MTHFS Human

What is MTHFS and what is its primary metabolic function?

MTHFS is an essential enzyme in the folate-mediated one-carbon pathway (FOCM) that catalyzes the irreversible ATP and Mg²⁺-dependent conversion of 5-formyltetrahydrofolate (5-FTHF) to 5,10-methenyltetrahydrofolate (5,10-MTHF) . It represents the only known enzyme capable of catalyzing both endogenous 5-FTHF and exogenous folic acid into 5,10-MTHF, which is subsequently reduced to 5-methyltetrahydrofolate (5-MTHF) and other reduced folates . This conversion is critical for regulating the flow of carbon through the one-carbon metabolic network, which supplies essential components for cellular growth and proliferation . MTHFS plays a pivotal role in folate homeostasis by recycling 5-FTHF, a stable storage form of folate, back into the metabolically active folate pool, thereby supporting numerous biochemical processes including DNA synthesis and methylation reactions .

How does MTHFS deficiency present clinically in humans?

MTHFS deficiency (MIM: #618367) presents as a rare autosomal recessive neurodevelopmental disorder with characteristic clinical features becoming apparent after approximately four months of normal development . The clinical manifestations typically include global developmental delay (GDD), hypotonia progressing to hypertonia and spasticity, microcephaly, seizures (onset typically at 2-3 years of age), and cerebral hypomyelination . Additional features may include feeding difficulties, exaggerated startle response, short stature, progressive spasticity, and vocal fold paralysis in some cases . Brain MRI findings consistently demonstrate cerebral hypomyelination, cerebellar and vermis atrophy or hypoplasia, and a thin corpus callosum . Approximately one-third of affected children develop visual disturbances, sensorineural hearing loss, and progressive leukodystrophy by six years of age . Some patients also present with more unusual features such as macrocytic anemia, elevated neopterin levels, high-arched palate, and deep brain calcification .

What is the structural basis for MTHFS function?

The three-dimensional structure of human MTHFS (hMTHFS) provides critical insights into its catalytic mechanism and substrate specificity . Crystal structures have been determined for native hMTHFS, a binary complex with ADP, hMTHFS bound with the N5-iminium phosphate reaction intermediate, and an enzyme-product complex . The N5-iminium phosphate intermediate, captured in crystal structure analyses, reveals the unique strategy employed by hMTHFS for substrate recognition . The enzyme structure shows a channel connecting the ATP binding site with the substrate binding pocket where Y152 plays a key role in positioning the γ-phosphate for nucleophilic attack . This structural information elucidates how N10-substituted folate analogues can inhibit enzyme activity by preventing proper positioning of the γ-phosphate, which has significant implications for cancer therapy as inhibition of MTHFS in human MCF-7 breast cancer cells has been shown to arrest cell growth .

How do mutations in the MTHFS gene affect enzyme activity?

Mutations in the MTHFS gene result in altered enzyme activity, affecting the conversion of 5-FTHF to 5,10-MTHF. Functional studies conducted by transient transfection of wild-type and mutant MTHFS into HEK293T cells have demonstrated the impact of specific variants on enzyme activity . Biallelic variants, including homozygous and compound heterozygous mutations, significantly reduce enzyme function, leading to metabolic disturbances characterized by low-normal levels of 5-MTHF in cerebrospinal fluid (CSF) despite normal peripheral folate levels . The severity of enzyme activity impairment correlates with the biochemical phenotype, with homozygous loss-of-function variants causing more pronounced reduction in CSF 5-MTHF levels and corresponding increase in 5-FTHF concentration . This biochemical imbalance subsequently leads to disruption of numerous folate-dependent metabolic processes, particularly affecting central nervous system development and function.

What biochemical markers are characteristic of MTHFS deficiency?

MTHFS deficiency presents with a distinct biochemical profile that aids in diagnosis. The primary biochemical hallmark is a reduced level of 5-MTHF in cerebrospinal fluid (CSF), despite normal peripheral folate levels . This discrepancy between CSF and peripheral folate status is a critical diagnostic indicator. Additionally, affected individuals typically show markedly elevated levels of 5-FTHF in both blood and CSF, reflecting the enzymatic block in converting this substrate . Some patients demonstrate hyperhomocysteinemia, with reported values of up to 19 μmol/L (reference range: 2-9 μmol/L) . Macrocytic anemia may be present in a subset of patients, likely due to impaired DNA synthesis resulting from disrupted folate metabolism . These biochemical abnormalities reflect the central role of MTHFS in one-carbon metabolism and folate homeostasis, particularly in the central nervous system.

What are the recommended methods for measuring MTHFS activity in clinical samples?

Assessment of MTHFS activity in clinical samples involves specific enzymatic assays using patient-derived cells, typically fibroblasts. The methodology includes:

• Preparation of cell lysates by suspending fibroblast pellets in 100 mmol/L TRIS/HCl buffer (pH 7.3) and subjecting them to three freeze-thaw cycles

• Centrifugation of the cell suspension at 10,000g for 5 minutes to obtain the supernatant for enzyme assay

• Determination of total protein concentration using the bicinchoninic acid protein assay

• Incubation of 10 μg total protein at 37°C in 70 mmol/L TRIS/HCl buffer (pH 7.3) containing 20 mmol/L MgCl₂

This enzymatic assay measures the conversion of 5-FTHF to 5,10-MTHF, allowing quantification of MTHFS activity. Reduced activity compared to control samples supports the diagnosis of MTHFS deficiency, particularly when correlated with genetic findings and clinical presentation.

