Cerebral Folate Deficiency (FOLR1) and Other Genetic Cerebral Folate Deficiencies

FOLR1-related cerebral folate transport deficiency (folate receptor α deficiency), hereditary folate malabsorption (SLC46A1), SLC19A1-related folate transport deficiency, DHFR, MTHFD1 and MTHFS deficiencies, and secondary cerebral folate deficiencies.

Literature and regulatory information updated: 6 November 2026.

0. Reference Summary Table

1. Introduction

1.1 Definition of the syndrome and the entities

Cerebral folate deficiency (CFD) is defined, in its most widely used formulation, as a low concentration of 5-methyltetrahydrofolate (5-MTHF), the main circulating folate species, in the cerebrospinal fluid (CSF), in the presence of normal total peripheral folate [24]. The same term is often used to describe situations in which CSF 5-MTHF is low but peripheral folate is also low or has not been measured, which corresponds to a different pathophysiological situation; therefore, a result reported as “cerebral folate deficiency” requires confirmation that peripheral folate was in fact normal [15, 24]. GeneReviews stresses that the term is generic: it implies neither an aetiology nor a clinical picture, it does not define a CSF folate value below which neurocognitive deficits arise, and it does not define a value that is a sufficient treatment target [2]. FOLR1-related cerebral folate transport deficiency (FOLR1-CFTD) refers specifically to CFD caused by loss of function of folate receptor α (FRα) [2]. The 2022 French protocol defines CFD as a neurological syndrome with very low CSF 5-MTHF and normal red cell concentration, and covers only the FOLR1 form, as it is the only one with biological and genetic confirmation available in France [17].

This article treats FOLR1-CFTD as the main entity and the other genetic causes of CFD as a class with a common structure. It groups three hereditary defects of folate transport — FOLR1-CFTD, hereditary folate malabsorption (HFM, due to SLC46A1) and SLC19A1-related folate transport deficiency (SLC19A1-FTD) — and the inborn errors of folate metabolism accompanied by CFD, such as dihydrofolate reductase (DHFR), methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), 5,10-methenyltetrahydrofolate synthetase (MTHFS) and methylenetetrahydrofolate reductase (MTHFR) deficiencies [2, 6, 7]. Severe MTHFR deficiency is part of the differential, and “folinic acid-responsive seizures” now correspond to pyridoxine-dependent epilepsy due to ALDH7A1 [25, 26].

1.2 Classification

The review by Ramaekers and Quadros offers a different view of the aetiological hierarchy: it considers that the most frequent primary cause of the CFD syndrome is serum autoantibodies against FRα, which impair transport in the choroid plexus, and that mitochondrial diseases, inborn errors and loss-of-function variants in FOLR1 account for a minority of cases [28]. GeneReviews places autoantibodies in the differential diagnosis, with variable and generally only modestly reduced CSF folate, and notes that they have been detected in several other conditions [2]. The two readings coexist in the literature and do not change the diagnostic approach to the genetic forms.

1.3 History and milestones

Hereditary folate malabsorption was the first disease of the class to be described: Luhby and colleagues observed two sisters in 1965, and Lanzkowsky described in 1970 a sporadic case in a 20-year-old man, with an isolated defect of intestinal folic acid absorption and of transport across the blood–brain barrier, recurrent megaloblastic anaemia, intellectual disability, seizures, movement disorders and basal ganglia calcification [5]. In 2006, Qiu and colleagues identified the proton-coupled folate transporter (PCFT, encoded by SLC46A1), a high-affinity intestinal transporter that functions at low pH, and showed that its loss of function is the molecular basis of HFM in a Puerto Rican family [10, 29]. DHFR deficiencies, proposed since 1967 on the basis of responses to folinic acid but not to folic acid, were only confirmed molecularly in 2011, when Banka and colleagues and Cario and colleagues simultaneously described homozygous DHFR variants; the historical cases of Tauro (1976) were reclassified as methionine synthase reductase deficiency and transcobalamin II deficiency [11, 30, 31].

FOLR1-CFTD was described in 2009 by Steinfeld and colleagues, in Göttingen, as a brain-specific folate transport defect: three patients with FOLR1 variants developed a progressive movement disorder, psychomotor decline and epilepsy, with markedly reduced CSF folate, profound hypomyelination on magnetic resonance imaging (MRI) and combined depletion of choline and inositol in the white matter on magnetic resonance spectroscopy (MRS) [32]. In the same article, retroviral transfection of patient cells with FRα or FRβ restored folate binding, and folinic acid normalised CSF folate and glial choline and inositol before clinical improvement [32]. Also in 2009, Cario and colleagues published congenital null variants of FOLR1 with progressive neurological disease and its treatment [33]. In 2012, Grapp and colleagues screened 72 children with low CSF 5-MTHF and neurological symptoms arising after infancy and identified FOLR1 alterations in 10, all with regression, ataxia, profound hypomyelination and cerebellar atrophy [34]. These were followed by the description of the transcytosis mechanisms in the choroid plexus (2013), the diagnostic synthesis by Pope and colleagues (2019), the dedicated GeneReviews chapter (2024), the French national protocol (2022) and, on 9 March 2026, the first regulatory approval of a medicine for FOLR1-CFTD, with the extension of indication for leucovorin tablets approved by the United States Food and Drug Administration (FDA) [2, 17, 19, 24, 35].

2. Molecular Genetics and Pathophysiology

2.1 Physiology of folate transport into the central nervous system

Folates are cofactors for DNA synthesis and repair, for the methylation reactions that regulate gene expression, for amino acid metabolism, for myelin formation and for neurotransmitter synthesis [2, 17]. Folate reaches the brain by two routes: through the choroid plexus into the CSF, which bathes the ventricles and the periventricular tissue, and directly across the vascular blood–brain barrier [2, 6]. In the choroid plexus, FRα is the most highly expressed transporter, and Steinfeld and colleagues concluded that it is the main route of cerebral folate supply; FOLR1 also has significant expression in the kidney [8]. FRα is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that binds folate and its reduced derivatives at neutral pH, internalises 5-MTHF by endocytosis and releases it when the endosome acidifies [8, 15]. Transport takes place by transcytosis in the choroid plexus and through exosomes that carry folate to the parenchyma [35, 36]. PCFT is also required for transport from blood into the CSF, so that loss of either transporter causes CFD; the reduced folate carrier (RFC, SLC19A1) mediates folate entry into all systemic and neural cells and probably participates in passage across the vascular blood–brain barrier [2, 6]. In healthy adults, CSF folate is two to three times serum folate, and normal CSF values are higher in childhood: about 100 nmol/L up to 2 years, 75 nmol/L at 5 years and 65 nmol/L at 19 years [4, 37].

2.2 FOLR1 and folate receptor α

FOLR1 lies at 11q13.4 (GRCh38 11:72,189,709–72,196,323), has seven exons spanning 6.7 kb and at least two promoters with tissue specificity; it encodes a 257-amino-acid protein with a signal peptide and a GPI-anchoring domain, identical to the ovarian cancer-associated antigen MOv18 [8]. The MANE Select transcript is NM_016729.3, as confirmed in VariantValidator; some reports and databases use RefSeq NM_016725, with the same coding coordinates for the variants described in this article [9, 38, 39]. Ablation of the homologous gene in the mouse (Folbp1) causes severe morphogenetic anomalies and embryonic death around day 10, reversed by maternal folinic acid supplementation [8].

The disease mechanism is loss of function [2]. Pathogenic variants may prevent synthesis of a complete protein, give rise to an unstable and rapidly degraded protein, impair trafficking of the protein–folate complex to or across the membrane, or reduce folate binding [2]. Grapp and colleagues showed that the missense variants studied reduced protein expression only slightly but prevented localisation to the cell surface, with retention in intracellular compartments and absence of folic acid binding [34]. Loss of FRα deprives of folate the tissue nourished by the CSF, especially the periventricular regions, where neural stem cells differentiate; the vascular blood–brain barrier probably remains functional, and variability in this route may explain differences in severity [2].

2.3 Spectrum of FOLR1 variants

Sequencing detects about 99% of known pathogenic variants and targeted deletion/duplication analysis about 1% [2]. Intronic variants close to the splice junction have been described, and other intronic variants may escape standard sequencing [2]. The December 2025 GeneReviews revision removed the list of specific variants [2]. The variants in the following table were normalised in ClinVar and VariantValidator.

