Identification of a new gain-of-function variant p.N536Y of KCNMA1 associated with PNKD3 in the Chinese population

Abstract

The high-conductance calcium- and voltage-dependent K⁺ potassium channels (BK channel) are important for the electrical excitation of nervous and muscular tissues.¹ In 2005, we identified the first disease-causing variant in the gene encoding the α-subunit KCNMA1 (p.D434G) in this channel and showed that the gain-of-function (GOF) variant of KCNMA1 causes paroxysmal nonkinesigenic dyskinesia with or without generalized epilepsy (PNKD3, MIM #609446).² In 2019, we showed that loss of function variants cause Liang-Wang syndrome (MIM #618729) with neurodevelopmental dysfunction of developmental delay, impaired intellectual development, poor or absent language ability, epilepsy, ataxia, and other multi-organ defects.³ Both PNKD3 and Liang-Wang syndrome are phenotypically heterogeneous, and detailed genotype–phenotype correlation is critical to genetic testing and counseling.

To date, only three KCNMA1 variants were shown to cause PNKD3, including p.D434G, p.N995S, and p.N536H.^2-10 Here, we report KCNMA1 variants in two new Chinese patients, including a novel variant p.N536Y in the same amino acid as p.N536H and the previously reported variant N995S. In the first patient, PNKD was diagnosed at an age of 2 years and 10 months (Figure 1A), but bilateral hydronephrosis was found at birth. Her developmental milestones were normal during the first 6 months, but then began to slow, and were manifested as low alertness, delayed language acquisition, and poor ability to communicate. When 6 months old, she was admitted to an intensive care unit (ICU) due to high potassium and low sodium, and she developed paroxysmal dyskinesia at the age of one that was successfully treated with carbamazepine, topiramate, and nitrazepam that reduced the frequency and severity of attacks. Whole exome sequencing, also of her parents, identified the de novo KCNMA1 variant c.1606A>T, p.N536Y (NM_002247) (Figure 1A,B). The N536 amino acid is highly conserved among different species through evolution (Figure 1C) and located in the Ca²⁺-sensing RCK1 domain (Figure 1D).

Patch-clamping revealed that variant p.N536Y significantly increases the mean amplitude of I_BK (Figure 1E) and shifts the G-V curves to more negative potentials by −67 mV at 10 μM [Ca²⁺] (Figure 1E,F), demonstrating that KCNMA1 variant p.N536Y is a functional gain-of-function variant. Compared with BK-WT channels, the G-V curves of mutant channels shifted to more negative potentials at all Ca²⁺ concentrations. The shift was approximately −105 mV in the presence of 0 μM intracellular [Ca²⁺], −78 mV in the presence of 1 μM intracellular [Ca²⁺], and approximately −67 mV in the presence of 10 μM intracellular [Ca²⁺] (Figure 1G). The data suggest that variant p.N536Y causes a small degree of reduced sensitivity of the mutant BK channels to Ca²⁺-dependent activation (Figure 1G–J). According to the ACMG guidelines, we classified the p.N536Y variant as a pathogenic variant.

The second patient was an 8-year-old boy with onset of PNKD at the age of one and epilepsy at 8 years. The boy was born at 40 weeks of gestation from non-consanguineous parents with a history of hypoxia. Both intellectual and motor development were delayed, and his academic performance was poor. He was admitted to a hospital for treatment of convulsions. Repeated video EEG revealed bilateral frontal and central discharges during sleep, suggestive of epilepsy. In the past 7 years, levetiracetam, nitrazepam, carbamazepine, and tobiate were used, but did not work well. The de novo p.N995S variant was identified by whole exome sequencing and confirmed by Sanger sequencing (Figure 1H,I). Electrophysiological studies of the p.N995S variant showed enhanced voltage-dependent activation of BK channels but minimal effect on the Ca²⁺-dependent activation of BK channels.^{5, 8} Previous studies also defined p.N995S as a pathogenic variant based on ACMG guidelines.^{5, 8}

Phenotypically, the onset of PNKD in patients with variant p.D434G occurred later than in the other three variants.^{2, 4-10} The age of onset for epilepsy did not differ much among the patients carrying KCNMA1 gain-of-function variants. Patients with p.N995S showed the most severe clinical features, whereas those with p.D434G showed less severe features. Thirteen patients in one family carrying variant p.D434G were reported; one showed epilepsy, seven showed PNKD, and five had both.² Fourteen reported patients carrying variant p.N995S showed the most heterogeneous phenotypes, including PNKD (10/14), epilepsy (9/14), developmental delay/intellectual disability/cognitive disorders (8/14), and cataplexy (3/14). The onset age of PNKD was between 7 months and 2 years.^{2, 4-10} The four patients with variant p.N536Y or p.N536H also showed similar phenotypic heterogeneity as those with p.N995S, including PNKD, epilepsy, and developmental delay/intellectual disability/cognitive disorders, as well as dystonia and autism spectrum disorder (Figure 1K).

All four gain-of-function KCNMA1 variants had increased voltage-dependent activation, but different calcium sensitivities. Variant p.D434G is located in the RCK1 Ca²⁺-binding domain and enhances both voltage-dependent activation and Ca²⁺ sensitivity.^{2, 5} Variant p.N995S is located in RCK2 and increases voltage-dependent activation without affecting Ca²⁺-dependent activation or Ca²⁺ sensitivity.^{2, 5} Variants p.N536Y and p.N536H are located at the same amino acid residue in the Ca²⁺-sensing RCK1 domain, increase voltage sensitivity but slightly decrease Ca²⁺-dependent activation (Figure 1J). The effects of variant p.N536Y on Ca²⁺ sensitivity are more pronounced than those of variant p.N536H (G-V curve shift: −105.06 mV vs. −91.00 mV at 0 μM [Ca²⁺], −78.44 mV vs. −78.68 mV at 1 μM [Ca²⁺], and −67.46 mV vs. −58.96 mV at 10 μM [Ca²⁺]) (Figure 1J and Liang et al.⁸). Analysis of the 3D structure of the BK channel showed that D434 is located close to the Ca²⁺-binding site in RCK1, whereas N536 and N995 are located far away from the Ca²⁺-binding sites in RCK1 and RCK2, respectively.⁹ The spatial location to the Ca²⁺-binding sites may explain the differences in the four variants' effects on Ca²⁺-sensitivity of BK channels.

The identification of the fourth KCNMA1 variant causing PNKD3, p.N536Y, significantly expands the spectrum of the genotypes associated with PNKD3. The identification of two different variants at the N536 residue suggests that N536 is a mutation hotspot in the BK channel. There are three patients reported to carry the p.N536H variant.^8-10 The three patients showed phenotypic heterogeneity (Figure 1K). Our patient with the p.N536Y variant had bilateral hydronephrosis, high potassium, and low sodium. These data significantly expand the spectrum of phenotypes associated with KCNMA1 gain-of-function variants. The genotype–phenotype correlation studies provide important input to genetic testing and counseling of carriers with KCNMA1 variants.

Yufeng Huang: Writing – original draft; methodology; data curation; formal analysis. Lina Liang: Methodology; data curation. Xuebin Xu: Data curation; resources. Yin Liu: Data curation; resources. Feng Gao: Data curation; resources. Xuelian He: Conceptualization. Qing K. Wang: Conceptualization; writing – review and editing.

This study was supported by the National Key R&D Program of China (2023YFA1800901), the China National Natural Science Foundation grant (No. 32270662 and 82302107) and Wuhan Municipal Health Commission Young Talents “Chen Xing” Project (2023).