A large consanguineous family with a homozygous Metabotropic Glutamate Receptor 7 (mGlu7) variant and developmental epileptic encephalopathy: Effect on protein structure and ligand affinity

Epileptic Encephalopathies (EE) are a group of heterogeneous epileptic syndromes associated with severe cognitive stagnation or regression and behavioral disturbances due to frequent epileptiform activity [1]. The cognitive and behavioral impairments are caused by the epileptic activity itself above and beyond what might be expected from the underlying pathological one [1]. Many epileptic encephalopathies are known to have an identifiable molecular genetic basis. The genetic cause often leads to developmental delay on its own, with epilepsy worsening the development. In the latest ILAE classification, the term was changed to developmental encephalopathies and epilepsy (DEE) [2].

Recent advances in high-throughput parallel sequencing technologies allowed the identification of variants in more than 100 genes associated with DEE. Those encoding synaptic proteins include AMPA ionotropic receptor GluA2 subunit [3] and glutamate receptor ionotropic NMDA Type subunit 1(GRIN1) [4], subunit 2A (GRIN2A) [5], or subunit 2B (GRIN2B) [6] and recently the GRM4 and GRM7 genes encoding metabotropic glutamate receptor 4 (mGlu4) and 7 (mGlu7), respectively [7, 8]. The mGlu receptors are G-protein coupled receptors that modulate neurotransmission and synaptic plasticity throughout the central nervous system [9]. Particularly, the mGlu7 protein is a GTP-binding protein–coupled receptor (GPCR) with a heterodimeric structure exclusively expressed in the central nervous system (CNS) with a relatively high expression in the cortex, amygdala, hippocampus, and hypothalamus [10, 11]. The mGlu7 dimer contains two large extracellular domains called the Venus flytrap domains (VFD) containing the glutamate-binding site and cysteine-rich domains (CRDs), seven transmembrane-spanning domains called heptahelical domains (HD) and a C-terminal intracellular domain. Conformational changes induced by ligand binding to the mGlu7 allowed the propagation of signals from the VFD via CRDs to the HD domain and the C-terminal tail [9]. Indeed, mGlu7 plays a critical role in synaptic transmission in neurons where it can act as an auto- or hetero-receptor by inhibiting further release of excitatory neurotransmitter glutamate and inhibitory neurotransmitter GABA, respectively [9, 12, 13].

The GRM7 gene encodes the mGlu7 receptor belonging to mGlu's type III receptors, which are G-protein coupled receptors that modulate neurotransmission and synaptic plasticity throughout the central nervous system. Indeed, recent studies showed that mGlu's type III including mGlu4 and mGlu7 receptors are associated with neurodevelopmental disorders. De novo duplication in GRM4 gene was found in a patient with a severe psychomotor retardation, epilepsy, mild dysmorphic features and behavioral disturbances [20]. The GRM4 gene variants were also described as associated with juvenile myoclonic epilepsy, characterized by myoclonic jerks, absence and generalized seizures [21, 22]. Nevertheless, pathogenic GRM7mutations were reported in patients diagnosed with neurodevelopmental disorders [23‐25]. Recently, Marafi et al. described biallelic variants of GRM7 in 11 patients belonging to six families with developmental and epileptic encephalopathy (DEE). Most patients in their cohort had a severe developmental impairment, early-onset seizures and frequent abnormal epileptiform activity revealed by EEG and severe neurological phenotype. Based on the clinical presentation of our patients and those described by Marafi et al. (Table 2), we noticed similar features including an early onset of seizure, microcephaly and cortical atrophy, developmental delay and intellectual disability [7]. Epilepsy was polymorphic and could be generalized or focal with age-dependent electro-clinical syndromes (West syndrome then evolves into a Lennox Gastaut syndrome for the index case). For all the family members and 6/9 patients from the cohort studied by Marafi et al., epilepsy is drug resistant. Two of our patients (index case and case VII) had no seizure after the first decade but with a worsened cognitive deficit and behavioral disorders. All these elements are consistent with the diagnosis of developmental encephalopathy and epilepsy. Serial EEG showed a slowing background with focal or multifocal epileptiform activity and a less frequently generalized slow spike and slow wave pattern ([7]; Tables 1, 2). Stereotypes of the hands are neither constant nor specific. Facial dysmorphia was present in only one patient and was similar to that in our index patient. Brain imaging was also nonspecific in the patients described by Marafi et al. where most frequent signs were cortical atrophy, hypomyelination and hypoplasia of the corpus callosum, whereas MRI results showed cortical and sub-cortical atrophy, a thin corpus callosum without white matter abnormality in the index case of the studied family but hypomyelinisation was noticed for her cousin.

In addition to this phenotypic heterogeneity associated to GRM7 mutations described in the Marafi et al. series, an allelic variability was also noticed [7]. In fact, these mutations could be located anywhere in the gene including the transmembrane domain and the ligand-binding domain [7]. In the present study, the c.1411G>A p. (Gly471Arg) variant is located in the conserved VFTD N-terminal sub-domain of the protein which plays a crucial role in glutamate binding [9, 26]. According to the bioinformatic tools, the p.Gly471Arg missense variant leads to conformational changes of the N-terminal domain and the modification of hydrogen bonds that probably disturb the mGlu7 protein folding and stability. Furthermore, docking analysis performed in the large cavity that forming the glutamate-binding site in the mutated protein showed that the p.Gly471Arg variant caused complete burial of ligand inside the binding pocket of the mGlu7 protein. Indeed, the replacement of hydrophobic (Glycine) by hydrophilic (Arginine) amino acid caused by the p.Gly471Arg variant might decrease glutamate binding [27]. Thus, based on our bioinformatic and docking analyses and according to recently reported functional studies, we suggest that the misfolded mGlu7-Gly471Arg protein might be degraded via the proteasomal and/or autophagosomal-lysosomal pathway. Indeed, functional studies were performed on mGlu7 proteins mutated in the VFTD extracellular dimerization domain [28, 29], comparable with the mutation observed in our case. These studies showed that the p.I154T mutation disrupted the mGlu7 receptor dimerization, caused a post-transcriptionally reduced expression level of mGlu7 I154T and impaired its trafficking towards the neuronal cell surface to bind to the ligand [28, 29].

The binding of glutamate to the VFTD domain is crucial to initiating conformational changes through extracellular cysteine-rich domains (N-terminal domain) and then in the transmembrane and cytoplasmic domains of the mGlu7 receptor leading to correct synaptic transmission [9, 26]. In fact, the c.1411G>A (p.Gly471Arg) mutation in the N-terminal domain of the mGlu7 could therefore disturb the signaling pathway and subsequently alter synaptic transmission. This is consistent with the reported results of Song et al. who demonstrated a marked decrease of the axon outgrowth of GRM7 variants in the primary cultures of neurons compared to WT, consequently leading to a decrease of presynaptic terminations in mature neurons [28]. On the other hand, Fisher et al. investigated the mechanistic links between mutations located in the VFTD domain of mGlu7 and the NDD phenotypes using mGlu7-I154T knock in mice. Indeed, GRM7^I154T/I154T mice exhibited a significant loss in body weight, locomotor disorders, convulsive seizures, and brain weight and corpus callosum reductions [29]. Overall, these clinical and functional data demonstrate that mutations in the VFTD domain of the mGlu7 receptor should be considered as a potential cause of developmental and epileptic encephalopathy disease phenotypes.