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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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MTOR Mutations and Clinical Photographs of Patients With MTOR Mutations, Megalencephaly, and Cutis Tricolor of the Blaschko-Linear Type
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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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Brain Imaging and Histopathologic Analysis in Patient LR13-389 With MTOR p.Ser2215Phe Mutation
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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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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
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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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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
Table.
Clinical, Neuroimaging, Neuropathologic, and Molecular Findings of MTOR and DEPDC5 Mutation–Positive Patients

Abbreviations: cDNA, complementary DNA; fb, skin fibroblasts; fb+, fibroblasts grown from hyperpigmented streaks; fb–, fibroblasts grown from hypopigmented streaks; FCD, focal cortical dysplasia; NA, not applicable; PIGM, pigmentary mosaicism of skin; PQD, posterior quadrantic dysplasia; s, saliva; wbc, white blood cells; +, seizures; ++, intractable seizures leading to epilepsy surgery; –, no data.

a Further details regarding the specific alternative allele levels in various tissues are provided in eTable 3 and eTable 4 in the Supplement.

b Skin color pattern consistent with cutis tricolor of the Blaschko-linear type, which consists of diffuse alternating hypopigmented and hyperpigmented streaks.

c a1 and a2 denote 2 identical twin brothers. Both had autism at 17 years of age with severe impairment of social reciprocity, no eye contact, and absent speech. They met the criteria for autism spectrum disorders on formal longitudinal neurodevelopmental assessments.

d This girl has early autistic features at 3 years of age, a capillary malformation on the scalp that has faded with age, and a small hemangioma on her chest wall that completely involuted in infancy.

e This boy also has a small liver hemangioma that measured 2 × 1 cm.

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Original Investigation
July 2016

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

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
Abstract

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.

Introduction

Focal cortical dysplasia (FCD) is a malformation of cortical development found in almost half of children referred for epilepsy surgery, making it the most frequent known cause of intractable focal epilepsy.1 The histopathologic changes that define FCD type 2 (FCD2) include cortical dyslamination and large dysmorphic neurons without (type 2a) or with (type 2b) balloon cells.2 Functional defects in the phosphatidylinositol 3-kinase (PI3K)–AKT (serine/threonine kinase, also known as protein kinase B)–mammalian target of rapamycin (mTOR) pathway have been demonstrated by Western blot, immunohistochemical staining, and AKT kinase and RNA expression analyses,3-5 and mutations of DEPDC5 (OMIM 614191), PTEN (germline) (OMIM 601728), and PIK3CA (postzygotic or mosaic) (OMIM 171834) have been found in FCD, classified as FCD1 or FCD2.3,6-13 Furthermore, 3 previous studies14-16 reported mosaic mutations in MTOR in patients with FCD2. Many studies2,3,6,17-19 have found links among diffuse megalencephaly, hemimegalencephaly, and FCD2, which have historically been considered distinct disorders. A link is further supported by discovery of germline or mosaic mutations of the same genes in the PI3K-AKT-mTOR pathway in patients with megalencephaly, hemimegalencephaly, or FCD. These data suggest that further genetic studies of FCD, as well as hemimegalencephaly and megalencephaly, are warranted to define additional genetic causes and explore links between the phenotypes and the levels and distribution of mosaic mutations.

We used massively parallel sequencing, histopathologic analysis, and several functional assays of PI3K-AKT-mTOR activity in brain and peripheral tissues in FCD, hemimegalencephaly, and megalencephaly. These experiments led us to discover low-level mutations of MTOR in FCD2a, a compelling demonstration of the pattern of mosaicism in affected brain tissues, a broad spectrum of brain overgrowth phenotypes distinguished by different mutations and levels of mosaicism, and a link between mosaic mutations of MTOR and pigmentary mosaicism.

Box Section Ref ID

Key Points

  • Question What are the genetic causes of segmental cortical dysplasia and diffuse brain overgrowth, and, of the mosaic causes, what is the level of mosaicism across these disorders?

  • Findings In this whole-exome sequencing study, constitutional and mosaic mutations of MTOR are associated with a broad spectrum of brain disorders, ranging from megalencephaly to focal cortical dysplasia (FCD) and hypomelanosis of Ito, with a mutation gradient identified in MTOR-related focal cortical dysplasia.

  • Meaning MTOR-activating mutations in the megalencephaly, hemimegalencephaly, and FCD2 spectrum of malformations have important clinical implications and may constitute treatable causes of intractable epilepsy associated with FCD.

