WWOX Human

What is the WWOX gene and what is its basic function in humans?

The WWOX (WW domain-containing oxidoreductase) gene is located in the chromosomal common fragile site FRA16D and encodes a tumor suppressor protein containing WW domains and an oxidoreductase domain. It plays critical roles in maintaining central nervous system homeostasis and contributes to genomic stability . The WWOX protein is highly conserved evolutionarily, with human WWOX showing 93% identity and 95% similarity to mouse Wwox at the protein sequence level . The gene functions in multiple cellular processes including regulation of neuronal differentiation and migration in the brain, with its dysfunction leading to serious neurodevelopmental disorders in humans .

What clinical syndromes are associated with WWOX mutations in humans?

Germline biallelic mutations in the WWOX gene lead to two major clinical phenotypes:

• WOREE syndrome (WWOX-related epileptic encephalopathy, also known as developmental and epileptic encephalopathy 28 or DEE28, OMIM 616211): Characterized by early-onset severe epilepsy with variable seizure manifestations (tonic, clonic, tonic-clonic, myoclonic, infantile spasms, and absence seizures), severe developmental delay, and profound disability. Most affected patients cannot make eye contact, sit, speak, or walk .

• SCAR12 (spinocerebellar ataxia, autosomal recessive 12, OMIM 614322): A milder phenotype typically caused by missense mutations in WWOX, featuring ataxia and epilepsy that may be responsive to antiepileptic drugs, though patients still display ataxia and intellectual disability .

Additionally, WWOX mutations have been documented in patients with West syndrome, characterized by epileptic spasms with hypsarrhythmia .

What animal models are available for studying WWOX function and pathology?

Several animal models have been developed to study WWOX function and dysfunction:

• Wwox-knockout mice: Complete ablation of Wwox leads to postnatal lethality and recapitulates many of the severe phenotypes seen in WOREE syndrome, including intractable epilepsy, hypomyelination, and early death . These models demonstrate that targeted disruption of the Wwox gene causes neurodevelopmental disorders including abnormal neuronal differentiation and migration in the brain, cerebral malformations such as microcephaly, neuronal disorganization, and defective cerebellar midline fusion .

• Neuron-specific Wwox-knockout mice: These models allow researchers to study the specific effects of WWOX loss in neurons while maintaining expression in other tissues, helping to isolate neuronal contributions to the observed phenotypes .

• Rat models: Rat models with Wwox deficiency have also been developed and show similar neurological phenotypes to mouse models, providing complementary systems for research .

The striking phenotypic similarity between rodent models and human patients (both showing severe epileptic seizures and growth retardation) makes these models particularly valuable for translational research .

How can cellular models be used to study WWOX function in DNA repair pathways?

Cellular models have been instrumental in elucidating WWOX's role in DNA repair:

• MEF (Mouse Embryonic Fibroblast) lines: Comparison of Wwox-knockout and wild-type MEFs reveals that absence of Wwox is associated with mild genome instability, including chromosomal alterations and specific copy number variations (CNVs). Karyotype analysis of knockout MEFs showed near tetraploidy with structural abnormalities including del(7)(A1B4) and del(4)(C4) not present in wild-type cells .

• Human cancer cell lines with modified WWOX expression:

  • MDA MB-231 breast cancer cells with inducible WWOX expression

  • MCF10A cells with stably silenced WWOX (shWWOXA and shWWOXB clones)

  • 293/HW1 cells for NHEJ (Non-Homologous End Joining) studies

  • H1299 EJ2 cells for Alt-NHEJ assessment

  • HeLa Sa26 cells for SSA (Single-Strand Annealing) repair studies

These models enable detailed investigation of how WWOX affects different DNA repair pathways through techniques such as comet assays (for detecting DSBs), immunofluorescence assays for DSB markers (53BP1 and γH2AX), and pathway-specific reporter assays .

What are the neuroimaging findings in patients with WWOX mutations?

Brain MRI studies in patients with WWOX mutations reveal several consistent abnormalities:

• Hypoplasia of the corpus callosum

• Progressive cerebral atrophy

• Delayed myelination

• Optic nerve atrophy

These neuroimaging findings correlate with the severe neurological phenotypes observed in affected individuals, particularly those with WOREE syndrome. The specific pattern of myelination defects seen in human patients is mirrored in mouse models, where hypomyelination is a characteristic feature . This consistency between human and mouse phenotypes supports the validity of mouse models for studying the molecular mechanisms underlying these developmental abnormalities.

How effective are current treatment approaches for WWOX-related disorders?

Current treatment approaches for WWOX-related disorders show limited efficacy:

• Antiepileptic drugs (AEDs): WOREE syndrome is generally refractory to current AEDs, representing a significant unmet medical need. In contrast, epilepsy in SCAR12 patients can be partially managed with available AEDs, though the ataxia and intellectual disability persist .

