De novo somatic mutations in components of the PI3K-AKT3-mTOR pathway cause hemimegalencephaly

Abstract

De novo somatic mutations in focal areas are well documented in diseases such as neoplasia but are rarely reported in malformation of the developing brain. Hemimegalencephaly (HME) is characterized by overgrowth of either one of the two cerebral hemispheres. The molecular etiology of HME remains a mystery. The intractable epilepsy that is associated with HME can be relieved by the surgical treatment hemispherectomy, allowing sampling of diseased tissue. Exome sequencing and mass spectrometry analysis in paired brain-blood samples from individuals with HME (n = 20 cases) identified de novo somatic mutations in 30% of affected individuals in the PIK3CA, AKT3 and MTOR genes. A recurrent PIK3CA c.1633G>A mutation was found in four separate cases. Identified mutations were present in 8-40% of sequenced alleles in various brain regions and were associated with increased neuronal S6 protein phosphorylation in the brains of affected individuals, indicating aberrant activation of mammalian target of rapamycin (mTOR) signaling. Thus HME is probably a genetically mosaic disease caused by gain of function in phosphatidylinositol 3-kinase (PI3K)-AKT3-mTOR signaling.

Figure 1

MRI and mutation analysis in hemimegalencephaly. (a) Axial T2-weighted brain MRI of cases identified with mutations. Arrows indicate the affected hemispheres, showing thickened cortical mantle, changes in white matter signal and alterations in ventricular shape, resulting in increased hemispheric size and midline shift of falx cerebri. (b) Sequencing counts from exome sequencing of each of three brain-blood paired samples. Mut, mutation; ref, reference. (c) Mass spectrometry validation of mutations. Wild-type sequences (blue) and de novo mutations (red) are correlated with results from next-generation sequencing.

Figure 2

Consistent de novo mutation burden across affected hemispheres. (a) HME-1573 sampled during surgery in left orbital (Orb), frontal (Fr), central operculum (Co) and occipital (Occ) regions. (b) HME-1656 sampled in parietal (Par), central operculum and temporal (Tem) regions. (c) HME-1563 was sampled multiple independent times in the central operculum region (yellow circles), but samples could not be precisely aligned anatomically. Left, MRI scans; circles indicate brain areas examined by mass spectrometry analysis. Right, each sample aliquot was analyzed by mass spectrometry for mutation burden, describing variation in mutation burden across anatomical locations.

Figure 3

The de novo mutations identified in HME correlate with hyperactive mTOR signaling. (a) Schematic of the PI3K-AKT3-mTOR pathway and downstream phosphorylated ribosomal protein S6 kinase (P-S6K) and phosphorylated ribosomal protein S6 (P-S6). (b) HME pathological samples show an increased percentage of cells with positive staining for P-S6 and MAP2. Left P-S6, biotin-streptavidin DAB staining; right, P-S6, fluorescence-conjugated staining. MAP2 was used to identify neurons. Scale bars, 100 µm. (c) Quantification of P-S6–positive cells from 5–7 representative cortical areas per case. **P < 0.01 (relative to non-HME samples, one-way ANOVA with Bonferroni posttest, n > 50 cells per region). Error bars, s.e.m.