CIC de novo loss of function variants contribute to cerebral folate deficiency by downregulating FOLR1 expression.

Background Cerebral folate deficiency (CFD) syndrome is characterised by a low concentration of 5-methyltetrahydrofolate in cerebrospinal fluid, while folate levels in plasma and red blood cells are in the low normal range. Mutations in several folate pathway genes, including FOLR1 (folate receptor alpha, FRα), DHFR (dihydrofolate reductase) and PCFT (proton coupled folate transporter) have been previously identified in patients with CFD. Methods In an effort to identify causal mutations for CFD, we performed whole exome sequencing analysis on eight CFD trios and identified eight de novo mutations in seven trios. Results Notably, we found a de novo stop gain mutation in the capicua (CIC) gene. Using 48 sporadic CFD samples as a validation cohort, we identified three additional rare variants in CIC that are putatively deleterious mutations. Functional analysis indicates that CIC binds to an octameric sequence in the promoter regions of folate transport genes: FOLR1, PCFT and reduced folate carrier (Slc19A1; RFC1). The CIC nonsense variant (p.R353X) downregulated FOLR1 expression in HeLa cells as well as in the induced pluripotent stem cell (iPSCs) derived from the original CFD proband. Folate binding assay demonstrated that the p.R353X variant decreased cellular binding of folic acid in cells. Conclusion This study indicates that CIC loss of function variants can contribute to the genetic aetiology of CFD through regulating FOLR1 expression. Our study described the first mutations in a non-folate pathway gene that can contribute to the aetiology of CFD.

First case report of cerebral folate deficiency caused by a novel mutation of FOLR1 gene in a Chinese patient.

BACKGROUND: Cerebral folate deficiency (CFD) is a neurological disease, hallmarked by remarkable low concentrations of 5-methyltetrahydrofolic acid (5-MTHF) in cerebrospinal fluid (CSF). The primary causes of CFD include the presence of folate receptor (FR) autoantibodies, defects of FR encoding gene FOLR1, mitochondrial diseases and congenital abnormalities in folate metabolism. CASE PRESENTATION: Here we first present a Chinese male CFD patient whose seizure onset at 2 years old with convulsive status epilepticus. Magnetic Resonance Imaging (MRI) revealed the development of encephalomalacia, laminar necrosis in multiple lobes of the brain and cerebellar atrophy. Whole Exome Sequencing (WES) uncovered a homozygous missense variant of c.524G > T (p.C175F) in FOLR1 gene. Further laboratory tests demonstrated the extremely low level of 5-MTHF in the CSF from this patient, which was attributed to cerebral folate transport deficiency. Following the intravenous and oral treatment of calcium folinate, the concentrations of 5-MTHF in CSF were recovered to the normal range and seizure symptoms were relieved as well. CONCLUSIONS: One novel variation of FOLR1 was firstly identified from a Chinese male patient with tonic-clonic seizures, developmental delay, and ataxia. The WES and laboratory results elucidated the etiology of the symptoms. Clinical outcomes were improved by early diagnosis and proper treatment.

Treatment of Folate Metabolism Abnormalities in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder that currently has no approved medical therapy to address core symptoms or underling pathophysiological processes. Several compounds are under development that address both underlying pathophysiological abnormalities and core ASD symptoms. This article reviews one of these treatments, d,l-leucovorin calcium (also known as folinic acid) for treatment of folate pathway abnormalities in children with ASD. Folate is a water-soluble B vitamin that is essential for normal neurodevelopment and abnormalities in the folate and related pathways have been identified in children with ASD. One of these abnormalities involves a partial blockage in the ability of folate to be transported into the brain utilizing the primary transport mechanism, the folate receptor alpha. Autoantibodies which interfere with the function of the folate receptor alpha called folate receptor alpha autoantibodies have been identified in 58%-76% of children with ASD and independent studies have demonstrated that blood titers of these autoantibodies correlate with folate levels in the cerebrospinal fluid. Most significantly, case-series, open-label, and single and double-blind placebo-controlled studies suggest that d,l-leucovorin, a reduced folate that can bypass the blockage at the folate receptor alpha by using the reduced folate carrier, an alternate pathway, can substantially improve particular symptoms in children with ASD, especially those positive for folate receptor alpha autoantibodies. This article reviews the current evidence for treating core and associated symptoms and underlying pathophysiological mechanisms in children with ASD with d,l-leucovorin.