• Emerging gene therapy approaches: Recent preclinical research has demonstrated that AAV9-mediated delivery of WWOX gene therapy (AAV-SynI-WWOX) can rescue multiple phenotypes in Wwox-null mice:

  • Rescue of brain hyperexcitability and seizures

  • Correction of hypoglycemia

  • Improvement of myelination deficits

  • Prevention of premature lethality

  • Amelioration of behavioral deficits

This proof-of-concept research suggests that neuronal restoration of WWOX expression through gene therapy could potentially treat both WOREE syndrome and SCAR12. The research was conducted using adeno-associated viral vectors (AAV9) carrying either murine Wwox or human WWOX cDNA under the control of the human neuronal Synapsin I promoter, ensuring neuron-specific expression .

How does WWOX influence DNA repair pathway choice?

WWOX plays a complex role in regulating DNA double-strand break (DSB) repair pathway choice, with significant implications for genome stability and cancer therapy resistance:

This pathway regulation suggests that WWOX status might predict tumor response to radiation therapy and certain chemotherapeutic agents, with potential applications in personalized cancer treatment strategies.

What is the relationship between WWOX and genome stability?

WWOX contributes to genome maintenance through several mechanisms:

• Chromosomal stability: Karyotype analysis of Wwox-knockout MEFs revealed specific structural abnormalities not present in wild-type cells, including del(7)(A1B4) and del(4)(C4) .

• Copy number variations (CNVs): Array comparative genomic hybridization analysis identified three distinct deletions shared by different Wwox-knockout MEF lines at chromosome locations 1 H6, 4 B3 and 8 C2, suggesting that Wwox absence leads to specific patterns of genomic instability .

• Response to DNA damage: While Wwox deficiency doesn't appear to cause spontaneous DNA damage (as measured by comet assays and immunofluorescence for DSB markers), it does alter the cellular response to induced damage through its effects on repair pathway choice .

• Tumor suppressor function: The location of WWOX in the chromosomal common fragile site FRA16D suggests its involvement in preventing genomic instability in regions prone to breakage under replication stress .

Understanding these mechanisms is crucial for interpreting how WWOX mutations contribute to both neurodevelopmental disorders and cancer predisposition.

What are the technical considerations for WWOX gene therapy development?

Development of WWOX gene therapy requires addressing several technical considerations:

• Vector design and selection:

  • AAV9 serotype has shown efficacy in crossing the blood-brain barrier and targeting neurons

  • The human neuronal Synapsin I promoter (SynI) provides neuron-specific expression, preventing off-target effects in other tissues

  • Vector capacity must accommodate the WWOX cDNA (~1.1 kb) along with regulatory elements

• Delivery optimization:

  • Timing of intervention appears critical - neonatal administration was effective in mouse models, suggesting early intervention may be necessary for human patients

  • Route of administration (intracranial vs. intravenous) affects distribution and efficacy in the central nervous system

  • Dosing studies are needed to determine the minimum effective dose and therapeutic window

• Safety considerations:

  • Potential immune responses to the viral vector

  • Risk of insertional mutagenesis (though minimal with AAV vectors)

  • Potential off-target effects of WWOX overexpression

  • Long-term stability of transgene expression

The proof-of-concept success in mouse models provides encouragement for further development, but translation to human patients will require additional preclinical studies in larger animal models and careful safety assessment.

How might genetic testing strategies be optimized for WWOX-related disorders?

Optimizing genetic testing strategies for WWOX-related disorders requires consideration of several factors:

• Clinical predictors for targeted testing:

  • The incorporation of epilepsy genetics into clinical practice suggests that clinician prediction of specific genetic causes has limited sensitivity, arguing for parallel gene testing approaches rather than sequential single-gene testing .

  • For patients with early-onset epileptic encephalopathy, severe developmental delay, and brain abnormalities consistent with WOREE syndrome, WWOX should be included in first-line testing panels.

• Testing methodologies:

  • Next-generation sequencing (NGS) panels for epilepsy genes, including WWOX

  • Whole exome sequencing (WES) for cases where targeted panels are negative

  • Copy number variation (CNV) analysis, as some WWOX-related disorders result from partial or complete gene deletions

  • RNA sequencing may be valuable for detecting splicing variants

• Age-specific considerations:

  • Highest diagnostic yield is observed in neonatal and infantile-onset epilepsies

  • Different testing approaches may be appropriate for SCAR12 (later onset) versus WOREE syndrome (early onset)

• Genetic counseling integration:

  • Comprehensive genetic counseling is essential both pre- and post-test

  • Family testing is important as 31% of families with pathogenic variants had an additional affected relative diagnosed after genetic counseling

A multidisciplinary approach involving neurologists, geneticists, genetic counselors, and epileptologists provides the most comprehensive care pathway for patients with suspected WWOX-related disorders.