c. Function-based therapies
Mutations falling upon genes involved in ion channel functions including sodium (Na+), calcium (Ca+), and potassium K+) collectively are responsible for a considerable proportion of infantile and childhood onset drug resistant genetic epilepsies and DEE (El Achkar et al 2015; Parrini et al 2017; Stödberg et al 2020; Alsubaie et al 2020; Willimsky et al 2021). The functional mechanisms underlying Na+ channel ion genes including SCN1A, the most relevant epilepsy gene, SCN8A and SCN2A, and their implications for treatment selection has been highlighted in the previous section especially in relation to sodium-channel blockers ASM.
Potassium channels play an important role in regulation of neuronal excitability. Pathogenic variants have been identified in several potassium channel genes including KCNA2, KCNB1, KCNC1, KCND2, KCND3, KCNH1, KCNH2, KCNH5, KCNJ10, KCNMA1, KCNQ2, KCNQ3, and KCNT1 and have been associated with a variety of epilepsy phenotypes from mild to severe conditions (Weckhuysen et al 2012; Marini et al 2017; Corbett et al 2016; McTague A. et al 2018; Symonds JD, McTague 2020; Nikitin, Vinogradova 2021)
In the first section we discussed how, seizures related to KCNQ2mutations, might be suppressed by ASM acting as sodium channel blockers. Here we review novel therapeutic strategies based upon better understanding KCNQ2-DEE pathophysiological mechanisms. For instance retigabine is a selective Kv7.2/3 (voltage-dependent neuronal potassium channel) activator and antagonizes the functional impairment caused byKNCQ2 mutations leading to a channel loss of function (Orhan et al.2014, Miceli et al 2018). A multicentre study including 11 patients proved the clinical efficacy and usefulness of retigabine in the treatment of KCNQ2 encephalopathy (Millichap et al., 2016). Unfortunately, the retigabine preparation originally approved for the treatment of focal seizures has been withdrawn from the market. Recently, a case report showed that gabapentin might also be regarded as a precision therapy for epileptic encephalopathy due to KCNQ2 loss-of-function mutations (Soldovieri et al 2020). It should be emphasized that there are KCNQ2-DEE caused by gain-of-function variants (Devaux et al., 2016; Millichap et al., 2017). Therefore, evaluation of the specific variant is important for treatment selection, because Kv7.2/3 activators such as retigabine and gabapentin could be aggravating when used in patients with gain-of- function mutations. Extensive research is ongoing to identify novel molecules endowed with different actions on potassium channels.
Heterozygous, mostly de novo, variants in KCNT1 gene, encoding the potassium channel KNa1.1, play a causative role in a wide spectrum of seizure disorders including epilepsy of infancy with migrating focal seizures (EIMFS), severe ADSHE (Barcia et al 2012; Heron et al 2012). All gene variants evaluated to date seem to lead to a gain-of-function phenotype, irrespective of the type of associated epilepsy (Barcia et al 2019; McTague et al 2018). Seizures associated with KCNT1-related epilepsies are often severe and drug-resistant. Quinidine is a class Ia antiarrhythmic and antimalarial drug which operates as a partial antagonist of KCNT1 sodium-activated potassium channel. In 2014 Bearden et al reported the first clinical evidence that quinidine may be an effective medication in mutations causing a gain of function leading to constitutive activation with a pharmacologic inhibition of the channel (Bearden et al 2014). A patients with EIMFS carrying an activating mutation in KCNT1 during treatment with quinidine had a dramatic reduction in seizure frequency. Additional two patients carrying KCNT1 gain of function mutations were treated with quinidine that was able to reverse channel function leading to a seizure reduction (Milligan et al 2014). Furthermore, Mikati et al in 2015, described a reduction of 80% of seizure frequency in a 3-years –old male with EIMFS due to a de novo, gain of function, KCNT1 mutation (Mikati et al 2015). In the same paper they described a second patient, 11-years-old female with a de novo, gain of function KCNT1 mutation and nocturnal seizures, who did not respond to quinidine. Further reports of patients carrying KCNT1 mutations treated with quinidine without success are on record (Chong et al 2016; Barcia et al 2019; Mullen et al 2018; Borlot et al 2020). Thus, since the efficacy of quinidine is unclear and controversial its use in daily practice is at present not recommended and needs further evaluation.
Beside voltage-gated ion channel genes and mutations disrupting ion influx and efflux through cell membrane ultimately leading to an abnormal neuronal excitability, an unbalance between excitation and inhibition can also be caused by mutations of genes involved in neurotransmitter functions and homeostasis including their release, re-uptake and production.
