Mutations in CRADD Result in Reduced Caspase-2-Mediated Neuronal Apoptosis and Cause Megalencephaly with a Rare Lissencephaly Variant ARTICLE Mutations in CRADD Result in Reduced Caspase-2-Mediated Neuronal Apoptosis and Cause Megalencephaly with a Rare Lissencephaly Variant Nataliya Di Donato,1,2,17,* Ying Y. Jean,3 A. Murat Maga,4,5 Briana D. Krewson,6 Alison B. Shupp,6 Maria I. Avrutsky,3 Achira Roy,2 Sarah Collins,2 Carissa Olds,2 Rebecca A. Willert,6 Agnieszka M. Czaja,6 Rachel Johnson,6 Jessi A. Stover,6 Steven Gottlieb,7 Deborah Bartholdi,8 Anita Rauch,9 Amy Goldstein,10 Victoria Boyd-Kyle,6 Kimberly A. Aldinger,2 Ghayda M. Mirzaa,2,11 Anke Nissen,12 Karlla W. Brigatti,6,13 Erik G. Puffenberger,6,13 Kathleen J. Millen,2 Kevin A. Strauss,6,13,14 William B. Dobyns,2,11,15,17 Carol M. Troy,3,16,17 and Robert N. Jinks6,17,* Lissencephaly is a malformation of cortical development typically caused by deficient neuronal migration resulting in cortical thick- ening and reduced gyration. Here we describe a ‘‘thin’’ lissencephaly (TLIS) variant characterized by megalencephaly, frontal predomi- nant pachygyria, intellectual disability, and seizures. Trio-based whole-exome sequencing and targeted re-sequencing identified recessive mutations of CRADD in six individuals with TLIS from four unrelated families of diverse ethnic backgrounds. CRADD (also known as RAIDD) is a death-domain-containing adaptor protein that oligomerizes with PIDD and caspase-2 to initiate apoptosis. TLIS variants cluster in the CRADD death domain, a platform for interaction with other death-domain-containing proteins including PIDD. Although caspase-2 is expressed in the developing mammalian brain, little is known about its role in cortical development. CRADD/caspase-2 signaling is implicated in neurotrophic factor withdrawal- and amyloid-b-induced dendritic spine collapse and neuronal apoptosis, sug- gesting a role in cortical sculpting and plasticity. TLIS-associated CRADD variants do not disrupt interactions with caspase-2 or PIDD in co-immunoprecipitation assays, but still abolish CRADD’s ability to activate caspase-2, resulting in reduced neuronal apoptosis in vitro. Homozygous Cradd knockout mice display megalencephaly and seizures without obvious defects in cortical lamination, supporting a role for CRADD/caspase-2 signaling in mammalian brain development. Megalencephaly and lissencephaly associated with defective programmed cell death from loss of CRADD function in humans implicate reduced apoptosis as an important pathophysiological mech- anism of cortical malformation. Our data suggest that CRADD/caspase-2 signaling is critical for normal gyration of the developing human neocortex and for normal cognitive ability. Introduction Folding of the human cerebral cortex into its complex pattern of gyri and sulci and the precise layering of neu- rons within the cortex (cortical lamination) depend upon a finely tuned program of neuronal proliferation, migration, connectivity, and pruning.1–5 Lissencephaly is a cortical malformation characterized by abnormally broad or absent gyri with unusually thick cortex (10– 20 mm, normal range 1–4.5 mm6) that is classically caused by deficient neuronal migration.7–10 From review of phenotypes in more than 1,400 subjects with lissence- phaly, we identified a relatively mild variant characterized by anterior-predominant pachygyria with shallow and unusually wide sulci, mildly thick cortex (5–7 mm) in at least the frontal regions, and no non-cortical malforma- 1Institute for Clinical Genetics, Faculty of Medicine Carl Gustav Carus, TU Dre attle Children’s Research Institute, Seattle, WA 98105, USA; 3Department of Pa NY 10032, USA; 4Center for Developmental Biology and Regenerative Medicin Craniofacial Medicine, Department of Pediatrics, University of Washington, S Behavior Program, Franklin & Marshall College, Lancaster, PA 17604, USA; 7D dren, Wilmington, DE 19803, USA; 8Department of Human Genetics, Inselspit ich, 8952 Schlieren, Switzerland; 10Department of Pediatrics, School of Medicin burgh, PA 15224, USA; 11Department of Pediatrics, University of Washingt Germany; 13Clinic for Special Children, Strasburg, PA 17579, USA; 14Lancaste University of Washington, Seattle, WA 98195, USA; 16The Taub Institute for Res ical Center; Department of Neurology, Columbia University Medical Center, N 17These authors contributed equally to this work *Correspondence: nataliya.didonato@uniklinikum-dresden.de (N.D.D.), rjinks http://dx.doi.org/10.1016/j.ajhg.2016.09.010. The American Jou � 2016 The Author(s). This is an open access article under the CC BY-NC-ND tions in 13 families, a pattern we refer to as ‘‘thin’’ lissen- cephaly (TLIS). Several families had multiple affected siblings, indicative of autosomal-recessive inheritance. Similar phenotypes have been reported a few times, but with no underlying cause identified (MIM: 610279).11–14 We performed trio-based whole-exome sequencing in two families with TLIS and detected a homozygous muta- tion of CRADD (GenBank: NM_003805.3; c.382G>C [p.Gly128Arg] [MIM: 603454]) in two siblings from a Mennonite family. Targeted re-sequencing of an addi- tional 18 unrelated individuals with TLIS identified two other homozygous missense mutations in CRADD (c.508C>T [p.Arg170Cys] and c.509G>A [p.Arg170His]) in families of Turkish and Finnish ancestry, respectively, and a fourth missense change (c.491T>G [p.Phe164Cys]) in trans with a 3.07 Mb deletion of chromosome 12q22 sden, 01307 Dresden, Germany; 2Center for Integrative Brain Research, Se- thology and Cell Biology, Columbia University Medical Center, New York, e, Seattle Children’s Research Institute, Seattle, WA 98105, USA; 5Division of eattle, WA 98105, USA; 6Department of Biology, Biological Foundations of ivision of Pediatric Neurology, Nemours Alfred I. DuPont Hospital for Chil- al, 3010 Bern, Switzerland; 9Institute of Medical Genetics, University of Zur- e, University of Pittsburgh, Children’s Hospital of Pittsburgh of UPMC, Pitts- on, Seattle, WA 98195, USA; 12Medical Genetics Center, 80335 Munich, r General Hospital, Lancaster, PA 17602, USA; 15Department of Neurology, earch on Alzheimer Disease and the Aging Brain, Columbia University Med- ew York, NY 10032, USA @fandm.edu (R.N.J.) rnal of Human Genetics 99, 1117–1129, November 3, 2016 1117 license (http://creativecommons.org/licenses/by-nc-nd/4.0/). mailto:nataliya.didonato@uniklinikum-dresden.de mailto:rjinks@fandm.edu http://dx.doi.org/10.1016/j.ajhg.2016.09.010 http://crossmark.crossref.org/dialog/?doi=10.1016/j.ajhg.2016.09.010&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ that includes CRADD in an affected child of a fourth family. CRADD encodes caspase-recruitment-domain and death domain (DD)-containing protein (also known as RAIDD, receptor-interacting protein-associated ICH-1/CED-3 ho- mologous protein with a death domain), which is required for activation of caspase-2-mediated apoptosis.15,16 In general, CRADD serves as an adaptor between PIDD (p53-induced death domain-containing protein 1, PIDD1 [MIM: 605247]) and caspase-2 (CASP2 [MIM: 600639]) during formation of the apoptosis-promoting PIDDosome.15–18 Phosphorylation of PIDD by signaling associated with cellular or genotoxic stress stimulates bind- ing of the PIDD-DD to the CRADD-DD, which in turn al- lows the caspase recruitment domain (CARD) of CRADD to bind and activate caspase-2.15–20 Dendritic spine collapse and neuronal apoptosis induced by neurotrophic factor withdrawal or amyloid-b aggregation can be initiated by CRADD/caspase-2 signaling in the absence of PIDD, suggesting that formation of the PIDDosome may not be required for caspase-2-driven synaptic remodeling