Other published variants include homozygous p.(Cys105Arg), predicted to abolish a disulphide bridge required for correct folding [44], homozygous p.(Glu50Lys) in a Chinese family [45], p.(Cys65Trp) with p.(Trp143Arg), the latter of uncertain significance, in Greek siblings [46], and c.45G>T with c.493+2T>C in an Italian adult [18]. ClinVar also lists deletions of one or more exons and copy number variants encompassing the gene [38].

2.4 Genotype–phenotype correlations, penetrance and allelic disorders

There are no clinically relevant genotype–phenotype correlations, and only two studies have examined the functional consequences of five variants in expressed proteins [2, 17]. In the Grapp series, three patients with a milder phenotype had biochemical and imaging data very similar to those of the severe patients and shared alleles with them, which indicates that severity does not depend solely on residual receptor function and that other factors contribute [34]. Manco and colleagues attributed the paucisymptomatic adult-onset picture of their patient to reduced FRα expression and function without complete loss [18]. Heterozygotes are asymptomatic [2]. No allelic germline phenotypes of FOLR1 other than FOLR1-CFTD are known [2]. Study of FOLR1 in neural tube defects showed no association in three case–control studies, and another study described gene conversions from a pseudogene in 4 of 50 patients with sporadic neural tube defects and in one of 150 controls [8].

2.5 SLC46A1 and hereditary folate malabsorption

SLC46A1 (17q11.2) encodes PCFT, a transporter of the solute carrier superfamily with 12 transmembrane domains, confirmed by cryo-electron microscopy structure, high affinity for folic acid, reduced folates and antifolates, and a low pH optimum [4]. PCFT is highly expressed at the apical membrane of the duodenum and proximal jejunum, where it is required for intestinal folate absorption, and is present, together with FRα, in the choroid plexus, both being required for folate transport into the CSF [4]. Transport is sodium-independent, absent at near-neutral pH and inhibited by methotrexate; the gene was initially described as a haem transporter (HCP1) [10]. The mechanism is loss of transport function: missense variants in the transmembrane domains give rise to unstable proteins, with markedly reduced or absent function, some reaching the membrane and others not, and three mutated isoforms retained residual activity [4].

No large deletions or duplications of SLC46A1 have been described; the Japanese deep intronic variant requires methods that detect splice variants outside the canonical junctions [4]. There are no established genotype–phenotype correlations; an individual with homozygous nonsense variants and total absence of PCFT, treated from infancy with low-dose intramuscular 5-formyl-THF, had a completely benign phenotype at 45 years [4].

2.6 SLC19A1 and the inborn errors of folate metabolism

RFC transports folates into all cells; the severity of SLC19A1-FTD depends on the degree of loss of function [6]. The variant NM_194255:c.1042G>A p.(Gly348Arg), homozygous in four individuals, is associated with severe early anaemia, mucositis, immunodeficiency and neurocognitive deficits; c.634_636del p.(Phe212del), in a conserved internal loop, caused only late megaloblastic anaemia, responsive to oral folate [6, 48]. RFC also transports 2',3'-cGAMP, which activates the STING pathway, and this defect is not corrected by folate; its clinical role is unknown [6]. RFC-null mice die in utero, which suggests human embryonic loss when loss of function is complete [6]. The SLC19A1 polymorphisms that modify antifolate pharmacokinetics belong to pharmacogenetics and are outside the scope of this article [6].

DHFR (5q14.1) reduces dihydrofolate to tetrahydrofolate (THF) and, more slowly, folic acid to dihydrofolate; in its deficiency, plasma and red cell folate is normal, but the oxidised forms are relatively elevated and the proportion of THF derivatives is reduced [7]. The variants p.(Leu80Phe) and p.(Asp153Val) were described in the homozygous state in the two 2011 series, and the disease is accompanied by reduced tetrahydrobiopterin (BH4) in the CSF, because DHFR participates in the pterin salvage pathway [11, 49]. Folic acid is inappropriate in DHFR deficiency because it needs the enzyme itself to be activated [15]. MTHFD1 is a trifunctional cytoplasmic enzyme that synthesises 10-formyl-THF from THF and formate and converts it into 5,10-methylene-THF; deficiency impairs thymidylate synthesis and homocysteine remethylation, while sparing purine synthesis [7]. The common MTHFD1 polymorphism c.1958G>A p.(Arg653Gln) is a susceptibility factor, not a disease [15]. MTHFS converts 5-formyl-THF (folinic acid) into 5,10-methenyl-THF in an ATP-dependent manner; its deficiency traps folinic acid, which was increased 30-fold in the fibroblasts of one patient and may act as a toxic metabolite [7, 12]. This step explains why the United States leucovorin label advises against it in MTHFS deficiency: MTHFS is the primary enzyme of leucovorin metabolism [21].

2.7 Secondary cerebral folate deficiency

In mitochondrial diseases, transport in the choroid plexus is secondarily impaired, presumably through altered energy metabolism; CSF folate is variably reduced and may be normal, and there is no specific transporter defect [2, 4]. In Kearns-Sayre syndrome (mitochondrial DNA deletion, usually de novo), the CSF/serum folate ratio is low (0.6, with control values of 1.5 to 3) and 5-MTHF is profoundly reduced [27]. In AADC deficiency, methylation of accumulated L-DOPA consumes methyl groups, reduces S-adenosylmethionine in the central nervous system and sometimes 5-MTHF [50]. In DHPR deficiency, central folate deficiency can cause rapid neurological deterioration despite good phenylalanine control [49]. These secondary causes generally have less depletion than receptor deficiency [7].

3. Clinical Spectrum and Manifestations by System

3.1 FOLR1-CFTD: untreated natural history

The initial signs of FOLR1-CFTD are subtle and appear during the first year: delay mainly in cognition, speech and walking, and behavioural changes, sometimes autistic in type [2]. Overt signs usually appear after the first year, although earlier presentations have been described [2]. The French protocol places the onset of delay or regression in the first three years, after an asymptomatic period, with epilepsy appearing later and in the background [17]. In the review by Potic and colleagues, which brought together 31 published patients and two new ones, age at onset ranged from 3 months to 3 years 2 months, mostly between 1 and 2.5 years [39]. Early unilateral strabismus and nystagmus have been described as first signs [2].

Without treatment, the disease progresses to developmental regression, ataxia, tremor and hypotonia, and then to severe cognitive decline, increasing immobility and intractable seizures [2]. Drop attacks provoked by specific movements, such as washing the face or hands, have been described [2, 40]. Despite severe neurological disability, untreated patients may live into adulthood, and there are reports of untreated patients in adolescence and up to 30 years of age, with established neurological damage [2].

These frequencies derive from case reports, without a population denominator, and mainly reflect patients diagnosed late [2]. In the Turkish series of nine patients from six families, the mean age at first complaints was 3.7 years and at diagnosis 10.4 years; the most frequent first complaints were febrile convulsions and attention deficit, hyperactivity and learning difficulties, epilepsy was the most common prior diagnosis, and the first seizure occurred at a mean age of 5.2 years [51].

3.2 Manifestations by system

Central nervous system and development. The French protocol lists regression and motor and language delay after an asymptomatic period, ataxia, spasticity, dyskinesia, hypotonia, dystonia, acquired microcephaly and epilepsy [17]. The epilepsy is characterised by resistance to anticonvulsants, frequent seizures, possible status epilepticus, focal and generalised, myoclonic and tonic seizures, and possibly absences, myoclonic-astatic and myoclonic-atonic seizures and spasms; one patient had no epilepsy [17]. The first seizures may be associated with fever [2]. In the report by Papadopoulou and colleagues, the older brother had drug-resistant epileptic spasms after the first year [46]. Pérez-Dueñas and colleagues described a healthy infant who developed an ataxic syndrome in the second year, followed by choreic movements and progressive myoclonic epilepsy [44]. Saudubray includes FOLR1 variants among the metabolic causes of prominent chorea [52].

Electroencephalogram. The background rhythm is slow, with multifocal or generalised paroxysmal activity; it may be normal [17].

