Cerebral folate deficiency: A report of two affected siblings

Abstract

Cerebral folate deficiency (CFD) is a rare progressive neurological condition characterized by normal blood folate level and low 5-methyltetrahydrofolate (5-MTHF) levels in the cerebrospinal fluid. Patients present with different neurological findings including hypotonia and microcephaly. Later, patients develop ataxia, seizures, para or quadri-plagia. Herein, we report two siblings; born to consanguineous parents; who had normal neurological development in early childhood. Subsequently they developed drug-resistant seizures, neurological regression, and spastic quadriplegia. After thorough investigations patients had brain MRI which showed abnormal white matter signals and ventricular dilatation, CSF with low 5-MTHF, and whole exome sequencing (WES) revealed a novel homozygous variant in FOLR1 (c.245A > G; p.Tyr82Cys) consistent with the diagnosis of cerebral folate deficiency. They were treated with folinic acid in addition to standard anti-seizure medications. WES aids in reaching CFD diagnosis due to FOLR1 pathogenic variants. These results can be used for future counselling to prevent recurrence in future pregnancies by preimplantation genetic testing prior to implanting the embryo in the uterus. Treatment with folinic acid was shown to improve the neurological symptoms namely reduced the seizures and spasticity.

1. Introduction

Folic acid is one of the B-complex water-soluble vitamins. Folate is absorbed from the gastrointestinal system into the blood circulation by two mechanisms: the reduced folate carrier (RFC1 encoded by the SLC19A1 gene) and the proton coupled folate transporter (PCFT encoded by the SLC46A1 gene) [1]. From the blood, folate is transported via the folate receptor alpha (FRα encoded by the FOLR1 gene) which is expressed on the basal side of the choroid plexus to the blood-brain-barrier by endocytosis and transcytosis into the cerebrospinal fluid (CSF) [2]. Folate has vital roles in the human body, one of which is the role in the central nervous system which includes the synthesis of neurotransmitters and myelin formation [3]. This is accomplished when folic acid is converted to L-methylfolate which can pass the blood-brain barrier and assist in the formation of tetrahydrobiopterin (BH4) which is an essential cofactor for the synthesis of the three monoamine neurotransmitters: serotonin, dopamine and norepinephrine. BH4 in turn activates two enzymes that are essential for these neurotransmitter's synthesis namely tyrosine hydroxylase which functions in the synthesis of dopamine and norepinephrine and tryptophan hydroxylase which functions in the synthesis of serotonin [4].

For over five decades, the deficiency of intake of folic acid in the preconception period have been associated with the development of neural tube defects in the fetus [5]. However the association between folic acid deficiency and neurological sequelae was first described in 1973 [6]. It was followed for the first time to report low CSF folate in 1981 associated with low serum folate [7]. Cerebral folate deficiency (CFD) is a rare progressive neurological condition characterized by normal blood and red blood cell folate level and low 5-methyltetrahydrofolate (5-MTHF) levels in the CSF. Patients present with different neurological findings including hypotonia and microcephaly. Later, patients develop different neurological manifestations including ataxia, seizures, para or quadri-plagia. The first report of CFD was in 2002 were it was investigated in a group of children who presented with normal serum folate level but low 5-methyltetrahydrofolate (5-MTHF) in the CSF. The five reported children had normal development until four to six months of age, followed by deceleration of the head growth, neurological regression, cerebellar ataxia, choreoathetosis, and seizures [8]. Mutations in the FOLR1 gene were identified to cause CFD which is a rare disease that is inherited as autosomal recessive condition. Few cases have been reported in the literature and the prevalence of this condition is not known [9] [10] [11] [12]. Recently, mutations in the capicua (CIC) gene which was shown to dysregulate the folate transport genes have been identified in patients with CFD [13].

In this report we present two siblings with CFD. The clinical description, molecular results, neuroimaging findings, and the response to folinic acid therapy are discussed.

