FARS2 Human

What is FARS2 and what is its primary function in cellular metabolism?

FARS2 (OMIM# 611592) encodes the mitochondrial phenylalanyl-tRNA synthetase (mtPheRS), which is responsible for charging mitochondrial tRNA^Phe with phenylalanine for mitochondrial translation. The protein contains four major functional domains: an N-terminal domain, a catalytic domain, a linker region (residues 290–322), and an anticodon-binding domain (ABD). These domains work together through complex interactions and conformational changes that enable mtPheRS to function as a monomer during the aminoacylation process . This activity is essential for mitochondrial protein synthesis and, consequently, for the biogenesis of the mitochondrial oxidative phosphorylation system (OXPHOS) that produces the majority of cellular ATP.

What are the major clinical presentations associated with FARS2 mutations?

FARS2 mutations have been linked to two major clinical presentations:

• Early-onset epileptic mitochondrial encephalopathy - This more severe phenotype occurs in approximately two-thirds of cases and typically has a poorer prognosis. These patients present with developmental delay and may exhibit a wide range of brain abnormalities. Most previously reported patients with early-onset FARS2-linked encephalopathy died before two years of age, though there are exceptions with prolonged survival .

• Spastic paraplegia - This less severe phenotype is associated with longer survival. Patients typically present with weakness of the lower extremities, spasticity, and difficulties with walking. Intellectual disability or developmental delay may be observed in these patients as well .

The epileptic seizures observed in FARS2 patients can have variable features, including neonatal multifocal seizures, infantile spasms, or myoclonic epilepsy with onset later in infancy .

How do researchers assess mitochondrial tRNA aminoacylation in FARS2 deficiency models?

Assessment of mitochondrial tRNA aminoacylation is a critical methodological approach in FARS2 research. In Drosophila models, researchers use the following procedure:

• Isolation of total RNA from tissues with preservation of the aminoacyl-tRNA bond

• Separation of charged and uncharged tRNAs using acid-urea polyacrylamide gel electrophoresis

• Northern blot analysis with specific tRNA probes

• Quantification of the ratio between aminoacylated and non-aminoacylated forms of tRNA^Phe

This methodology reveals that dFARS2 (Drosophila homologue) is required for mitochondrial tRNA aminoacylation. In studies, wild-type flies show efficient aminoacylation of mt-tRNA^Phe, while dFARS2 knockout mutants demonstrate significantly reduced levels of aminoacylated mt-tRNA^Phe . This approach allows researchers to quantitatively assess the impact of specific FARS2 mutations on aminoacylation efficiency.

What animal models are most effective for studying FARS2 deficiency and associated pathologies?

Drosophila melanogaster has emerged as a particularly valuable model organism for studying FARS2 deficiency. Researchers have successfully developed:

• Knockout models - Using CRISPR/Cas9 genome editing to generate loss-of-function dFARS2 mutants. For example, a specific mutant allele with a 1 bp deletion followed by an 11 bp insertion in exon 1 of dFARS2 causes a frameshift at codon 87 and a premature stop codon at position 101 .

• Knockdown models - Using RNAi approaches with the GAL4/UAS binary expression system. This allows tissue-specific knockdown of dFARS2 when combined with tissue-specific GAL4 drivers (e.g., ubiquitous Da-GAL4 or neuronal elav-GAL4) .

• Humanized fly models - Expressing human wild-type FARS2 or disease-associated variants (e.g., p.G309S, p.D142Y) in dFARS2 mutant backgrounds to study variant-specific phenotypes .

These Drosophila models effectively recapitulate key phenotypic features of human FARS2-associated diseases, including developmental delay and seizure susceptibility. Importantly, they allow for precise genetic manipulation and comprehensive biochemical analysis of mitochondrial function .

How can researchers distinguish genotype-phenotype correlations in FARS2-related disorders?

