FOLR1 Gene Variation With Adult-Onset Cerebral Folate Deficiency and Stable Clinical and MRI Features up to 2 Years

Abstract

Objectives
The objective of this case report was to describe the first report of FOLR1 variants associated with adult-onset paucisymptomatic leukoencephalopathy associated with cerebral folate deficiency (CFD).

Methods
Considering the patient's symptoms, a nonprogressive leukoencephalopathy was suspected. CSF 5-methyltetrahydrofolate levels were low (10 nmol/L, normal range 41–117). With no other identifiable causes, a genetic analysis was conducted, revealing a compound heterozygous FOLR1 variation (c.45G>T and c. 493+2T>C).

Results
A 47-year-old man with a history of drug and alcohol abuse was admitted to the hospital for double vision and postural instability. MRI of the brain was performed, which showed bilateral leukoencephalopathy. Diffusion tensor imaging revealed a diffuse reduction in fractional anisotropy, suggesting microstructural changes. MRI of the brain and overall clinical picture were stable on subsequent serial examinations.

Discussion
Scientific evidence supports the deleterious effect of c.45G>T and c.493+2T>C variations on the folate receptor-α (FRα) protein structure and function. The weakness of the expression and function of FRα without elimination of its function caused by specific compound heterozygous variations may explain the atypical features observed in our patient. Although rare, CFD should be considered in paucisymptomatic adult patients with stable diffuse MRI white matter changes.

Full text

NEUROIMAGE OPEN ACCESS
FOLR1 Gene Variation With Adult-Onset Cerebral
Folate Deficiency and Stable Clinical and MRI Features
up to 2 Years
Carlo Manco, MD, Rosa Cortese, MD, PhD, Manfredi Alberti, Dr, Silvia Bianchi, PhD, Lucia Monti, MD,
Nicola De Stefano, MD, PhD, and Carla Battisti, MD, PhD
Neurol Genet 2023;9:e200104. doi:10.1212/NXG.0000000000200104
Correspondence
Dr. Cortese
rosa.cortese@unisi.it
Abstract
Objectives
The objective of this case report was to describe the first report of FOLR1 variants associated
with adult-onset paucisymptomatic leukoencephalopathy associated with cerebral folate de-
ficiency (CFD).
Methods
Considering the patient’s symptoms, a nonprogressive leukoencephalopathy was suspected.
CSF 5-methyltetrahydrofolate levels were low (10 nmol/L, normal range 41–117). With no
other identifiable causes, a genetic analysis was conducted, revealing a compound heterozygous
FOLR1 variation (c.45G>T and c. 493+2T>C).
Results
A 47-year-old man with a history of drug and alcohol abuse was admitted to the hospital for
double vision and postural instability. MRI of the brain was performed, which showed bilateral
leukoencephalopathy. Diffusion tensor imaging revealed a diffuse reduction in fractional an-
isotropy, suggesting microstructural changes. MRI of the brain and overall clinical picture were
stable on subsequent serial examinations.
Discussion
Scientific evidence supports the deleterious effect of c.45G>T and c.493+2T>C variations on
the folate receptor-α(FRα) protein structure and function. The weakness of the expression and
function of FRαwithout elimination of its function caused by specific compound heterozygous
variations may explain the atypical features observed in our patient. Although rare, CFD should
be considered in paucisymptomatic adult patients with stable diffuse MRI white matter changes.
Introduction
FOLR1 gene variations are commonly associated with cerebral folate deficiency (CFD), a rare
neurologic syndrome characterized by low CSF concentration of 5-methyltetrahydrofolate (5-
MTHF) despite normal peripheral folate metabolism.
1
CFD typically manifests in early infancy
with symptoms, such as irritability, sleep disturbances, and subsequently progresses to severe
epilepsy, cerebellar ataxia, and psychomotor retardation.
1
MRI of the brain usually shows
diffuse, leukodystrophy-like, white matter changes. In children, treatment with oral calcium
folinate or folinic acid has shown improvement in clinical symptoms, as well as MRI and EEG
abnormalities.
1
To date, clinical and imaging features associated with adult-onset CFD and
FOLR1 gene variations have not been described.
From the Centre for Precision and Translational Medici ne (C.M., R.C., S.B., N.D.S., C.B.), Department of Medici ne, Surgery and Neuroscience, University of Siena; Ne urology Unit
(M.A.), Department of Neurology and Huma n Movement Sciences, University Hospital of Siena; Department of Medica l, Surgical and Neurological Science (M.A.), University of Siena;
and Diagnostic and Functional Neuroimaging Unit (L.M.), Department of Neurology and Huma n Movement Sciences, University Hospit al of Siena, Italy.
Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article.
The Article Processing Charge was funded by the authors.
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivativesLicense 4.0 (CC BY-NC-ND), which permits downloading
and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1