Cerebral folate deficiency: Analytical tests and differential diagnosis.

Cerebral folate deficiency is typically defined as a deficiency of the major folate species 5-methyltetrahydrofolate in the cerebrospinal fluid (CSF) in the presence of normal peripheral total folate levels. However, it should be noted that cerebral folate deficiency is also often used to describe conditions where CSF 5-MTHF is low, in the presence of low or undefined peripheral folate levels. Known defects of folate transport are deficiency of the proton coupled folate transporter, associated with systemic as well as cerebral folate deficiency, and deficiency of the folate receptor alpha, leading to an isolated cerebral folate deficiency associated with intractable seizures, developmental delay and/or regression, progressive ataxia and choreoathetoid movement disorders. Inborn errors of folate metabolism include deficiencies of the enzymes methylenetetrahydrofolate reductase, dihydrofolate reductase and 5,10-methenyltetrahydrofolate synthetase. Cerebral folate deficiency is potentially a treatable condition and so prompt recognition of these inborn errors and initiation of appropriate therapy is of paramount importance. Secondary cerebral folate deficiency may be observed in other inherited metabolic diseases, including disorders of the mitochondrial oxidative phosphorylation system, serine deficiency, and pyridoxine dependent epilepsy. Other secondary causes of cerebral folate deficiency include the effects of drugs, immune response activation, toxic insults and oxidative stress. This review describes the absorption, transport and metabolism of folate within the body; analytical methods to measure folate species in blood, plasma and CSF; inherited and acquired causes of cerebral folate deficiency; and possible treatment options in those patients found to have cerebral folate deficiency.

Cerebral folate deficiency in adults: A heterogeneous potentially treatable condition.

OBJECTIVE: To describe the phenotype and the response to folinic acid supplementation of cerebral folate deficiency (CFD) in adults, a disorder diagnosed on low 5-methyltetrahydro-folate (5MTHF) in cerebrospinal fluid (CSF), which can correspond to a inherited disorder of folate metabolism (IDFM) or to a metabolic consequence of various neurological diseases. METHODS: We conducted a retrospective study on 224 adult patients with neurological symptoms who had a 5MTHF CSF dosage, collecting their neurologic and neuroimaging data. RESULTS: 69 patients had CFD (CSF 5MTHF level < 41 nmol/L), 25 of them had severe CFD (sCFD; ≤25 nmol/L) with adult onset neurological symptoms in 41%. 56% of sCFD patients had an underlying identified neurologic disorder, mainly mitochondrial diseases, hepatic encephalopathy and primary brain calcifications (no identified IDFM), the others were classified as undiagnosed. sCFD patients presented most frequently pyramidal syndrome (75%), movement disorders (56%), cerebellar syndrome (50%) and intellectual disability (46%). MRI findings mostly showed white matter abnormalities (WMA; 32%) and calcifications (12%), and were normal in 23%. The clinico-radiological phenotype of sCFD patients was not clearly different from non CFD patients in terms of manifestations frequency. However, their neurological picture was more complex with a higher number of combined neurological symptoms (4.7±1.6 vs 3.4±1.7, p = .01). In Magnetic Resonance Spectroscopy (MRS), Choline/Creatine (Cho/Cr) ratio was lower in sCFD patients (n = 7) compared to non-CFD patients (n = 73) (p = .005), with good sensitivity (71%) and excellent specificity (92%). Among twenty-one CFD patients treated with folinic acid, nine had a sustained improvement, all with sCFD but one (50% of sCFD patients improved). In two undiagnosed patients with extremely low 5MTHF CSF values, MRI WMA and low Cho/Cr ratios, folinic acid treatment leaded to a dramatic clinical and radiological improvement. CONCLUSION: CSF 5MTHF dosage should be considered in patients with mitochondrial diseases, primary brain calcifications and unexplained complex neurological disorders especially if associated with WMA, since folinic acid supplementation in patients with sCFD is frequently efficient.

