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Original Investigation |

Association of MTOR Mutations With Developmental Brain Disorders, Including Megalencephaly, Focal Cortical Dysplasia, and Pigmentary Mosaicism

Ghayda M. Mirzaa, MD1,2; Catarina D. Campbell, PhD3; Nadia Solovieff, PhD3; Carleton P. Goold, PhD3; Laura A. Jansen, MD, PhD4; Suchithra Menon, PhD3; Andrew E. Timms, PhD5; Valerio Conti, BSc, PhD6,7; Jonathan D. Biag, MS3; Carissa Olds, MSc2; Evan August Boyle, BS8; Sarah Collins, MS2; Gisele Ishak, MD9; Sandra L. Poliachik, PhD9; Katta M. Girisha, MD10; Kit-San Yeung, BSc, MPhil11; Brian Hon Yin Chung, MD11; Elisa Rahikkala, MD, PhD12,13; Sonya A. Gunter, MS4; Sharon S. McDaniel, MD14; Colleen Forsyth Macmurdo, DO15; Jonathan A. Bernstein, MD, PhD15; Beth Martin, BS16; Rebecca J. Leary, PhD3; Scott Mahan, MT(ASCP)3; Shanming Liu, MSc3; Molly Weaver, BS17; Michael O. Dorschner, PhD17; Shalini Jhangiani, MS18,19; Donna M. Muzny, MSc18,19; Eric Boerwinkle, PhD19,20; Richard A. Gibbs, PhD18,19; James R. Lupski, MD, PhD, DSc(Hon)18,19,21,22; Jay Shendure, MD, PhD16; Russell P. Saneto, DO, PhD23,24; Edward J. Novotny, MD2,23; Christopher J. Wilson, PhD25; William R. Sellers, MD3; Michael P. Morrissey, PhD3; Robert F. Hevner, MD, PhD2,26; Jeffrey G. Ojemann, MD26; Renzo Guerrini, MD6,7,27; Leon O. Murphy, PhD3; Wendy Winckler, PhD3; William B. Dobyns, MD1,2
[+] Author Affiliations
1Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle
2Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, Washington
3Novartis Institutes for BioMedical Research Inc, Cambridge, Massachusetts
4Department of Neurology, University of Virginia, Charlottesville
5Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, Washington
6Paediatric Neurology, Neurogenetics, and Neurobiology Unit and Laboratories, A. Meyer Children’s Hospital, Florence, Italy
7Department of Neuroscience, Pharmacology and Child Health, University of Florence, Florence, Italy
8Department of Genetics, Stanford University School of Medicine, Stanford, California
9Department of Radiology, Seattle Children's Hospital, Seattle, Washington
10Department of Medical Genetics, Kasturba Medical College, Manipal University, Manipal, Karnataka, India
11Department of Pediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
12PEDEGO Research Group and Medical Research Center Oulu, University of Oulu, Oulu, Finland
13Department of Clinical Genetics, Oulu University Hospital, Oulu, Finland
14Pediatric Neurology and Epilepsy, Kaiser Permanente San Francisco Medical Center, San Francisco, California
15Division of Medical Genetics, Department of Pediatrics, Stanford University School of Medicine, Stanford, California
16Department of Genome Sciences, University of Washington, Seattle
17Department of Pathology, University of Washington, Seattle
18Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas
19Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas
20Human Genetics Center, University of Texas Health Science Center at Houston, Houston
21Department of Pediatrics, Baylor College of Medicine, Houston, Texas
22Department of Pediatrics, Texas Children’s Hospital, Houston
23Division of Neurology, Pediatrics, and Radiology, University of Washington, Seattle
24Center for Developmental Therapeutics, Seattle Children's Research Institute, Seattle, Washington
25Editas Medicine, Cambridge, Massachusetts
26Department of Neurological Surgery, University of Washington, Seattle
27IRCCS Stella Maris Foundation, Pisa, Italy
JAMA Neurol. 2016;73(7):836-845. doi:10.1001/jamaneurol.2016.0363.
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Published online

Importance  Focal cortical dysplasia (FCD), hemimegalencephaly, and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. These disorders are associated with significant childhood morbidity and mortality.

Objective  To identify the underlying molecular cause of FCD, hemimegalencephaly, and diffuse megalencephaly.