Methods and Results
We report on a 47-year-old man who presented at the emer-
gency department with a four-day history of slight double vi-
sion and postural instability. Inhis medical history, he reported
psoriasis, drug abuse up to age 21 years and alcohol addiction
for many years. The neurologic examination showed walking
ataxia and vertical diplopia in the left/upper left gaze position. A
first brain MRI showed diffuse, bilateral, and symmetric
supratentorial hyperintensity on FLAIR images, without en-
hancement after gadolinium injection (Figure, A). MRI of the
spinal cord was unremarkable. Diffusion-tensor imaging (DTI)
was also acquired and showed a lower fractional anisotropy in
both abnormal and normal-appearing white matter in our pa-
tient (Figure, C) when compared with the same brain regions
of a sex-matched and age-matched healthy control (Figure, D).
During the hospitalization, the patient was tested for blood
and urine biochemical routine, autoimmunity screening, the
dosage of folate and lysosomal enzymes, β-galactocere-
brosidase, pyruvate and lactate, and the assay of serum heavy
metal: All measures were within normal ranges.
CSF analyses revealed a mild increase in protein levels
(51,70 mg/dL, n.v. 20–40) with normal cell count. At 2 yearsof
follow-up, the neurologic examination and brain MRI
performed were stable (Figure, B) than a nonprogressive leu-
koencephalopathy was hypothesized.
2
A further CSF analysis
showed reduced 5-MTHF levels (10 nmol/L, normal values:
41–117), and genetic testing with next-generation sequencing
of selected genes revealed the heterozygous variation of the
FOLR1 double gene (c.45G>T and c.493+2T>C); therefore,
CFD was diagnosed. The patient’s father, mother, and 2
daughters were also assessed. All were symptom-free, had
normal neurological examination, and unremarkable blood
tests. MRI results showed no abnormalities. The genetic
analysis was performed also in the asymptomatic father,
mother, and 2 daughters, with the father being a carrier of the
variant c.45G > T and 2 daughters and mother being carriers of
the c.493+2T>C variation. Written informed consent was
obtained from the patient for the case presentation.
Discussion
FOLR1 gene variations are associated with CFD, an autoso-
mal recessive disorder characterized by low CSF 5-MTHF
concentrations, normal plasma folate values, and late infantile
onset with severe developmental regression, epilepsy, and
leukodystrophy.
3,4
Folic acid and folates are essential for
neurodevelopment and myelin formation
5,6
after intestinal
Figure MRI FLAIR Axial Images Performed at Symptoms Onset and 2 Years Later and Correspondent Baseline Diffusion
Tensor Imaging (DTI) Analysis Comparing the Patient With a Sex-Matched and Age-Matched Healthy Control (HC)
Subject
MRI of the brain showed diffuse, bilateral, and symmetric supratentorial hyperintensities on FLAIR images, without enhancement after gadolinium injection
(A) stable 2 years later (B). DTI revealed a diffuse decrease in fractional anisotropy in the CFD patient (C) when compared with a HC (D), suggesting a
widespread microstructural damage beyond the lesional tissue.
Glossary
5-MTHF = 5-methyltetrahydrofolate; CFD = cerebral folate deficiency; DTI =diffusion tensor imaging; FRα= folate receptor-α.
2Neurology: Genetics | Volume 9, Number 6 | December 2023 Neurology.org/NG