Neuroimaging. MRI shows non-specific white matter hyperintensity, indicative of diffuse hypomyelination, cerebral and cerebellar atrophy and calcifications [2]. The French protocol adds cerebellar hypoplasia, cortical atrophy, gyration anomalies and reduced choline and inositol peaks on MRS [17]. FOLR1-CFTD is included in the group of early-onset encephalopathies with hypomyelination [36]. Bilateral basal ganglia calcifications [40] and periventricular calcifications [53] have been described, as well as cortical involvement associated with hypomyelination [41].

Peripheral nervous system, vision and hearing. There may be peripheral neuropathy [17]; Kobayashi and colleagues documented polyneuropathy with axonal and demyelinating components, which improved with treatment [41]. The French protocol recommends ear, nose and throat and ophthalmological assessment after diagnosis [17]. Wang and colleagues report visual and hearing impairment in some patients [45].

Haematology and other organs. The full blood count and peripheral folate are normal, which distinguishes FOLR1-CFTD from HFM and DHFR deficiency [4, 7]. FRα participates in renal folate retention, but its loss is not associated with clinically significant systemic deficiency or anaemia [6].

3.3 Late-onset and adult forms

Ferreira and colleagues described late diagnosis in adult siblings, with fewer seizures after folinic acid; at the time there were 18 individuals diagnosed in childhood [54]. Karin and colleagues reported marked improvement in an adult patient [55]. Manco and colleagues described the first case of paucisymptomatic adult-onset leukoencephalopathy: a 47-year-old man with a history of alcohol and drug use, admitted for diplopia and postural instability, with bilateral leukoencephalopathy, diffuse reduction in fractional anisotropy, CSF 5-MTHF of 10 nmol/L (normal 41–117) and compound heterozygosity c.45G>T and c.493+2T>C, clinically and radiologically stable for two years [18]. The French protocol accepts 5-MTHF values up to 15–20 nmol/L in late forms [17].

3.4 Treated FOLR1-CFTD

In younger siblings diagnosed with minimal or absent signs, early treatment with 5-formyl-THF substantially improved, completely reversed or prevented all manifestations [2]. In the founding article, the younger sister of the German boy was treated soon after the first motor signs, at 2 years 3 months, recovered completely and had no further symptoms; the brother, severely disabled and wheelchair-bound, with resistant seizures, had a reduction in seizure frequency and severity and began walking with support; the Italian girl, severely disabled at 5 years, improved slowly [1]. In the 13-year follow-up of seven patients from Göttingen, five improved, one treated before symptoms remained asymptomatic and one with irregular treatment deteriorated; CSF 5-MTHF and MRS parameters normalised soon after initiation, whereas magnetisation transfer and diffusion tensor imaging tracked the gradual clinical improvement and progressive myelination [56]. In patients with long-standing deficits, seizures may decrease and cognitive function may improve, but responses are frequently subjective, anecdotal and inconsistently documented [2]. In the Potic review, almost all patients had incomplete improvement; four did not improve (one with a concomitant POLG1 variant and three Algerian siblings treated 13 to 17 years after onset), and complete clinical and radiological recovery was documented only with treatment started before 3 years of age [39]. Brunetti and colleagues observed no appreciable improvement in three siblings treated orally and intravenously after more than 15 years of disease [57]. The French protocol summarises that early treatment halts regression, improves motor skills, language and interaction, reduces or abolishes seizures and reverses white matter abnormalities, and that it appears ineffective when involvement is already severe [17]. The review that supported the FDA label identified 46 treated patients in 26 publications up to 2024; of the 27 treated orally only, 24 had improvements and 3 remained unchanged or without progression [21].

3.5 Hereditary folate malabsorption

Infants with HFM may be born with adequate stores, but they do not absorb folate from milk and rapidly become deficient; serum and CSF folate are low before clinical signs, in the first month of life [4]. The presentation includes anorexia, poor weight gain, diarrhoea or oral mucositis, megaloblastic anaemia that is often trilineage, thrombocytopenia in at least 30%, with no haemorrhages described, and immunodeficiency with hypoimmunoglobulinaemia documented in about 25%, probably underestimated, Pneumocystis jirovecii and cytomegalovirus infections and possible T-cell and B-cell abnormalities [4]. Infants may die of infection before diagnosis [4]. In Jebnoun's Tunisian family, the first five of eight affected children died in the first months with diarrhoea, vomiting, drowsiness, pallor and glossitis [5]. Neurological manifestations include subtle delays, which with absent or inadequate treatment progress to progressive delay, movement disorders, peripheral neuropathy and behavioural and cognitive deficits; seizures occur in about 40%, typically between 6 and 12 months, and become intractable if treatment is delayed [4]. Intracranial calcifications, especially of the basal ganglia, are frequent, and MRI shows hypomyelination with cerebral and cerebellar atrophy [4]. Lubout and colleagues described bilateral occipital cortical calcifications in a 1-year-old boy [58]. Some patients have no neurological manifestations despite very low CSF folate, for unknown reasons [4]. Geller's Puerto Rican sisters developed normally on folate and were well at 9 and 6 years [5].

3.6 Other entities of the class

In untreated SLC19A1-FTD, megaloblastic anaemia was observed in 5 of 5 patients, recurrent infections, leucopenia or thrombocytopenia, low immunoglobulins, mucositis and diarrhoea in 4 of 5, developmental delay in 4 of 5, seizures in 1 of 5 and delayed myelination with cerebral or cerebellar calcifications in the 3 patients with MRI; birth weight and head circumference in the low centiles suggest in utero onset in the severe forms, and two patients had curly grey hair [6]. DHFR deficiency usually manifests in the first months with developmental delay, macrocytic anaemia, pancytopenia and neurodegeneration of variable intensity, but may be limited to macrocytosis without anaemia [2]; the 2011 series range from severe delay with generalised seizures and cerebral and cerebellar atrophy to absence epilepsy with eyelid myoclonia and learning difficulties, or absence of symptoms at 11 years [11]. MTHFD1 deficiency combines megaloblastic anaemia, severe combined immunodeficiency and atypical haemolytic uraemic syndrome, with seizures, developmental delay and neuroimaging abnormalities; two untreated patients died at 9 weeks [7]. GeneGuidance adds microangiopathy with retinopathy, sensorineural deafness, autoimmunity and hepatic fibrosis [15], and a 2023 report described a presentation as severe combined immunodeficiency [59]. MTHFS deficiency, in the review of 12 patients from 10 families, always began in the neonatal or infantile period, with severe global developmental delay, microcephaly, hypotonia, growth delay, mild dysmorphism and epilepsy; two died in early childhood and the survivors retained severe impairment [15]. Rodan's two patients also had an exaggerated startle response, short stature, progressive spasticity and episodes of hyperthermia [12].

4. Clinical Recognition and Red Flags

4.1 When to suspect FOLR1-CFTD

The diagnosis should be considered in a child with refractory epilepsy associated with psychomotor regression and a neurodevelopmental disorder of secondary onset [17]. More broadly, CFD of any aetiology should be considered in patients with unexplained, multisymptomatic neurological disease, with symmetrical hypomyelination-type white matter involvement or with a reduced choline/creatine ratio on MRS [17]. The Turkish series suggests considering it in the presence of febrile convulsions, developmental delay, ataxia, autism spectrum disorder, acquired microcephaly and MRI with white matter involvement and cerebellar atrophy [51]. Girgis and colleagues propose suspecting it in the presence of global delay, intractable myoclonic epilepsy and ataxia with cerebellar atrophy and cerebral calcifications [53]. Brunetti and colleagues insist on including FOLR1 early in the genetic testing of developmental and epileptic encephalopathies, because the picture overlaps with that of many of them [57]. A family history of affected siblings or consanguinity supports the diagnosis, but its absence does not exclude it [2].

4.2 Red flags

4.3 Urgent situations and therapeutic trial

In refractory neonatal seizures without an evident cause, the French protocol recommends a vitamin combination (pyridoxine, biotin and folinic acid, possibly followed by pyridoxal phosphate) in parallel with sample collection, and justifies folinic acid in the neonatal period by its potentiating effect on pyridoxine [17]. Saudubray recommends systematically assessing the response to vitamins in intractable seizures and, in adults, a short trial of high-dose B vitamins, because transporter defects can produce non-specific encephalopathies without biochemical markers [60]. When FOLR1-CFTD is suspected, the French protocol proposes collecting blood and CSF before the trial and starting folinic acid 3–5 mg/kg/day until the 5-MTHF result is received, with clinical improvement expected within two months if treatment is early (before 6 years of age) [17]. GeneGuidance recommends starting oral folinic acid as soon as the CSF shows low 5-MTHF with normal peripheral folate, without waiting for the genetic result, and never replacing it with folic acid [15].

