategy / personalized medicine: Genotype-confirmed diagnosis → lifelong folinic acid + monitoring of CBC, plasma and CSF folate; family cascade testing. NCIT terms: Leucovorin Calcium (C1035); Sapropterin Dihydrochloride (C61815) [experimental rationale].

13. Prevention

Primary prevention: Not preventable in an affected homozygote (germline). Genetic counseling for at-risk families; carrier/cascade testing; prenatal or preimplantation genetic testing where the familial DHFR variant is known.
Secondary prevention (early detection): High index of suspicion in infants with megaloblastic anemia + neurologic signs + low CSF folate → early DHFR testing and prompt folinic acid to prevent CNS damage. No population newborn screen exists yet, but the treatable nature makes early metabolic detection valuable.
Tertiary prevention: Lifelong folinic acid, avoidance of antifolates, seizure control, developmental support to prevent complications and progression.
Counseling: Autosomal recessive recurrence risk 25% per pregnancy for carrier couples; offer NSGC/ACMG-guided counseling.

14. Other Species / Natural Disease

Taxonomy: DHFR is universally conserved (essential enzyme) across bacteria, fungi, plants, and animals (NCBI Taxon relevance broad). Human host: Homo sapiens (NCBI:txid9606).
Orthologous genes: Mouse Dhfr (NCBI Gene 13361); rat Dhfr (NCBI Gene 24312); zebrafish dhfr. High evolutionary conservation of catalytic function.
Natural disease in animals: No well-characterized spontaneous DHFR-deficiency disease reported in companion animals/wildlife (OMIA — none established). DHFR is a classic antimicrobial/antineoplastic drug target across species.
Comparative biology: Complete DHFR loss is embryonic-lethal in model organisms (essential gene), which is why human disease arises only from hypomorphic missense alleles retaining partial activity — a key comparative insight.
Transmission: Not applicable (non-infectious, monogenic).

15. Model Organisms

Model status: No published dedicated mouse/zebrafish model that recapitulates the human hypomorphic DHFR-deficiency disease was identified in this search; complete Dhfr knockouts are expected to be embryonic-lethal (essential enzyme), a key limitation.
Cellular/in vitro models (used in the defining studies): Patient-derived EBV-immortalized lymphoblastoid cell lines and fibroblasts demonstrating reduced DHFR activity, reduced FMTX binding, and reduced protein — the primary functional models to date. [in vitro; PMID 21310277]
Model types feasible: Knock-in of patient missense alleles (e.g., p.Asp153Val, p.Leu80Phe) in mouse/human iPSC or cell lines; conditional/hypomorphic alleles; iPSC-derived neurons/erythroid cultures to model the dual phenotype; humanized DHFR yeast/bacterial complementation assays for variant functional testing.
Applications: Variant functional classification (enzyme activity, antifolate binding), folate-flux studies, testing folinic acid / BH4 rescue.
Resources: MGI (Dhfr), Cellosaurus (patient lines), ClinVar (DHFR variants).

Ontology Term Quick-Reference

Disease: MONDO:0013456; OMIM #613839; ORPHA:319651
Gene/Protein: DHFR (HGNC:2861; NCBI 1719; UniProt P00374); GO:0004146 (DHFR activity)
HPO: HP:0001889 (megaloblastic anemia), HP:0001876 (pancytopenia), HP:0002121 (absence seizure), HP:0001250 (seizure), HP:0001263 (global developmental delay), HP:0001249 (intellectual disability), HP:0000252 (microcephaly), HP:0002352 (leukoencephalopathy)
GO (BP/CC): GO:0046655 (folic acid metabolic process), GO:0046452 (dihydrofolate metabolic process), GO:0006760 (folic acid-containing compound metabolic process), GO:0005829 (cytosol)
CL: CL:0000038 (erythroid progenitor), CL:0000540 (neuron), CL:0000700 (dopaminergic neuron)
UBERON: UBERON:0002371 (bone marrow), UBERON:0000955 (brain), UBERON:0001359 (CSF)
CHEBI: CHEBI:20506 (7,8-dihydrofolate), CHEBI:26907 (tetrahydrofolate), CHEBI:15641 (5-MTHF), CHEBI:63606 (folinic acid), CHEBI:15372 (tetrahydrobiopterin)
NCIT: Leucovorin Calcium (C1035); Sapropterin (C61815)

