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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 2  |  Page : 72-77

Homozygous mutation in the MTHFS gene may contribute to the development of cerebral folate deficiency syndrome

1 Center for Precision Environmental Health; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030-3411, USA
2 Center for Precision Environmental Health; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030-3411, USA
3 Center for Precision Environmental Health; Department of Molecular and Human Genetics; Department of Molecular and Cellular Biology; Department of Medicine, Baylor College of Medicine, Houston, Texas 77030-3411, USA

Date of Submission01-Nov-2019
Date of Decision01-Feb-2020
Date of Acceptance20-Apr-2020
Date of Web Publication26-Jun-2020

Correspondence Address:
Richard H Finnell
Center for Precision Environmental Health, Baylor College of Medicine, One Baylor Plaza, BCM 946, Houston, Texas 77030-3411
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2096-2924.288022

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Objective: The purpose of this study was to examine the role of rare variants in the one-carbon metabolic pathway in the etiology of the cerebral folate deficiency (CFD) syndrome. The CFD syndrome is a neurometabolic syndrome identified by low concentrations of 5-methyltetrahydrofolate (5-MTHF) in the cerebrospinal fluid (CSF) in spite of near-normal peripheral folate levels resulting in neurodevelopmental disorders.
Methods: The localized folate metabolism impairments in CFD are thought to be either the result of mutations in genes responsible for folate transport or folate turnover through degradation. Genes that have been previously implicated in the etiology of CFD include folate receptor alpha-1 (FOLR1), dihydrofolate reductase, proton-coupled folate transporter, and capicua. We performed whole-exome sequencing (WES) analysis of a CFD patient that revealed 99 novel missense mutations, of which 21 were classified as damaging mutations through the Poly-Phen2 prediction algorithm. In vitro functional studies were conducted by transient transfection of wild-type and mutant MTHFS into HEK293T cells to determine the impact of the variants on enzyme activity.
Results: Of the damaging variants identified in the WES studies, we focused on the gene coding for the enzyme 5,10-methenyl-tetrahydrofolate synthetase (MTHFS). This enzyme catalyzes the production of methenyl THF which is subsequently converted to 5-MTHF. The CFD patient described within was found to carry a homozygous mutation, c.101G>T (p.R34L, rs200058464) in MTHFS, while the parents of the proband are heterozygotes for the MTHFS gene, and the healthy sibling is not a carrier.
Conclusion: The mutant allele displayed a 50% reduction in luciferase activity (P < 0.05), suggesting that homozygous loss of the MTHFS gene may play a significant role in the development of CFD.

Keywords: Cerebral Folate Deficiency Syndrome; MTHFS; Intellectual Disability

How to cite this article:
Sakthivel D, Lei Y, Cao X, Finnell RH. Homozygous mutation in the MTHFS gene may contribute to the development of cerebral folate deficiency syndrome. Reprod Dev Med 2020;4:72-7

How to cite this URL:
Sakthivel D, Lei Y, Cao X, Finnell RH. Homozygous mutation in the MTHFS gene may contribute to the development of cerebral folate deficiency syndrome. Reprod Dev Med [serial online] 2020 [cited 2021 Jun 22];4:72-7. Available from: https://www.repdevmed.org/text.asp?2020/4/2/72/288022

  Introduction Top

Cerebral folate deficiency (CFD) is an autosomal recessive disorder associated with low 5-methyltetrahydrofolate (5-MTHF) in the cerebrospinal fluid (CSF), while circulating folate concentrations are within the low normal clinical range.[1] In humans, the folate concentration in the CSF usually is two-fold higher than in serum.[2] As folate participates in the de novo synthesis of purines and thymidine, and along with cobalamin, it represents a class of indispensable cofactors for S-adenosylmethionine production by the brain. Therefore, folate deficiencies are often associated with a variety of neurological disorders.[3] Affected children develop normally until four months of age and then display the onset of symptoms secondary to the folate deficiency in the cerebral spinal fluid. Typical features include marked unrest, psychomotor regression, deceleration of head growth, cerebellar ataxia, spastic paraplegia, dyskinesias (chorea–athetosis, hemiballismus), and chronic epilepsy.[4] One-third of the children also develop signs of visual disturbances, sensorineural hearing loss, and progressive leukodystrophy by six years of age.[3]

The molecular mechanism underlying CFD has previously been attributed to mutations in folate receptor alpha-1 (FOLR1), which regulates active folate transport into the CSF through receptor-mediated FOLR1 endocytosis.[5],[6] The patient reported here was found to be a heterozygote for a FOLR1 9bp deletion in the exon1–intron1 boundary; however, her healthy asymptomatic father also had the same deletion, suggesting that this was not causative of the patient's CFD, leading us to search for other potential candidate gene variants. Whole-exome sequencing (WES) of the proband and her family identified a biallelic suspected loss-of-function variant in the MTHFS gene.