4.4 Newborn screening

There is no biochemical newborn screening for FOLR1-CFTD [2]. Dreha-Kulaczewski and colleagues concluded that presymptomatic therapy appears to ensure normal development and justifies newborn screening, and Akgun and Tas propose including FOLR1 in genomic screening programmes [51, 56]. For HFM, Mahadeo and colleagues proposed newborn screening in high-risk populations, such as the Puerto Rican population [16]. Glutamate formiminotransferase deficiency is detected on tandem mass spectrometry screening, because formiminoglutamate overlaps with C4 acylcarnitine, but it is generally asymptomatic [7].

5. Diagnosis: Criteria, Testing and Algorithms

5.1 Diagnostic criteria

No published consensus clinical criteria exist for any of the genetic CFDs; the operational criteria come from GeneReviews and the French protocol [2, 6, 17].

The two 5-MTHF thresholds do not coincide: GeneReviews, drawing on Pope and colleagues, typically uses ≤10 nmol/L, and the French protocol gives <5 nmol/L in neonatal forms and accepts values up to 15–20 nmol/L in late forms [2, 17]. In the United States label, 17 of 21 patients with available values had pretreatment 5-MTHF <10 nmol/L, against reference ranges of 40 to 240 nmol/L depending on the laboratory [21]. Papadopoulou and colleagues found only moderately low values (19 and 49 nmol/L, against normal ranges of 44–122 and 63–122 nmol/L) in siblings with biallelic variants [46]. An intermediate value does not exclude the disease, especially in late forms or under supplementation.

5.2 Biomarkers and tests in order

CSF 5-MTHF with simultaneous peripheral folate. This is the central test and can only be interpreted with plasma and red cell folate collected simultaneously; without peripheral values, the CSF value is inconclusive [15]. The CSF/plasma folate ratio helps: Pérez-Dueñas and colleagues found 5-MTHF of 2 nmol/L (normal 48–127) with a CSF/plasma ratio of 0.4 (normal 1.5–3.5) and recommended FOLR1 testing in profound deficiency with a reduced ratio [44]. Reference values depend on age (about 100, 75 and 65 nmol/L at 2, 5 and 19 years) and on technical factors [4, 37]; Akiyama and colleagues published reference values based on 600 paediatric cases [61]. Pre-analytical handling is critical: 5-MTHF is sensitive to light and oxidation, and CSF should be collected into an appropriate tube, protected from light and frozen immediately, with transport on dry ice [15, 17]. The result takes one to three weeks [17]. Prior folic acid supplementation can alter interpretation, because unmetabolised folic acid raises total serum folate without raising 5-MTHF and, in excess, may inhibit transport into the CSF [61].

Neuroimaging. MRI and MRS guide the diagnosis and serve as a baseline for follow-up [2, 56]. Computed tomography may reveal calcifications not visible on MRI [40, 53].

Genetic testing. Single-gene testing is rarely useful and is not recommended; GeneReviews proposes a multigene neurodevelopmental, neurometabolic or epilepsy panel that includes FOLR1, or exome sequencing, genome sequencing being able to detect variants outside the coding region [2]. In HFM, by contrast, SLC46A1 sequencing is recommended, preceded by targeted testing for founder variants in patients of Puerto Rican or Japanese ancestry [4]. In SLC19A1-FTD, sequencing of the gene or a panel are the first options, with deletion/duplication analysis if only one variant is found [6].

Tests and markers in HFM. Very low basal serum folate in untreated patients (typically <1.5 nmol/L, against normal values of 10–45 nmol/L in countries with fortification), low red cell folate, usually macrocytic anaemia with neutrophil hypersegmentation (normocytic if there is malnutrition or iron deficiency), leucopenia, thrombocytopenia or pancytopenia in at least 30%, low immunoglobulins in about 25% and a bone marrow with megaloblastic erythropoiesis [4]. After an oral load of 5 mg of folic acid, serum folate barely rises within four hours, whereas in unaffected individuals it reaches at least 200 nmol/L [4]. Basal CSF folate is typically <5 nmol/L and remains well below serum levels even after intramuscular 5-formyl-THF [4]. Elevated CSF homocysteine is the most sensitive indicator of folate deficiency and of insufficient CSF folate [4]. Other inconsistent findings are increased urinary formiminoglutamate, orotic aciduria, increased plasma sarcosine and cystathionine, and low methionine [7].

Other entities. In DHFR deficiency, serum folate, vitamin B12 and ferritin are normal, CSF 5-MTHF and BH4 are low and there is no methylmalonic aciduria [11]; enzyme activity and binding of fluorescein-labelled methotrexate in immortalised lymphoblasts discriminate patients and carriers, but only in a research setting [15]. In MTHFD1 deficiency, total homocysteine is elevated with normal or low methionine, and diagnosis depends on sequencing [7]. In MTHFS deficiency, CSF 5-MTHF is low or low-normal [12].

5.3 Algorithm

5.4 Test limitations

FOLR1 sequencing detects about 99% of known variants, but this proportion refers to the variants described and is not a clinical sensitivity [2]. The exome does not reliably identify single-exon deletions and duplications, which targeted analysis detects better, and does not cover deep intronic variants; the genome detects variants outside the coding region [2]. Apparent homozygosity may result from an undetected deletion on the other allele or from uniparental isodisomy [2]. In HFM, the deep intronic variant c.1166-284T>G requires methods that detect splice variants [4]. Low CSF 5-MTHF identifies a syndrome with many causes, not a gene, and may be falsely low because of inappropriate handling [15]. A normal 5-MTHF does not exclude the diagnosis if the sample was collected on folinic acid [46]. The genetic report should state the gene, the transcript with version (NM_016729.3 for FOLR1), the HGVS nomenclature, the zygosity and the classification according to the criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) [2, 9]. Biallelic variants of uncertain significance, or a pathogenic variant with one of uncertain significance, neither establish nor exclude the diagnosis; parental testing confirms the phase, and the French protocol recommends discussing these cases at a multidisciplinary meeting with a specialist centre [2, 17].

5.5 Prenatal and preimplantation diagnosis

Once both pathogenic variants in the family are known, prenatal diagnosis by amniocentesis or chorionic villus sampling and preimplantation genetic diagnosis are possible; for FOLR1-CFTD and HFM, the aim of prenatal testing may also be to start 5-formyl-THF at birth [2, 4]. In France, requests are discussed at a multidisciplinary prenatal diagnosis centre and, given the severity, are generally accepted for FOLR1 anomalies [17].

6. Differential Diagnosis

6.1 Comparison table

6.2 Main differential

Systemic versus isolated deficiencies. The most useful distinction is between FOLR1-CFTD, in which peripheral folate and the full blood count are normal, and the diseases in which the deficiency is also systemic: HFM, with very low serum folate and megaloblastic anaemia in the first months, and SLC19A1-FTD, with normal serum but low red cell folate [4, 6]. The neurological deficits of FOLR1-CFTD appear later and may not be recognised before the second or third year, unlike HFM and SLC19A1-FTD, in which they manifest in the first months [4]. Saudubray distinguishes DHFR deficiency from FOLR1-CFTD by the megaloblastic anaemia [7]. In the assessment of macrocytic anaemia, nutritional folate deficiency, intestinal disease with malabsorption and vitamin B12 deficiency must be excluded, as they may be primary or aggravating factors [6]; in HFM, phenytoin and valproate must also be excluded [4].

Secondary CFDs. Saudubray recommends distinguishing the primary defect from acquired causes (perinatal asphyxia, central nervous system infection) and genetic causes (Rett syndrome, Kearns-Sayre syndrome, MTHFR deficiency, white matter diseases), which generally have less depletion than receptor deficiency [7]. In Kearns-Sayre syndrome, the reduced CSF/serum folate ratio is part of the major phenotypic expression, alongside progressive external ophthalmoplegia, ptosis, pigmentary retinopathy, conduction block, ataxia, elevated CSF protein and ragged red fibres [27]. The review by Pope and colleagues also includes serine deficiency and pyridoxine-dependent epilepsy among the secondary causes, and drugs, immune activation, toxins and oxidative stress among the acquired causes [24]. In MTHFR deficiency, plasma folate is low without anaemia, and CSF 5-MTHF only became measurable with mefolinate (5-MTHF) and not with folic or folinic acid in three patients [2, 7].

