Epilepsy and BRAF Mutations: Phenotypes, Natural History and Genotype-Phenotype Correlations

Abstract

Objective: Cardiofaciocutaneous syndrome (CFCS) is a rare developmental disorder caused by upregulated signaling through the RAS-mitogen-activated protein kinase (MAPK) pathway, mostly resulting from de novo activating BRAF mutations. Children with CFCS are prone to epilepsy, which is a major life-threatening complication. The aim of our study was to define the natural history of epilepsy in this syndrome and exploring genotype-phenotype correlations.

Methods: We performed an observational study, including 34 patients with molecularly confirmed diagnosis (11 males, mean age: 15.8 years). The mean follow-up period was 9.2 years. For all patients, we performed neurological examination, cognitive assessment when possible, neuroimaging, electrophysiological assessment and systematic assessment of epilepsy features. Correlation analyses were performed, taking into account gender, age of seizure onset, EEG features, degree of cognitive deficits, type of mutation, presence of non-epileptic paroxysmal events and neuroimaging features.

Results: Epilepsy was documented in 64% of cases, a higher prevalence compared to previous reports. Patients were classified into three groups based on their electroclinical features, long-term outcome and response to therapy. A genotype-phenotype correlation linking the presence/severity of epilepsy to the nature of the structural/functional consequences of mutations was observed, providing a stratification based on genotype to improve the clinical management of these patients.

Keywords: BRAF; cardiofaciocutaneous syndrome; epilepsy; genotype–phenotype correlations; hyperekplexia; status epilepticus.

Figure 1

Group1: Ictal EEG in a 6-year-old boy (Pt5, Lys601Gln). (A) Left hyperkinetic seizure: Ictal discharge characterized by rapid spikes, starting on the left middle and posterior areas and spreading to the same right regions, followed by diffuse slow waves. After 10 s, a discharge of rapid SW started on the left occipital regions, lasting 20 s. (B) Right focal seizure characterized by sustained right deviation of the eyes and head, followed by eye clonic movements. Ictal discharge, starting in the right posterior regions, characterized by rapid spikes, then by SW and then again by rapid spikes. (C) Muscle artifacts associated with a startle response without paroxysmal abnormalities.

Figure 2

Group 2: Ictal EEG in a 9-year-old girl (Pt16, Trp531Cys). (A) Subclinical discharge of diffuse and irregular SW. (B) Bursts of diffuse polyspike and polyspike-waves, with anticipated onset over the frontal or posterior areas, accompanied by myoclonic jerks of both arms (arrows) while awake. (C) The same polyspike and polyspike-waves recorded during sleep. Note the spindles on the fronto-central regions (*). (D) Muscle artifacts associated to a startle response without paroxysmal abnormalities.

Figure 3

Group 1: MRI of a 11-year-old boy (Pt1, Lys 601Gln). (A) Inversion Recovery (IR) Turbo Spin Echo (TSE) Image, coronal plane. (B) TSE T2 weighted image, sagittal plane. The MRI shows a small left hippocampus and enlarged temporal horn and collateral sulcus, consistent with left-sided hippocampal sclerosis. Note the mild widening of left sylvian fissure and of the left fronto-temporal cerebrospinal fluid (CSF) spaces, consistent with cortical atrophy.

Figure 4

Domain organization of BRAF and location of residues mutated in CFCS. (A) Cartoon showing the domain organization of BRAF. The three conserved regions, CR1, CR2 and CR3, are highlighted in cyan, green and orange, respectively. The cysteine-rich domain (CRD, pink), located within CR1, contacts both subunits of the 14-3-3 zeta dimer in the inactive BRAF/MEK1/14-3-3 complex. Binding of the 14-3-3 dimer to phosphorylated Ser365 (CR2) and Ser729 (CR3) maintains BRAF in an autoinhibited state through an intramolecular interaction occurring between CR1 and the kinase domain CR3. In the active state, dephosphorylation of Ser365 results in an “open” conformation of the kinase, which stabilizes its binding with activated RAS proteins. In this activated con formation, a 14-3-3 dimer rearranges to bridge pSer729 of two BRAF proteins, thus driving the formation of the active BRAF dimer (21). The autoinhibited state is stabilized by interaction of residues 598–602 in the activation segment (the so-called ‘inhibitory turn’) with hydrophobic residues in the “GxGxxG” consensus sequence within the glycine-rich loop (residues 464–469) [21]. (B) Three-dimensional structure of the BRAF/MEK1/14-3-3 complex from cryo-electron microscopy in its autoinhibited conformation (PDB 6nyb). BRAF, MEK1 and the two monomers of 14-3-3 zeta are highlighted in blue, red, gray and yellow, respectively. The positions of the affected residues are highlighted in green. Two major mutation clusters are apparent: class I (1, ellipse), including residues located in/close to the CRD contacting the 14-3-3 protein (i.e., Thr241, Leu245, Ala246, Gln257, Asp565, Gln709); class II (2, rectangle) including residues mapped in the inhibitory turn region (i.e., Phe468, Gly469, Thr470, Lys483, Leu485, Val487, Lys499, Glu501, Leu525, Trp531, Asn581, Phe595, Thr599, Lys601). The arrow indicates residue Asp638, which is located in the protein core and has been assigned to class III (3). (C) In the complex, Thr241 is closer than 5 Å to Ser57 in 14-3-3 zeta monomer 1 and Glu17 in monomer 2. Gln257 is close to Arg60 in 14-3-3 zeta monomer 1. (D) Leu245 is close to Lys212 in 14-3-3 zeta monomer 1. The 3D position of residues 360–370 is also indicated. Residues 282–359 and 371–448 are not resolved. (E) Lys212 in 14-3-3 zeta monomer 1 is also close to Asp565. Gln709 does not have any 14-3-3 zeta residues closer than 5 Å in this experimental structure, but its substitution to arginine likely alters the interaction with the molecular partner. The 3D position of Asp638 is also shown. This variant is nearly equidistant from CRD and the inhibitory turn region. (F,G) BRAF variants in the active site region. Note that Phe595 is part of the DFG motif (594–596) in the inhibitory turn region, which plays an essential role in the correct positioning of ATP also in the active open conformation of BRAF. Thr599 and Lys601 are part of the TVKS motif; residues 469–470 are part of the p-loop, responsible for BRAF autophosphorylation.