absorption and are reduced, methylated, and enters the cir-
culation as 5-MTHF. The FOLR1 gene encodes for the folate
receptor-α(FRα), which is essential in allowing 5-MTHF to
cross the blood-brain barrier.
7
Our patient is the first reported case in the literature of a
compound heterozygous variation of FOLR1 associated with
adult-onset leukoencephalopathy and clinical paucisympto-
matic picture. Based on the available data, patient’s conditions
have remained stable over time in the absence of treatment.
Despite the mild clinical involvement, MRI changes were
diffuse and DTI analysis showed the presence of diffuse mi-
crostructural changes beyond lesional white matter.
From a genetic standpoint, in our patient, we identified 2
genetic variations. The first variation is the c.45G>T variation,
which is categorized as a missense variation. The second
variation is the c.493+2T>C variation, affecting the donor site
of the splice. In the ClinVar database, the c.45G>T variation
has been reported with supporting evidence indicating its
deleterious effects on protein structure and function, as sug-
gested by silicon analysis. In addition, Grapp et al. conducted a
study using FRαexpression model cell systems and fibroblasts
from a cohort of patients with missense variations in the
FOLR1 gene. Their findings revealed that although FRαwas
expressed, it was not properly localized to the cell membrane.
Instead, it was misdirected to various intracellular compart-
ments, leading to a reduction in folic acid binding, thus
compromising its primary target.
7
The c.493+2T>C variation, despite having a total frequency
of 0.3123% in the general population according to the ge-
nome aggregation database, could potentially lead to the loss
of protein function and contribute to the development of the
disease. However, conflicting data regarding the true patho-
genicity of this variation have been reported in the
literature.
8,9
Prediction software for splice alterations, such as
BDGP and ESEfinder, suggests that the c.493+2T>C varia-
tion may disrupt or weaken the native splice donor site.
Therefore, one possible explanation for the atypical features
observed in our patient could be that the presence of these specific
compound heterozygous variations weakens the expression and
function of FRαwithout completely eliminating its function. This
partial impairment could result in a milder phenotype with late-
onset symptoms and a relatively limited number of symptoms
(pauci-symptomatic phenotype). The variations may cause a
blurred picture of the disease presentation, deviating from the
typical pattern seen in cases with a complete loss of function. This
phenomenon highlights the complexity of genotype-phenotype
correlations and suggests that variations in the degree of protein
impairment can lead to diverse clinical manifestations.
In conclusion, this clinical case is an example of how variations in
the FOLR1 gene can be present in adults with limited clinical
findings and a stable disease course. Thus, it highlights the im-
portance of considering the diagnosis of CFD in adult patients
presenting with neurologic symptoms and exhibiting a leukodys-
trophy pattern on MRI. This case underscores the need for in-
creased awareness and recognition of CFD in the adult population
to ensure appropriate diagnosis and management of this condition.
Study Funding
The authors report no targeted funding.
Disclosure
The authors report no relevant disclosures. Go to Neurology.
org/NG for full disclosures.
Publication History
Received by Neurology: Genetics July 21, 2023. Accepted in final form
September 1, 2023. Submitted and externally peer reviewed. The
handling editor was Editor Stefan M. Pulst, MD, Dr med, FAAN.
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Appendix Authors
Name Location Contribution
Carlo
Manco, MD
Centre for Precision and
Translational Medicine,
Department of Medicine,
Surgery and Neuroscience,
University of Siena, Italy
Drafting/revision of the
manuscript for content,
including medical writing for
content; major role in the
acquisition of data; study
concept or design; analysis
or interpretation of data
Rosa
Cortese,
MD, PhD
Centre for Precision and
Translational Medicine,
Department of Medicine,
Surgery and Neuroscience,
University of Siena, Italy
Drafting/revision of the
manuscript for content,
including medical writing for
content; analysis or
interpretation of data
Manfredi
Alberti, Dr
Neurology Unit, Department of
Neurology and Human
Movement Sciences, University
Hospital of Siena; Department
of Medical, Surgical and
Neurological Science,
University of Siena, Italy
Major role in the acquisition
of data, analysis or
interpretation of data
Silvia
Bianchi,
PhD
Centre for Precision and
Translational Medicine,
Department of Medicine,
Surgery and Neuroscience,
University of Siena, Italy
Major role in the acquisition
of data; analysis or
interpretation of data
Lucia
Monti, MD
Diagnostic and Functional
Neuroimaging Unit,
Department of Neurology and
Human Movement Sciences,
University Hospital of Sie na, Italy
Analysis or interpretation of
data
Nicola De
Stefano,
MD, PhD
Centre for Precision and
Translational Medicine,
Department of Medicine,
Surgery and Neuroscience,
University of Siena, Italy
Drafting/revision of the
manuscript for content,
including medical writing for
content; study concept or
design
Carla
Battisti,
MD, PhD
Centre for Precision and
Translational Medicine,
Department of Medicine,
Surgery and Neuroscience,
University of Siena, Italy
Drafting/revision of the
manuscript for content,
including medical writing for
content; study concept or
design
Neurology.org/NG Neurology: Genetics | Volume 9, Number 6 | December 2023 3

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4Neurology: Genetics | Volume 9, Number 6 | December 2023 Neurology.org/NG

DOI 10.1212/NXG.0000000000200104
2023;9; Neurol Genet
Carlo Manco, Rosa Cortese, Manfredi Alberti, et al.
Clinical and MRI Features up to 2 Years
Gene Variation With Adult-Onset Cerebral Folate Deficiency and StableFOLR1
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