2. Case description

Two siblings born to first cousins couples with uneventful pregnancy. The older sibling, a 15-year-old male, had normal development during the first two years of life. At the age of three years, speech delay was noticed and at the age of four years he developed generalized tonic clonic seizures which was resistant to anti-seizure medications and required multiple hospitalizations. The child progressively lost his motor skills and language abilities, developed spastic quadriplegia, and became bed ridden. Physical examination revealed microcephaly, generalized weakness, and spasticity. Brain MRI showed ventricular system dilatation and abnormal white matter signals (Image 1). At the age of 11 years, he had genetic evaluation and whole exome sequencing revealed a novel homozygous variant in the FOLR1 gene (c.245A > G; p.Tyr82Cys) (Image 2). This novel variant has not been previously reported, however, it affects a highly conserved position, and predicted to be deleterious in various in silico programs including PolyPhen, SIFT, and mutationTaster. The diagnosis of CFD was further confirmed biochemically by identifying low 5-MTHF in CSF of <10 nmol/L (control 40–120 nmol/L). He was started on folinic acid (1.5 mg/kg/day) in 2 divided doses, in addition to Lamotrigine and Clobazam. His seizures became less frequent and a repeat lumbar puncture showed improved in 5-MTHF levels to 26 nmol/L. His folinic acid dose was increased to 3 mg/kg/day. His spasticity slightly improved. At the time of this report, he is bed ridden and has no speech or vocal abilities.

Image 1.

Brain imaging of patient 1 with 1. evidence of ventricular system dilatation and 2. abnormal white matter in the form of high signal intensity in the cortical and subcortical area.

Image 2.

The known FOLR1 variant c.245A > G p.(Tyr82Cys) causes an amino acid change from Tyrosine to Cysteine at position 82 on Exon 4 at chromosome 11.

The younger sister who was 13 years old had normal development during early childhood. She presented at the age of five years with absence seizures and later developed drug-resistant generalized tonic-clonic epilepsy. She progressively lost all her motor and speech abilities and developed spastic quadriplegia. Her physical examination revealed weakness and spasticity in all her limbs. Brain MRI showed thinning of the corpus callosum, dilatation of the ventricular system and cerebellar atrophy (Image 3). At the age of nine years, when her brother was diagnosed with CFD, she had genetic evaluation and target mutation testing revealed the same homozygous FOLR1 gene variant in her brother (Image 2). She underwent lumbar puncture which showed low levels of 5-MTHF in CSF (<10 nmol/L; control 40–120 nmol/L). She was commenced on folinic acid (1 mg/kg/day) in two divided doses. He seizure became more controlled on folinic acid, in addition to Sodium Valproate and Clobazam. Repeat LP showed 5-MTHF to be 51 nmol/L. Her spasticity was reduced and at the time of this report she was able to say <5 words and could be assessed into setting position only, which is an improvement in her status.

Image 3.

MRI brain of patient 2 showing 1. Thinning of corpus callosum 2. Ventricular system dilatation 3. Cerebellar atrophy.

They have three unaffected siblings who were tested and none was found to be homozygous for the FOLR1 variant. Both parents were confirmed to be heterozygous carriers for the FOLR1 variant.

3. Discussion

There are five underlying mechanisms of CFD that have been identified. Firstly, folate transport reduction across the blood-brain barrier into the central nervous system. This can be due to disorders of the FRα which includes autoantibodies against the receptor, functional loss of the FRα due to genetic defects leading to dysregulation of the receptor expression, defects with FRα endocytosis or pathology with cellular membrane as in Smith-Lemli-Opitz syndrome [14]. Secondly, reduction in the intracellular pool leading to reduced folate storage. Another mechanism is through increased usage of folic acid in the brain which could be due to hereditary conditions such as aromatic amino acid decarboxylase deficiency or infectious e.g. reactivated herpes infection or immune mediated conditions like subacute sclerosing pan encephalitis. Also, increased catabolism of folic acid in the nervous system which in turn can be due to oxidative stress or inflammation. Finally, CFD can be due to conditions affecting folic acid metabolism in the brain which can be subdivided into hereditary condition such as enzyme deficiencies e.g. dihydrofolate reductase deficiency or acquired as in hyperhomocysteinemia due to B2,B6, B9, and B12 deficiencies combined [14].