and neuronal apoptosis.21,22 The caspases are intracellular proteases that maintain homeostasis through regulation of inflammation (cas- pase-1, -4, -5, -12) and apoptosis (caspase-2, -3, -6, -7, -8, -9).4 During mammalian brain development, caspase-3 (CASP3 [MIM: 600636]) is the primary executioner of syn- aptic and neuronal pruning and is activated by the mito- chondrial (intrinsic) apoptotic cascade through APAF1 (apoptotic protease-activating factor 1 [MIM: 602233])/ caspase-9 (CASP9 [MIM: 602234]) activation.2,4,23,24 Although caspase-2 is expressed in neurons in the embry- onic and adult mammalian brain,25–28 its role in brain development has been elusive.4 Here we show that the human CRADD variants associ- ated with TLIS abrogate CRADD’s ability to activate cas- pase-2 and drive neuronal apoptosis. The megalencephaly, lissencephaly variant, and intellectual disability associated with loss of CRADD/caspase-2-mediated apoptosis imply a role for CRADD/caspase-2 signaling in development of the human cerebral cortex. Subjects and Methods Research Subjects W.B.D. has ascertained ~1,400 children with lissencephaly or subcortical band heterotopia over the last 30 years. Clinical infor- mation and results of genetic testing were obtained from medical records submitted together with MRI scans by referring physicians or parents of the subjects. Genetic testing included results of chro- mosome analysis, fluorescence in situ hybridization for deletion 17p13.3 (MIM: 247200), chromosome microarrays, MLPA for LIS1 (PAFAH1B1 [MIM: 601545]) and DCX (MIM: 300121), Sanger sequencing of single genes (LIS1, DCX, ARX [MIM: 300382], TUBA1A [MIM: 602529], TUBB2B [MIM: 612850], RELN [MIM: 600514], VLDLR [MIM: 192977], ACTB [MIM: 102630], and ACTG1 [MIM: 102560]), targeted gene panels, and whole-exome 1118 The American Journal of Human Genetics 99, 1117–1129, Nove sequencing. We re-reviewed scans of 188 subjects ascertained be- tween 2009 and 2015, when high-resolution scans were available for most individuals. All were reviewed with particular attention to the severity and gradient of the gyral malformation, cortical thickness, and presence of associated brain malformations. Thin undulating LIS with normal cerebellum (TLIS) was seen in 9 of 188 subjects (5%). We also searched W.B.D.’s large research database for subjects with frontal pachygyria and normal cere- bellum and corpus callosum and identified 12 additional individ- uals with the specific TLIS pattern. Several had large or relatively large occipitofrontal circumference (OFC), which we subdivide into true (OFC > 3 SD above the mean), mild (OFC 2–3 SD above the mean), and relative (OFC more than 2 SD greater than height) megalencephaly.29 Institutional review boards at The University of Chicago and Seattle Children’s Hospital approved this study. Seven members of an additional Mennonite family carrying CRADD c.382G>C and receiving medical care at the Clinic for Special Children (Stras- burg, PA) were added to the study (see Results). Their participation was approved by the Institutional Review Board at Lancaster Gen- eral Hospital. Informed consent signed by the parents or legal guardians was obtained for all participating individuals. Whole-Exome Sequencing Exome capture and sequencing were done using Agilent SureSelect Human All Exon V5 enrichment and Illumina HiSeq 2500 sequencing (paired-end 2 3 100 nt). The sequencing data were processed using CLC Biomedical Genomics Workbench (QIAGEN) including read alignment to UCSC Genome Browser hg19, variant calling (SNPs and indels), annotation, and filtering against three public databases (ExAC, dbSNP, and 1000 Genomes Project). For each sample, the average coverage through the exome was 80- fold and more than 90% of the exome was covered at least 20- fold. We focused on protein-altering and splice-site variants