Mutations in MYO9B are associated with Charcot–Marie–Tooth disease type 2 neuropathies and isolated optic atrophy

**Background:** Charcot–Marie–Tooth disease (CMT) is a heterogeneous group of inherited peripheral neuropathies caused by mutations in at least 100 genes, yet approximately 60% of axonal forms (CMT2) remain genetically undiagnosed. Optic atrophy (OA) can co-occur with CMT, but 50–60% of OA cases lack a genetic diagnosis. MYO9B encodes an unconventional myosin motor protein with RhoGAP activity, but it had not been associated with human disease. This study aimed to identify novel disease genes for CMT2 and OA.

**Methods:** Whole exome sequencing (WES) was performed on 15 unrelated Italian families with autosomal recessive HMN/CMT2. Additional screening was conducted via the Inherited Neuropathy Consortium database (1790 CMT2 and 161 distal HMN families) and a custom NGS panel of 72 patients with isolated OA of unknown origin. Variants were confirmed by Sanger sequencing. Functional studies included Western blot analysis of patient fibroblasts, live cell imaging of NIH/3T3 cells expressing mutant MYO9B constructs, and filopodia tip accumulation assays. Myo9b knockout mice were analyzed by light and electron microscopy of sciatic and optic nerves.

**Key Results:** In Family A, two siblings with CMT2 carried a homozygous c.526T>C (p.Tyr176His) variant in MYO9B. In Family B, two sisters with CMT2 were compound heterozygous for c.188A>G (p.Tyr63Cys) and c.241_243del (p.Arg81del). All four patients had onset before age 20, slow progression to moderate–severe disability (CMTNSv2 scores 16–31), and electrophysiological findings of sensory–motor polyneuropathy with reduced amplitudes and intermediate conduction slowing. In a 28-year-old woman with isolated OA, compound heterozygous variants c.2972G>A (p.Arg991Gln) and c.3082A>G (p.Ile1028Val) were identified in trans. Functional analysis of the p.Tyr176His variant showed significantly reduced MYO9B protein expression in patient fibroblasts (p = 0.0009) and impaired motor activity: the mutant failed to accumulate at Rac1-induced lamellipodia and filopodial tips, similar to a known motor-dead mutant (G244R). Myo9b knockout mice exhibited degenerating myelinated axons in sciatic nerves at 12 months (p = 0.0089) and a decreased density of myelinated axons in optic nerves (p = 0.0145), with no significant change in g-ratio (p > 0.9999). MYO9B was expressed in Schwann cells, axons, spinal cord glia, retinal ganglion cells, and photoreceptors.

**Clinical Implications:** This study establishes MYO9B as a novel disease gene for autosomal recessive CMT2 and isolated OA. The findings provide a genetic diagnosis for previously undiagnosed patients and expand the spectrum of inherited neuropathies. The association with OA suggests that MYO9B should be considered in genetic testing for isolated optic atrophy, especially when common causes (OPA1, LHON) are excluded. The functional data indicate that loss of MYO9B motor activity and reduced protein expression underlie the pathogenesis, likely through dysregulation of RhoA signaling and cytoskeletal remodeling in neurons and glia. Further studies are needed to confirm the role of MYO9B in OA and to explore potential therapeutic targets.