Epileptic encephalopathies and leukodystrophies. FOLR1-CFTD shares features with many developmental and epileptic encephalopathies and with hypomyelinating leukodystrophies; for this reason GeneReviews and Brunetti and colleagues recommend panels or exomes that include FOLR1 [2, 57]. The French protocol recommends excluding structural, infectious, immune and other genetic causes [17]. Neonatal “folinic acid-responsive seizures”, described by Torres in 1999 as seizures in the first hours or days that initially respond to phenobarbital and cease within 24 hours with folinic acid, are the same disease as pyridoxine-dependent epilepsy due to ALDH7A1 [25, 62].

Anti-FRα autoantibodies. Autoantibodies that bind strongly to FRα have been found in children with developmental delay, progressive irritability, sleep disturbances, psychomotor delay and ataxia, with variable and generally only modestly reduced CSF folate [2]. Ramaekers and Quadros consider them the main cause of the CFD syndrome and describe age-dependent pictures, including autism spectrum disorders [28]. Testing for them is only justified when genetic testing is negative or the picture is atypical for a genetic cause.

7. Treatment and Risk Management Strategies

7.1 Principles

Treatment of the genetic CFDs is replacement with a reduced folate that bypasses the defect, with the dose guided by CSF 5-MTHF and by clinical course [2, 4]. There are no controlled clinical trials in any of the entities of the class; the evidence comes from case reports and series, from expert opinion in GeneReviews and the French protocol, and from the systematic literature review that supported the United States label [2, 17, 21]. GeneReviews states that there are no established clinical practice guidelines for FOLR1-CFTD or for HFM [2, 4]. The French protocol is, to date, the only national diagnosis and care document addressing FOLR1-CFTD [17].

7.2 Folate formulations

Folinic acid (5-formyltetrahydrofolate, 5-formyl-THF; calcium folinate in the Portuguese nomenclature; leucovorin) is a racemic mixture with equal amounts of the active L-isomer and the inactive D-isomer [2, 21]. Orally, the L-isomer is metabolised in the intestine by MTHFS and MTHFR to 5-MTHF, which is the main active metabolite in plasma; leucovorin as such is not observed in the CSF [21]. Absorption is saturable: in adults, the apparent bioavailability of the L-isomer is 97% with 25 mg, 75% with 50 mg and 37% with 100 mg, which justifies dividing the daily dose and preferring single doses of up to 25 mg [21]. The plasma half-life is 0.5 to 1.3 hours for the L-isomer and 3 to 7 hours for 5-MTHF, with renal excretion [21].

Levofolinate (levoleucovorin, (6S)-5-formyl-THF) contains only the natural isomer, is the preferred substrate of the enzymes and transporters and is given at half the dose of the racemic form; GeneReviews considers it the preferred folate, but in the United States it exists only in parenteral form, whereas in Europe an oral form also exists [2, 4]. In Portugal, Levofolinato de Cálcio Farmoz is registered as a 10 mg/ml solution for injection [63]. (6S)-5-MTHF (levomefolate) is the physiological folate of blood and exists only in oral formulations; the HFM GeneReviews chapter notes that Metafolin® has strengths of 1 mg or less, impractical in practice, and Deplin® 15 mg tablets [4]. In France, 5-MTHF in 15 mg gastro-resistant tablets is only accessible through compassionate access authorisation, and the protocol warns that its very low stability and bioavailability limit efficacy [17]. The FOLR1-CFTD GeneReviews chapter notes that there is as yet no published clinical experience with 5-MTHF in this disease or in HFM [2].

Folic acid is not recommended: it binds strongly to FRα and may interfere with residual receptor function; there is also emerging evidence that high-dose folic acid may be harmful to the developing nervous system [2, 64]. Akiyama and colleagues documented, in a patient with Kearns-Sayre syndrome, that high-dose folic acid did not normalise CSF 5-MTHF, that the addition of folinic acid did not do so either and that withdrawal of folic acid normalised the CSF [61]. The French protocol classifies folic acid as of little or no efficacy and recommends stating explicitly on the prescription the risk of substituting folic acid for folinic acid, with direct contact between the hospital pharmacy and the community pharmacy [17]. In Saudubray, FOLR1-related CFD is described as responding exclusively to folinic acid and not to folic acid [7].

7.3 FOLR1-CFTD: doses and routes

The dose reference in this article is that of the recommendation sources: the French protocol, which proposes 3–5 mg/kg/day, and GeneReviews, which proposes lower initial doses (1.75–3.5 mg/kg/day) with titration according to CSF levels [2, 17]. The United States label, which serves as a comparison, starts at 1–2 mg/kg/day, below both sources, and sets maxima (8.5 mg/kg/day or 330 mg/day) that no guideline defines; the French protocol dose is within the range authorised in the USA [21]. In the review of 25 orally treated patients with dose data, the initial dose ranged from 0.5 to 3 mg/kg/day, was 2 mg/kg/day in 14, and, among the 20 with escalation, 17 had a maximum of ≤6 mg/kg/day (range 1.7–8.5 mg/kg/day), with no evident relationship to age or severity [21]. In individual reports, Delmelle and colleagues used 5 mg/kg/day orally [66], the Turkish series a mean of 5.2 mg/kg/day [51], Rahma and colleagues 5 mg/kg/day [42] and Potic and colleagues oral levofolinate 2 mg/kg/day escalated up to 7–8 mg/kg/day [39]; Ferreira and colleagues refer to high doses of 2–5 mg/kg/day described as effective [54].

The intravenous route was used when the oral response was insufficient despite normal CSF levels. Delmelle and colleagues treated two sisters with 5 mg/kg/day orally, with CSF 5-MTHF already normal (60.1 nmol/L; normal 53–182) but persistent severe epilepsy and regression in the younger one; a three-day intravenous course (6 mg/kg/day for 24 hours and then 12 mg/kg/day for 48 hours) raised CSF levels to 180 nmol/L and improved the clinical picture and epilepsy, with maintenance by monthly infusions of 20–25 mg/kg and improvement confirmed at two years [66]. Mafi and colleagues, Kobayashi and colleagues and Jaafar and Obeid described responses to high-dose oral folinic acid combined with intravenous or intramuscular administrations [41, 43, 67]. In the Göttingen follow-up, lack of effect prompted escalation to the intravenous and intrathecal routes, and the authors recommend sufficient doses from the outset and considering early intrathecal administration [56]. These approaches derive from case reports, not from controlled comparisons, and GeneReviews classifies improvement with the intravenous route as anecdotal [2].

The aim of treatment is to bring CSF folate into the normal range for age, which the oral route usually achieves when the dose is sufficient [2]. The available observations suggest that CSF levels normalise more easily in FOLR1-CFTD than in HFM [2]. Treatment is lifelong [17].

7.4 Hereditary folate malabsorption: doses and routes

The aim is to prevent, reverse or attenuate the haematological, immunological and neurological deficits and to optimise cognitive development [4]. Systemic correction is easy, but neurological correction is difficult; severe neurological complications and seizures are invariably due to diagnostic delay and to doses insufficient to achieve adequate CSF folate [4]. The main error is to take the rapid correction of anaemia, gastrointestinal manifestations and immunodeficiency with low parenteral doses as a sign of success: these doses do not reach the central nervous system and allow progressive neurocognitive deterioration and seizures [4].

The maximum attainable CSF folate appears to lie between 40 and 50 nmol/L, and normal values for age are unlikely to be reached by any route [4]. Lubout and colleagues achieved normalisation of CSF folate with intramuscular folinic acid, and not with oral folinic acid, in a 1-year-old boy, with resolution of almost all systemic and neurological signs [58]. Torres and colleagues used serial monitoring of CSF 5-MTHF to guide intramuscular treatment, which prevented new signs even with relatively low values [68]. When available and cost is not a limitation, levofolinate is preferable, especially in refractory neurological disease, because it has twice the biological effect of the racemic form at the same dose [4]. Infants diagnosed before symptoms should be treated immediately with sufficient folate to prevent systemic and cerebral deficiency [4].