CFD is a progressive neurological disorder that are usually present as early as four months of age up-to three years of age in the infantile form with neurological regression. Symptoms range from sleep disturbance, irritability, visual disturbance and seizures in the form of absence seizure, myoclonic fits and tonic-clonic episodes. Visual and auditory disturbance are followed between three to six years of age. Other clinical presentation include ballism, ataxia and choreoathetosis [8]. Later affected individuals could present with gait disturbance and difficulty in speech [15]. Patients with CFD exhibit different neurological finding on physical examination which include hypotonia, microcephaly, ataxia and gait abnormalities. Radiological investigations namely brain MRI which is the most widely reported shows evidence of hypomyelination and atrophy of different parts of the brain, particularly the white matter (leukodystrophy). Electroencephalography (EEG) when performed may show hypsarrhythmia [16] [17]. Milder forms of CFD between 3 and 5 years of age have been reported with learning difficulties and attention deficit and hyperactivity disorder. On physical examination, these children have ataxic gait [14]. The presence of FRα antibodies during adolescence and adulthood can predispose individuals to refractory schizophrenia and treatment-resistant depression [[18], [19], [20]].

The diagnosis of CFD is established based on low levels of 5-methyletetrahydrofolate (5-MTHF) in the CSF despite normal blood and RBC folate levels [8]. The diagnosis of the genetic inherited form of CFD can be confirmed by identifying biallelic pathogenic variants in the FOLR1 gene. The autoimmune CFD can be diagnosed by testing autoantibodies which are two forms: the blocking antibody and the binding antibody [21].

FOLR1 mutations can result in decreasing protein expression, impairing folate biding, or mistargeting. Nonsense and splice mutations typically result in truncated proteins and failure of protein expression. In contrast, missense mutations usually result in protein mistargeting and impaired folate binding. Therefore, it is expected that the missense variant Tyr82Cys described in this report results in protein mistargeting and/or impaired folate binding leading to the inability of the transport of folate compounds [2].

In addition to FOLR1 gene, other genes have been described in the literature to be associated with CFD. Six rare missense variant of lysin demethylase 6B (KDM6B) have been shown to downregulate protein levels of FOLR1 [22]. Also, de novo stop gain mutation in the capicua (CIC) gene were identified. This CIC nonsense variant (p.R353X) was found to downregulate FOLR1 expression in cell lines which was found also to decrease cellular binding of folic acid in cells on folate binding assays [13].

CFD due to FOLR1 mutation is inherited as autosomal recessive condition were both parents are required to be carriers. Consanguinity poses a specific risk for inherited recessive diseases [23]. For the siblings described here, parents were first degree cousins and they both were confirmed to be carriers of the FOLR1 variant. In addition to confirming the diagnosis, genetic testing can aid in the prevention of recurrence of inherited genetic disease. This is achieved by preimplantation genetic testing (PGT) for the fertilized embryo to select the unaffected one to be transferred to be implanted in the uterus [24]. PGT can be used for inherited conditions to prevent the recurrence of the concerned disease when parents are known to be carriers for these conditions [25] [26].

Individuals affected with CFD are treated with folinic acid which was shown to have better outcome if started early on. Doses starts at <1 mg/kg/day to reach therapeutic dose at 1–3 mg/kg/day which can be administered orally, although intravenous administration of the drug have been reported as well with good clinical response [27] [28] [29] [30]. Treatment with ketogenic diet have also shown to improve the condition especially in patients with drug resistant epilepsy [28].

In summary, we present two siblings diagnosed with CFD after the development of neurological regression and generalized tonic-clonic seizures which were poorly controlled by anti-seizure medications. Both patients showed evidence of neurological improvement and better control of their seizures and the spasticity also improved when treated with folinic acid. The importance of exome sequencing testing has a role through PGT in preventing the recurrence of CFD in the tested embryo.

Authors contribution

Rabah Almahmoud and Ayman Elhattab conceived the research concept. Mohammed Mekki reviewed and commented on the MRI brain, Rabah Almahmoud, and Ayman Elhattab, performed data collection. Finally, the Rabah Almahmoud prepared the first draft and all reviewers reviewed and approved final draft of the manuscript.

Funding

The study was not funded by any grant.

Data availability statement

The entire data is available with the principal investigator (RA).

Declaration of Competing Interest

The authors declare that they have no conflict of interest related to the study.

Data availability

The data that has been used is confidential.