Genetic assessment and folate receptor autoantibodies in infantile-onset cerebral folate deficiency (CFD) syndrome.

INTRODUCTION: Cerebral folate deficiency (CFD) syndromes are defined as neuro-psychiatric conditions with low CSF folate and attributed to different causes such as autoantibodies against the folate receptor-alpha (FR) protein that can block folate transport across the choroid plexus, FOLR1 gene mutations or mitochondrial disorders. High-dose folinic acid treatment restores many neurologic deficits. STUDY AIMS AND METHODS: Among 36 patients from 33 families the infantile-onset CFD syndrome was diagnosed based on typical clinical features and low CSF folate. All parents were healthy. Three families had 2 affected siblings, while parents from 4 families were first cousins. We analysed serum FR autoantibodies and the FOLR1 and FOLR2 genes. Among three consanguineous families homozygosity mapping attempted to identify a monogenetic cause. Whole exome sequencing (WES) was performed in the fourth consanguineous family, where two siblings also suffered from polyneuropathy as an atypical finding. RESULTS: Boys (72%) outnumbered girls (28%). Most patients (89%) had serum FR autoantibodies fluctuating over 5-6 weeks. Two children had a genetic FOLR1 variant without pathological significance. Homozygosity mapping failed to detect a single autosomal recessive gene. WES revealed an autosomal recessive polynucleotide kinase 3´phosphatase (PNKP) gene abnormality in the siblings with polyneuropathy. DISCUSSION: Infantile-onset CFD was characterized by serum FR autoantibodies as its predominant pathology whereas pathogenic FOLR1 gene mutations were absent. Homozygosity mapping excluded autosomal recessive inheritance of any single responsible gene. WES in one consanguineous family identified a PNKP gene abnormality that explained the polyneuropathy and also its contribution to the infantile CFD syndrome because the PNKP gene plays a dual role in both neurodevelopment and immune-regulatory function. Further research for candidate genes predisposing to FRα-autoimmunity is suggested to include X-chromosomal and non-coding DNA regions.

The proton-coupled folate transporter (PCFT-SLC46A1) and the syndrome of systemic and cerebral folate deficiency of infancy: Hereditary folate malabsorption.

The proton-coupled folate transporter (PCFT-SLC46A1) is the mechanism by which folates are absorbed across the brush-border membrane of the small intestine. The transporter is also expressed in the choroid plexus and is required for transport of folates into the cerebrospinal fluid. Loss of PCFT function, as occurs in the autosomal recessive disorder "hereditary folate malabsorption" (HFM), results in a syndrome characterized by severe systemic and cerebral folate deficiency. Folate-receptor alpha (FRα) is expressed in the choroid plexus, and loss of function of this protein, as also occurs in an autosomal recessive disorder, results solely in "cerebral folate deficiency" (CFD), the designation for this disorder. This paper reviews the current understanding of the functional and structural properties and regulation of PCFT, an electrogenic proton symporter, and contrasts PCFT properties with those of the reduced folate carrier (RFC), an organic anion antiporter, that is the major route of folate transport to systemic tissues. The clinical characteristics of HFM and its treatment, based upon the thirty-seven known cases with the clinical syndrome, of which thirty have been verified by genotype, are presented. The ways in which PCFT and FRα might interact at the level of the choroid plexus such that each is required for folate transport from blood to cerebrospinal fluid are considered along with the different clinical presentations of HFM and CFD.

Neurometabolic Disorders: Potentially Treatable Abnormalities in Patients With Treatment-Refractory Depression and Suicidal Behavior.

