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J Med Genet
2019 Feb 01;562:113-122. doi: 10.1136/jmedgenet-2018-105322.
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De novo pathogenic variants in neuronal differentiation factor 2 (NEUROD2) cause a form of early infantile epileptic encephalopathy.
Sega AG
,
Mis EK
,
Lindstrom K
,
Mercimek-Andrews S
,
Ji W
,
Cho MT
,
Juusola J
,
Konstantino M
,
Jeffries L
,
Khokha MK
,
Lakhani SA
.
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BACKGROUND: Early infantile epileptic encephalopathies are severe disorders consisting of early-onset refractory seizures accompanied often by significant developmental delay. The increasing availability of next-generation sequencing has facilitated the recognition of single gene mutations as an underlying aetiology of some forms of early infantile epileptic encephalopathies.
OBJECTIVES: This study was designed to identify candidate genes as a potential cause of early infantile epileptic encephalopathy, and then to provide genetic and functional evidence supporting patient variants as causative.
METHODS: We used whole exome sequencing to identify candidate genes. To model the disease and assess the functional effects of patient variants on candidate protein function, we used in vivo CRISPR/Cas9-mediated genome editing and protein overexpression in frog tadpoles.
RESULTS: We identified novel de novo variants in neuronal differentiation factor 2 (NEUROD2) in two unrelated children with early infantile epileptic encephalopathy. Depleting neurod2 with CRISPR/Cas9-mediated genome editing induced spontaneous seizures in tadpoles, mimicking the patients' condition. Overexpression of wild-type NEUROD2 induced ectopic neurons in tadpoles; however, patient variants were markedly less effective, suggesting that both variants are dysfunctional and likely pathogenic.
CONCLUSION: This study provides clinical and functional support for NEUROD2 variants as a cause of early infantile epileptic encephalopathy, the first evidence of human disease caused by NEUROD2 variants.
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30323019
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Figure 3
CRISPR/Cas9-mediated knockdown induces seizures in Xenopus tropicalis tadpoles. (A) Uninjected control tadpole displays normal swimming behaviour with straight tail (top image), while neurod2 CRISPR tadpole displays seizure behaviour (C-shaped convulsion), with tail bending towards the head (bottom image). (B) Quantification of seizure behaviour in seconds per minute, showing increase in severity of seizures in tadpoles injected with both non-overlapping CRISPRs (neurod2-1A and neurod2-1B) over the course of development. t-Test reveals significant differences (p<0.0001) between seizure time of CRISPR and uninjected control (UIC) tadpoles at each stage of development. Data are a compilation of 7 independent experiments. (C) Neurod2-1A is 100% efficient (n=10). Pie charts show total efficiency, divided into inframe, out-of-frame, other and no edit. Bar graphs show percentage of sequences with specific amount of nucleotides gained or lost. (D) Neurod2-1B is 97% efficient. Pie charts show total efficiency, divided into inframe, out-of-frame, other and no edit. Bar graphs show percentage of sequences with specific amount of nucleotides gained or lost.
Figure 4
Overexpression of NEUROD2 in Xenopus laevis suggests patient variants are dysfunctional. (A) Normal neuronal -tubulin expression in a stage 28 wild-type X. laevis embryo. Expression extends along the neural tube, from the anterior/posterior axis on the dorsal side of the tadpole. Strong expression is also seen in the brain and eye. (B) Overexpression of wild-type NEUROD2 in X. laevis results in varying degrees of ectopic neuronal induction. Severe induction (top image) is categorised by ectopic neurons extending down the full length of the anterior/posterior axis of the tadpole, with signal at least halfway down the side of the tadpole, extending dorsally to ventrally. Mild induction (bottom image) is classified by showing ectopic neuronal induction but not meeting severe parameters (ie, neuronal -tubulin signal not extending down the length of the anterior/posterior axis and/or not extending at least halfway down dorsally/ventrally. (C) 1250pg of human NEUROD2 (wild-type, p.M134T and p.E130Q) mRNA was injected in one of two cells at the two-cell stage of X. laevis embryos, and ectopic neuron induction severity was categorised as severe, mild or none. NEUROD2, neuronal differentiation factor 2.
tubb3 (tubulin beta 3 class III) gene expression in X. tropicalis embryo, NF stage 28, assayed via inset hybridization, lateral view dorsal up and anteriorright.