Methods
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 with institutional review board approval. Informed consent and research samples were obtained from all patients and selected family members. Brain tissue was obtained during clinically indicated epilepsy surgery. Data analysis was performed from July 2014 to July 2015.

Magnetic Resonance Imaging

Patients in our epilepsy surgery cohort underwent brain magnetic resonance imaging (MRI) performed on Siemens 1.5-T (Symphony or Avanto) or 3-T (TrioTim) systems. Preoperative structural data included axial, coronal, and sagittal T1- and T2-weighted and axial fluid-attenuated inversion recovery images. We performed skull stripping with the brain extraction tool in FSL and then made 3-dimensional renderings with BioImage Suite.20,21 Coordinates for brain tissue samples were created using the image-guided surgery system. Intraoperative tissue coordinates were downloaded from the image-guided surgery system and transformed into MRI space using BioImage Suite.21 Tissue coordinates were overlaid onto 3-dimensional renderings of the brain to visualize sample locations. Patients in our megalencephaly cohort underwent standard brain MRI, including T1-weighted, T2-weighted, and fluid-attenuated inversion recovery sequences in axial, sagittal, and coronal planes at their respective centers.

Molecular Methods

Genomic DNA was isolated with standard protocols. We hypothesized that FCD and hemimegalencephaly are caused by low-level mosaic mutations; therefore, we designed a tetrad-based whole-exome sequencing (WES) approach that consisted of standard-depth (approximately 50-60X) sequencing in peripheral samples (blood, saliva, or skin) from the affected child and both parents plus deep (approximately 150-180X) WES in DNA isolated from affected brain tissue. Data were analyzed using a standard trio approach with the peripheral sample, confirmation with the affected brain sample, and paired analysis between unaffected and affected brain sequence data (eFigure 1 in the Supplement). Mutations were confirmed using several targeted methods, including single-molecule molecular inversion probes, amplicon sequencing, and Sanger sequencing. The same methods were used to screen our cohort of children with FCD, hemimegalencephaly, or megalencephaly. Details regarding the sequencing methods are provided in the eMethods in the Supplement.

Neuropathologic and Functional Analysis

Histologic sections of resected brain tissue were scored for features of FCD. Individuals with FCD2 or hemimegalencephaly were selected for further study. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections by standard techniques using antibodies directed against mouse monoclonal Map2 (Millipore), rabbit monoclonal Thr308-phospho-Akt, and rabbit monoclonal phospho-S6 ribosomal protein (both from Cell Signaling Technology).3 Western blot analysis was performed on 100 to 200 mg of frozen cortical tissue following standard protocols published previously.3

Neuronal Culture Models

Wild-type or mutant hemagglutinin-tagged MTOR constructs were expressed by electroporation in embryonic day 18 cultured rat cortical neurons. Neurons aged 7 to 14 days were treated with vehicle, amino acid and growth factor starvation conditions, or mTOR inhibitors and then fixed and immunostained using standard techniques. Cultures were imaged with an automated confocal microscopy imaging system (InCell 6000; General Electric) and high-content automated analysis performed with InCell Analyzer software (General Electric) and Tibco Spotfire (Tibco). Antibodies against phospho-S6 (Serine 240/244), hemagglutinin tag, NeuN, or Map2 were used.

Results
Exome Sequencing

We performed tetrad-based WES in 8 children with FCD (FCD2a, 5 children; hemimegalencephaly-FCD2a, 1 child; and FCD2b, 2 children) to search for de novo germline and mosaic mutations. To identify de novo germline mutations, we filtered for variants observed in the patient but not the parents. To identify mosaic mutations, we looked for variants present in brain DNA that were not found in the matched peripheral sample. We successfully generated sequencing data for all 8 samples from affected brain, 7 of 8 peripheral samples, and 15 of 16 parents (eTable 1 and eResults in the Supplement). We sequenced brain samples to a mean read depth of 171X coverage across the targeted loci (range, 130-204X) to facilitate identification of mosaic mutations at low allelic fractions, peripheral samples from 7 of 8 probands to a mean of 88X (range, 69-103X), and parental samples to a mean of 82X (range, 59-101X).

De Novo Deletion in DEPDC5

We performed variant identification and filtering for de novo single-nucleotide variants (SNVs) and indels and manually reviewed all filtered variants to remove artifacts not captured in our filtering scheme. In the 7 patients with complete parental data, we identified 8 putative de novo missense SNVs, 1 de novo in-frame insertion, and 1 de novo deletion in DEPDC5, leading to a frameshift (c.4187delC) (eTable 2 and eFigure 2 in the Supplement). This girl with hemimegalencephaly also had copy number neutral loss of heterozygosity on chromosome 22, suggesting possible loss of the other allele (eFigure 3 in the Supplement). Several other somatic indels were not deemed to be pathogenic based on multiple lines of evidence (eTable 3 in the Supplement).