The protein encoded by the MTHFS gene is an essential enzyme in one-carbon metabolism. De facto, MTHFS is the only enzyme known to catalyze the irreversible ATP and Mg2+-dependent conversion of 5-formyltetrahydrofolate (5-FTHF) to 5,10-methenyltetrahydrofolate (5,10-MTHF), which is further reduced to 5MTHF [Figure 1], a crucial intermediate in the one-carbon metabolic pathway that supplies carbon units for DNA synthesis and DNA methylation.[7],[8] In a developing rabbit fetus, MTHFS activity is low until mid-gestation, after which the activity of the MTHFS enzyme escalates to ensure an adequate supply of 5,10-MTHF to meet the metabolic demands of the rapidly growing fetus during the late-term and postnatal period.[9] The lack of MTHFS activity due to acquired or inherited disorders result in decreased 5,10-MTHF bioavailability, compromising 5MTHF-dependent biological processes. The central nervous system, in particular, relies on 5MTHF for various activities, including the synthesis of monoamine neurotransmitters such as serotonin, dopamine, and norepinephrine.[10] Furthermore, the increase in 5-FTHF as a consequence of decreased MTHFS activity inhibits de novo purine synthesis and cell growth in mouse embryonic fibroblast cells.[11] Collectively, alterations in MTHFS enzyme activity may serve as a critical link between folate deficiency, growth retardation, and neurological disorders.
Figure 1: 5-Formyltetrahydrofolate serves as the stable storage unit of THF cofactors. MTHFS catalyzes the irreversible ATP and Mg2+-dependent conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate, a metabolically active form of folate which is further reduced to 5-methyltetrahydrofolate. 5,10-Methenyltetrahydrofolate supplies carbon for thymidine production and, thereby, DNA synthesis. 5-Methyltetrahydrofolate is essential for the production of methyl donors for DNA, RNA, protein, and lipid methylation.

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The human MTHFS gene maps to 15q25.1 containing four splice variants encoding three different isoforms. A compound heterozygote variant, c.434G>A (p. 145Q) and c.107T>C (p.L36P) in the MTHFS gene, has been previously identified as the cause of some of the established clinical features of CFD syndrome, such as epilepsy, spasticity, and cerebral hypomyelination.[12] In this study, we hypothesize that the homozygous mutation in the MTHFS gene may be a contributing factor to the etiology of the CFD syndrome in our patient.

  Methods Top

Study approval and sample collection

The proband in our study (II-1) was identified by self-referral through the mother of the patient. The folate concentrations in CSF and serum were measured by the treating hospital. This study was approved by the Institution Review Board of the University of Texas at Austin. All samples were procured with written informed consent from the parents.

DNA extraction and whole-exome sequencing

Archive-quality genomic DNA was extracted from whole blood samples using the Gentra Puregene Blood Kit (Qiagen). DNA sequencing libraries were established using NEBNext Ultra™ DNA Library Kit (New England Biolabs Inc., USA) following protocols described by the manufacturer (ver 3.0). Whole exomes, including the 3'-untranslated regions, were captured using SureSelectXT Human All Exon v4 kit (Agilent) and subjected to sequencing on Illumina HiSeq 2000 platform at University of Texas at Austin. The generated libraries were sequenced to obtain an average depth coverage of 20×.

In silico analysis

Next-generation sequencing results were analyzed following GATK best practices (https://software.broadin stitute.org/gatk/best-practices/). Briefly, reads were mapped to hg19 using BWA alignment software,[13] which were then sorted and indexed by SAMtools,[14] base recalibrated by GATK,[15] and duplicate reads removed by Picard software (https://broadinstitute.github.io/picard/). Variant calling was done by GATK HaplotypeCaller methods.[15]De novo variant analysis was performed with TrioDeNovo software[16] and variant annotations were performed with Annovar.[17] Sanger sequencing validation of MTHFS c.101G>T (p.R34L) was performed according to a previously established protocol[18] using the primers: 5'-AGGGCCAGAGGCGAGGTAAG-3' (forward) and 5'-GCGTCCAGACCACGACTAGG-3' (reverse).

