A Pleiotropic and Functionally Divergent RAC3 Variant Disrupts Neurodevelopment and Impacts Organogenesis

Area of Science:

• Genetics and Molecular Biology
• Developmental Biology
• Neuroscience

Background:

• The RAC3 gene encodes a Rho-family GTPase crucial for cytoskeletal regulation and neurodevelopment.
• De novo RAC3 variants are typically gain-of-function mutations causing severe neurodevelopmental disorders.

Purpose of the Study:

• To investigate the pathogenicity of a novel de novo RAC3 p.(T17R) variant identified in a fetus with multisystem congenital anomalies.
• To elucidate the molecular mechanisms underlying the observed developmental phenotypes caused by the RAC3 p.(T17R) variant.

Main Methods:

• Genome sequencing to identify the RAC3 p.(T17R) variant.
• In silico analyses including variant prioritization, structural modeling, and pathogenicity prediction.
• In vitro biochemical assays (GDP/GTP exchange, GTP hydrolysis, effector pull-down, reporter analyses) and neuronal morphology studies.
• In vivo mouse in utero electroporation to assess cortical neuron development.

Main Results:

• The RAC3-T17R variant showed increased GDP/GTP exchange, impaired GTP hydrolysis, and reduced binding to canonical effectors.
• Transcriptional activation via SRF, NFκB, and AP1 pathways was not observed with the mutant.
• Neuronal overexpression of RAC3-T17R impaired axon formation, cortical neuron migration, and dendritic development.
• The affected fetus exhibited corpus callosum agenesis, microcephaly, organomegaly, and limb contractures.

Conclusions:

• The RAC3 p.(T17R) variant represents a signaling-deficient allele disrupting corticogenesis and organogenesis through variant-specific mechanisms.
• This study expands the understanding of RAC3-related disorders, demonstrating that noncanonical variants can cause complex, multisystem developmental phenotypes.