Mosaic Mutations in MTOR

To identify mosaic SNVs, we used an algorithm sensitive to low allelic fractions. After filtering and manual review, we identified 8 SNVs present at allelic fractions less than 0.1 (eTable 3 in the Supplement), including 3 missense mutations in MTOR in 3 unrelated children with FCD2a. The fraction of reads supporting the mutant allele was always less than 0.1 in brain and 0 in peripheral samples and parents. The p.Leu1460Pro mutation is located in the focal adhesion targeting domain of MTOR and the p.Ser2215Phe and p.Ser2215Tyr mutations in the kinase domain (Table and Figure 1A). We next reexamined targeted sequencing data generated by single-molecule molecular inversion probe in 42 individuals with FCD or hemimegalencephaly (including 21 with FCD2) and identified the p.Ser2215Phe mutation in 1 unrelated patient with FCD2a (LR13-389).

MTOR Mutations in Megalencephaly

Prompted by these findings, we reexamined sequencing data in 51 individuals with unexplained megalencephaly, including 2 with trio-based WES, and identified 6 additional patients with MTOR mutations, who segregated into 2 groups. The first group consisted of 3 children with asymmetric megalencephaly, cortical dysplasia (including polymicrogyria), and linear streaks of hypopigmented and hyperpigmented skin following the lines of Blaschko (Figure 1, B-D). All 3 had the same MTOR mutation, p.Thr1977Ile, with levels of mosaicism varying from 0.23 to 0.55 in affected skin and 0.07 to 0.20 in blood or saliva (Table). Their skin markings correspond to cutis tricolor of the Blaschko-linear type, a form of pigmentary mosaicism (Rudolf Happle, MD, Department of Dermatology, University of Freiburg, Freiburg, Germany, written communication, April 2015).22 This result is a more specific dermatologic diagnosis than hypomelanosis of Ito, which has been used for almost any form of pigmentary mosaicism. The second group included 4 children (including 2 identical twins) with symmetric megalencephaly, intellectual disability, autism (in the 2 oldest), and limited or no polymicrogyria. All 4 had the same de novo constitutional MTOR mutation, p.Glu1799Lys (Table). The alternative allele fractions of all mutations identified by various methods in these 3 groups are further detailed in eTable 4 in the Supplement. The neuroimaging and neuropathologic features for all 3 groups are summarized in eTable 5 and eTable 6 and represented in eFigures 4, 5, and 6 in the Supplement.

Mutation Gradient and Epileptogenesis

For 1 patient (LR13-389; p.Ser2215Phe), we had DNA from 10 different brain regions after parietotemporal lobectomy with intraoperative electrocorticography (Figure 2). The mutation level (alternate allele fraction) varied from 0 to 0.086 and demonstrated a striking gradient with an epicenter in the posterior temporal lobe and lower levels at the periphery. We were unable to detect the mutation in 6 of 7 samples from the anterior edge of the resected area with a mean read depth of 269X (range, 178-351X), even though all tissue samples showed clear FCD2a pathology. The patient underwent a functional hemispherectomy 6 months later because of recurrent epilepsy. Another patient (LR12-245, p.Ser2215Tyr) had several surgical resections. The patient initially underwent temporal and occipital lobectomy with intraoperative electrocorticography performed at 7 months, followed by a staged additional craniotomy for left parietal resection with electrocorticography at 5 years of age, with resolution of epilepsy after the second procedure (Figure 3, A-E). Western blots from highly epileptogenic posterior temporal-occipital cortex resected during the first surgery had higher phospho-S6 expression than parietal cortex resected during the second surgery (Figure 3, F and G, and eFigure 7 in the Supplement). Intracranial electroencephalography by depth electrodes and grids before the second surgery revealed seizure onset from the mesial parietal region, which exhibited elevated phospho-S6 levels, but not from the lateral parietal region, which exhibited low phospho-S6 expression (Figure 3, H and I). Strikingly, the mutation was not detected in either sample (eTable 4 in the Supplement).

