Gain-of-function FHF1 mutation causes early-onset epileptic encephalopathy with cerebellar atrophy

Abstract

Objective: Voltage-gated sodium channel (Nav)-encoding genes are among early-onset epileptic encephalopathies (EOEE) targets, suggesting that other genes encoding Nav-binding proteins, such as fibroblast growth factor homologous factors (FHFs), may also play roles in these disorders.

Methods: To identify additional genes for EOEE, we performed whole-exome sequencing in a family quintet with 2 siblings with a lethal disease characterized by EOEE and cerebellar atrophy. The pathogenic nature and functional consequences of the identified sequence alteration were determined by electrophysiologic studies in vitro and in vivo.

Results: A de novo heterozygous missense mutation was identified in the FHF1 gene (FHF1AR114H, FHF1BR52H) in the 2 affected siblings. The mutant FHF1 proteins had a strong gain-of-function phenotype in transfected Neuro2A cells, enhancing the depolarizing shifts in Nav1.6 voltage-dependent fast inactivation, predicting increased neuronal excitability. Surprisingly, the gain-of-function effect is predicted to result from weaker interaction of mutant FHF1 with the Nav cytoplasmic tail. Transgenic overexpression of mutant FHF1B in zebrafish larvae enhanced epileptiform discharges, demonstrating the epileptic potential of this FHF1 mutation in the affected children.

Conclusions: Our data demonstrate that gain-of-function FHF mutations can cause neurologic disorder, and expand the repertoire of genetic causes (FHF1) and mechanisms (altered Nav gating) underlying EOEE and cerebellar atrophy.

Figure 1. Heterozygous missense mutation (C>T) in FHF1 in 2 siblings with early-onset epileptic encephalopathy (EOEE) with cerebellar atrophy

(A) Whole-exome sequencing on a family quintet with 2 affected siblings (EOEE with cerebellar atrophy) and unaffected parents and sibling identified a heterozygous missense mutation in FHF1 (p.R114H in A-isoform, p.R52H in B-isoform). (B) Alignment of fibroblast growth factor homologous factor (FHF) sequences in the region of mutation. The FHF1 amino acid sequence contributing to the β4 and β5 strands and the β4/β5 loop is highly conserved among the 4 FHF core domains (shaded residues), including the β4/β5 loop arginine (blue) that is mutated to histidine (orange) in the affected siblings. (C) Ribbon diagram of the FHF1 core structure. The affected arginine residue side chain (blue) in the β4/β5 loop projects to the protein surface. (D) Ribbon diagram of the FHF/voltage-gated sodium channel (Nav) interface. The FHF β4/β5 loop arginine (blue) interacts with aspartate and histidine residues (red) in the Nav cytoplasmic tail.

Figure 2. Cerebellar atrophy in 2 siblings with a gain-of-function FHF1 mutation

MRI of the brain was normal at infant age in both affected siblings. However, repeat MRI in their further disease course, which was degenerative, showed the emergence of cerebellar atrophy in both children: (A) proband at age 6 years: axial and sagittal image show prominent fissures between shrunken cerebellar folia; (B) younger brother of proband at age 3 years: coronal image shows cerebellar atrophy.

Figure 3. Substitutions at FHF1B-R52/FHF1A-R114 are gain-of-function for inactivation gating of voltage-gated sodium channel (Na_v) 1.6

(A, B) Voltage dependence of Na_v1.6 steady-state inactivation in the presence of FHF1B_WT, FHF1B_R52H, or no fibroblast growth factor homologous factor (FHF). Sodium currents from noninactivated channels were measured at the reporting voltage (0 mV) following 60 ms conditioning voltages ranging from −110 to −25 mV. Superimposed sodium currents (A, above) recorded during the reporting voltage from several conditioning voltages (A, below) show that there is larger percentage of maximal sodium current after a given conditioning voltage in presence of FHF1B compared to no FHF, and the percentage of maximal current is larger still in the presence of FHF1B_R52H. Na_v1.6 inactivation in cells without FHF (n = 7, V_1/2 −81.4 ± 1.6 mV), with FHF1B_WT (n = 8, V_1/2 −74.9 ± 1.3 mV), and with FHF1B_R52H (n = 9, V_1/2 −65.8 ± 1.3 mV, p < 0.0002 vs FHF1B_WT) (B). Dotted lines indicate the color-coded conditioning voltages in A. (C) Voltage dependence of steady-state Na_v1.6 inactivation in cells without FHF (n = 7, V_1/2 −81.4 ± 1.6 mV), with FHF1A_WT (n = 7, V_1/2 −63.4 ± 1.6 mV), and with FHF1A_R114H (n = 8, V_1/2 −56.4 ± 0.9 mV, p < 0.004 vs FHF1A_WT). (D) Na_v1.6 inactivation in presence of FHF1B_WT (n = 8, V_1/2 −74.9 ± 1.3 mV), FHF1B_R52H (n = 9, V_1/2 −65.8 ± 1.3 mV), FHF1B_R52G (n = 9, V_1/2 −70.7 ± 0.7 mV, p < 0.02 vs FHF1B_WT), or FHF1B_R52A (n = 8, V_1/2 −67.1 ± 1.5 mV, p < 0.002 vs FHF1B_WT). All substitutions at FHF1B-Arg52 are gain-of-function. (B–D) Percentage of channels available after each conditioning voltage expressed and graphed as mean ± SEM.

