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Case Reports
. 2019 Nov 15;28(22):3755-3765.
doi: 10.1093/hmg/ddz194.

Somatic double-hit in MTOR and RPS6 in hemimegalencephaly with intractable epilepsy

Cristiana Pelorosso  1 , Françoise Watrin  2 , Valerio Conti  1 , Emmanuelle Buhler  2 , Antoinette Gelot  3 , Xiaoxu Yang  4 , Davide Mei  1 , Jennifer McEvoy-Venneri  4 , Jean-Bernard Manent  2 , Valentina Cetica  1 , Laurel L Ball  4 , Anna Maria Buccoliero  5 , Antonin Vinck  2 , Carmen Barba  1 , Joseph G Gleeson  4 , Renzo Guerrini  1   6 , Alfonso Represa  2
Affiliations
Case Reports

Somatic double-hit in MTOR and RPS6 in hemimegalencephaly with intractable epilepsy

Cristiana Pelorosso et al. Hum Mol Genet. .

Abstract

Single germline or somatic activating mutations of mammalian target of rapamycin (mTOR) pathway genes are emerging as a major cause of type II focal cortical dysplasia (FCD), hemimegalencephaly (HME) and tuberous sclerosis complex (TSC). A double-hit mechanism, based on a primary germline mutation in one allele and a secondary somatic hit affecting the other allele of the same gene in a small number of cells, has been documented in some patients with TSC or FCD. In a patient with HME, severe intellectual disability, intractable seizures and hypochromic skin patches, we identified the ribosomal protein S6 (RPS6) p.R232H variant, present as somatic mosaicism at ~15.1% in dysplastic brain tissue and ~11% in blood, and the MTOR p.S2215F variant, detected as ~8.8% mosaicism in brain tissue, but not in blood. Overexpressing the two variants independently in animal models, we demonstrated that MTOR p.S2215F caused neuronal migration delay and cytomegaly, while RPS6 p.R232H prompted increased cell proliferation. Double mutants exhibited a more severe phenotype, with increased proliferation and migration defects at embryonic stage and, at postnatal stage, cytomegalic cells exhibiting eccentric nuclei and binucleation, which are typical features of balloon cells. These findings suggest a synergistic effect of the two variants. This study indicates that, in addition to single activating mutations and double-hit inactivating mutations in mTOR pathway genes, severe forms of cortical dysplasia can also result from activating mutations affecting different genes in this pathway. RPS6 is a potential novel disease-related gene.