What is the spectrum of pathogenic variants identified in the MTHFS gene?

To date, eleven pathogenic variants in the MTHFS gene have been identified across nine unrelated cases with MTHFS deficiency . These include four novel variants reported in three Chinese patients: c.504del (p.Y169Tfs*17), c.158C>T (p.S53F), c.117+1del, and c.182A>G (p.E61G) . Other reported pathogenic variants include c.434G>A (p.Arg145Gln), which was identified in a homozygous state in siblings presenting with a neurometabolic phenotype . The variants encompass missense mutations, frameshift mutations, and splice site alterations, leading to various degrees of enzyme dysfunction . Whole-exome sequencing (WES) has been the primary method for identifying these variants, often revealing biallelic suspected loss-of-function variants that significantly impair MTHFS function . The identification of these genetic variants, combined with functional studies demonstrating their impact on enzyme activity, has established MTHFS deficiency as a distinct genetic disorder.

How does genetic analysis contribute to MTHFS deficiency diagnosis?

Genetic analysis plays a pivotal role in diagnosing MTHFS deficiency, particularly through the following approaches:

• Whole-exome sequencing (WES) to identify biallelic variants in the MTHFS gene

• SNP array analysis to detect long runs of homozygosity (ROH) segments that include the MTHFS gene, which can indicate potential ancestral mutations in consanguineous families

• Segregation analysis within families to confirm the inheritance pattern and pathogenicity of identified variants

• Functional validation studies of novel variants through transient transfection of wild-type and mutant MTHFS into cell lines (e.g., HEK293T cells) to assess the impact on enzyme activity

These genetic approaches are particularly valuable given the rarity of MTHFS deficiency and its clinical overlap with other neurological disorders. Genetic confirmation provides a definitive diagnosis, enables appropriate genetic counseling for families, and guides targeted therapeutic interventions.

What in vitro models are used to study MTHFS function and pathogenic variants?

Researchers employ several in vitro models to investigate MTHFS function and the impact of pathogenic variants:

• Transient transfection of wild-type and mutant MTHFS constructs into HEK293T cells to assess enzyme activity and functional consequences of specific variants

• Fibroblast cultures derived from patients with MTHFS deficiency to study enzyme activity in native cellular contexts

• HeLa cell models to investigate the role of MTHFS in purinosome formation and its SUMO protein-dependent interactions

• Crystallographic studies using purified human MTHFS protein to determine three-dimensional structure and mechanisms of substrate binding and catalysis

These in vitro systems provide valuable platforms for studying the molecular consequences of MTHFS variants, evaluating potential therapeutic approaches, and understanding the broader impacts of MTHFS dysfunction on cellular metabolism and growth.

What animal models inform our understanding of MTHFS biology?

Animal models have provided significant insights into MTHFS biology, particularly regarding its essential role in development and metabolism:

• Mouse models have demonstrated that MTHFS is essential for embryonic development, as homozygous knockout embryos are not viable

• Mouse embryonic fibroblasts hemizygous for MTHFS exhibit decreased de novo purine biosynthesis, highlighting the critical role of MTHFS in this metabolic pathway

• Studies in mice have shown that MTHFS functions as a component of the purinosome in a small ubiquitin-like modifier (SUMO) protein-dependent manner, revealing its involvement in macromolecular complex formation for metabolic channeling

These animal models collectively indicate that MTHFS plays non-redundant roles in folate metabolism and embryonic development, which aligns with the severe developmental phenotypes observed in human patients with MTHFS deficiency.

What treatment strategies show efficacy for MTHFS deficiency?

Current treatment strategies for MTHFS deficiency focus on addressing the biochemical imbalances and managing clinical symptoms:

How might MTHFS inhibition be leveraged in cancer research?

MTHFS inhibition represents a promising approach in cancer research, building on several key observations:

• MTHFS regulates the flow of carbon through the one-carbon metabolic network, which supplies essential components for cellular growth and proliferation

• Inhibition of MTHFS in human MCF-7 breast cancer cells arrests cell growth, suggesting therapeutic potential in cancer treatment

• The crystal structure of human MTHFS provides a rational basis for the design and optimization of specific inhibitors targeting this enzyme

• N10-substituted folate analogues have been shown to inhibit MTHFS by placing Y152 in the middle of the channel connecting the ATP binding site with the substrate binding pocket, preventing the positioning of γ-phosphate for nucleophilic attack

Future research could focus on developing more specific and potent MTHFS inhibitors based on structural insights, evaluating their efficacy across various cancer types, and investigating potential synergistic effects with existing chemotherapeutic agents.

What remains unknown about the pathophysiology of MTHFS deficiency?

Despite advances in understanding MTHFS deficiency, several knowledge gaps persist:

• The precise mechanisms by which reduced 5-MTHF levels in CSF lead to the neurological manifestations observed in patients remain incompletely understood

• The role of MTHFS in purinosome formation and its implications for disease pathogenesis require further investigation

• The relationship between specific MTHFS variants and clinical phenotype severity (genotype-phenotype correlations) needs more comprehensive characterization

• Optimal treatment protocols, including dosing regimens for 5-MTHF supplementation and potential combination therapies, remain to be established

• Long-term outcomes and natural history of MTHFS deficiency are largely unknown due to the rarity of the condition and relatively recent recognition as a distinct disorder

Addressing these knowledge gaps through continued research will be crucial for improving diagnosis, treatment, and outcomes for patients with this rare genetic disorder.