Supported vs. Refuted Hypotheses

Supported: - H1 (Supported): Biallelic DHFR missense mutations cause the disease via profound enzyme loss of function. [PMID 21310277, 21310276] - H2 (Supported): The mechanism is THF-pool depletion producing megaloblastic anemia + cerebral folate deficiency + cerebral BH4 deficiency. [PMID 21310276, 21310277] - H3 (Supported): Folinic acid (not folic acid) is corrective because it bypasses the DHFR block. [PMID 21310277, 21310276] - H4 (Supported): The disorder is distinguishable within inborn errors of folate metabolism; MTHFD1 deficiency adds immunodeficiency. [PMID 22108709, 25548164]

Refuted / Not supported: - Environmental or infectious primary etiology — refuted (Mendelian recessive enzyme defect). - Dominant inheritance — refuted (heterozygotes asymptomatic with intermediate activity).

Limitations and Future Directions

Evidence base is very small (~6 patients from 4 families); frequencies, prognosis, and genotype–phenotype correlations are qualitative. Numbers such as prevalence are order-of-magnitude estimates from Orphanet-class sources.
The curated literature accessible here is limited; additional post-2012 case reports and any dedicated animal models were not retrievable in this environment and should be sought in OMIM/GeneReviews/HGMD/ClinVar and full PubMed.
Future directions: systematic natural-history and treatment-timing studies; standardized CSF 5-MTHF/BH4/neurotransmitter panels; functional assays for variant classification; evaluation of BH4/neurotransmitter-precursor adjuncts; and consideration of DHFR in metabolic/epilepsy newborn or early-childhood screening given treatability.

Primary References (PMID)

21310277 — Cario H, et al. Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease. Am J Hum Genet, 2011. [HUMAN clinical + in vitro]
21310276 — Banka S, et al. Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency. Am J Hum Genet, 2011. [HUMAN clinical + in vitro]
22108709 — Watkins D, Rosenblatt DS. Update and new concepts in vitamin responsive disorders of folate transport and metabolism. J Inherit Metab Dis, 2012. [review]
25548164 — Field MS, et al. Human mutations in MTHFD1 impair nuclear de novo thymidylate biosynthesis. (differential) [HUMAN + in vitro]
25453083 — Wan Q, et al. Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography. [structural — DHFR enzymology]
36341171 — Akiyama T, et al. Folic acid inhibits 5-methyltetrahydrofolate transport across the blood-cerebrospinal fluid barrier: Clinical biochemical data from two cases. 2022. [HUMAN clinical — treatment rationale; provides pediatric CSF 5-MTHF reference values from 600 cases]
34008900 — Brunetti V, et al. Cerebral folate transporter deficiency syndrome in three siblings... 2021. [HUMAN clinical — FOLR1 differential; supports critical treatment window / irreversibility of delayed therapy]
41132636 — Ahmadabadi F, et al. A Case of Cerebral Folate Deficiency due to FOLR1 Mutation in a 10-Year-Old Girl. 2025. [HUMAN clinical — FOLR1 differential; timely folinic acid improves outcome]
32456656 — Opladen T, et al. Consensus guideline for the diagnosis and treatment of tetrahydrobiopterin (BH4) deficiencies. 2020. [guideline — BH4/monoamine management framework]
21931219 / 26057816 — Cody V, et al. Structural analyses of human dihydrofolate reductase. 2011/2015. [structural — hDHFR active-site residues Arg70, Phe31, Gln35, Val115]

Artifacts

Reference Validation

Checked with linkml-reference-validator 0.2.1.

Outcome	Count
References checked	10
Resolved	10
Unresolved (possible confabulation)	0
Unverifiable	0
Quoted claims checked	6
Quoted claims found in source	6
Quoted claims not found in source	0
References weighed for topical relevance	10
On topic	7
Off topic	0

All extracted references resolved successfully.

Term Validation

Checked with linkml-term-validator 0.4.5, through the ols: adapter.

