UNC13B variants associated with partial epilepsy with favourable outcome

Abstract

The unc-13 homolog B (UNC13B) gene encodes a presynaptic protein, mammalian uncoordinated 13-2 (Munc13-2), which is highly expressed in the brain-predominantly in the cerebral cortex-and plays an essential role in synaptic vesicle priming and fusion, potentially affecting neuronal excitability. However, the functional significance of the UNC13B mutation in human disease is not known. In this study, we screened for novel genetic variants in a cohort of 446 unrelated cases (families) with partial epilepsy without acquired causes by trio-based whole-exome sequencing. UNC13B variants were identified in 12 individuals affected by partial epilepsy and/or febrile seizures from eight unrelated families. The eight probands all had focal seizures and focal discharges in EEG recordings, including two patients who experienced frequent daily seizures and one who showed abnormalities in the hippocampus by brain MRI; however, all of the patients showed a favourable outcome without intellectual or developmental abnormalities. The identified UNC13B variants included one nonsense variant, two variants at or around a splice site, one compound heterozygous missense variant and four missense variants that cosegregated in the families. The frequency of UNC13B variants identified in the present study was significantly higher than that in a control cohort of Han Chinese and controls of the East Asian and all populations in the Genome Aggregation Database (gnomAD). Computational modelling, including hydrogen bond and docking analyses, suggested that the variants lead to functional impairment. In Drosophila, seizure rate and duration were increased by Unc13b knockdown compared to wild-type flies, but these effects were less pronounced than in sodium voltage-gated channel alpha subunit 1 (Scn1a) knockdown Drosophila. Electrophysiological recordings showed that excitatory neurons in Unc13b-deficient flies exhibited increased excitability. These results indicate that UNC13B is potentially associated with epilepsy. The frequent daily seizures and hippocampal abnormalities but ultimately favourable outcome under anti-epileptic therapy in our patients indicate that partial epilepsy caused by UNC13B variant is a clinically manageable condition.

Keywords: Drosophila knockdown model; UNC13B; electrophysiology; loss of function; partial epilepsy.

Figure 1

Genetic data of cases with UNC13B variants. (A) Pedigrees and DNA sequencing chromatogram of the eight cases with UNC13B variants and their corresponding phenotypes. (B) Amino acid sequence alignment of the six missense variants with protein substitutions show that Arg221, Arg661 and Gly794 are highly conserved across species. Thr103 and Gly882 are highly conserved in vertebrates but less conserved in lower animals, while Ser397 shows a low degree of conservation. (C) Schematic illustration of the Munc13-2 protein and the location of the UNC13B variants or protein substitutions identified in this study.

Figure 2

Schematic illustration of hydrogen bonds in Munc13-2. (A) Left: Thr103 (red) forms two hydrogen bonds (yellow) with Gly102. Right: Thr103Met destroys the two hydrogen bonds. (B) Left: Arg661 (green) forms five hydrogen bonds (yellow)—one each with Tyr492, Ser659 and Lys663 and two with Glu493. Right: Arg661Cys destroys three of the five hydrogen bonds without affecting those with Ser659 and Lys663. (C) Left: Gly794 (blue) forms one hydrogen bond (yellow) with Ala790. Right: Gly794Asp does not affect hydrogen bonding in Munc13-2. (D) Gly882 (cyan) does not form hydrogen bonds with any other residue, and Gly882Trp does not affect hydrogen bonding in Munc13-2.

Figure 3

Model of calmodulin docking on Munc13-2 and the effect of variants. (A) Complex of wild-type (WT) Munc13-2 and calmodulin. (B–D) Complex of mutant Munc13-2 and calmodulin. (C) Gly794Asp alters the docking site, interface area and Gibbs free energy of Munc13-2.

Figure 4

Representative EEG recordings and MRI from patients with UNC13B variants. (A) Interictal EEG in Case 1 showed left occipital slow spike waves. (B) Interictal EEG in Case 3 showed left central-temporal slow spike waves. (C and D) Interictal EEG in Case 8 showed spikes or sharp waves in the left or right temporal lobe, or of slow spike waves in the right frontal lobe. (E and F) Coronal and axial T₂-FLAIR MRI of Case 8 revealed structural asymmetry in the hippocampus; the right hippocampus was smaller than the left one, with a slightly higher signal. The boundary between the left hippocampus and surrounding tissues was indistinct.

Figure 5

Knockdown of Unc13b induces seizure-like behaviour in Drosophila. (A) Behaviour in the BS paralysis test; the three stages observed in Unc13b knockdown flies were seizure (manifesting as vibrating wings, circled in red), paralysis and recovery. (B) Seizures occurred at a higher rate in Unc13b knockdown flies (tub-Gal4>Unc13b-RNAi) than in wild-type (WT) flies (Canton-S) and tub-Gal4>Chd3-RNAi and other control groups. The tub-Gal4>Scn1a-RNAi positive controls had a higher rate of seizures than the tub-Gal4>Unc13b-RNAi group. (C) The recovery time from seizure was longer in tub-Gal4>Unc13b-RNAi than in Canton-S but shorter than in tub-Gal4>Scn1a-RNAi.

Figure 6

Unc13b knockdown induces increased neural excitability in projection neurons. (A) Representative trace of extracellular electrical activity in Canton-S wild-type (WT) projection neurons showing sporadic action currents. (B) Regular high-frequency burst firing was recorded in projection neurons of tub-Gal4>Unc13b-RNAi flies. (C) Action current frequency was significantly higher in tub-Gal4>Unc13b-RNAi than in Canton-S. (D) There was no difference in amplitude of action currents between tub-Gal4>Unc13b-RNAi and Canton-S.

Figure 7

Brain morphology in Unc13b knockdown flies. (A, C, E and G) Serial images of the brain of a wild-type fly from anterior to posterior showing the structure of antennal lobes (A), ellipsoid body (C), fan-shaped body (E), and protocerebral bridge (filled arrowhead) and mushroom body calyx (open arrowhead) (G). (B, D, F and H) Serial images of the brain of a Unc13b knockdown fly from anterior to posterior showing the structure of antennal lobes (B), ellipsoid body (D), fan-shaped body (F), and protocerebral bridge (white arrowhead) and mushroom body calyx (open arrowhead) (H). Scale bar = 100 μm. (I) Cell body of neurons (open arrowhead) in the mushroom body of a wild-type fly. (J) Cell body of neurons (open arrowhead) in the mushroom body of a Unc13b knockdown fly. Scale bar = 10 μm.