7.5 Other entities

In SLC19A1-FTD, the aim is a supranormal blood folate level that normalises cellular folate, and even higher levels for the CSF [6]. The proposed initial oral doses are 2.5–5.0 mg/kg/day of racemic 5-formyl-THF or 1.5–3.0 mg/kg/day of 5-MTHF; the dose required for the CSF is unknown and probably much higher, and without the possibility of monitoring the CSF a higher initial dose increases the likelihood of physiological levels [6]. The cousins homozygous for p.(Gly348Arg) improved with folinic acid, and Svaton's patient responded to folic acid, although GeneReviews advises against folic acid because it is a poor RFC substrate and may block the choroid plexus [6, 48, 69]. In DHFR deficiency, folinic acid normalised red cell volume and the bone marrow and improved neurological function, with transient improvement of seizures but no ultimate control in severe cases, and there was no response to folic acid [2, 7]; in the Banka series, folinic acid improved the anaemia and seizures, but CSF BH4 continued to fall [11]. In MTHFD1 deficiency, oral folinic acid normalised homocysteine and the bone marrow and improved neurological function; two patients treated long term had normal development at 8 and 22 years [7]. In MTHFS deficiency, folinic acid and folic acid failed and, in Rodan's first patient, folinic acid worsened the CFD; oral L-5-MTHF with intramuscular methylcobalamin, or 5-MTHF up to 12 mg/day with hydroxocobalamin 5 mg/day, raised 5-MTHF and normalised homocysteine with slight or subjective improvement [12, 15]. The United States label does not recommend leucovorin in MTHFS deficiency [21]. Treatment of MTHFR deficiency is based on betaine and 5-MTHF; Saudubray indicates 15–60 mg/day of oral 5-MTHF, as calcium mefolinate [65].

In secondary CFDs, folinic acid is used as an adjunct. In Kearns-Sayre syndrome, folinic acid 2.5 mg/kg/day enabled a patient who had lost the ability to walk to walk again with an ataxic gait, normalised CSF 5-MTHF within one year and improved myelination [27]. Saudubray describes improvement in seizures and other neurological problems with folinic acid in mitochondrial disease with CFD [70]. In AADC deficiency, folinic acid 10–20 mg/day is recommended, and in DHPR deficiency 15–20 mg/day in one or two doses [49, 50].

7.6 Adverse effects, contraindications and interactions

The United States label contraindicates leucovorin in FOLR1-CFTD in patients with a history of any hypersensitivity reaction to leucovorin, levofolinate, folic acid or a component of the tablet; anaphylaxis and urticaria have been described, and treatment should be interrupted or permanently discontinued according to severity [21]. Post-marketing reactions include pruritus, rash, dyspnoea, chills and temperature changes, and safety data specific to FOLR1-CFTD are limited to case reports [21]. Folinate is contraindicated in pernicious anaemia and other megaloblastic anaemias due to vitamin B12 deficiency, because it can induce haematological remission with progression of the neurological manifestations [17, 20, 21].

Antiepileptic drugs. High-dose folic acid may reduce the effect of phenobarbital, phenytoin and primidone and increase seizures in susceptible children; it is not known whether folinic acid has the same effect, but they share metabolic pathways, and the United States label recommends increased seizure monitoring [21]. The Portuguese SmPC of Lederfoline® mentions the risk of increased seizures with phenobarbital, phenytoin, primidone and succinimides owing to reduced plasma concentrations and recommends clinical monitoring, possibly of levels, and adjustment of the antiepileptic during supplementation and after its discontinuation [20]. The French protocol makes the same recommendation [17]. In HFM, phenytoin and valproate should be avoided, because of their effects on intestinal absorption and on folate metabolism, respectively [4]. In FOLR1-CFTD, GeneReviews warns that some antiepileptic drugs, in particular valproate, interfere with one-carbon metabolism [2, 71]. No antiepileptic drug has shown specific efficacy; with the optimal folinic acid dose, antiepileptic drugs can in some patients be reduced or withdrawn [2, 4]. Papadopoulou and colleagues described a relative response of epileptic spasms to the ketogenic diet, with topiramate and valproate, and further improvement with folinic acid [46]; Akgun and Tas suggest vagus nerve stimulation as an adjunct [51].

Trimethoprim-sulfamethoxazole. Leucovorin reduces the efficacy of trimethoprim-sulfamethoxazole, with more treatment failures and higher mortality in Pneumocystis jirovecii pneumonia in patients with HIV infection, and the label recommends avoiding the combination [21]. In HFM, however, trimethoprim-sulfamethoxazole is the treatment of Pneumocystis pneumonia, and it was used as prophylaxis before starting folate because of reports of infection at the start of therapy [4]. The sequence and the need for folate in these situations require a specialist decision, because the two sources diverge.

Others. Leucovorin increases the toxicity of fluorouracil [20, 21]. In SLC19A1-FTD, methotrexate should not be used to treat cancer or leukaemia, and anionic compounds that inhibit RFC, such as salicylates at sustained therapeutic levels, should be avoided if there is residual function; pemetrexed may retain activity [6]. In HFM, transfusion is rarely necessary and, if there is selective IgA deficiency, washed red cell concentrates should be used [4].

7.7 Supportive treatment

The initial assessment includes neurological examination (gait, hypotonia, tremor, strabismus, nystagmus, seizure type and frequency), assessment of motor, adaptive, cognitive and language development, cognitive testing, screening for behavioural and sleep disturbances from 12 months of age, assessment by physical and rehabilitation medicine, physiotherapy and occupational therapy, genetic counselling and assessment of family support needs [2]. The French protocol adds ear, nose and throat, ophthalmological, neuropsychological, occupational therapy and speech and language therapy assessments, to be repeated to measure benefit [17]. Supportive measures include early intervention, physiotherapy for mobility and prevention of contractures, scoliosis and hip dislocation, baclofen, tizanidine, botulinum toxin or antiparkinsonian drugs for hypertonia or dystonia, occupational therapy, swallowing assessment with nasogastric tube or gastrostomy in severe cases, augmentative and alternative communication and the behavioural interventions used in autism spectrum disorder [2]. In HFM, folate replacement rapidly corrects the immunodeficiency, and developmental and educational services address residual deficits [4].

7.8 Regulatory status

Sources: Drugs@FDA and FDA labels [19, 21, 72]; GeneGuidance for Khapzory® [15]; SmPC and INFARMED search [20, 63, 73]; GeneReviews and French protocol [4, 17]; ANVISA open data [74].

The United States approval followed its own path. In September 2025, the FDA announced that it was working with GSK on revising the leucovorin label for CFD in adults and children, in an announcement publicly associated with autism; the March 2026 approval limited the indication to FOLR1-CFTD, without including autism spectrum disorder, and the agency justified the choice by the quality of the data in that subpopulation [75, 76]. The decision was based on a systematic literature review, without a clinical trial [21, 75]. There is no centrally authorised European Medicines Agency medicine with an indication in the genetic CFDs, and EU folinates are nationally authorised. The French protocol records that, in 2022, folinic acid 5 and 25 mg tablets were available in France, and that compounded folinic acid preparations are reimbursable in paediatric dilutions or in dysphagia [17].

7.9 Clinical evidence and trials

None of these studies has a control group, and all depend on reports with heterogeneous documentation; comparison with the progressive natural history is the basis for inferring efficacy [21]. Negative results are concentrated in patients treated late, which makes it impossible to separate the effect of dose from the effect of age at initiation [39, 57]. A search of ClinicalTrials.gov in November 2026 found no trials targeting FOLR1-CFTD, HFM, DHFR deficiency or SLC19A1-FTD; the registered trials of folinic acid or leucovorin focus on autism spectrum disorder, such as NCT02839915, NCT04060017 and NCT04060030 (phase 2), NCT07630506 (phase 2, recruiting since May 2025, n=184) and NCT07642661 (placebo-controlled phase 2, started in June 2026, n=150), and one of them, NCT01602016, was terminated [77]. These autism trials do not apply to the genetic CFDs.

7.10 Developments in the past 12 months

No gene therapy, gene editing or cell therapy programmes for FOLR1-CFTD or the other genetic CFDs were found in the sources consulted; current treatment is metabolic replacement, and the research objective closest to practice is presymptomatic diagnosis [56, 77].