To establish genotype-phenotype correlations in FARS2-related disorders, researchers should employ a multi-faceted approach:

• Comprehensive genetic analysis - Document all compound heterozygous or homozygous variants in patients with similar clinical presentations.

• Structural analysis of variants - Determine whether mutations affect specific domains of the FARS2 protein (N-terminal domain, catalytic domain, linker region, or anticodon-binding domain).

• In vitro functional studies - Measure aminoacylation activity of recombinant mutant FARS2 proteins.

• Animal model validation - Test specific variants in model organisms and assess phenotypic outcomes.

• Biochemical assessment of patient samples - Analyze OXPHOS complex assembly and activity in patient-derived cells.

Research has revealed that different FARS2 mutations can produce distinct phenotypes. For example, in Drosophila models, expression of human FARS2 with the p.G309S variant induces seizure behaviors, while the p.D142Y variant primarily causes locomotion defects . These findings align with clinical observations where p.G309S is associated with early-onset epileptic encephalopathy and p.D142Y with spastic paraplegia .

What biochemical assays are most informative for evaluating mitochondrial dysfunction in FARS2 deficiency?

A comprehensive biochemical analysis of FARS2 deficiency should include the following methodologies:

In Drosophila dFARS2 knockout models, these assays have revealed reduced aminoacylation of mt-tRNA^Phe, decreased levels of mitochondrially-encoded proteins, impaired assembly of OXPHOS complexes, and reduced enzymatic activities of Complexes I and IV .

How should researchers design rescue experiments to validate pathogenicity of FARS2 variants?

A robust experimental design for validating FARS2 variant pathogenicity includes:

• Generation of expression constructs:

  • Wild-type human FARS2

  • FARS2 variants identified in patients

  • All constructs should use identical vectors and expression systems

• Integration into model systems:

  • For Drosophila, use site-specific integration to ensure equivalent expression levels

  • Express constructs in a dFARS2 knockout background

  • Use appropriate controls (empty vector, wild-type rescue)

• Phenotypic assessment:

  • Developmental timeline (pupal development, eclosion rates)

  • Behavioral assays (seizure susceptibility, climbing ability)

  • Survival analysis

• Biochemical validation:

  • Aminoacylation efficiency of mt-tRNA^Phe

  • Steady-state levels of mitochondrial tRNAs

  • OXPHOS complex assembly and activity

This approach has successfully demonstrated that human FARS2 can partially rescue dFARS2 knockout phenotypes in Drosophila, while disease-associated variants (p.G309S and p.D142Y) show differential rescue abilities and distinct phenotypic consequences .

What is the significance of compound heterozygosity in FARS2 mutations and how should it be investigated?

Compound heterozygosity (carrying two different mutations, one on each allele) is common in FARS2-related disorders and presents unique research challenges:

• Clinical significance:

  • Most patients with FARS2-related disorders are compound heterozygotes

  • Phenotypic differences may result from different combinations of compound heterozygous variants

  • The interaction between different mutant proteins may influence disease severity

• Experimental approaches:

  • Express both variants simultaneously in model systems to mimic patient genotypes

  • Perform in vitro studies to determine whether mutant proteins interact functionally

  • Analyze aminoacylation activity of individual and combined mutant enzymes

• Case example analysis:

  • A patient carrying the p.(Arg419His) variant compound heterozygous with p.(His84Pro) showed a more severe phenotype compared to patients with other combinations including p.(Arg419His)

  • This suggests that specific combinations of variants may produce unique clinical presentations

• Data integration:

  • Correlate in vitro enzymatic activities with clinical severity

  • Build predictive models based on structural impacts of different variant combinations

Researchers should consider that "phenotypic differences in these cases may result from different genetic modifiers and/or the presence of different compound heterozygous FARS2 variants" .

How does FARS2 deficiency impact the broader mitochondrial translation machinery?