Transient transfection assay

pGL3 reporter system (Promega, Madison, WI, USA) was used to study the variant effect on MTHFS promoter transcription activity. The MTHFS promoter region (−729~+275, +1:TTS) containing MTHFS c.101G>T (p.R34L) was inserted to pGL3-Basic vector by subclone using primers: 5'-CACTACCTCGAGTGCTTTA GCAGT GTTCCCGATT-3' (forward) and 5'-CAGCGCAAGCTTGGA CCATGCACAGATCAGCA-3' (reverse) which contains restriction enzyme sites for XhoI and HindIII, respectively. HEK293T cells were seeded onto a 24-well plate and cultured in DMEM with 10% heat-inactivated fetal bovine serum and 1% antibiotic. Twenty-four hours later when the cells were entirely adherent, Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) reagent was used to transfect the cells with 1 μg of each of the following plasmids: pGL3-MTHFS containing the SNP (reporter plasmid denoted as TT), pGL3-basic (negative control), and pRL-TK (positive control that provides constitutive expression of luciferase gene indicated as GG). Twenty-four hours posttransfection, rapid lysis of the cells was achieved with Passive Lysis Buffer (Promega), and the cells were subjected to Dual-Luciferase Reporter Assay System (Promega) to measure luciferase activity. Three independent experiments were performed with triplicate wells for each of the cell lines.

  Results Top

Clinical outcomes

The Caucasian proband (II-1) genetically and clinically evaluated in this study belongs to a family with no known consanguinity [Figure 2]. The proband showed signs of seizures at three years of age, at which time postelectroechogram and brainstem auditory evoked response evaluations resulted in having a vagus nerve stimulator implant. At nine years of age, the patient developed a significant speech and language disorder and presented with vocal fold paralysis necessitating vocal cord repair surgery. At 11.5 years of age, the patient was hospitalized for acute repetitive cluster seizures indicating a prolonged epileptic crisis. The patient was administered 250 mg of Zarontin three times a day to control her epileptic seizures. She required placement of a G-tube for poor feeding associated with dysphagia. Neurological evaluation revealed generalized hypertonia, epileptic aphasia, and clonis. Hearing and ophthalmological examinations revealed central visual processing and auditory processing disorder. She was given 15 mg L-methyl folate in an effort to treat the signs of severe CFD (CSF 5-MTHF: 35 nmol/L, while the normal value is 40–150 nmol/L for teenagers). Systemic examination revealed several other complications such as obesity, immune deficiency, chronic kidney infections, chronic urinary tract infection, chronic sinusitis, persistent dehydration adrenal crisis coma, unexplained bleeding in trachea, shingles, and aspiration pneumonia. Sadly, the patient died secondary to hepatic failure at 18 years of age.
Figure 2: Pedigree of the reported family in our study depicting an autosomal inheritance pattern. The arrow indicates the deceased proband (II-1), who is a homozygous carrier of the MTHFS c.101G>T p.R34L mutation, diagnosed with cerebral folate deficiency. The healthy parents of the propositus (I-1, I-2) are a heterozygous carrier of the MTHFS mutation.

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Genetic variants determined by whole-exome sequencing

Given the many unknowns concerning the genetic etiology of this disease, we performed WES hoping to find candidate genes in an unbiased fashion. Sequencing analysis was performed on the proband, parents, and the healthy sibling. The analytical platform primarily focused on nonsense, missense, indels, and de novo mutations. Our variant filtering strategy limited our search to 572 recessive missense mutations for which the healthy sibling was a heterozygote. Causal variants involved in folate metabolism were initially evaluated. The patient was a compound heterozygote for the CFD associated capicua gene: paternally inherited 1360_32G > A and maternally inherited 3796_15C > T. Neither of these variants was predicted to be pathogenic. Genes related to mitochondrial disorders were also considered. COQ6 (coenzyme Q6 homolog, monooxygenase) V406M (rs8500) was the only gene with a known recessive inheritance pattern. This variant has a very high minor allele frequency of 40% and is predicted to be benign. Therefore, this variant was not considered for further assessment. We subsequently identified 99 recessive missense mutations for which the healthy sibling had a wild-type genotype. Of these, there were 69 benign (<0.6), nine possibly damaging (0.6–0.9), and 12 probably damaging (>0.9) mutations classified according to the polymorphism phenotype (PolyPhen-2) prediction score [Supplementary Table 1].

