Evidence for a Dual-Pathway, 2-Hit Genetic Model for Focal Cortical Dysplasia and Epilepsy

Abstract

Background and objectives: The 2-hit model of genetic disease is well established in cancer, yet has only recently been reported to cause brain malformations associated with epilepsy. Pathogenic germline and somatic variants in genes in the mechanistic target of rapamycin (mTOR) pathway have been implicated in several malformations of cortical development. We investigated the 2-hit model by performing genetic analysis and searching for germline and somatic variants in genes in the mTOR and related pathways.

Methods: We searched for germline and somatic pathogenic variants in 2 brothers with drug-resistant focal epilepsy and surgically resected focal cortical dysplasia (FCD) type IIA. Exome sequencing was performed on blood- and brain-derived DNA to identify pathogenic variants, which were validated by droplet digital PCR. In vitro functional assays of a somatic variant were performed.

Results: Exome analysis revealed a novel, maternally inherited, germline pathogenic truncation variant (c.48delG; p.Ser17Alafs*70) in NPRL3 in both brothers. NPRL3 is a known FCD gene that encodes a negative regulator of the mTOR pathway. Somatic variant calling in brain-derived DNA from both brothers revealed a low allele fraction somatic variant (c.338C>T; p.Ala113Val) in the WNT2 gene in 1 brother, confirmed by droplet digital PCR. In vitro functional studies suggested a loss of WNT2 function as a consequence of this variant. A second somatic variant has not yet been found in the other brother.

Discussion: We identify a pathogenic germline mTOR pathway variant (NPRL3) and a somatic variant (WNT2) in the intersecting WNT signaling pathway, potentially implicating the WNT2 gene in FCD and supporting a dual-pathway 2-hit model. If confirmed in other cases, this would extend the 2-hit model to pathogenic variants in different genes in critical, intersecting pathways in a malformation of cortical development. Detection of low allele fraction somatic second hits is challenging but promises to unravel the molecular architecture of FCDs.

Figure 1. Genetic Analysis, Imaging and Histopathology of Family With FCDIIA

(A) Pedigree of family and segregation of NPRL3 and WNT2 pathogenic variants. The NPRL3 mutant allele was detected in both blood and brain samples for brothers II.2 and II.3. No DNA was available from their sister (II.1). (B–D) Preoperative MRI scans (right is on the reader’s left). (B and C) MRI from II.2 showing an abnormal longitudinally oriented region of gray matter with blurred gray-white boundaries in the right posteromesial frontal region (arrows). (D) MRI from II.3 shows a globular region of abnormal gray matter in the left anteromesial frontal region (arrows). (E–J) Histopathology images showing disorganized gray matter and dysmorphic neurons for II.2 (panels E, G, and I) and II.3 (panels F, H, and J). (E and F) Phosporylated neurofilament staining of dysmorphic neurons. (G and H) Hemotoxylin and eosin staining showing dysmorphic neurons. (I and J) NeuN staining showing dyslamination. Magnification of 400× (E-H) or 40× (I and J); scale bars represent 20 µm (F), 100 µm (G and H), 1 mm (I), and 0.5 mm (J). FCDIIA = focal cortical dysplasia type IIA.

Figure 2. Validation of NPRL3 and WNT2 Variants

(A) Sanger sequence chromatograms showing the germline NPRL3 c.48delG (p.Ser17Alafs*70) variant in individuals I.2, II.2, and II.3. (B) ddPCR readout showing WNT2 mutant template (c.338C>T, p.Ala113Val) in blue (arrow), wild-type template in green, and droplets without DNA template in gray. Individual II.2's brain sample contains low-level somatic variant with variant allele fraction of 0.3%. (C) ddPCR shows that individual II.2's blood sample contains only wild-type template (green). (D) The limit of detection of the ddPCR assay was established by serially diluting mutant gBlocks into wild-type gBlocks to obtain different mutant/(mutant + wild-type) ratios: 2.5%, 1%, 0.5%, 0.25%, 0.1%, 0.05%, and 0.025%. An amplitude of 5,000 was set as the positive mutant droplet threshold. Mutant allele at a frequency ≥0.025% could be consistently detected with at least 3 droplets.

Figure 3. The WNT2 c.338C>T Variant Affects Canonical WNT Signaling

(A) Relative luciferase activity measured using the TOPFlash assay in HEK293T cells transfected with expression vectors for active WNT2-V5 (WNT2 WT), mutant WNT2-V5 (WNT2 c.338C>T), or an equal mix of both (WNT2 Mix). The TOPFlash assay measures canonical WNT signaling through a reporter with functional TCF/LEF responsive promoter (gray bars; TOP) or a control FOPFlash reporter with mutated, nonfunctional TCF/LEF binding sites (black bars; FOP) by standard dual-luciferase assay. Error bars indicate SDs between 3 independent experiments. Differences assessed using the Student t test. (B) Detection of V5-tagged wild-type and mutant WNT2 proteins transfected into HEK293T cells for dual-luciferase reporter assay, and endogenous levels of β-actin to show equal loading, by Western blot.

Figure 4. Diagram Showing Key Upstream Regulators and Activators of the mTOR and WNT Signaling Pathways