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. 2015 May;52(5):330-7.
doi: 10.1136/jmedgenet-2014-102813. Epub 2015 Feb 27.

De novo gain-of-function and loss-of-function mutations of SCN8A in patients with intellectual disabilities and epilepsy

Maxime G Blanchard  1 , Marjolein H Willemsen  2 , Jaclyn B Walker  3 , Sulayman D Dib-Hajj  4 , Stephen G Waxman  4 , Marjolijn C J Jongmans  2 , Tjitske Kleefstra  2 , Bart P van de Warrenburg  5 , Peter Praamstra  5 , Joost Nicolai  6 , Helger G Yntema  2 , René J M Bindels  1 , Miriam H Meisler  3 , Erik-Jan Kamsteeg  2
Affiliations

De novo gain-of-function and loss-of-function mutations of SCN8A in patients with intellectual disabilities and epilepsy

Maxime G Blanchard et al. J Med Genet. 2015 May.

Abstract

Background: Mutations of SCN8A encoding the neuronal voltage-gated sodium channel NaV1.6 are associated with early-infantile epileptic encephalopathy type 13 (EIEE13) and intellectual disability. Using clinical exome sequencing, we have detected three novel de novo SCN8A mutations in patients with intellectual disabilities, and variable clinical features including seizures in two patients. To determine the causality of these SCN8A mutations in the disease of those three patients, we aimed to study the (dys)function of the mutant sodium channels.

Methods: The functional consequences of the three SCN8A mutations were assessed using electrophysiological analyses in transfected cells. Genotype-phenotype correlations of these and other cases were related to the functional analyses.

Results: The first mutant displayed a 10 mV hyperpolarising shift in voltage dependence of activation (gain of function), the second did not form functional channels (loss of function), while the third mutation was functionally indistinguishable from the wildtype channel.

Conclusions: Comparison of the clinical features of these patients with those in the literature suggests that gain-of-function mutations are associated with severe EIEE, while heterozygous loss-of-function mutations cause intellectual disability with or without seizures. These data demonstrate that functional analysis of missense mutations detected by clinical exome sequencing, both inherited and de novo, is valuable for clinical interpretation in the age of massive parallel sequencing.

Keywords: Epilepsy and seizures; Movement disorders (other than Parkinsons); encephalopathy; intelectual disability; sodium channel.

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Figures

Figure 1
Figure 1
Localisation and evolutionary conservation of three newly identified de novo mutations in SCN8A encoding the sodium channel NaV1.6. (A) The D58 residue in the N-terminus. (B) N984 in the intracellular loop close to the sixth transmembrane helix of domain 2 (D2S6). (C) G1451 in the sixth transmembrane helix of domain 3 (DIIIS6), in close proximity to the channel pore. h, Homo sapiens; m, Mus musculus; a, Anolis carolinensis (reptile); f, Takifugu rubripes (fish); dpara, Drosophila melanogaster ‘paralytic’ (alternative name NaV1). Amino acids are indicated by the single-letter code and dots represent identity to the human amino acid. Multiple sequence alignment was carried out using ClustalW V.2.0.12.
Figure 2
Figure 2
Voltage-evoked currents of mutant proteins in transfected HEK293 cells. (A) Wild type (wt), D58N and N984K gave rise to substantial voltage-evoked currents while the G1451S expressing cells demonstrated current densities similar to non-transfected cells. (B) Average current–voltage curve for non-transfected cells (squares), wt (full circles), D58N (upward triangles), N984K (downward triangles) and G1451S (stars). N=3 for non-transfected cells, and N≥10 for all other conditions.
Figure 3
Figure 3
Voltage dependence of activation and steady-state inactivation of mutant proteins in transfected HEK293 cells. (A) Individual current–voltage (I–V) curves were converted to normalised conductance–voltage (g-V) curves and fitted with a Boltzmann equation yielding the voltage of half-maximal activation (V0.5) and slope factor (k). (B) Average normalised conductances of wild type (wt), D58N and N984K as a function of voltage. Lines represent Boltzmann fit to the average conductance voltage curve. The N984K mutant V0.5 is significantly shifted towards hyperpolarised voltages values (p<0.05, n=10). N≥10 for each conditions. (C) The protocol used to study steady-state inactivation is shown. The fraction of non-inactivated channels after a 500 ms conditioning period is indicated as the ratio of the current after the conditioning period (I2) to the current obtained before conditioning (I1). (D) The average current ratio (I2/I1) for wt, D58N and N984K as a function of the conditioning voltage. The dotted line represents a modified Boltzmann fit to the average wt steady-state inactivation curve. Fit to D58N and N984 is not shown for the purpose of clarity. There is no difference in voltage of half-maximal inactivation between wt and mutants (p>0.05, n≥8).
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