absent in databases or present with allele frequencies below 0.01. We filtered variants to systematically identify homozygous variants that were heterozygous in both parents, compound-heterozygous variants inherited from one parent, as well as de novo variants. Targeted Panel Sequencing with Single-Molecule Molecular Inversion Probe Using an established MIPGEN pipeline,30 we designed a pool of 673 single-molecule molecular inversion probes (smMIP) targeting the coding regions of 16 known LIS-associated genes—ACTB, ACTG1, ARX, DCX, DYNC1H1 (MIM: 600112), KIF2A (MIM: 602591), KIF5C (MIM: 604593), LIS1, RELN, TUBA1A, TUBA8 (MIM: 605742), TUBB (MIM: 191130), TUBB2B (MIM: 612850), TUBB3 (MIM: 602661), TUBG1 (MIM: 191135), and VLDLR— and our candidate gene, CRADD. In total, smMIPs were tiled across 47,975 bp of coding sequence. Capture reactions (100 ng) were performed in parallel using previously published protocols.30,31 Massively parallel sequencing was done on the Illumina HiSeq and MiSeq platforms. The average collapsed coverage (number of single-molecule consensus sequences or ‘‘smc-reads’’)31 across all samples was 21. Cradd Knockout Mice Cradd�/� mice on a C57BL/6 background were obtained from the Jackson Laboratories with permission from Prof. Tak Mak (University of Toronto).17 Wild-type (WT) C57BL/6 mice were also obtained from the Jackson Laboratories and all mice were mber 3, 2016 maintained under pathogen-free conditions. All animal experi- ments were approved by the Institutional Animal Care and Use Committees (IACUC) of Franklin & Marshall College and Columbia University Medical Center. Whole-Animal Perfusion Fixation Transcardial perfusion fixation was used to prepare mouse heads/ brains (as described elsewhere32) for micro-CT and histological analyses. In brief, mice were anesthetized by intraperitoneal (i.p.) injection of ketamine/xylazine mixture (up to 80 mg/kg body weight ketamine and 10 mg/kg body weight xylazine). When a surgical plane of anesthesia was achieved, the heart was exposed, a 23G cannula inserted into the ascending aorta via the left ventricle, and a perfusate exit hole placed in the right atrium. The circulation was slowly flushed with 20–30 mL of phosphate buffered saline (PBS; pH 7.4) followed by perfusion of 30–40 mL of 0.22 mm sterile-filtered, freshly prepared 4% paraformaldehyde in PBS. Upon complete blanching of the liver and mesentery, mice were decapitated and intact skulls and brains were postfixed over- night in 4% paraformaldehyde in PBS at 4�C. Cortical/Brain Histology Dissected brains were infiltrated with 30% sucrose in PBS, embedded in optimum cutting temperature (OCT) compound, and sectioned at 20 mm on a cryostat. Every 10th section was placed on a slide and stained in 0.1% cresyl violet solution for 10 min, rinsed quickly in PBS, dehydrated in 95% ethanol, and left in xylene before being coverslipped with Permount (Fisher Scientific). Brightfield images were taken using a Leica MZFLIII microscope with a Leica DFC425 camera and LAS V3.8 software. Volumetric Mouse Skull CT A total of 17 skulls were imaged at the Small Animal Tomographic Analysis Lab at Seattle Children’s Research Institute using a Skyscan 1076C microtomograph (Bruker) with settings optimized for mouse skulls (voltage, 55 kV; current, 180 mA; 0.5 mm Al filter; 100 ms exposure; 0.6� rotation steps; three frames averaged per rotation; 360� degree full rotation). Scan duration was approxi- mately 10 min per sample. 