Cerebral Folate Deficiency via the FOLR1 Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1595 FOLR1$440.00 81479 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

FOLR1 was sequenced in 72 patients with low levels (<40nmol/l) of 5MTHF in cerebrospinal fluid. Pathogenic FOLR1 variants were identified in 10 (~14%) patients (Grapp et al. 2012). The authors noted that of the 14 patients that had extremely low CSF 5MTHF levels (<5nmol/l), 10 (~70%) had pathogenic FOLR1 variants.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 FOLR1$990.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Features

Cerebral folate deficiency (CFD) is a neurometabolic disorder caused by reduced levels of folate in the brain. CFD patients typically present with global developmental delay around 2 years of age. Common symptoms seen in CFD patients include hypotonia, ataxia, myoclonic seizures, and athetoid movements (Grapp et al. 2012). Moderate to severe psychomotor regression is observed following disease onset. Patients are intellectually disabled, lack language, and have an increased incidence of aggressive behavior. If left untreated, patients develop frequent refractory seizures and are often wheelchair-bound due to worsening motor symptoms. MRI reveals abnormal myelination of the periventricular and subcortical white matter as well as cerebellar atrophy. Metabolite analysis reveals low 5-methyl-tetrahydrofolate levels in cerebrospinal fluid (CSF), but a normal folate metabolism profile in plasma. This CSF-plasma difference helps distinguish CFD from other folate deficiency syndromes (Pérez-Dueñas et al. 2010).

Oral folinic acid supplementation was found to greatly improve symptoms, particularly seizures and motor abnormalities, in affected individuals (Pérez-Dueñas et al. 2010; Grapp et al. 2012). Early folinic acid treatment at first clinical onset was reported to completely reverse CFD symptoms (Steinfeld et al. 2009).


Cerebral folate deficiency is inherited in an autosomal recessive manner and can be caused by variants in the FOLR1 gene. Nonsense, missense, and splice site variants as well as small duplications in FOLR1 have been reported to cause cerebral folate deficiency (Grapp et al. 2012). Other reported causes of CFD include: autoantibodies directed against the FOLR1 protein, mitochondrial disorders, and Rett syndrome (Ramaekers et al. 2013).

Folate derivatives are essential cofactors required for many biological processes, including DNA synthesis. Dietary folate is metabolized in the liver to the physiologically active form 5-methyl-tetrahydrofolate (5MTHF) which is then distributed by the bloodstream (Hyland et al. 2010). Humans possess multiple folate receptors, and folate receptor-alpha appears to be the primary receptor responsible for transporting 5MTHF across the blood-CSF barrier in adults (Steinfeld et al. 2009). FOLR1 encodes the cerebral folate receptor-alpha. Loss of FOLR1 results in greatly reduced levels of 5MTHF in the brain. Supplementation with folinic acid increases serum levels of 5MTHF and increases the amount of 5MTHF transported to the CSF via other low affinity folate transporters (Ramaekers et al. 2013).

Testing Strategy

Testing involves PCR amplification from genomic DNA and bidirectional Sanger sequencing of the coding exons and ~10bp of adjacent noncoding sequences. This testing strategy will reveal coding sequence changes, splice site mutations and small insertions or deletions in the FOLR1 gene, but will not detect large deletions in the FOLR1 locus. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Indications for Test

FOLR1 sequencing should be considered in patients with onset of seizure and developmental regression after two years of age who have normal blood folate levels, but have severely reduced 5MTHF in cerebrospinal fluid. One study suggests that CSF 5MTHF levels < 5 nmol/l are a strong indication for FOLR1 sequencing (Grapp et al. 2012).  As mentioned above, FOLR1 variants are not the sole cause of CFD; Ramaekers et al. have published guidelines for molecular diagnosis in suspected CFD patients (Ramaekers et al. 2013).


Official Gene Symbol OMIM ID
FOLR1 136430
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Cerebral Folate Deficiency 613068


Genetic Counselors
  • Grapp M, Just IA, Linnankivi T, Wolf P, Lucke T, Hausler M, Gartner J, Steinfeld R. 2012. Molecular characterization of folate receptor 1 mutations delineates cerebral folate transport deficiency. Brain 135: 2022–2031. PubMed ID: 22586289
  • Hyland K, Shoffner J, Heales SJ. 2010. Cerebral folate deficiency. Journal of Inherited Metabolic Disease 33: 563–570. PubMed ID: 20668945
  • Pérez-Dueñas B, Toma C, Ormazábal A, Muchart J, Sanmartí F, Bombau G, Serrano M, García-Cazorla A, Cormand B, Artuch R. 2010. Progressive ataxia and myoclonic epilepsy in a patient with a homozygous mutation in the FOLR1 gene. Journal of Inherited Metabolic Disease 33: 795–802. PubMed ID: 20857335
  • Ramaekers V, Sequeira JM, Quadros EV. 2013. Clinical recognition and aspects of the cerebral folate deficiency syndromes. Clinical Chemistry and Laboratory Medicine 51: PubMed ID: 23314536
  • Steinfeld R, Grapp M, Kraetzner R, Dreha-Kulaczewski S, Helms G, Dechent P, Wevers R, Grosso S, Gärtner J. 2009. Folate Receptor Alpha Defect Causes Cerebral Folate Transport Deficiency: A Treatable Neurodegenerative Disorder Associated with Disturbed Myelin Metabolism. The American Journal of Human Genetics 85: 354–363. PubMed ID: 19732866
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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