The proband (II-1) had a homozygous mutation c.101G>T p.R34L (rs200058464) in the folate pathway gene, MTHFS. The p.R34L variant is reported at a frequency of 0.01471, with the highest allele count in the European (non-Finnish) population according to the gnomAD and dbSNP databases. This variant is multiallelic; it falls on five transcripts in two genes: coding sequence variants in the MTHFS gene and intronic variants in the ST20-MTHFS, an important paralog of the MTHFS.[19] PolyPhen predicts the variant to be benign, and the SIFT algorithm predicts that the amino acid substitution is tolerated. Ensembl genome browser 97 (https://ensembl.org) was then used to predict the approximate location and identify any regulatory feature consequence of the single-nucleotide variant (SNV) [Figure 3]a. The SNV is located in the promoter region, which generally serves as the binding site for the following transcription factors: TEA domain factor 4 (TEA4), ETS domain-containing proteins (ELK1, ELF1), Spi-B PU-box-binding proteins (SPIB), detected through Ensembl genome browser 97 (https://ensembl.org). Further research is warranted to delineate how dysregulation of the above transcription factors affects folate metabolism. Interestingly, structural and functional characterization of Mycoplasma pneumoniae MTHFS revealed that Arg34 does not participate in the active site of either ADP-MG2+ or 5-FTHF-ADP complex.[20] However, sequence comparison of MTHFS with its orthologs across different species shows that Arg34 is specific to humans, suggesting a yet to be unraveled role for Arg34 in humans[21] [Figure 3]b. Of note, there were no de novo variants detected in the patient.
Figure 3: Rare homozygous MTHFS SNV identified in cerebral folate deficiency patient. (a) Schematic representation of the approximate location of SNV, c.101G>T using Ensemble genome Browser 97. (b) A partial alignment of human MTHFS with yeast orthologue as per established sequence alignment using the pole Bio-Informatique Lyonnais Network Protein Sequence Analysis Server (Holmes and Appling, 2002). Nucleotides highlighted in green show sequence conservation. The mutation site is indicated with an arrow. Blue box denotes conservative substitutions.

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C. Transcriptional activity of mutated MTHFS gene

To explore the functional impact of c. 101G>T p.R34L variant on the promoter activity of MTHFS, HEK293 cells were subjected to a luciferase reporter gene assay. For each of the three expression constructs (GG: pRL-TK provides constitutive expression of the wild-type Renilla luciferase. TT: pGL3-MTHFS with c.101G>T SNP, mutation of interest. pGL3-Basic: empty vector serving as the negative control), three independent reporter assays were conducted. To eliminate variation in cell density and transfection efficiency, the firefly luciferase fluorescence in the pGL3 vector was normalized by the Renilla fluorescence. GG showed a consistently high luciferase expression compared to the basal expression seen with pGL3- Basic. TT suppressed luciferase activity nearly three-fold (P < 0.05), suggesting that G to T polymorphism has deleterious effects on MTHFS promoter activity. Thus, c.101G>T (rs200058464) is a crucial regulatory SNV, which, in the homozygous state, may result in an aggressive phenotype. The raw data of relative luciferase expression are presented in [Figure 4].
Figure 4: Quantitative analysis of the impact of MTHFS SNP (rs200058464) in the luciferase activity. The vector constructs shown are as follows: - GG: pRL-TK provides constitutive expression of the wild-type Renilla luciferase. TT: pGL3-MTHFS with c.101G>T SNP, mutation of interest. pGL3-Basic: empty vector serving as the negative control. The luciferase activity was measured 24 hours posttransfection, normalized relative to the protein content in the cell lysates. pGL3 mutant vector (TT) shows a three-fold reduction in luciferase expression (P < 0.05), close to the basal expression of pGL3-Basic vector suggesting a loss of function phenotype for c.101G>T SNP.

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  Discussion Top

Compound heterozygous mutations in MTHFS gene have been previously reported as a cause of neurometabolic symptoms such as microcephaly, epilepsy, and cerebral hypomyelination.[12] Herein, we report the first case of a rare homozygous mutation in the MTHFS gene manifesting as global developmental regression, grand mal seizures, focal motor impairments, and vocal fold paralysis, among other neurological complications. Our study identifies MTHFS SNV, c.101G>T as a contributing factor to the CFD disorder. To assess the biological relevance of the SNV, Ensembl genome browser 97 was used to pinpoint the SNV. c.101G>T is located in the promoter region in proximity to the transcription start site. In line with our biallelic loss of function speculation, transient transfection reporter assay showed a three-fold reduction in promoter activity. Gene promoters are directly involved in transcription and regulation of gene expression through the binding of various transcription factors with high sequence specificity. Thus, sequence variation alters the binding capacity of transcription factors resulting in gene dysregulation. The binding site of the Elf-1 transcription factor encompasses the SNV location. Mammalian Elf-1/Drosophila grainy head (grh) is an essential gene in Drosophila neurulation. Grh null mutants develop neural tube defects and are resistant to folate supplementation,[22] implying that functional derailments in Elf-1 can cause folate resistance perhaps underlying the failed treatment with L-methyl folate in our patient.