600 shadow images were acquired per specimen, which were reconstructed as image stacks at 34.42-micron resolution using the manufacturer’s proprietary software. All reconstructed image stacks were brought into stan- dardized anatomical orientation using Dataviewer (Bruker) and converted to NIfTI volumetric format to be loaded into 3D Slicer to visualize the skull morphology. Skull measurements were also taken using 3D Slicer.33 Soft tissue, such as brain, cannot be effec- tively imaged via micro-CT. Instead, we chose to look at the en- docranial volume, which is tightly correlated with brain size. Although segmenting the endocranial space using automated watershed algorithms from the skull is straightforward, it requires all cranial openings to be sealed digitally, so that segmentation does not leak into other regions. To reduce the amount of manual segmentation and avoid rater bias on measurements, we chose to construct an unbiased atlas of our study population, segment the endocranial space, and warp it back to the constituent samples us- ing deformable registration. We used the open-source DRAMMS deformable registration software and atlas-building pipeline in this study.34,35 The DRAMMS registration software consists of two major components: attribute matching and mutual-saliency weighting. Attribute matching characterizes each voxel by a high dimensional vector of multi-scale and multi-orientation Ga- The American Jou bor attributes. This method provides more information than the intensity information traditionally used (see Developmental Brain ADC Atlas in Web Resources).34–37 Because the reliability and accessibly of correspondence varies across anatomical structures, mutual-saliency upweights regions of the volume where corre- spondence can be reliably established between the source and target images. The preferred use of reliable correspondence reduces the negative impact of outlier regions on the registration quality (see Developmental Brain ADC Atlas in Web Resources).34–37 Atlas construction was performed using the DRAMMS deformable regis- tration in a classic unbiased population registration framework.37 The atlas construction framework iteratively finds a virtual space that resides in the centroid of the study population (centroid meaning that the deformations needed to transform all subjects into this virtual space sum up to zero everywhere in this virtual space). Therefore, the constructed atlas is unbiased to any subject in the population and is hence representative of the mean anat- omy/geometry of the population (see Developmental Brain ADC Atlas in Web Resources).37 The constructed atlas was segmented using the editor module of 3D Slicer. This reference segmentation was then mapped onto the individual samples by inversing their transformation into the atlas (Figure S7). Individual volumes of endocranial space were calculated after this transformation and are reported in Table S2. cDNA Cloning and Mutagenesis Full-length human CRADD coding cDNA (GenBank: NM_003805. 3) and the coding sequence for the PIDD death domain (PIDD- DD) (PIDD1 [GenBank: NM_145886.3]) were amplified by PCR from cDNA reverse transcribed from human ARPE-19 cell mRNA using Moloney Murine Leukemia Virus (M-MuLV) reverse tran- scriptase (NEB). PCR products were ligated into pENTR/D- TOPO (ThermoFisher), and CRADD TLIS mutations (c.382G>C, c.491T>G, c.508C>T, c.509G>A) were introduced with site- directed mutagenesis (QuikChange II, Agilent Technologies). LR recombination (LR Clonase II, ThermoFisher) was used to generate FLAG (pCAGIG/RFC-A/N-FLAG) or V5 (pcDNA3.1/N- V5) mammalian expression plasmids from pENTR clones. All clones were verified with Sanger sequencing. Transduction of Pen1-CRADD CRADD cDNAs—WT and CRADD TLIS variants described above— were subcloned into the Novagen pET15b expression system in- frame with an N/C-terminal polyhistidine sequence downstream of the T7 promoter and expressed in BL21 cells that express the T7 RNA polymerase under an IPTG-inducible promoter. The expressed proteins were purified on a Ni2þ resin as previously described.38 Penetratin 1 (Pen1) was mixed at a 2:1 molar ratio with purified CRADD and incubated 2 hr at 37�C to generate disul- fide-linked