Design, Setting, and Participants  Patients with FCD, hemimegalencephaly, or megalencephaly (mean age, 11.7 years; range, 2-32 years) were recruited from Pediatric Hospital A. Meyer, the University of Hong Kong, and Seattle Children’s Research Institute from June 2012 to June 2014. Whole-exome sequencing (WES) was performed on 8 children with FCD or hemimegalencephaly using standard-depth (50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and their parents and deep (150-180X) sequencing in affected brain tissue. Targeted sequencing and WES were used to screen 93 children with molecularly unexplained diffuse or focal brain overgrowth. Histopathologic and functional assays of phosphatidylinositol 3-kinase–AKT (serine/threonine kinase)–mammalian target of rapamycin (mTOR) pathway activity in resected brain tissue and cultured neurons were performed to validate mutations.

Main Outcomes and Measures  Whole-exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders.

Results  Low-level mosaic mutations of MTOR were identified in brain tissue in 4 children with FCD type 2a with alternative allele fractions ranging from 0.012 to 0.086. Intermediate-level mosaic mutation of MTOR (p.Thr1977Ile) was also identified in 3 unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin. Finally, a constitutional de novo mutation of MTOR (p.Glu1799Lys) was identified in 3 unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in 2 children with FCD2a from whom multiple affected brain tissue samples were available revealed a mutation gradient with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all MTOR mutations identified here drive constitutive activation of mTOR complex 1 and enlarged neuronal size.

Conclusions and Relevance  In this study, mutations of MTOR were associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2a to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations may be sufficient to cause cellular hypertrophy in cultured neurons and may provide a demonstration of the pattern of mosaicism in brain and substantiate the link between mosaic mutations of MTOR and pigmentary mosaicism in skin.

Figures in this Article


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Figure 1.
MTOR Mutations and Clinical Photographs of Patients With MTOR Mutations, Megalencephaly, and Cutis Tricolor of the Blaschko-Linear Type

A, Positions of MTOR coding mutations identified in this report relative to their locations within the MTOR protein domain structure. The height of the bar indicates the number of patients with mutations at that amino acid (AA) position, and the color of the square indicates the phenotype of the patient(s). Patient LR13-310 (B) and LR14-326 (C and D) show alternating, predominantly linear streaks of hyperpigmented and hypopigmented skin, which correspond to cutis tricolor of the Blaschko-linear type. FAT indicates focal adhesion targeting; FATC, FRAP, ATM, TRRAP C-terminal domain; and FCD2, focal cortical dysplasia type 2.

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Figure 2.
Brain Imaging and Histopathologic Analysis in Patient LR13-389 With MTOR p.Ser2215Phe Mutation

A-C, An area of cortical infolding and thickening in the left posterior temporal and parietal lobes (arrowheads) on preoperative T2- (A) and T1-weighted (B) images that has been excised on a postsurgical T1-weighted image (C). D and E, Preoperative deep (D) and superficial (E) 3-dimensional images show the locations and levels of mosaicism (alternate allele fractions) of specimens collected during surgery. These specimens include the amygdala (a), hippocampus (b), deep anterior temporal lobe (c), frontal operculum (d), anterior temporal lobe and superior temporal gyrus (e and f), anterior temporal lobe and middle temporal gyrus (g), posterior temporal lobe (h), inferior parietal lobe (i), and superior parietal lobe (j). The levels of mosaicism, although all low, are highest in the center of the dysplasia (h at 9%), intermediate along the posterior border (i and j at 3%), and too low to detect consistently along the anterior border of the lesion (a-g at 0%-1%), even though all sections show changes of focal cortical dysplasia type 2a. F-I, Brain sections stained with NeuN, which all show loss of cortical lamination, excessive tall vertical columns of neurons (especially prominent in F), numerous maloriented large neurons, and blurring of the cortical-white matter boundary. The proportion of large dysplastic neurons appears higher in sections with higher mutation levels (F and I) compared with regions with undetected mutations (G and H). In the section from the center of the dysplasia with the highest level of mutation, a transition can be seen with less severe dysplasia on the left of the image and more severe dysplasia with more numerous large dysplastic neurons on the right (F). J, Same section as F stained with Map2 at a higher power showing several large dysplastic neurons with disorganized processes, excessive cytoskeletal elements within cell bodies, and abnormally oriented dendrites that often crowd together.