Functional Effects

To evaluate the functional effect of identified mutations, we queried the Catalogue of Somatic Mutations in Cancer database and found that 4 of 5 MTOR mutations (plus Thr1977Arg but not Thr1977Ile) have been observed in tumors.23 All activate mTOR complex 1 (mTORC1) signaling in vitro.24 We assessed PI3K-AKT-mTOR activation by Western blot for mutations in MTOR, DEPDC5, PIK3CA, AKT, and control brain tissue (Figure 4, A-C). All samples with mutations had elevated phospho-S6 expression, whereas T308 phospho-AKT was elevated only in samples with PIK3CA and AKT3 mutations. Immunohistochemical analysis in MTOR p.Ser2215Tyr and DEPDC5 samples revealed increased expression of phospho-S6 in large dysmorphic neurons (Figure 4D).

We next electroporated constructs that contained wild-type or mutant MTOR into cultured rodent neurons and measured mTORC1 activation using high-content imaging of phospho-S6 (serine 240/244) immunofluorescence. Neurons expressing mutant MTOR constructs revealed hyperactivation of mTORC1 compared with wild type (eFigure 8 in the Supplement). To determine whether these mutations affect regulation of mTORC1 signaling by nutrients and growth factors, we subjected mutant and wild-type neurons to starvation with phosphate-buffered saline and glucose solution. Although starvation completely suppressed mTORC1 activity in wild-type neurons, neurons that expressed the MTOR mutations had constitutive activation (Figure 4, E and F), consistent with the effect of these mutations in human cell lines (eFigure 9 in the Supplement).24 Of interest, mutants found in patients with megalencephaly and hemimegalencephaly induced an intermediate level of mTORC1 hyperactivation between wild-type and FCD mutations. To assess whether the FCD or megalencephaly MTOR mutations induced cellular hypertrophy, a hallmark of FCD histopathologic findings, we measured neuronal cell size using automated analysis of a combination of nuclei staining (Hoechst) and the NeuN neuronal marker that outlines the cell soma. Consistent with mTORC1 hyperactivation, we observed significant increases in cell size in mutant neurons (Figure 4, G and H, and eFigure 8 in the Supplement) that could be reversed by 7 days of mTOR inhibition with the rapamycin analogue RAD001 (Figure 4G).

Discussion

In our cohort, we have identified 5 de novo MTOR mutations at 4 amino acid residues in 10 unrelated patients with FCD, hemimegalencephaly, and megalencephaly. Our data confirm the occurrence of low-level mosaic MTOR mutations in a subset of patients with FCD2 and provide evidence of (1) an association of MTOR with a spectrum of brain malformations, ranging from FCD2a to diffuse megalencephaly; (2) a causal link between MTOR mutations and induction of elevated mTORC1 activity and neuronal hypertrophy; (3) a gradient of mutation in the brain in which levels of mosaicism below the detection level of next-generation sequencing are still sufficient to cause FCD2 disease; (4) a strong link between functional expression of the mutation and epilepsy; (5) a new association between MTOR mutations and autism; (6) a new association between mosaic MTOR mutations and pigmentary mosaicism; and (7) variation in phospho-AKT and phospho-S6 expression in brain tissue that correlates with mutations of different core components of the PI3K-AKT-mTOR pathway.

MTOR and DEPDC5

MTOR is a serine/threonine protein kinase that integrates many intracellular and extracellular signals and serves as a central regulator of cell proliferation, growth, metabolism, and survival.25,26 Upregulation of MTOR mediates the cellular and molecular changes, leading to cortical malformation and epilepsy in FCD and tuberous sclerosis complex.3,5DEPDC5 is a repressor of mTORC1 signaling so that loss-of-function mutations of DEPDC5 also upregulate MTOR activity.27,28 Adding to our report, mutations of DEPDC5 have been observed with hemimegalencephaly, FCD2a, FCD2b, and probably FCD1.6,7 Loss of heterozygosity of chromosome 22 was observed in the patient with the DEPDC5 alteration, which suggests inactivation of both copies of the gene in affected (brain) tissue.

Three MTOR-Related Phenotypes

Our 10 patients with MTOR mutations segregate into 3 distinct groups with emerging genotype-phenotype correlations (Table). The first consists of FCD2a. All 4 children with FCD2a had low-level mosaic mutations detectable only in the brain at levels ranging from 0.012 to 0.086 of reads. The second consists of asymmetric megalencephaly, cortical dysplasia, and pigmentary mosaicism. The skin phenotype could be designated as hypomelanosis of Ito, but the more specific diagnosis of cutis tricolor of the Blaschko-linear type reflects the association with hypopigmented and hyperpigmented streaks.22 Indeed, review of photographs of children with hemimegalencephaly or FCD and hypomelanosis of Ito usually reveal hypopigmented and hyperpigmented streaks.29-33 All 3 children in this group had the p.Thr1977Ile MTOR mutation at intermediate mutation levels. The third group includes children with diffuse megalencephaly, intellectual disability, and (when old enough to be evaluated) autism. None had dysmorphic features or altered skin pigmentation, but 3 were noted to have patchy areas of cortical dysplasia. All 3 had the recurrent, de novo, constitutional MTOR mutation, p.Glu1799Lys.