FARS2 deficiency extends beyond direct aminoacylation defects to influence the entire mitochondrial translation apparatus:

• Primary effects on tRNA^Phe charging:

  • Reduced aminoacylation of mt-tRNA^Phe directly impairs incorporation of phenylalanine during mitochondrial translation

  • This creates a bottleneck in the synthesis of all 13 mitochondrially-encoded proteins

• Secondary effects on mitochondrial protein stability:

  • Decreased steady-state levels of mitochondrially-encoded proteins

  • Imbalance between nuclear-encoded and mitochondrially-encoded OXPHOS subunits

  • Potential proteostatic stress and activation of mitochondrial quality control systems

• Tertiary effects on OXPHOS complex assembly:

  • Impaired assembly of respiratory chain complexes (particularly those with multiple mitochondrially-encoded subunits)

  • Reduced enzymatic activities of Complexes I and IV

  • Metabolic adaptations in response to OXPHOS deficiency

• Tissue-specific consequences:

  • Neuronal tissues show particular vulnerability to FARS2 deficiency

  • This may reflect the high energy demands of neurons and their reliance on mitochondrial function

Experimental evidence from Drosophila models indicates that dFARS2 deficiency leads to reduced levels of aminoacylated mt-tRNA^Phe, decreased mitochondrial protein synthesis, and impaired assembly and activity of OXPHOS complexes .

What are the neurological mechanisms underlying seizures in FARS2-deficient models?

The mechanisms underlying seizure susceptibility in FARS2 deficiency involve multiple neurobiological pathways:

• Energy deficit hypothesis:

  • Impaired mitochondrial translation leads to OXPHOS dysfunction

  • Neurons have high energy demands and limited metabolic flexibility

  • Energy deficit may disrupt membrane potential maintenance and neurotransmission

• Excitation-inhibition imbalance:

  • GABAergic inhibitory neurons may be particularly sensitive to mitochondrial dysfunction

  • Differential vulnerability of neuronal subtypes could lead to network hyperexcitability

• Experimental evidence from models:

  • Drosophila dFARS2 mutants demonstrate bang-sensitive seizure phenotypes

  • Expression of human FARS2 p.G309S variant (associated with epileptic encephalopathy) induces seizure behaviors

  • In contrast, p.D142Y variant (associated with spastic paraplegia) does not cause seizures but affects locomotion

• Developmental considerations:

  • FARS2 deficiency causes developmental delay in Drosophila models

  • Altered neurodevelopment may contribute to seizure susceptibility

  • Early developmental periods may represent critical windows for intervention

Researchers investigating seizure mechanisms should consider both acute bioenergetic dysfunction and long-term developmental impacts of FARS2 deficiency.

What experimental approaches show promise for treating FARS2-related disorders?

While current treatments for FARS2-related disorders remain largely supportive, several experimental approaches warrant investigation:

• Gene therapy strategies:

  • AAV-mediated delivery of functional FARS2 to affected tissues

  • Targeting approaches for efficient blood-brain barrier crossing

  • Evaluation of minimum effective dosage to prevent immunological complications

• Metabolic bypass therapies:

  • Ketogenic diet to provide alternative energy substrate (ketone bodies)

  • Supplementation with compounds that support mitochondrial function (CoQ10, riboflavin, L-carnitine)

  • Evaluation of timing and duration of intervention

• Novel small molecule approaches:

  • Compounds that may enhance residual FARS2 activity

  • Molecules that stabilize partially functional mutant FARS2 proteins

  • Screens using Drosophila models to identify suppressors of FARS2 deficiency phenotypes

• Mitochondrial transplantation:

  • Experimental delivery of functional mitochondria to affected tissues

  • Assessment of functional integration and therapeutic durability

The Drosophila models described in the search results provide valuable platforms for therapeutic screening, as they recapitulate key disease phenotypes and allow for rapid testing of candidate interventions .

How can researchers effectively model adult-onset FARS2-related neurological symptoms?

