Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis

Abstract

In severe early-onset epilepsy, precise clinical and molecular genetic diagnosis is complex, as many metabolic and electro-physiological processes have been implicated in disease causation. The clinical phenotypes share many features such as complex seizure types and developmental delay. Molecular diagnosis has historically been confined to sequential testing of candidate genes known to be associated with specific sub-phenotypes, but the diagnostic yield of this approach can be low. We conducted whole-genome sequencing (WGS) on six patients with severe early-onset epilepsy who had previously been refractory to molecular diagnosis, and their parents. Four of these patients had a clinical diagnosis of Ohtahara Syndrome (OS) and two patients had severe non-syndromic early-onset epilepsy (NSEOE). In two OS cases, we found de novo non-synonymous mutations in the genes KCNQ2 and SCN2A. In a third OS case, WGS revealed paternal isodisomy for chromosome 9, leading to identification of the causal homozygous missense variant in KCNT1, which produced a substantial increase in potassium channel current. The fourth OS patient had a recessive mutation in PIGQ that led to exon skipping and defective glycophosphatidyl inositol biosynthesis. The two patients with NSEOE had likely pathogenic de novo mutations in CBL and CSNK1G1, respectively. Mutations in these genes were not found among 500 additional individuals with epilepsy. This work reveals two novel genes for OS, KCNT1 and PIGQ. It also uncovers unexpected genetic mechanisms and emphasizes the power of WGS as a clinical tool for making molecular diagnoses, particularly for highly heterogeneous disorders.

Figure 1.

Paternal isodisomy in Patient 2. (A) We observed multiple Mendelian errors on chromosome 9 which led us to suspect uniparental disomy (UPD). All variants in the patient (OTH_5) appeared to have been inherited from his father (OTH_6). The KCNT1 variant is illustrated here as an example. Grey bars represent individual sequencing reads from the sample indicated on the left, and colored letters divergences from the reference sequence. The grey ‘pile-up’ along the top indicates the sequence coverage. The genotype of each individual is shown. (B) These plots show the proportion of variants that are homozygous in 500 kb windows across chr9. OTH_5 was completely homozygous, apart from a few spurious calls; the pattern is similar to that seen on chromosome X in males. His father, OTH_6, is shown for comparison. Note that the dip in the middle represents the centromere.

Figure 2.

Electrophysiological and channel expression analysis of KCNT1 mutation found in Patient 2. WT or A945T mutant Slack channel was expressed in Xenopus laevis oocytes, and two-electrode voltage clamping (TEVC) performed. (A) A representative trace of current activity recorded from an oocyte expressing WT or A945T is shown on top, with the voltage-clamping protocol displayed underneath. (B) Averaged quantitation of the peak current is compared at +60 mV (P < 0.001, n = 5, Student's t-test; representative of three independent experiments). (C) The quality of RNA injected into Xenopus oocytes was checked on a 1% formaldehyde agarose gel. (D) Current–voltage relations for the WT or A945T channels. Channel activity as measured at peak current amplitude and normalized to the value at +60 mV is plotted against voltage (**P < 0.01, ***P < 0.001, n = 5, two-way ANOVA, Sidak's multiple comparisons test).

Figure 3.

PIGQ splicing mutation in Patient 4. (A) The variant causes skipping of exon 3. This image shows the Bioanalyzer gel from an RT-PCR (see Materials and Methods) and demonstrates the presence of two PIGQ transcripts in the heterozygous parents (OTH_13, OTH_14). The blue arrow indicates the band expected from the annotated transcript, and the red arrow that expected from the skipping of exon 3. (B) Severely decreased functional activity of the mutant PIGQ. PIGQ-deficient CHO cells were transiently transfected with WT or mutant PIGQ cDNA (lacking exon 3). Restoration of the surface expression of CD59, a GPI-anchored protein, was assessed by flow cytometry after staining with anti-CD59 antibody. The mutant PIGQ did not restore the surface expression of CD59 as efficiently as the WT. X axis, fluorescence intensity corresponding to CD59 expression level per cell; Y axis, relative cell number. (C) The expression of mutant protein was greatly decreased and could not be detected by western blotting.