KCNMA1 genotype-phenotype correlation and functional validation of novel sites

Objective: To analyze genotype-phenotype correlations in patients with KCNMA1 variants and functionally characterize two unreported missense variants: c.853A>T (p.I285F) and c.3196G>A (p.G1066R). Methods: We retrospectively reviewed clinical data from six pediatric patients with KCNMA1 variants. Protein structural modeling and cross-species conservation analysis were performed. HEK293T cells were transfected with wild-type or mutant BK channel constructs. Channel function was assessed using whole-cell patch-clamp electrophysiology to measure current amplitude, current density, and voltage/Ca²⁺sensitivity. Protein expression was evaluated by Western blot analysis of total and membrane fractions. Results: (1) Five patients presented with epileptic seizures, including myoclonic, focal motor/non-motor, generalized tonic-clonic, and atonic seizures; two exhibited drug resistance. Paroxysmal nonkinesigenic dyskinesia (PNKD) was observed in two patients. All five patients with epilepsy demonstrated language impairment or global developmental delay. (2) The residues affected by the mutations, I285 and G1066, are highly conserved across species. Protein three-dimensional structure predictions revealed alterations in the number and distance of hydrogen bonds for both mutations. (3) Under low Ca²⁺ (1 μM), the p.I285F variant significantly reduced BK current amplitude (p < 0.05) and current density across the 50–150 mV range. At high Ca²⁺ (10 μM), current density was increased in the hyperpolarized range (-50 to 0 mV) compared to wild-type. The half-activation voltage (V₁/₂ ) was significantly left-shifted at 1 μM Ca²⁺ (p < 0.05), while the slope factor (k) remained unchanged. (4) At both 1 μM and 10 μM Ca²⁺, p.G1066R did not alter BK current amplitude. However, at 10 μM Ca²⁺, current density at 0 mV was significantly lower than wild-type (p < 0.05). The I-V curve was right-shifted, while V₁/₂ and k values remained unchanged. (5) Total protein levels of both p.I285F and p.G1066R were significantly lower than wild-type (p < 0.05), whereas membrane protein expression did not differ significantly (p > 0.05). Conclusion: KCNMA1 -related disorders commonly present with epilepsy, developmental delay, and PNKD3. p.I285F and p.G1066R are loss-of-function variants, impairing channel activity through reduced current amplitude, altered voltage sensitivity, and lower total protein expression, supporting their pathogenicity.