Other groups reported low-level mosaic MTOR mutations in the brain in 12 (16%) of 77 individuals with FCD214 or 6 of 13 individuals with FCD2b,15 similar to our data. In addition, MTOR mutations have been reported in the brain but not blood-derived DNA in 2 patients with hemimegalencephaly.6,19

Patient Phenotypes and the PI3K-AKT-mTOR Pathway

Our results combined with published data indicate that FCD2 and hemimegalencephaly are disorders of the PI3K-AKT-mTOR pathway. Mutations of pathway genes PIK3CA, PTEN, AKT3, MTOR, DEPDC5, and TSC2 have been identified in 19 of 53 patients (36%) with hemimegalencephaly3,6,19 and 23 of 116 (20%) with FCD2 (including this report).14-16 The most frequently mutated genes so far have been PIK3CA in hemimegalencephaly (9 of 53 [17%]) and MTOR in FCD2 (22 of 116 [19%]).3,6,14,16,18,19

Based on pathological features, FCD was separated into 9 groups, including FCD2a and FCD2b, based on the absence or presence of balloon cells. Adding our data to other reports, mutations of PTEN, MTOR, and DEPDC5 have been associated with FCD2a or FCD2b (and possibly FCD1) in different patients, observations that weaken the rationale for separating them. We propose instead that separation using functional assays and mutation analysis is more useful. We found that PIK3CA, PTEN, and AKT3 mutation–positive samples had elevations of T308 phospho-AKT expression, whereas MTOR and DEPDC5 samples did not. This finding is consistent with upstream AKT activation in patients with pathway mutations upstream of mTORC1. Expression of phospho-S6 was increased in all mutation-positive specimens, possibly correlating with the level of mosaicism.

With our rodent functional expression system, we evaluated MTOR mutants and found hyperactivation patterns that segregated by patient groups: mutations found in patients with megalencephaly or hemimegalencephaly with broad distributions of MTOR mutant neurons (Glu1799Lys, Thr1977Ile, and Cys1483Tyr) exhibited a level of elevated mTORC1 signaling that was intermediate between wild-type levels and those observed in mutants found in patients with FCD2a (Ser2215Phe, Ser2215Tyr, and Leu1460Pro). These data suggest that small lesions of FCD2a can be epileptogenic because of strong elevation of mTORC1 activity. Only weaker mutations may be observed in patients with a broad distribution of mutant cells in brain and peripheral tissues because more severe mutations may be lethal. Common to all mutations, however, was the finding that MTOR inhibitors decreased pathologic neuronal enlargement.

Conclusions

Our discovery of MTOR-activating mutations in the megalencephaly, hemimegalencephaly, and FCD2 spectrum of malformations has important clinical implications. Everolimus, an inhibitor of mTORC1 activity, is an approved treatment for tuberous sclerosis complex–associated subependymal giant cell astrocytomas and is currently in a phase 3 clinical trial as an adjunctive therapy in patients with tuberous sclerosis complex and refractory partial-onset epilepsy (Efficacy and Safety of 2 Trough Ranges of Everolimus as Adjunctive Therapy in Patients With Tuberous Sclerosis Complex and Refractory Partial-Onset Seizures). Several other molecules that inhibit PI3K-AKT-mTOR components are undergoing clinical development for cancer and alone or in combination offer a new approach to the treatment of intractable epilepsy that involves direct correction of the underlying molecular defect rather than simply reducing membrane excitability. Multiple studies1,34-36 have found that 25% to 40% of intractable epilepsy that requires resective surgery is caused by FCD. Although the contribution of MTOR and other PI3K-AKT pathway genes to intractable epilepsy not amenable to surgical treatment is unknown, our data suggest that it may be high. Thus, mutations of the PI3K-AKT-mTOR pathway, including MTOR itself, may prove to be a common and treatable cause of intractable focal epilepsy.