SIFT (0.41) and PolyPhen (0) programs predict the p.R34L mutation to be tolerated and benign. A missense variant that causes a significant change in the protein backbone conformation creates mutant proteins impeding enzymatic activity resulting in disease states.[23] Based on free energy simulations, Wright and Lim developed a comprehensive mathematical model to predict changes in protein state based on amino acid substitutions.[24] In an analysis of 94 single-point mutations in 47 different proteins, arginine to leucine changes retains the wild-type folded protein state.[24] It is important to recall that arginine is a hydrophilic positively charged amino acid present at the surface of the proteins. Frequently, arginine molecules are involved in salt-bridges where they pair up with negatively charged aspartate/glutamate to stabilize hydrogen bonds, vital for protein stability. Arginines are predominantly found in the binding sites of active proteins wherein they interact with negatively charged phosphates enabling posttranslational modification of proteins.[25] Contrary to the computational predictions, functional characterizations demonstrate that only arginine substitutions for another positively charged amino acid such as lysine are tolerated. Leucine is an aliphatic hydrophobic neutral amino acid and prefers to be buried in the hydrophobic core, suggesting that arginine to leucine substitution might interfere with protein stability, thereby the catalytic activity of the enzyme.[26]

Low 5MTHF levels in CSF relative to systemic levels together with the clinical findings confirmed a CFD diagnosis for our proband. 5MTHF is one among the five one-carbon THF derivatives. Folate-mediated one-carbon pathway (FOCM) is a highly intertwined metabolic network wherein THF supplies one-carbon moieties for de novo purine synthesis, DNA methylation, and neurotransmitter synthesis. MTHFS is required to convert both exogenous folic acid and endogenous 5-FTHF to reduced folates. In mice, MTHFS is an essential gene, as MTHFS null mice are not viable.[11] Mice in which the MTHFS gene was ablated using a gene trap for exon1 (MTHFSgt/+) were fed a diet lacking folate which produced a 60% reduction in MTHFS protein levels. MTHFS perhaps protects cellular folate pools by suppressing its expression in response to folate deficiency. In neuroblastoma cell lines, overexpression of MTHFS accelerates folate catabolism and curtails cellular concentrations of 5MTHF and 5-FTHF,[7] indicating the possibility that MTHFS catalyzes the oxidation of reduced folate coenzymes secondary to its catalytic activity in the synthesis of 5,10-MTHF. Alternatively, MTHFS could alter the relative distribution of certain one-carbon folates, favoring the accumulation of more chemically unstable forms of folate that are highly susceptible to degradation.[27] Although the molecular mechanism remains to be fully elucidated, it is clear that MTHFS plays a crucial regulatory role in determining tissue folate levels.

Mounting evidence links impairments in FOCM to neurological sensorimotor deficits and neuropsychiatric diseases, including cerebral ischemia.[28] GWAS study identifies MTHFS as a gene significantly associated with cerebellar ischemic stroke, possibly due to its role in regulating homocysteine synthesis through 5-FTHF.[29] Ischemia and other vasculopathies are associated with low folate levels resulting in hyperhomocysteinemia.[30],[31] It is possible that tissue-specific low activity folates due to debilitated MTHFS enzyme activity as seen in our patient might contribute to hyperhomocysteinemia. In particular, elevated homocysteine levels compromise the blood–brain barrier (BBB) integrity.[32] Since the permeability of nutrients is stringently controlled in BBB, microvessel endothelium has higher density of tight junctions for increased cell adhesion and minimal paracellular diffusion. Homocysteine causes cell detachment either through superoxide-mediated endothelial dysfunction or repressing claudin-5, a highly expressed member of the BBB tight junctions.[33],[34] Attenuated BBB and increased permeability provide a passage for toxin-mediated tissue injury, resulting in cerebral ischemia-associated seizures overlapping some of the symptoms seen in our patient.[35] The microbiota is known to produce folate in the gut. However, no study on the association of microbiota and CFD has yet been performed. Overall, the novel genetic finding reported here opens new avenues for the investigation of CFD etiology concerning MTHFS mutation.

Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website.


The authors are grateful to the family of the proband for their cooperation and assistance with this study. Dr. Finnell receives support from the Baylor College of Medicine endowment from the William T. Butler, M.D. Distinguished Chair.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]


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