For instance, glutamate-mediated excitatory neurotransmission is partly mediated by activation of NMDA receptors. These cation channel-receptors are made up of several subunits. Mutations of GRIN1, GRIN2A, GRIN2B, and GRIN2D genes, which encode the NMDA receptors subunits, have associated with epileptic phenotypes (Carvil et al 2013; Lemke et al 2013; Lesca et al 2013; Lemke et al 2014). Memantine is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist currently used for the treatment of Alzheimer’s disease, the drug seems to also have an anticonvulsant effect of in some epilepsy animal models (Ghasemi M, Schachter 2011) mechanism of action seems to be related to NMDA receptor activity. In 2014 Pierson and colleagues, reported a 6, 5 years old patient with epileptic encephalopathy due to a de novo variant inGRIN2A gene (Pierson et al 2014). Mutant NMDA receptors were expressed in Xenopus laevis oocytes and treated with memantine. Exposure to memantine was shown to restore receptor proper function. Following this successful experimental testing the boy was treated with memantine which to led to a dramatic reduction of seizure and an improvement in the interictal EEG recordings (Pierson et al 2014). Further patients carrying GRIN2D and GRIN2B mutations were also treated with memantine without the same exciting results (Li et al 2016; Platzer et al 2017). Similarly to quinidine, the efficacy of memantine is unclear and its use in patients is at present not indicated.
Genes related to inhibitory GABAA neurotransmitters also play a key role in genetic epilepsies including genetic generalized epilepsies (GGE) and several DEE. Mutations in genes coding for subunits of GAABA includingGABRG2; GABRA1; GABRB3, GABRD are included amongst the long list of epilepsy genes (Dibbens et al 2004; El Achkar et al 2015; Parrini et al 2018; May et al 2018; Stödberg et al 2020; Alsubaie et al 2020; Willimsky et al 2021). Vinpocetine is an alkaloid derived from the periwinkle plant its pharmacological effects of include vasodilation antioxidation, anti-inflammation, synaptic modulation, and antithrombosis properties (Kiss, Karpati 1996). A single patient with Lennox-Gastaut syndrome carrying a GABRB3 (c.905A>G; Y302C) missense mutation was supplemented with Vinpocetine leading to reduction of epileptiform activity on EEG recordings and improvement of cognitive, behavioral and language functions (Billakota et al 2019). Vinpocetine passes through the blood-brain barrier and inhibits voltage–gated calcium channels, glutamate receptors, and voltage-dependent Na-channels (Bönöczk et al 2000). Antiepileptiform effects of vinpocetine could also derive from the above, plus its inhibition of sodium channels and potentiation of GABA activity (Billakota et al 2019).
Lafora disease is a severe, autosomal recessive, progressive myoclonus epilepsy, caused by loss of function mutations in EPM2A or NHLRC1 genes, encoding laforin and malin (Minassian et al 1998; Chan et al 2003). Lafora disease is invariably fatal, typically within 10 years. The absences or either proteins results in abnormal glycogen metabolism, abnormally long branches are generated by glycogen synthase, leading to glycogen insolubility that precipitate, aggregates and accumulates to form the Lafora Bodies (Nitschke et al 2018). Metformin, the most commonly prescribed drug for type 2 diabetes mellitus, is an activator of AMP-induced kinase (AMPK). In mice and rats, metformin was shown to have positive effects on neuronal survival and seizure termination (Dulovic et al 2014; Ashabi et al 2014; Yang et al 2017). Studies in a mouse model of Lafora disease showed that metformin ameliorated neuropathological symptoms, reduced seizure susceptibility and slightly reduced the numbers of Lafora bodies (Berthier et al 2016; Sanchez- Elexpuru et al 2017). In 2019 Bisulli et al described the efficacy of metformin in Lafora Disease in reduction of seizure frequency and global clinical improvement (of twelve treated patients, three had a clinical response, which was temporary in two) underlining the neuroprotective action of metformin (Bisulli et al 2019).
‘Girls clustering epilepsy’, originally described as ‘female only epilepsy with cognitive impairment’, is a rare disorder combining late infantile onset of clusters of focal seizures, usually occurring during febrile episodes, with cognitive impairment and frequent autistic traits (Depienne et al 2009, Marini et al 2012; Kolc et al 2020). In 2008, Dibbens et al linked this disorder to PCDH19 gene mutations (Dibbens et al 2008). Functional studies showed that PCDH19 gene mutations lead to deficiency of the neurosteroid allopregnanolone, one of the most potent GABA-receptor modulators (Marini et al 2012; Tan et al 2015). These findings provide evidence for a role of neurosteroids in PCDH19-related epilepsy and create realistic opportunities for targeted therapeutic interventions. Ganaxolone, a synthetic methyl analogue of allopregnanolone, has already been used to treat epilepsy and status epilepticus related to various aetiologies in open-label pilot studies, with encouraging results in some patients (Pieribone et al 2007; Kerrigan et al 2000) however, no randomized studies are available (Lattanzi et al 2021).
Bi-allelic loss-of-function variants in GAD1 cause a syndromic infantile onset developmental and epileptic encephalopathy presenting around the second months of life with epileptic spasms or myoclonic seizures (Chatron et al 2020). The GAD1 gene encodes an enzyme that catalyses the conversion of glutamic acid to GABA. Mutations cause a loss the enzyme of function thus the impaired GABAergic inhibitory function and excess of excitation might underlie neuronal hyperexcitability and seizures. A combination of KD and GVG both enhancing GABA function and availability have been shown to improve clinical outcome in these patients (Chatron et al 2020, von Hardenberg et al 2020 and Neuray et al 2020).
Table 2, here below, summarizes the above reported evidences