Figure 4. R56H fhf1b1 overexpression (OE) causes epileptiform discharges in transgenic zebrafish larvae

(A) The Tol2 system was used to transiently overexpress wild-type (WT) and R56H mutant zebrafish fhf1b1. WT and R56H zebrafish fhf1b1 cDNA was expressed under the control of the zebrafish CNS-specific her4 promoter. A fluorescent reporter mCherry was used to identify larvae with fhf1b1 expression. Self-cleaving peptide, P2A, enabled the separation between fhf1b1 and mCherry in order to avoid possible disadvantages of a fusion protein. Representative images of 5 dpf larvae microinjected with zebrafish cDNA encoding WT and mutated fhf1b1, selected for tectal field recordings and mRNA analysis. Red fluorescence depicts the brain-specific expression of Fhf1b. Scale bar, 500 μm. (B) Percentage of larvae with abnormal epileptiform activity after overexpression of WT and R56H fhf1b1. Overexpression of mutant Fhf1b led to a significant increase of the number of larvae with seizure-like events in comparison to WT-fhf1b1-OE and control larvae (27/54 larvae with R56H-fhf1b1-OE vs 15/54 larvae with WT-fhf1b1-OE and 7/40 control larvae; *p = 0.0293 and **p = 0.0012, respectively, Fisher exact test). Overexpression of mCherry alone resulted in 17.5% of seizing larvae, which was not statistically different from 27.8% for WT-fhf1b1-OE larvae (p = 0.3261, Fisher exact test). (C) Occurrence of epileptiform events/recording in WT-fhf1b1-OE (5.4 ± 0.9), R56H-fhf1b1-OE (6.0 ± 0.6), and control larvae (4.1 ± 0.4) (p = 0.3237, one-way analysis of variance [ANOVA]). Results are mean ± SEM. (D) Mean duration of epileptiform events in WT-fhf1b1-OE (129.4 ± 7.1 ms), R56H-fhf1b1-OE (141.9 ± 7.0 ms), and control larvae (148.2 ± 16.0 ms) (p = 0.4112, one-way ANOVA). Results are mean ± SEM. (E) Cumulative duration of epileptiform events in WT-fhf1b1-OE (743.6 ± 172.1 ms), R56H-fhf1b1-OE (836.5 ± 78.3 ms), and control larvae (629.3 ± 102.1 ms) (p = 0.5556, one-way ANOVA). Results are mean ± SEM. (F) Representative spontaneous epileptiform activity recorded from 5 dpf R56H-fhf1b1-OE larvae. Top trace represents typical epileptiform pattern as seen in gap-free recordings. Bottom trace shows high-resolution magnification of the selected epileptiform events. Next to the traces, an agar immobilized 5 dpf zebrafish with the recording electrode placed in the optic tectum (OT) is shown. FB = forebrain. (G) fhf1b1 mRNA expression in WT and R56H-fhf1b1-OE larvae. fhf1b1 was expressed approximately 3-fold higher in WT and mutant-OE larvae in comparison to fhf1b1 baseline level in control larvae. **p = 0.0080 WT-fhf1b1-OE vs control; *p = 0.0293 R56H-fhf1b1-OE vs control (2-tailed Student t-test). β-2 microglobulin and β-actin were used as normalizing controls. Values are mean ± SEM (triplicate samples with triplicate qPCR experiments).