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Using whole exome sequencing, Fassio et al. identify de novo mutations in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathies and epilepsy. Functional and expression studies demonstrate impaired lysosomal homeostasis, defective neurite elongation and loss of excitatory inputs in cultured neurons., V-type proton (H+) ATPase (v-ATPase) is a multi-subunit proton pump that regulates pH homeostasis in all eukaryotic cells; in neurons, v-ATPase plays additional and unique roles in synapse function. Through whole exome sequencing, we identified de novo heterozygous mutations (p.Pro27Arg, p.Asp100Tyr, p.Asp349Asn, p.Asp371Gly) in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathy with epilepsy. Early manifestations, observed in all patients, were developmental delay and febrile seizures, evolving to encephalopathy with profound delay, hypotonic/dyskinetic quadriparesis and intractable multiple seizure types in two patients (p.Pro27Arg, p.Asp100Tyr), and to moderate delay with milder epilepsy in the other two (p.Asp349Asn, p.Asp371Gly). Modelling performed on the available prokaryotic and eukaryotic structures of v-ATPase predicted p.Pro27Arg to perturb subunit interaction, p.Asp100Tyr to cause steric hindrance and destabilize protein folding, p.Asp349Asn to affect the catalytic function and p.Asp371Gly to impair the rotation process, necessary for proton transport. We addressed the impact of p.Asp349Asn and p.Asp100Tyr mutations on ATP6V1A expression and function by analysing ATP6V1A-overexpressing HEK293T cells and patients’ lymphoblasts. The p.Asp100Tyr mutant was characterized by reduced expression due to increased degradation. Conversely, no decrease in expression and clearance was observed for p.Asp349Asn. In HEK293T cells overexpressing either pathogenic or control variants, p.Asp349Asn significantly increased LysoTracker® fluorescence with no effects on EEA1 and LAMP1 expression. Conversely, p.Asp100Tyr decreased both LysoTracker® fluorescence and LAMP1 levels, leaving EEA1 expression unaffected. Both mutations decreased v-ATPase recruitment to autophagosomes, with no major impact on autophagy. Experiments performed on patients’ lymphoblasts using the LysoSensor™ probe revealed lower pH of endocytic organelles for p.Asp349Asn and a reduced expression of LAMP1 with no effect on the pH for p.Asp100Tyr. These data demonstrate gain of function for p.Asp349Asn characterized by an increased proton pumping in intracellular organelles, and loss of function for p.Asp100Tyr with decreased expression of ATP6V1A and reduced levels of lysosomal markers. We expressed p.Asp349Asn and p.Asp100Tyr in rat hippocampal neurons and confirmed significant and opposite effects in lysosomal labelling. However, both mutations caused a similar defect in neurite elongation accompanied by loss of excitatory inputs, revealing that altered lysosomal homeostasis markedly affects neurite development and synaptic connectivity. This study provides evidence that de novo heterozygous ATP6V1A mutations cause a developmental encephalopathy with a pathomechanism that involves perturbations of lysosomal homeostasis and neuronal connectivity, uncovering a novel role for v-ATPase in neuronal development.

Recently, de novo mutations in the gene KCNA2, causing either a dominant-negative loss-of-function or a gain-of-function of the voltage-gated K+ channel Kv1.2, were described to cause a new molecular entity within the epileptic encephalopathies. Here, we report a cohort of 23 patients (eight previously described) with epileptic encephalopathy carrying either novel or known KCNA2 mutations, with the aim to detail the clinical phenotype associated with each of them, to characterize the functional effects of the newly identified mutations, and to assess genotype-phenotype associations. We identified five novel and confirmed six known mutations, three of which recurred in three, five and seven patients, respectively. Ten mutations were missense and one was a truncation mutation; de novo occurrence could be shown in 20 patients. Functional studies using a Xenopus oocyte two-microelectrode voltage clamp system revealed mutations with only loss-of-function effects (mostly dominant-negative current amplitude reduction) in eight patients or only gain-of-function effects (hyperpolarizing shift of voltage-dependent activation, increased amplitude) in nine patients. In six patients, the gain-of-function was diminished by an additional loss-of-function (gain-and loss-of-function) due to a hyperpolarizing shift of voltage-dependent activation combined with either decreased amplitudes or an additional hyperpolarizing shift of the inactivation curve. These electrophysiological findings correlated with distinct phenotypic features. The main differences were (i) predominant focal (loss-of-function) versus generalized (gain-of-function) seizures and corresponding epileptic discharges with prominent sleep activation in most cases with loss-of-function mutations; (ii) more severe epilepsy, developmental problems and ataxia, and atrophy of the cerebellum or even the whole brain in about half of the patients with gain-of-function mutations; and (iii) most severe early-onset phenotypes, occasionally with neonatal onset epilepsy and developmental impairment, as well as generalized and focal seizures and EEG abnormalities for patients with gain- and loss-of-function mutations. Our study thus indicates well represented genotype-phenotype associations between three subgroups of patients with KCNA2 encephalopathy according to the electrophysiological features of the mutations.