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Article Information

Corresponding Author: Ghayda M. Mirzaa, MD, Center for Integrative Brain Research, Seattle Children’s Research Institute, 1900 Ninth Ave, Mailstop C9S-10, Seattle, WA 98101 (gmirzaa@uw.edu).

Accepted for Publication: February 1, 2016.

Published Online: May 9, 2016. doi:10.1001/jamaneurol.2016.0363.

Author Contributions: Drs Mirzaa and Campbell contributed equally to this work as first authors, and Drs Solovieff and Goold contributed equally as second authors. Drs Mirzaa and Dobyns had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Mirzaa, Solovieff, Goold, Jansen, Liu, Lupski, Wilson, Morrissey, Ojemann, Guerrini, Winckler, Dobyns.

Acquisition, analysis, or interpretation of data: Mirzaa, Campbell, Solovieff, Goold, Jansen, Menon, Timms, Conti, Biag, Olds, Boyle, Collins, Ishak, Poliachik, Girisha, Yeung, Chung, Rahikkala, Gunter, McDaniel, Macmurdo, Bernstein, Martin, Leary, Mahan, Liu, Weaver, Doerschner, Jhangiani, Muzny, Boerwinkle, Gibbs, Shendure, Saneto, Novotny, Wilson, Sellers, Morrissey, Hevner, Ojemann, Guerrini, Murphy, Winckler, Dobyns.

Drafting of the manuscript: Mirzaa, Campbell, Solovieff, Goold, Conti, Boyle, Ishak, Lupski, Wilson, Sellers, Murphy, Dobyns.

Critical revision of the manuscript for important intellectual content: Mirzaa, Campbell, Solovieff, Goold, Jansen, Menon, Timms, Biag, Olds, Collins, Ishak, Poliachik, Girisha, Yeung, Chung, Rahikkala, Gunter, McDaniel, Macmurdo, Bernstein, Martin, Leary, Mahan, Liu, Weaver, Doerschner, Jhangiani, Muzny, Boerwinkle, Gibbs, Shendure, Saneto, Novotny, Wilson, Morrissey, Hevner, Ojemann, Guerrini, Murphy, Winckler, Dobyns.

Statistical analysis: Mirzaa, Solovieff, Jansen, Timms, Boyle, Boerwinkle.

Obtained funding: Jansen, Chung, Gibbs, Guerrini, Murphy, Dobyns.

Administrative, technical, or material support: Goold, Menon, Collins, Poliachik, Yeung, Chung, Rahikkala, Gunter, Macmurdo, Martin, Leary, Liu, Doerschner, Jhangiani, Muzny, Gibbs, Shendure, Saneto, Novotny, Wilson, Ojemann, Guerrini, Murphy, Winckler, Dobyns.

Study supervision: Goold, Ishak, Leary, Gibbs, Lupski, Shendure, Wilson, Sellers, Morrissey, Ojemann, Murphy, Winckler, Dobyns.

Conflict of Interest Disclosures: Mr Boyle and Dr Shendure reported having a patent and copyright for systems, algorithms, and software for molecular inversion probe design with royalties paid by Roche. Drs Campbell, Solovieff, Goold, Menon, Leary, Sellers, Morrissey, Murphy, and Winckler and Messrs Biag, Mahan, and Liu reported being employees of Novartis Inc. Novartis Institutes for BioMedical Research funded exome sequencing and neuronal culture experiments. Dr Lupski reported owning stock in 23andMe, working as a paid consultant for Regeneron Pharmaceuticals, having stock options in Lasergen Inc, being a member of the Scientific Advisory Board of Baylor Miraca Genetics Laboratories, and being a coinventor on multiple US and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, and bacterial genomic fingerprinting. The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from the chromosomal microarray analysis and clinical exome sequencing offered in the Baylor Miraca Genetics Laboratory. No other disclosures were reported.

Funding/Support: This study was funded by grants K08NS092898 (Dr Mirzaa), NS058721 (Dr Dobyns), NS092772 (Dr Dobyns), NS072162 (Dr Jansen), and HG006542 (Baylor-Hopkins Center for Mendelian Genomics Research Program) from the National Institute of Neurological Disorders and Stroke, Citizens United for Research in Epilepsy (Dr Jansen); grant N602531 from the European Union Seventh Framework Program under project DESIRE (Dr Guerrini); E-Rare JTC 2011 (Dr Guerrini); and the SK Yee Medical Research Fund (Mr Yeung and Dr Chung).

Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We thank Rudolf Happle, MD, for reviewing the cutaneous phenotype. We also thank the patients and their families for their important contribution to our research.

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