Deep histopathology genotype-phenotype analysis of focal cortical dysplasia type II differentiates between the GATOR1-altered autophagocytic subtype IIa and MTOR-altered migration deficient subtype IIb

Abstract

Focal cortical dysplasia type II (FCDII) is the most common cause of drug-resistant focal epilepsy in children. Herein, we performed a deep histopathology-based genotype-phenotype analysis to further elucidate the clinico-pathological and genetic presentation of FCDIIa compared to FCDIIb. Seventeen individuals with histopathologically confirmed diagnosis of FCD ILAE Type II and a pathogenic variant detected in brain derived DNA whole-exome sequencing or mTOR gene panel sequencing were included in this study. Clinical data were directly available from each contributing centre. Histopathological analyses were performed from formalin-fixed, paraffin-embedded tissue samples using haematoxylin-eosin and immunohistochemistry for NF-SMI32, NeuN, pS6, p62, and vimentin. Ten individuals carried loss-of-function variants in the GATOR1 complex encoding genes DEPDC5 (n = 7) and NPRL3 (n = 3), or gain-of-function variants in MTOR (n = 7). Whereas individuals with GATOR1 variants only presented with FCDIIa, i.e., lack of balloon cells, individuals with MTOR variants presented with both histopathology subtypes, FCDIIa and FCDIIb. Interestingly, 50% of GATOR1-positive cases showed a unique and predominantly vacuolizing phenotype with p62 immunofluorescent aggregates in autophagosomes. All cases with GATOR1 alterations had neurosurgery in the frontal lobe and the majority was confined to the cortical ribbon not affecting the white matter. This pattern was reflected by subtle or negative MRI findings in seven individuals with GATOR1 variants. Nonetheless, all individuals were seizure-free after surgery except four individuals carrying a DEPDC5 variant. We describe a yet underrecognized genotype-phenotype correlation of GATOR1 variants with FCDIIa in the frontal lobe. These lesions were histopathologically characterized by abnormally vacuolizing cells suggestive of an autophagy-altered phenotype. In contrast, individuals with FCDIIb and brain somatic MTOR variants showed larger lesions on MRI including the white matter, suggesting compromised neural cell migration.

Fig. 1

Histopathology findings of FCD ILAE Type IIa and IIb. A 42-year-old male individual with frontal lobe epilepsy since age 5 years (ID3). The MRI was suspicious for a bottom-of-sulcus FCD with transmantel sign. Histopathology confirmed, however, FCDIIa and a pathogenic DEPDC5 mosaicism. The arrow points to the sharp border between the cortical FCDIIa and the normal-appearing white matter (WM). NCx—adjacent normal 6-layered neocortex. Neurofilament SMI32 immunohistochemistry. Scale bar = 2.5mm (applies also to B). Higher magnification in C reveals dysmorphic neurons with a predominant vacuolizing phenotype (red arrow) suggesting accumulation of lipofuscins and an altered autophagy pathway. Scale bar = 100 µm (applies also to D). Same neurons were also labeled with antibodies directed against the phospho-S6-Ser236 epitope (E). The black arrow in (E) points to a neuron with a vacuolizing phenotype. Scale bar in E = 100 µm, applies also to (F); B 19-year-old male individual with frontal lobe epilepsy since age nine years (ID15), histopathological confirmed FCDIIb at a bottom-of-sulcus (BoS; higher magnification in D) and a pathogenic MTOR mosaicism. Dysmorphic neurons and balloon cells were aggregated in the neocortex and white matter (arrow in B) compatible with a migration-deficient phenotype. F both, dysmorphic neurons and balloon cells were labelled with antibodies directed against the pS6 Ser236 epitope

Fig. 2

Autophagocytic phenotype in DEPDC5 altered FCDIIa. Images were taken from a 42-year-old male individual with drug-resistant focal epilepsy and FCDIIa, DEPDC5 altered (ID3). A juxtanuclear accumulation of autophagosomes can be anticipated from the displacement of neurofilaments (arrow in A as an example, NF-SMI32) and silver impregnation (arrow in B, Bielschowski silver staining, see also higher magnification in E). C p62-immunoreactivity highlighted the aggregation of autophagosomes in FCDIIa. D Further confirmation of the autophagocytic phenotype in FCDIIa by double immunofluorescence and laser scanning microscopy of NF-SMI32 (in red) and p62 (in green). Scale bar in A and B = 100 µm. The scale bar in C = 25 µm, applies also to (D) and (E)

Fig. 3

Representative FLAIR neuroimaging findings of FCDIIa, DEPDC5 altered and FCDIIb, MTOR altered. A individual ID4 with histopathologically confirmed FCDIIa and a two-hit DEPDC5 variant. The MRI showed no definite lesion and the right fronto-mesial lobe was surgically removed (arrow); B individual ID5, classified as bottom-of-sulcus FCD; C individual ID14 with a thickened neocortex and distinct signal intensity change in FLAIR; D individual ID15 with a distinct transmantle sign; Individual #14 and #15 both revealed somatic MTOR variants