Adult-onset FARS2-related neurological symptoms present unique modeling challenges that require specialized approaches:

• Conditional knockout/knockdown systems:

  • Temporal control of FARS2 expression using inducible systems

  • Allows distinction between developmental and adult-onset phenotypes

  • Can model progressive nature of some FARS2-related disorders

• Aging studies in model organisms:

  • Extended analysis of Drosophila models throughout lifespan

  • Assessment of progressive neurodegeneration patterns

  • Correlation of biochemical defects with symptom onset and progression

• Tissue-specific approaches:

  • Targeted FARS2 manipulation in specific neuronal populations

  • Differential vulnerability analysis across neuronal subtypes

  • Correlation with clinical neuroanatomical patterns

• Quantitative phenotypic assessments:

  • Standardized locomotor assays (e.g., climbing tests in Drosophila)

  • Neurophysiological measurements (electrophysiology, calcium imaging)

  • Structural neuroimaging in vertebrate models

• Biochemical trajectory analysis:

  • Temporal profiling of mitochondrial function

  • Identification of compensatory mechanisms that may delay symptom onset

  • Determination of thresholds for clinical manifestation

The climbing assay in Drosophila models expressing human FARS2 variants provides a quantitative measure of locomotor function that can model aspects of adult-onset spastic paraplegia .

What emerging technologies might advance FARS2 research beyond current methodologies?

Several cutting-edge technologies hold promise for advancing FARS2 research:

• Single-cell omics approaches:

  • Single-cell transcriptomics to identify cell type-specific responses to FARS2 deficiency

  • Spatial transcriptomics to map anatomical patterns of mitochondrial dysfunction

  • Integration of multi-omics data for systems-level understanding

• Advanced imaging technologies:

  • Live-cell imaging of mitochondrial translation using fluorescent reporters

  • Super-resolution microscopy of OXPHOS complex assembly

  • In vivo imaging of neuronal activity in model organisms

• CRISPR-based technologies:

  • Base editing or prime editing for precise correction of FARS2 mutations

  • CRISPR activation/interference for modulating FARS2 expression

  • High-throughput CRISPR screens to identify genetic modifiers

• Organoid and advanced culture systems:

  • Patient-derived brain organoids to model neurodevelopmental aspects

  • Microfluidic organ-on-chip systems for studying tissue interactions

  • 3D bioprinted tissues with controlled mitochondrial function

• Computational approaches:

  • Molecular dynamics simulations of mutant FARS2 proteins

  • AI-driven prediction of variant pathogenicity

  • Network modeling of mitochondrial disease progression

Integration of these technologies with established Drosophila and other model systems will provide unprecedented insights into FARS2 function and pathology.

How does FARS2 deficiency compare with deficiencies in other mitochondrial aminoacyl-tRNA synthetases?

Comparative analysis across mitochondrial aminoacyl-tRNA synthetase (mt-ARS) deficiencies reveals important insights:

• Phenotypic patterns:

  • Most mt-ARS deficiencies cause neurological phenotypes, but with variable tissue specificity

  • FARS2 deficiency shares features with other mt-ARS disorders but has distinct clinical presentations

  • Research suggests these phenotypes "are generally not specific to individual enzymes"

• Mechanistic similarities and differences:

  • All mt-ARS deficiencies impair mitochondrial translation

  • Differential tissue expression and regulation may contribute to tissue-specific phenotypes

  • Varying importance of specific amino acids in mitochondrial proteins

• Research approach unification:

  • Standardized functional assays across different mt-ARS deficiencies

  • Comparative multi-omics analysis between different disorders

  • Unified database of variants and phenotypes

• Therapeutic implications:

  • Common pathways may suggest shared therapeutic approaches

  • Unique features may necessitate tailored interventions

  • Understanding compensatory mechanisms across different mt-ARS deficiencies

Creating a comprehensive catalog of genotype-phenotype correlations across all mt-ARS disorders would advance understanding of both common disease mechanisms and unique aspects of FARS2 deficiency.