7.11 Special populations

Pregnancy. There are no adequate data on leucovorin in FOLR1-CFTD in pregnancy, nor lactation data [21]. Reproduction in patients with FOLR1-CFTD is not known to have occurred [2]. In HFM, a woman without PCFT, treated from birth, had two normal pregnancies with an increase in the intramuscular dose when planning pregnancy; women with HFM should be on the maintenance dose well before conception, may use prenatal vitamins containing 5-MTHF instead of folic acid, and their children do not appear to have an increased risk of neural tube defects if maternal intake is adequate [4]. In a pregnancy with a fetus affected by SLC19A1-FTD, 5-MTHF or 5-formyl-THF is recommended instead of folic acid; the folic acid in fortified flours is present in small amounts and is not a concern [6].

Newborns at risk. Newborn siblings should be tested for the familial variants soon after birth, if there was no prenatal testing, and treated immediately if affected [2, 4]. In HFM without known variants, serum and CSF folate and intestinal absorption are measured soon after birth [4].

Adults and older people. There are no data in FOLR1-CFTD above 65 years of age [21]. Adult patients diagnosed late may have fewer seizures and functional improvement [54, 55].

Renal and hepatic impairment. Leucovorin and 5-MTHF concentrations may increase, without clinical studies [21].

Transition. The French protocol recommends a planned transition to adult medicine, with renewed information for the patient, a joint transfer consultation or a detailed letter, neurocognitive reassessment, discussion of contraception, sexuality, genetic counselling, pregnancy and socio-professional integration, and adult follow-up by a neurologist, metabolic specialist and geneticist, with therapeutic education [17].

8. Reproductive Options and Family Counselling

8.1 Recurrence risks

All the genetic CFDs described in this article are autosomal recessive [2, 4, 6]. The parents of an affected individual are presumed to be heterozygous, and molecular confirmation in the parents is recommended to estimate the risk reliably [2]. If both are heterozygous, each sibling has, at conception, a 25% chance of being affected, a 50% chance of being an asymptomatic carrier and a 25% chance of being neither affected nor a carrier [2, 17]. If only one parent has the variant and parentage is confirmed, one of the variants may have arisen de novo in the proband or through a post-zygotic event in a mosaic parent; if the proband appears homozygous, a deletion of one or more exons not detected by sequencing or uniparental isodisomy should be considered [2, 4]. The proband's aunts and uncles have a 50% chance of being carriers [2].

FOLR1 heterozygotes are asymptomatic and are not at risk of developing the disease [2]. SLC46A1 heterozygotes have no clinically apparent folate deficiency; it is not known whether they may have slightly reduced serum folate and haemoglobin or manifestations with concomitant dietary deficiency or malabsorption [4].

8.2 Cascade testing and early diagnosis of siblings

Clarifying the genetic status of all the proband's siblings is recommended in order to identify as early as possible those who have inherited both variants and to start treatment, which can prevent or completely reverse the manifestations; late diagnosis is associated with irreversible neurological changes [2]. Carrier testing in relatives requires prior knowledge of the familial variants [2]. In HFM, newborn siblings and apparently asymptomatic younger siblings should be assessed immediately [4]. When the proband's molecular diagnosis is not confirmed, DNA banking allows future studies [4].

8.3 Prenatal and preimplantation diagnosis and family planning

The optimal time to determine risk and discuss prenatal and preimplantation diagnosis is before pregnancy, and genetic counselling should be offered to young adults who are affected, carriers or at risk of being carriers [2]. Prenatal testing for the familial variants by amniocentesis or chorionic villus sampling allows 5-formyl-THF to be started at birth [2, 4]. In France, prenatal and preimplantation diagnosis is only possible with ACMG class 4 or 5 variants in the proband and the parents, is discussed at a multidisciplinary prenatal diagnosis centre and, given the severity, is generally accepted for FOLR1 anomalies [17]. Views on the use of prenatal and preimplantation diagnosis vary among professionals and families, and the decision is personal [2]. As FOLR1-CFTD is treatable and early treatment has favourable outcomes, counselling should also present the option of immediate neonatal diagnosis with treatment from birth [2, 56].

8.4 Counselling of minors and consanguinity

Most patients with FOLR1-CFTD are children of consanguineous parents, and carrier testing of the partner of a carrier is particularly indicated in that context [2]. In the Turkish series, 88.9% of patients were children of consanguineous couples [51]. Testing of asymptomatic minor siblings is justified by the direct therapeutic benefit, since presymptomatic treatment prevents the disease [2].

9. Special Considerations: Population Diversity and Epidemiology

9.1 Frequency and what each source measures

There is no validated population prevalence estimate for FOLR1-CFTD; the count of more than 35 individuals from 32 families, plus four families known to the GeneReviews author, is a count of published cases and does not allow a rate to be calculated [2, 15]. Orphanet assigns the class <1/1,000,000 and keeps a validated count of three cases or families, outdated relative to the literature [3]. Prevalence is probably higher than known, because the signs are non-specific and because the disease is concentrated in consanguineous families from populations with limited access to molecular testing [2]. Mafi and colleagues cite a prevalence of <1/1,000,000, and Potic and colleagues counted about 30 published patients in 2023 [39, 43]. In the Göttingen network, screening of 72 children with low CSF 5-MTHF and symptoms after infancy identified FOLR1 variants in 10; this figure measures the proportion in a selected cohort and not a prevalence [34]. In an Indian cohort of 29 children with neurotransmitter disorders, seen between 2015 and 2024, three had FOLR1-CFTD [79].

For HFM, Orphanet records 30 cases or families and the class <1/1,000,000; GeneReviews counts 50 published families, all but five with molecular confirmation, plus six individuals, with a prevalence probably much higher because infants may die undiagnosed [4, 13]. For DHFR deficiencies, Orphanet validates six cases or families; for SLC19A1-FTD, five cases; for MTHFD1 deficiency, eight patients [14, 15].

9.2 Founder populations and geographical distribution

FOLR1-CFTD has been described worldwide [2]. In Europe, there are cases in Germany and Italy since the founding article, in Spain, Belgium, France, Greece and Serbia, and a Finnish patient with a concomitant POLG1 variant, with the first cases from South-Eastern Europe in 2023 [32, 39, 43, 44, 46, 66]. In North America there are reports from Canada [54]. Outside Europe, there are cases in Turkey, Iran, Tunisia, Egypt, India, China and Japan [41, 42, 45, 51, 53, 78, 79]. No FOLR1 founder variant was identified in the sources consulted.

In HFM, the SLC46A1 variant c.1082-1G>A is a founder variant in Puerto Rico: in a screening of 1582 newborns from 2005 (3% of births, with regional oversampling), three heterozygotes were identified, with a carrier frequency of 0.2% on the island and 6.3% in Villalba; six Puerto Rican patients with a clinical diagnosis were homozygous, and the variant spread with migration to the continental United States [16]. The deep intronic variant c.1166-284T>G is a possible Japanese founder variant [4]. An infant with HFM described by Zhao and colleagues in 2007 was of Spanish, Brazilian and Mexican origin [5].

No data on patients with genetic CFD in Portugal or Brazil were found in the sources consulted; this absence does not demonstrate that none exist.

9.3 Special populations and access

Dependence on CSF for diagnosis and follow-up limits access in settings without a neurotransmitter laboratory or without family consent for lumbar puncture, as in the Serbian case in which CSF was never collected [39]. Access to exome sequencing conditions the diagnosis; Potic and colleagues made the diagnosis by exome in a research setting and stress the need for universal access to genetic testing for treatable diseases [39]. The availability of formulations also varies: in France, 5-MTHF requires compassionate access; in the United States, oral levofolinate does not exist [2, 17].

9.4 Networks and associations

MetabERN, the European Reference Network for hereditary metabolic disorders, and EpiCARE, the European network for rare and complex epilepsies, are the relevant networks [22, 23]; the French protocol was drawn up by the rare epilepsy reference centres of Lille and Marseille [17]. GeneReviews lists Metabolic Support UK and MedlinePlus as resources [2, 4]. In France there is no specific patient association; the protocol lists the Maladies rares info services forum, Epilepsie France and Feux Follets [17].