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Figure 3.
Differential MTOR Mutation Burden and Phosphorylated Ribosomal Protein S6 Expression in a Girl (LR12-245) With Early- and Later-Onset Seizures Due to Focal Cortical Dysplasia Type 2

A and B, Magnetic resonance images (MRIs) performed at 6 months before the first surgical procedure. The findings include increased volume of the left midtemporal lobe, mild thickening, and irregularity of the cortex (arrowheads in A) and similar but more focal changes in the superior temporal lobe (arrowhead in B). C and D, MRIs performed before the second surgery at 5 years. The images show the surgical defect and subtle changes in the left parietal lobe. E, Locations of the 4 surgical specimens used for genetic and tissue analysis as indicated by colored circles: temporal lobe from the first surgery (a, yellow), occipital lobe seizure onset zone (SOZ) disconnected during the first surgery but was not removed until the second surgery (b, light blue), medial parietal SOZ from the second surgery (c, dark blue), and lateral parietal cortex that was also from the second surgery (d, red) that was not involved in seizure onset. The inset shows the locations in 3 dimensions. F and G, Western blots for phospho-S6 (pS6) from the occipital lobe (OL, location b in panel E), mesial parietal lobe (mesPL, c), and lateral parietal lobe (latPL, d). This analysis revealed a higher level of pS6 in the OL compared with the mesPL and latPL lobes. H, A 3-dimensional brain rendering trimmed to midline to display medial electrodes showing grid and strip placement for intracranial electroencephalographic (EEG) monitoring at 5 years. The inset provides a better 3-dimensional view of the locations. I, EEG tracings from intraoperative grids placed over the temporal, parietal, and occipital regions before the second resection. The tracings show several SOZs. The most active interictal discharges came from the most inferior leads on the mesPL grid (dark blue grid in H, leads 1-3 and 9-11), marking an SOZ. These events spread quickly to other mesPL leads and the latPL strip (red strip in E, leads 1-8). Several independent SOZ were seen in the OL, which had been disconnected several years before. The voltages are lower because the grid was not as closely apposed to the brain surface. Overall, the highest levels of pS6 expression were seen in the OL SOZ responsible for early-onset seizures, with lower expression in the medPL cortex SOZ responsible for later-onset seizures, and nearly undetectable levels in the latPL cortex that was not involved in seizure generation. These findings correlate with MTOR mutation burden determined from these same samples (panel E and eTable 4 in the Supplement).

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Figure 4.
Functional Consequences of Phosphatidylinositol 3-Kinase (PI3K)–AKT (Serine/Threonine Kinase, Also Known as Protein Kinase B)–Mammalian Target of Rapamycin (mTOR) Pathway Mutations in Patient Tissue and Rodent Neurons

A, Levels of T308 AKT phosphorylation (PI3K-PDK1 dependent) compared with ribosomal protein phospho-S6 (pS6) (mTOR complex 1 dependent) in control and dysplastic brain specimens containing mutations. Specimens with upstream pathway mutations (PIK3CA, AKT3) have the highest levels of AKT phosphorylation; specimens with downstream mutations (DEPDC5, MTOR) exhibit elevation of pS6 with lesser elevation of T308 pAKT. B and C, Mean results from 5 blots for T308 pAKT (B) and pS6 (C). D, pS6 expression at high power in a subset of neurons in dysplastic human cortex with DEPDC5 or MTOR mutations. Green indicates Map2 neuronal marker; red, pS6; blue, 4′,6-diamidino-2-phenylindole nuclear stain. Dysmorphic neurons coexpressing Map2 and pS6 (arrowheads) appear orange. E, Representative pS6 indirect immunofluorescence of rat neurons electroporated with wild-type or mutant MTOR constructs. F, Mean pS6 immunofluorescence intensity for DIV12 neurons transfected (NeuN positive, hemagglutinin-tagged/ZsGreen positive) with MTOR or empty vector constructs starved for 2 hours. Data are baseline subtracted (pS6 values in empty vector transfected neurons treated with 200 nM RAD001 [not shown]) and normalized to pS6 immunofluorescence intensities in wild-type MTOR neurons in normal media. Data points represent means per individual wells; columns and error bars, means across wells and SEMs. Within each group (megalencephaly and hemimegalencephaly [MEG/HMEG] vs focal cortical dysplasia type 2 [FCD2]), data for individual genotypes are significantly different from genotypes in each other group or controls (empty vector and wild-type MTOR electroporated neurons) (P < 10−4). G, Mean neuronal size (cell body area with NeuN stain) for transfected neurons treated from DIV7-DIV14 with dimethyl sulfoxide (DMSO) or 200 nM RAD001. One nM RAD001 intermediately reduced size (not shown). For every genotype, RAD001 significantly reduced size (P < 10−3). H, Representative immunostaining for Map2 and Hoechst.

aSignificant differences between genotypes (analysis of variance, Tukey test for multiple comparisons, comparing each condition to every other condition).

bSignificant difference of neuronal size for a given genotype compared with wild-type MTOR (analysis of variance, Dunnett test for multiple comparisons).

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