What laboratory studies should be ordered at an 18‑month well‑child visit to evaluate for folate metabolism disorders, including MTHFR deficiency and cerebral folate deficiency?

Laboratory Studies for Folate Metabolism Disorders at 18-Month Well-Child Visit

Order CSF 5-methyltetrahydrofolate (5-MTHF) measurement via lumbar puncture to definitively diagnose cerebral folate deficiency, as this is the hallmark test that distinguishes cerebral from systemic folate disorders and guides treatment decisions.

Primary Diagnostic Tests

Cerebrospinal Fluid Analysis (Gold Standard)

• CSF 5-MTHF measurement is essential for diagnosing cerebral folate deficiency, as low CSF 5-MTHF (<40 nmol/L) can occur even with normal peripheral folate levels 1, 2
• Normal CSF 5-MTHF should be >40 nmol/L; severe deficiency is defined as <13 nmol/L 1
• CSF analysis should also include biogenic amines and pterins to evaluate for secondary causes of cerebral folate deficiency 1
• This test requires lumbar puncture, which is justified given the severe neurological consequences of untreated cerebral folate deficiency 2

Peripheral Blood Tests

• Serum folate (normal ≥10 nmol/L or 4.4 ng/mL) to assess recent dietary intake 3, 4
• RBC folate (normal ≥340 nmol/L or 150 ng/mL) to evaluate long-term folate status over the preceding 3 months 3, 4
• Total homocysteine (tHcy) levels, as elevated homocysteine is consistently found in folate deficiency and MTHFR deficiency 3, 5
• Vitamin B12 (cobalamin) levels to rule out B12 deficiency, which presents with similar findings 3, 4
• Methylmalonic acid (MMA) to differentiate folate from B12 deficiency (MMA remains normal in isolated folate deficiency but is elevated in B12 deficiency) 3, 4

Complete Blood Count with Indices

• CBC with MCV and MCH to identify macrocytic anemia (high MCV) characteristic of folate deficiency 3
• Reticulocyte count to assess bone marrow response 6

Specialized Testing for MTHFR Deficiency

Genetic and Metabolic Studies

• MTHFR gene sequencing if severe hyperhomocysteinemia is present (tHcy >100 μmol/L suggests MTHFR deficiency) 5
• Plasma methionine levels, which are typically low in MTHFR deficiency 7
• Urine formiminoglutamic acid (FIGLU) if glutamate formiminotransferase deficiency is suspected 7

Additional Investigations for Cerebral Folate Deficiency

Folate Receptor and Transport Studies

• FOLR1 gene sequencing to identify folate receptor alpha defects causing isolated cerebral folate deficiency 2, 8
• CSF protein analysis for non-functional folate receptor protein 1 (FR1) 2
• Testing for folate receptor autoantibodies if FOLR1 sequencing is normal 2, 8

Secondary Cause Evaluation

• Mitochondrial disease workup (lactate, pyruvate, respiratory chain enzyme analysis) as Kearns-Sayre syndrome can cause cerebral folate deficiency 1, 8
• Serine biosynthesis pathway evaluation (plasma amino acids including serine) 8
• Review medication history for antifolate or anticonvulsant drugs 2, 8

Critical Clinical Context

Timing and Urgency

The 18-month timepoint is clinically significant because cerebral folate deficiency typically manifests from 4 months onward with irritability, sleep disturbances, and psychomotor retardation, progressing to cerebellar ataxia and spastic paraplegia 2. Early identification before age 6 years results in favorable clinical response to folinic acid treatment, whereas identification after age 6 years leads to only partial recovery with poorer outcomes 2.

Diagnostic Algorithm Priority

1. Start with peripheral blood tests (serum folate, RBC folate, homocysteine, B12, MMA, CBC) as these are non-invasive 3, 4
2. If peripheral folate is normal but clinical suspicion remains high (developmental delay, ataxia, movement disorders), proceed to lumbar puncture for CSF 5-MTHF 1, 2
3. If severe hyperhomocysteinemia is found (>100 μmol/L), pursue MTHFR genetic testing 5
4. If CSF 5-MTHF is low with normal FOLR1 sequencing, test for folate receptor autoantibodies 2

Common Pitfalls to Avoid

• Do not assume normal peripheral folate excludes cerebral folate deficiency, as the defining feature is low CSF 5-MTHF with normal systemic folate 1, 2, 8
• Always measure B12 and MMA alongside folate studies, as high folate can mask B12 deficiency while allowing irreversible neurological damage 3, 4
• Ensure proper sample handling for folate measurements, as folate is susceptible to degradation by light, temperature, pH, and oxygen 4
• The microbiological assay using Lactobacillus rhamnosus is the gold standard method for measuring folate 3, 4