Persistent toe walking is frequently labeled idiopathic; however, targeted genetic testing in selected cohorts can identify variants in genes implicated in neuromuscular disease. SBF1 is a known cause of autosomal recessive Charcot-Marie-Tooth disease type 4B3 (CMT4B3), whereas the clinical relevance of heterozygous SBF1 variants-particularly variants of uncertain significance (VUS)-remains unclear. We aimed to describe, in an exploratory manner, the clinical features of children with persistent toe walking in whom heterozygous SBF1 variants were identified, and to contextualize these observations using published CMT4B3 families and Human Phenotype Ontology (HPO) feature frequencies. We retrospectively analyzed children referred to a specialized toe-walking clinic who underwent a standardized blinded clinical assessment and targeted 49-gene next-generation sequencing. Individuals with alternative sequencing approaches or known non-genetic causes of toe walking were excluded. Heterozygous SBF1 variants were summarized using HGVS nomenclature, ACMG classification, population allele frequency, and report date. Phenotypic frequencies were compared with published SBF1-related CMT4B3 families and with HPO-reported feature frequencies for CMT4B3. The cohort comprised 86 children (mean age 9.5 years), all with persistent toe walking. Common findings included skeletal features (e.g., pes cavus and lumbar hyperlordosis), whereas muscle weakness and deep tendon reflex abnormalities were less frequent than reported in recessive CMT4B3 families. Genetic testing identified a spectrum of heterozygous SBF1 variants, predominantly classified as VUS. In this referral-based cohort, heterozygous SBF1 variants were observed in children with persistent toe walking and accompanying mild neuromotor/musculoskeletal features that partially overlap with reported CMT4B3 phenotypes; however, these findings are descriptive and do not establish causality or enrichment. Longitudinal follow-up, segregation/phase determination, and electrophysiological studies are needed to clarify clinical significance, potential biallelic configurations in some individuals, and possible gene-dosage or modifier effects.

Charcot-Marie-Tooth disease type 4B3 (CMT4B3) is a rare autosomal recessive neuropathy caused by biallelic MTMR5/SBF1 variants, which encode a catalytically inactive myotubularin involved in phosphoinositide metabolism and autophagy regulation. This study investigates the impact of MTMR5/SBF1 dysfunction on autophagy and mitophagy in patient-derived fibroblasts and examines the relationship between protein aggregates and autophagic machinery. Fibroblasts from a CMT4B3 patient with compound heterozygous MTMR5/SBF1 mutations were compared with a healthy control. Autophagic flux was analyzed via LC3B and SQSTM1; mitophagy was assessed through PINK1 and PRKN recruitment and by quantifying mitophagosomes and autolysosomes under mitochondrial stress. Protein aggregates were visualized using Proteostat and tested for colocalisation with autophagic structures. CMT4B3 fibroblasts showed normal basal macroautophagy but failed to increase autophagy in response to mitochondrial stress or protein aggregates. Conversely, mitophagy was strongly activated via the PINK1-PRKN pathway. These results reveal an uncoupling between mitophagy and macroautophagy, indicating that MTMR5/SBF1 mutations modify autophagic selectivity. Our findings provide new mechanistic insights into the pathogenesis of CMT4B3 and highlight the value of patient-derived fibroblasts for studying selective autophagy defects.

Biallelic loss of expression/function variants in MTMR5/SBF1 cause the inherited peripheral neuropathy Charcot-Marie-Tooth type 4B3. There is an incomplete understanding of the disease pathomechanism(s) underlying Charcot-Marie-Tooth type 4B3, and despite its severe clinical presentation, currently no disease-modifying therapies. A key barrier to the study of Charcot-Marie-Tooth type 4B3 is the lack of pre-clinical models that recapitulate the clinical and pathologic features of the disease. To address this barrier, we generated a zebrafish Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 mutant line with a full gene deletion of mtmr5. Resulting homozygous deletion zebrafish are born at normal Mendelian ratios and have preserved motor function. However, starting by 10 days post-fertilization, mutant zebrafish develop obvious morphometric changes in head size and brain volume. These changes are accompanied at the pathological level by abnormal axon outgrowths and by the presence of dysmyelination changes reminiscent of the nerve pathology in human Charcot-Marie-Tooth type 4B3. Importantly, RNA sequencing from brain-enriched samples identifies novel disease pathways including transcriptional changes in genes responsible for neurogenesis, chromatin remodelling/organization, and synaptic membrane homeostasis. Overall, our mtmr5 knockout zebrafish mirror genetic, clinical and pathologic features of human Charcot-Marie-Tooth type 4B3. As such, it represents a first pre-clinical model to phenocopy the disease, and an ideal tool for future studies on disease pathomechanism(s) and therapy development.

We present a case of autosomal dominant Charcot-Marie-Tooth disease type 4B3 (CMT4B3) in a family caused by a novel SBF1 missense mutation. Two patients, a mother and daughter, were recruited from our hospital. Both exhibited early-onset symptoms, including distal muscle atrophy of the limbs, without cranial nerve involvement. Electromyography was performed to assess nerve amplitudes and conduction velocities. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify genetic mutations. Electromyography revealed a significant decline in nerve amplitudes, while the nerve conduction velocities (NCVs) remained normal in the extremities. Sequencing identified a novel missense mutation (c.1398C > A, p.H466Q) in exon 13 of the SET binding factor 1 (SBF1) gene in both patients, indicating an autosomal dominant inheritance pattern. Pathogenicity and protein predictions suggest that the myotubularin-related protein 5 (MTMR5), encoded by the mutated SBF1, may possess an altered structure, resulting in disease. These findings will help expand the phenotypic and genetic spectrum of CMT4B3.

Myotubularin-Related Protein 5 (MTMR5) is an inactive, poorly characterized D3-phosphatidylinositol phosphatase. Mutations in MTMR5 have been linked to Charcot-Marie-Tooth Disease Type 4B3 (CMT4B3), a rare, early-onset, recessive peripheral neuropathy. Here, we describe the establishment and validation of three human induced pluripotent stem cell (iPSC) lines derived from unrelated CMT4B3 patients, each harboring homozygous MTMR5/Sbf1 mutations. Current MTMR5 -/- animal models do not clearly link Sbf1 mutations to severe neuropathy, so such a resource is highly desired to further elucidate the relationship between MTMR5 dysfunction and peripheral nerve degeneration.