What peripheral blood laboratory tests should be obtained to assess folate metabolism disorders in a well‑child with autism spectrum disorder or being evaluated for ASD?

Laboratory Testing for Folate Metabolism Disorders in Children with ASD

The American Academy of Pediatrics recommends folate receptor autoantibodies (FRAA) as the primary biomarker for identifying ASD patients who may benefit from leucovorin therapy, particularly when developmental regression or metabolic concerns are present. 1

First-Tier Screening Tests

For well-children with ASD or suspected ASD, the following peripheral blood tests should be obtained to assess folate metabolism:

Essential Metabolic Panel

• Serum folate – Low folate levels (particularly lowest quartile) correlate with increased ASD severity and should be measured as a baseline marker 1, 2
• Serum vitamin B12 – Decreased concentrations associate with poor adaptive behavior and developmental quotients 2
• Homocysteine – Elevated levels show strong linear association with ASD (AUC: 0.899) and serve as a novel diagnostic biomarker; levels are significantly higher in ASD children compared to controls 2
• Methylmalonic acid (MMA) – More sensitive than serum B12 alone for assessing functional B12 status and folate metabolism 1

Folate Pathway-Specific Markers

• Folate receptor autoantibodies (FRAA) – The primary recommended biomarker by the American Academy of Pediatrics for identifying treatment candidates 1
• Methionine – Part of the folate-methionine cycle assessment and included in discriminant models for ASD classification 3
• S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) ratio – Reflects methylation capacity and is a significant predictor in ASD classification models 3

Supporting Laboratory Tests

• Complete blood count (CBC) – To assess for anemia which may indicate metabolic disorders 4
• Serum amino acid analysis – Recommended for patients showing signs of metabolic disorders 4
• Ferritin and total iron binding capacity – Should be investigated when considering folate pathway abnormalities 1

Conditional Testing Based on Clinical Red Flags

When Cerebral Folate Deficiency (CFD) is Suspected

The American College of Medical Genetics identifies CFD as a "low incidence yet high impact" metabolic disorder requiring high clinical suspicion. 5 Test when the following are present:

• Developmental regression outside of typical ASD speech loss 5
• Seizures 5
• Hypotonia or dystonia 5

Additional testing in these cases:

• Genetic testing for MTHFR and folate metabolism pathway variants – Guides leucovorin therapy decisions 1
• Methionine synthase and transcobalamin II levels – These are significant predictors in folate-related ASD classification 3

When Mitochondrial Disease is Suspected

If constitutional symptoms, repeated regressions after age 3, or multiple organ dysfunctions are present, add: 4

• Lactate and pyruvate – Key indicators of mitochondrial dysfunction 1
• Serum metabolic profile – For detecting acidosis and other physiologic abnormalities 4

Important Clinical Considerations

Do not perform routine metabolic testing in all ASD cases. The American College of Medical Genetics emphasizes that metabolic or mitochondrial studies are not indicated unless multiple signs of these disorders are present, such as anemia, gastrointestinal dysfunction, cyclic vomiting, lactic acidosis, microcephaly, or seizures. 6

Key Clinical Indicators Warranting Expanded Testing

• True developmental regression (not just speech loss typical of ASD) 4
• Worsening neurological symptoms 4
• Lethargy and poor physical endurance 4
• Multisystem involvement (cardiac, hepatic, or renal manifestations) 4
• Seizures 4

Diagnostic Yield Context

The diagnostic yield of recommended genetic testing (chromosomal microarray as first-tier) in ASD is 40%, which is markedly higher than metabolic profiling in the absence of clear clinical concerns. 6 However, when specific metabolic red flags are present, folate metabolism testing becomes clinically indicated and potentially treatment-altering. 5, 1

Common Pitfalls to Avoid

• Don't delay metabolic screening if clinical indicators present – Pursue folate pathway and mitochondrial testing aggressively when metabolic abnormalities are suspected 1
• Don't rely on serum B12 alone – Homocysteine and methylmalonic acid are more sensitive for assessing functional status 1
• Don't overlook vitamin B12 deficiency – ASD probands frequently show vitamin B12 deficiency which correlates with poor developmental outcomes 2, 7