OBJECTIVE: Treatment-refractory depression is a devastating condition with significant morbidity, mortality, and societal cost. At least 15% of cases of major depressive disorder remain refractory to treatment. The authors previously identified a young adult with treatment-refractory depression and multiple suicide attempts with an associated severe deficiency of CSF tetrahydrobiopterin, a critical cofactor for monoamine neurotransmitter synthesis. Treatment with sapropterin, a tetrahydrobiopterin analogue, led to dramatic and long-lasting remission of depression. This sentinel case led the authors to hypothesize that the incidence of metabolic abnormalities contributing to treatment-refractory depression is underrecognized. METHOD: The authors conducted a case-control, targeted, metabolomic evaluation of 33 adolescent and young adult patients with well-characterized histories of treatment-refractory depression (at least three maximum-dose, adequate-duration medication treatments), and 16 healthy comparison subjects. Plasma, urine, and CSF metabolic profiling were performed by coupled gas chromatography/mass spectrometry and high-performance liquid chromatography electrospray ionization tandem mass spectrometry. RESULTS: CSF metabolite abnormalities were identified in 21 of the 33 participants with treatment-refractory depression. Cerebral folate deficiency (N=12) was most common, with normal serum folate levels and low CSF 5-methyltetrahydrofolate (5-MTHF) levels. All patients with cerebral folate deficiency, including one with low CSF levels of 5-MTHF and tetrahydrobiopterin intermediates, showed improvement in depression symptom inventories after treatment with folinic acid; the patient with low tetrahydrobiopterin also received sapropterin. None of the healthy comparison subjects had a metabolite abnormality. CONCLUSIONS: Examination of metabolic disorders in treatment-refractory depression identified an unexpectedly large proportion of patients with potentially treatable abnormalities. The etiology of these abnormalities remains to be determined.

Total folate and 5-methyltetrahydrofolate in the cerebrospinal fluid of children: correlation and reference values.

BACKGROUND: Cerebral folate deficiency (CFD) may be underdiagnosed, as it manifests with various non-specific neurological symptoms. The diagnosis of CFD requires a determination of 5-methyltetrahydrofolate (5MTHF) in the cerebrospinal fluid (CSF), which is available in a limited number of specialized laboratories. In clinical biochemistry laboratories, total folate (TF) determination in serum or plasma is routinely performed by automated analyzers. The aim of this study is to determine whether the automated assay of CSF TF is a helpful screening tool for CFD. METHODS: We analyzed CSF samples collected from 73 pediatric patients. We measured CSF TF, serum TF, and CSF 5MTHF in 73, 70, and 48 patients, respectively. The assay of 5MTHF was conducted by a newly developed system utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). We investigated the correlation between TF and 5MTHF in the CSF. RESULTS: There was a strong positive correlation between CSF TF and 5MTHF (ρ=0.930, p<0.0001, n=48). Age was negatively correlated with CSF TF (ρ=-0.557, p<0.0001, n=51), serum TF (ρ=-0.457, p=0.0008, n=51), and CSF 5MTHF (ρ=-0.387, p=0.0263, n=33), but not with the CSF/serum TF ratio. CONCLUSIONS: The automated assay of CSF TF is helpful to estimate CSF 5MTHF. The CSF TF assay may have a significant impact on the early diagnosis of CFD, because clinicians have better access to it than the 5MTHF assay.

Diagnosis and management of cerebral folate deficiency. A form of folinic acid-responsive seizures.

Folinic acid-responsive seizures (FARS) are a rare treatable cause of neonatal epilepsy. They have characteristic peaks on CSF monoamine metabolite analysis, and have mutations in the ALDH7A1 gene, characteristically found in pyridoxine-dependent epilepsy. There are case reports of patients presenting with seizures at a later age, and with folate deficiency due to different mechanisms with variable response to folinic acid supplementation. Here, we report 2 siblings who presented with global developmental delay and intractable seizures who responded clinically to folinic acid therapy. Their work-up included metabolic and genetic testing. The DNA sequencing was carried out for the ALDH7A1 gene, and the folate receptor 1 (FOLR1) gene. They had very low 5-methyltetrahydrofolate (5-MTHF) in CSF with no systemic folate deficiency and no characteristic peaks on neurotransmitter metabolite chromatogram. A novel mutation in the FOLR1 gene was found. The mutation in this gene is shown to affect CSF folate transport leading to cerebral folate deficiency. The response to treatment with folinic acid was dramatic with improvement in social interaction, mobility, and complete seizure control. We should consider the possibility of this treatable condition in appropriate clinical circumstances early, as diagnosis with favorable outcome depends on the specialized tests.