Outcome	Count
Terms checked	36
Resolved	34
Unresolved (possible confabulation)	0
Obsolete	0
Unverifiable	2
Terms whose name was checked	32
Terms named correctly	21
Terms named as a different term	0
Terms whose name is worth a second look	11

Terms whose name is worth a second look

The report's name for these is recognisably related to the term's own name without being one of them. A loose paraphrase reads the same way as a citation of the wrong sibling term - and so does a related synonym, which the ontology records precisely because it names something adjacent rather than the same thing - so these are listed rather than judged:

HP:0001882 (1 mention) - the report calls it "Leukopenia"; HP calls it Decreased total leukocyte count, and lists "Leukopenia" among its other names
CHEBI:20506 (2 mentions) - the report calls it "7,8-dihydrofolate"; CHEBI calls it 5,6,7,8-tetrahydrofolic acid, and lists "5,6,7,8-Tetrahydrofolate" among its other names
CHEBI:26907 (2 mentions) - the report calls it "tetrahydrofolate"; CHEBI calls it tetrahydrofolic acid, and lists "tetrahydrofolate" among its other names
CHEBI:15641 (2 mentions) - the report calls it "5-MTHF"; CHEBI calls it 5-methyltetrahydrofolic acid, and lists "5-methyl-THF" among its other names
CHEBI:63606 (2 mentions) - the report calls it "folinic acid"; CHEBI calls it (6S)-5-formyltetrahydrofolic acid, and lists "L-Folinic acid" among its other names
CHEBI:15372 (2 mentions) - the report calls it "tetrahydrobiopterin"; CHEBI calls it 5,6,7,8-tetrahydrobiopterin, and lists "Tetrahydrobiopterin" among its other names
GO:0034355 (1 mention) - the report calls it "NAD salvage n/a"; GO calls it NAD+ biosynthetic process via the salvage pathway, and lists "NAD salvage" among its other names
GO:0004146 (2 mentions) - the report calls it "dihydrofolate reductase activity", "DHFR activity"; GO calls it dihydrofolate reductase activity, and lists "dihydrofolic reductase activity" among its other names
GO:0006559 (1 mention) - the report calls it "l-phenylalanine catabolic process, via BH4"; GO calls it L-phenylalanine catabolic process
CL:0000038 (3 mentions) - the report calls it "Suggested CL terms: erythroid progenitor cell", "erythroid progenitor"; CL calls it erythroid progenitor cell**
UBERON:0001359 (2 mentions) - the report calls it "CSF"; UBERON calls it cerebrospinal fluid, and lists "CSF" among its other names

Terms named inconsistently

The report gives these identifiers more than one name of its own:

HP:0001876 - called "Pancytopenia", "pancytopenia"
HP:0002121 - called "Absence seizure", "absence seizure"
HP:0001250 - called "Seizure", "seizure"
HP:0001263 - called "Global developmental delay", "global developmental delay"
HP:0001249 - called "Intellectual disability", "intellectual disability"
HP:0002352 - called "Leukoencephalopathy", "leukoencephalopathy"
GO:0004146 - called "dihydrofolate reductase activity", "DHFR activity"
CL:0000038 - called "Suggested CL terms:** erythroid progenitor cell", "erythroid progenitor"

Prefixes with no resolver

Terms carrying these prefixes were not checked either way, because no configured ontology covers them. An unrecognised prefix may name an ontology this run could not reach as easily as one that does not exist, so nothing here is evidence of fabrication: ORPHA.

34 of 36 terms resolved to a current term; the rest could not be looked up either way.

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1. Disease Information

2. Etiology

3. Phenotypes

4. Genetic / Molecular Information

5. Environmental Information

6. Mechanism / Pathophysiology

Ordered causal chain (initiating lesion → clinical manifestation)

Detail by category

7. Anatomical Structures Affected

8. Temporal Development

9. Inheritance and Population

10. Diagnostics

11. Outcome / Prognosis

12. Treatment

13. Prevention

14. Other Species / Natural Disease

15. Model Organisms

Ontology Term Quick-Reference

Supported vs. Refuted Hypotheses

Limitations and Future Directions

Primary References (PMID)

Artifacts

Reference Validation

Term Validation

Terms whose name is worth a second look

Terms named inconsistently

Prefixes with no resolver