10. Illustrative Clinical Case

Potic and colleagues described, in 2023, two Serbian siblings with FOLR1-CFTD treated at different ages [39]. The older brother had normal development until 18 months, followed by progressive ataxic gait and speech regression; epilepsy began at 4 years, drug-resistant, with daily tonic, focal impaired-awareness, atonic and tonic-clonic seizures and frequent status epilepticus, with no response to ten antiepileptic drugs [39]. At 6 and a half years he had lost the ability to walk and sit, had poor head control, up to five words and autistic-type behaviour; at 7 and a half years he did not speak or interact, with axial and limb ataxia, axial hypotonia and mild pyramidal and bulbar signs [39]. MRI at 7 years showed diffuse supratentorial hypomyelination with relative sparing of the internal capsule and splenium, a thin corpus callosum, cerebellar hypomyelination and marked cerebral and cerebellar atrophy; the hypomyelination was stable from 2 to 7 years, whereas the atrophy progressed, and MRS showed reduced choline in the white matter [39]. Serum folate was normal (17.8 ng/mL; normal 3.1–20.5), and CSF was not collected owing to lack of parental consent and of local resources [39]. The younger sister developed normally until 22 months, when mild intention tremor of the upper limbs and mild axial ataxia appeared, without seizures; MRI at 2 years showed myelination insufficient for age, a slightly thin corpus callosum and no atrophy, with low choline on MRS [39].

Exome sequencing, performed in a research setting when the brother was 8 years old, identified the variant c.465_466delinsTG p.(Trp156Gly) in the homozygous state, confirmed by Sanger sequencing, with heterozygous parents [39]. The variant is classified in ClinVar as pathogenic or likely pathogenic, with a description on the MANE transcript NM_016729.3 [38]. Both siblings started oral levofolinate at 2 mg/kg/day [39]. In the boy, with no effect at that dose, it was increased to 5 mg/kg/day in the first month and then progressively up to 8 mg/kg/day; the bulbar signs resolved, weakness and ataxia decreased, independent walking returned within about eight months and seizures fell from dozens per day to zero to three brief seizures [39]. At 12 years he walked and performed simple tasks, retained milder cerebellar signs and hypotonia, the behavioural changes had decreased and expressive language had not improved; he remained on levetiracetam and lamotrigine, because attempts at reduction worsened the seizures [39]. MRI at 10 and 12 years showed incomplete progression of myelination and increased brain volume, and choline normalised [39]. In the sister, the dose was increased up to 7 mg/kg/day (after about three months according to the text, or after 12 months of 2–5 mg/kg/day according to the article's table); the neurological signs resolved within about three months, neurological examination and development were normal at 6 years and MRI, improved at 3 and 4 years, was normal at 6 [39]. The article's figures show the evolution of the brother's T2 MRI at 2, 5, 7, 10 and 12 years (https://pmc.ncbi.nlm.nih.gov/articles/PMC10339608/figure/Fig1/) and of the sister's at 2, 3, 4 and 6 years (https://pmc.ncbi.nlm.nih.gov/articles/PMC10339608/figure/Fig2/) [39].

The case illustrates the difference between treating a child at the onset of signs and treating a disease with six years of evolution, which is the conclusion of the French protocol and of GeneReviews [2, 17]. Two aspects of the report do not correspond to recommended practice: the diagnosis was made without measurement of CSF 5-MTHF, which the sources recommend to confirm CFD and to titrate the dose, and the dose was adjusted on clinical grounds alone [2, 17]. The use of oral levofolinate, available in Europe, corresponds to the formulation that GeneReviews considers preferable [2]. The final doses of 7–8 mg/kg/day of the L-isomer correspond to twice, in racemic form, the maximum doses of the French protocol and exceed the maximum of the United States label for the racemic form, which reinforces that these are approaches from a case report and not routine practice [17, 21].

11. Prognosis and Long-Term Follow-up

11.1 Prognosis

Without treatment, FOLR1-CFTD progresses to severe neurological disability, with immobility and intractable seizures, but compatible with survival into adulthood [2]. The prognosis under treatment depends mainly on age and degree of involvement at initiation: treatment of presymptomatic or mildly symptomatic siblings prevents or reverses the manifestations; treatment started before 3 years of age was the only one that produced complete clinical and radiological recovery; and treatment after more than 13 to 15 years of disease produced no appreciable improvement in several patients [2, 39, 57]. In adult patients diagnosed late, a reduction in seizures and functional improvement are possible [54, 55]. Irregular treatment was associated with deterioration [56], just as irregular folinic acid intake triggered seizures in DHFR deficiency [11]. In HFM, favourable neurological outcomes require adequate treatment soon after birth, and early mortality from infection occurs before diagnosis [4]. In MTHFS deficiency, there was no significant clinical improvement with metabolic treatment, and two of 12 patients died in early childhood [15].

11.2 Treatment monitoring in FOLR1-CFTD

GeneReviews recommends CSF 5-MTHF at diagnosis, repeated two to three weeks after initiation (before the next dose) to confirm the dose, a further measurement if there is a clinical change (delay, regression, new neurological signs, seizures) or doubt about adherence and, if possible, an annual measurement until 5 years of age [2]. The French protocol recommends monitoring CSF 5-MTHF during growth, after therapeutic adjustments, and in adults in the event of deterioration or insufficient response [17]. MRS normalises soon after the start of treatment, whereas magnetisation transfer and diffusion tensor imaging track the gradual recovery of myelin and correlate better with the clinical course, which makes them follow-up biomarkers [56].

11.3 Surveillance table

The CSF monitoring intervals in FOLR1-CFTD (two to three weeks after initiation and annually until 5 years) coincide with those in section 7, and the French protocol adds checks after each dose adjustment [2, 17].

12. Summary and Final Considerations

Cerebral folate deficiency is a biochemical syndrome, defined by low CSF 5-MTHF with normal peripheral folate, which has primary genetic causes, secondary causes and acquired causes [24]. FOLR1-CFTD is the genetic form that comes closest to an isolated CFD: loss of folate receptor α prevents folate transcytosis in the choroid plexus and deprives the periventricular tissue of folate, while blood and bone marrow remain normal [2, 35]. The disease is autosomal recessive and rare, with about 35 published patients, but is probably underdiagnosed [2]. It presents, after a normal or near-normal initial period, with regression, ataxia, tremor, myoclonus, drug-resistant myoclonic and tonic epilepsy and behavioural changes, with hypomyelination, cerebellar atrophy and sometimes calcifications [2, 17].

Diagnosis rests on very low CSF 5-MTHF, collected with pre-analytical care and interpreted with simultaneous plasma and red cell folate, and is confirmed by the identification of biallelic FOLR1 variants on a panel or exome [2, 15]. Treatment is lifelong oral folinic acid, at a dose titrated against CSF levels and clinical response: the French protocol proposes 3–5 mg/kg/day, up to 7 mg/kg/day, with monthly intravenous infusions in refractory cases, and GeneReviews proposes lower initial doses adjusted according to CSF levels; the United States label approved in March 2026 starts at 1–2 mg/kg/day and sets maxima of 8.5 mg/kg/day or 330 mg/day [2, 17, 21]. Folic acid should be avoided, and accidental substitution of folic acid for folinic acid is a real risk at dispensing [2, 17]. Age at initiation of treatment determines the outcome: presymptomatic treatment prevents the disease and treatment before 3 years of age can lead to complete clinical and radiological recovery, whereas treatment after many years of disease has limited results [2, 39, 56]. For this reason, immediate testing of siblings and, in at-risk populations, neonatal diagnosis are measures with direct benefit [2, 56].

The other diseases of the class are distinguished by peripheral folate and the full blood count. HFM causes systemic and cerebral deficiency in the first months, with megaloblastic anaemia, immunodeficiency and seizures, and requires intramuscular folinic acid or levofolinate at doses much higher than those that correct the blood [4]. SLC19A1-FTD reduces red cell folate with normal serum folate, DHFR deficiency combines macrocytic anaemia with CFD and responds to folinic acid but not to folic acid, MTHFD1 deficiency combines megaloblastic anaemia and combined immunodeficiency, and MTHFS deficiency is the exception in which folinic acid is not recommended and 5-MTHF is used [6, 7, 12, 21]. The absence of controlled trials, the heterogeneity of published doses and the dependence on lumbar puncture for titration are the main limitations of the current evidence [2, 21].

13. References

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