Genetic causes of cerebral folate deficiency: clinical, biochemical and therapeutic aspects.

Over the past decade, a syndrome consisting of low folate values in the cerebrospinal fluid (CSF) has been described. The syndrome has been associated with both genetic and acquired conditions that affect folate transport and metabolism and can result in severe neurological disorders. There is a wide range of underlying pathophysiological mechanisms, but a common feature in most patients is a good clinical response to folate therapy, especially when the syndrome is diagnosed early. In this review, we focus our attention on the genetic diseases leading to profound cerebral folate deficiency (CFD) and review current clinical, metabolic and therapeutic approaches.

Cerebral folate deficiency syndromes in childhood: clinical, analytical, and etiologic aspects.

BACKGROUND: Cerebral folate deficiency may be amenable to therapeutic supplementation. Diverse metabolic pathways and unrelated processes can lead to cerebrospinal fluid 5-methyltetrahydrofolate (5-MTHF) depletion, the hallmark of cerebral folate deficiency. OBJECTIVE: To analyze cerebral folate abundance in a large prospective series of children diagnosed with any neurologic disorder for which a diagnostic lumbar puncture was indicated. DESIGN: We studied the spectrum and frequency of disorders associated with cerebral folate deficiency by measuring cerebrospinal fluid 5-MTHF, biogenic amines, and pterins. Direct sequencing of the FOLR1 transporter gene was also performed in some patients. SETTING: Academic pediatric medical center. PARTICIPANTS: We studied 134 individuals free of neurometabolic disease and 584 patients with any of several diseases of the central nervous system. RESULTS: Of 584 patients, 71 (12%) exhibited 5-MTHF deficiency. Mild to moderate deficiency (n = 63; range, 19-63 nmol/L) was associated with perinatal asphyxia, central nervous system infection, or diseases of probable genetic origin (inborn errors of metabolism, white matter disorders, Rett syndrome, or epileptic encephalopathies). Severe 5-MTHF depletion (n = 8; range, 0.6-13 nmol/L) was detected in severe MTHF reductase deficiency, Kearns-Sayre syndrome, biotin-responsive striatal necrosis, acute necrotizing encephalitis of Hurst, and FOLR1 defect. A strong correlation was observed between cerebrospinal fluid and plasma folate levels in cerebral folate deficiency. CONCLUSIONS: Of the 2 main forms of cerebral folate deficiency identified, mild to moderate 5-MTHF deficiency was most commonly associated with disorders bearing no primary relation to folate metabolism, whereas profound 5-MTHF depletion was associated with specific mitochondrial disorders, metabolic and transporter defects, or cerebral degenerations. The results suggest that 5-MTHF can serve either as the hallmark of inborn disorders of folate transport and metabolism or, more frequently, as an indicator of neurologic dysfunction.

Folate analysis for the differential diagnosis of profound cerebrospinal fluid folate deficiency.

OBJECTIVE: To evaluate the automated determination of total cerebrospinal fluid (CSF) folates for the diagnosis of cerebral folate deficiency. METHOD: CSF and serum samples were analyzed in 60 children with different neurological disorders. RESULT: In all patients with genetic conditions leading to profound cerebral folate deficiency (impaired folate transport and metabolism), the automated folate determination showed altered values. CONCLUSION: CSF folate quantification provided profound CSF folate deficiency diagnosis caused either by folate transport or metabolism deficiencies.

Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency.

Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency. We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid. The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids. Moreover, this relationship provides insight into the role of folates in neurological conditions, including depression, Alzheimer disease, and Parkinson disease.

Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease.

The importance of intracellular folate metabolism is illustrated by the severity of symptoms and complications caused by inborn disorders of folate metabolism or by folate deficiency. We examined three children of healthy, distantly related parents presenting with megaloblastic anemia and cerebral folate deficiency causing neurologic disease with atypical childhood absence epilepsy. Genome-wide homozygosity mapping revealed a candidate region on chromosome 5 including the dihydrofolate reductase (DHFR) locus. DHFR sequencing revealed a homozygous DHFR mutation, c.458A>T (p.Asp153Val), in all siblings. The patients' folate profile in red blood cells (RBC), plasma, and cerebrospinal fluid (CSF), analyzed by liquid chromatography tandem mass spectrometry, was compatible with DHFR deficiency. DHFR activity and fluorescein-labeled methotrexate (FMTX) binding were severely reduced in EBV-immortalized lymphoblastoid cells of all patients. Heterozygous cells displayed intermediate DHFR activity and FMTX binding. RT-PCR of DHFR mRNA revealed no differences between wild-type and DHFR mutation-carrying cells, whereas protein expression was reduced in cells with the DHFR mutation. Treatment with folinic acid resulted in the resolution of hematological abnormalities, normalization of CSF folate levels, and improvement of neurological symptoms. In conclusion, the homozygous DHFR mutation p.Asp153Val causes DHFR deficiency and leads to a complex hematological and neurological disease that can be successfully treated with folinic acid. DHFR is necessary for maintaining sufficient CSF and RBC folate levels, even in the presence of adequate nutritional folate supply and normal plasma folate.

Cerebral folate deficiency.

Cerebral folate deficiency (CFD) is defined as any neurological syndrome associated with a low cerebrospinal fluid (CSF) concentration of 5-methyltetrahydrofolate (5MTHF) in the presence of normal peripheral folate status. CFD has a wide clinical presentation, with reported signs and symptoms generally beginning at around 4 months of age with irritability and sleep disturbances. These can be followed by psychomotor retardation, dyskinesia, cerebellar ataxia and spastic diplegia. Other signs may include deceleration of head growth, visual disturbances and sensorineural hearing loss. Identification of CFD is achieved by determining 5MTHF concentration in CSF. Once identified, CFD can in many cases be treated by administering oral folinic acid. Supplementation with folic acid is contraindicated and, if used, may exacerbate the CSF 5MTHF deficiency. Generation of autoantibodies against the folate receptor required to transport 5MTHF into CSF and mutations in the folate receptor 1 (FOLR1) gene have been reported to be causes of CFD. However, other mechanisms are probably also involved, as CFD has been reported in Aicardi-Goutiere's and Rett syndromes and in mitochondriopathies. Several metabolic conditions and a number of widely used drugs can also lead to a decrease in the concentration of CSF 5MTHF, and these should be considered in the differential diagnosis if a low concentration of 5MTHF is found following CSF analysis.

Cerebral folate deficiency.

Cerebral folate deficiency (CFD) is associated with low levels of 5-methyltetrahydrofolate in the cerebrospinal fluid (CSF) with normal folate levels in the plasma and red blood cells. The onset of symptoms caused by the deficiency of folates in the brain is at around 4 to 6 months of age. This is followed by delayed development, with deceleration of head growth, hypotonia, and ataxia, followed in one-third of children by dyskinesias (choreo-athetosis, hemiballismus), spasticity, speech difficulties, and epilepsy. The low level of 5-methyltetrahydrofolate in the CSF can result from decreased transport across the blood-brain barrier, which is most probably because of the blocking of folate transport into the CSF by the binding of folate receptor antibodies to the folate receptors in the choroid plexus. Treatment of the condition with folinic acid for prolonged periods can result in significant improvement of clinical symptoms and a return of 5-methyltetrahydrofolate levels in the CSF to normal. In view of this response to treatment in CFD and allied conditions, a case can be made for screening the CSF of patients with neurological disorders of unknown origin.