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Figures

Figure 1
Figure 1
Patient’s neuroimaging, cutaneous and anatomopathological findings. (A) and (B) Axial T2 and proton density (PD)-weighted MRI sections showing left HME, with enlarged ventricles, rightward deviation of the brain midline, coarse cortical gyri, with cortical thickening, abnormal with matter signal (decreased in T2 and increased in PD) blurring of the gray-white matter junction. (C) Hypochromic patches on the patient’s face (white arrows) and neck (white circle). (D) H&E and immunohistochemical staining of the dysplastic brain tissue. Dysmorphic neurons (white arrows) and balloon cells (black arrows) exhibit a marked hyperexpression of anti-PS6. Anti-NFs staining demonstrates neurofilament protein accumulation in dysmorphic neurons. Anti-GS and anti-GFAP immunoreactivity is present in balloon cells and reactive astrocytes but not in dysmorphic neurons. Anti-NEU-N staining shows cortical dyslamination. Scale bar for H&E, PS6, NFs, GS and GFAP = 50 μm. Scale bar for NEU-N = 500 μm.
Figure 2
Figure 2
Neuroprogenitors proliferation analysis on embryonic rat brains. (A) Confocal microphotographs of E16.5 brain coronal sections from rat embryos electroporated at E15 with IRES-GFP, RPS6 WT, RPS6 R232H, MTOR WT or MTOR S2215F plasmids. Immunostaining was performed with anti-PH3 and anti-GFP antibodies. Sections were counterstained with Hoechst. Arrows indicate PH3+ and GFP+ neuroprogenitors. Scale bar = 30 μm. (B) Quantification of the percentage of mitotic neuroprogenitors (PH3+, GFP+) among transfected cells (GFP+) in the VZ/SVZ of electroporated brains. Values are given as mean ± SEM. One-way ANOVA and Tukey’s multiple comparison tests, ****P < 0.0001; IRES-GFP, RPS6 WT, RPS6 R232H, MTOR WT, MTOR S2215F, n = 10; IRES-GFP/IRES-TOMATO, RPS6 WT/MTOR WT, RPS6R232H/MTOR S2215F, n = 8. Statistical elements are provided in Supplementary Material, Table S4. (C) Confocal microphotographs of E16.5 brain coronal sections from rat embryos electroporated at E15 with IRES-GFP, RPS6 WT, RPS6 R232H, MTOR WT or MTOR S2215F plasmids. Immunostaining was performed with anti-Ki67 and anti-GFP antibodies. Sections were counterstained with Hoechst. Arrows indicate Ki67+ and GFP+ neuroprogenitors. Scale bar: 30 μm. (D) Quantification of the percentage of cycling neuroprogenitors (Ki67+, GFP+) among transfected cells (GFP+) in the VZ/SVZ of electroporated brains. Values are given as mean ± SEM. One-way ANOVA and Tukey’s multiple comparison tests, ****P < 0.0001, ***P = 0.003; IRES-GFP, n = 7; RPS6 WT, MTOR WT, IRES-GFP/IRES-TOMATO, RPS6 WT/MTOR WT, RPS6 R232H/MTOR S2215F, n = 8; RPS6 R232H, MTOR S2215F, n = 9. Statistical elements are provided in Supplementary Material, Table S5.
Figure 3
Figure 3
Neuronal migration analysis on embryonic rat brains. (A) Upper panels: Confocal microphotographs of E20 brain coronal sections immunostained for GFP from rat embryos electroporated at E15 with IRES-GFP, RPS6 WT, RPS6 R232H, MTOR WT or MTOR S2215F plasmids. Lower panels: Confocal microphotographs of E20 brain coronal sections immunostained for GFP and TdTomato from rat embryos electroporated at E15 with IRES-GFP/IRES-TOMATO, RPS6 WT/MTOR WT or RPS6 R232H/MTOR S2215F plasmids. Sections were counterstained with Hoechst. White dotted lines delimitate CP, IZ and VZ/SVZ. Scale bars: 350 μm. (B) Quantification of the percentage of GFP+ neurons in VZ/SVZ (left), IZ (middle) and CP (right) of electroporated brains for each condition. Values are given as mean ± SEM. One-way ANOVA and Tukey’s multiple comparison tests, ****P < 0.0001; IRES-GFP, IRES-GFP/IRES-TOMATO, n = 9; RPS6 WT, RPS6 R232H, MTOR WT, n = 10; MTOR S2215F, RPS6 WT/MTOR WT, RPS6 R232H/MTOR S2215F, n = 11. Statistical elements are provided in Supplementary Material, Table S6.
Figure 4
Figure 4
Cell size quantification and morphological characterization of electroporated neurons at postnatal stages. (A) Confocal microphotographs of P28 brain coronal sections from rat embryos electroporated at E15 with IRES-GFP, RPS6 R232H, MTOR S2215F or RPS6 R232H/MTOR S2215F plasmids. All sections were immunostained for GFP and counterstained with Hoechst. RPS6 R232H/MTOR S2215F electroporated sections were additionally stained for tdTomato. Scale bar = 100 μm. (B) Quantification of the soma area (μm2) of electroporated neurons for each condition. Values are given as mean ± SEM. Kruskal Wallis and Dunn’s multiple comparisons tests, ****P < 0.0001; IRES-GFP, n = 356; RPS6 R232H, n = 256; MTOR S2215F, n = 294; RPS6 R232H/MTOR S2215F, n = 222. Statistical elements are provided in Supplementary Material, Table S7. (C) H&E and immunohistochemical staining of P28 brain coronal sections from rat embryos electroporated at E15 with RPS6 R232H, MTOR S2215F or RPS6 R232H/MTOR S2215F plasmids. Immunohistochemistry was performed with anti-PS6 and anti-GS antibodies. White arrows indicate cytomegalic neurons. Black and white asterisks indicate cytomegalic neurons with small cytoplasmic vacuoles/eccentric nuclei and binucleated cells, respectively. Control neurons from the contralateral hemisphere of a RPS6 R232H/MTOR S2215F electroporated brain are shown for comparison. Scale bars: 30 μm.
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