Pen1-CRADD. Linkage was assessed by 20% SDS-PAGE and by western blotting with anti-His antibody and anti-CRADD antibody as previously described.39 To ensure uniform protein transduction across cultures, Pen1-CRADD was used at the same concentration (27 nM). PC12 cells and mouse sympathetic neurons take up Pen1 proteins with near 100% efficiency. This approach is more efficient, less cytotoxic, and easier to titrate than transient transfection of cDNA constructs.40,41 Cell Lines and Transfection ARPE-19, HEK293T, and Neuro2a cells used in this study were purchased from the American Type Culture Collection (ATCC). rnal of Human Genetics 99, 1117–1129, November 3, 2016 1119 ARPE-19 cells were cultured in DMEM:F12 with 10% fetal bovine serum (FBS), and HEK293T and Neuro2a were cultured in DMEM with 10% FBS. Cells were transfected with FuGENE 6 (Roche) at a 3:1 FuGENE6:DNA ratio according to the manufacturer’s instruc- tions. Primary cultures of dermal fibroblasts from skin biopsies from CRADD TLIS-affected individuals (homozygous c.382G>C [p.Gly128Arg]) and their heterozygous parents were established by the Coriell Institute, and normal human dermal fibroblasts were acquired from Coriell. Fibroblasts were cultured at low pas- sage (%10) in DMEM with 10% FBS. Unless otherwise noted, all cells were maintained at 37�C and 5% CO2. To assess CRADD abundance in subject fibroblasts, cells were lysed in RIPA buffer (50 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris [pH 8.0] [Sigma]) containing protease inhib- itor cocktail (Roche), phosphatase inhibitor (PhosSTOP, Roche), and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 15 min at 4�C. Total protein concentration was normalized across affected subject, heterozygous parents, and normal fibroblast samples using the Bradford assay (Bio-Rad), and equal protein loading for immunoblotting was confirmed with mouse monoclonal anti-b-actin labeling (Sigma, 1:1,000,000). CRADD abundance was assessed with rabbit monoclonal anti-human RAIDD anti- bodies (Abcam cat# ab52621 and ab76465, RRID: AB_882269 and AB_1524296; 1:1,000). Goat anti-mouse IgG-HRP (Cell Signaling, 1:1,500) and goat anti-rabbit IgG-HRP (Cell Signaling, 1:1,500) were used as secondary antibodies. Blots were imaged with enhanced chemiluminescence using SignalFire Plus (Cell Signaling). PC12 Cell Culture and Viability PC12 cells obtained from the ATCC were grown as described pre- viously21 at 7.5% CO2 on rat-tail collagen-coated dishes in RPMI 1640 medium (Life Sciences) containing 1% penicillin/strepto- mycin (P/S), 5% fetal calf serum (FCS), and 10% heat-inactivated horse serum (HS, complete medium). PC12 cell survival was scored as reported previously.42 Culture medium was removed by aspira- tion and 100 mL of detergent-containing lysis solution was added to the well. This solution dissolves cell membranes, providing a suspension of intact nuclei that were quantified using a hemocy- tometer. Triplicate wells were scored and values reported as mean 5 SEM. Significance was calculated by Student’s t test. Primary Sympathetic Neuron Culture and Viability Neurons were cultured as described previously.22,43 In brief, sympathetic neurons were dissected from 1-day-old WT and CRADD-null17 (Cradd�/�) mouse pups. Cultures were maintained in RPMI 1640 medium supplemented with 10% horse serum and mouse nerve growth factor (NGF; 100 ng/ml) on collagen- coated 24-well dishes. At 1 day after plating, uridine (10 mM) and 5-fluorodeoxyuridine (10 mM) were added for 3 days to elimi- nate non-neuronal cells. Experiments were conducted after 6 days of culture. Viability was determined as previously reported.22 Each culture was scored as numbers of living phase-bright neurons counted in the same field at various times. Three replicate cultures were assessed for each condition; data were normalized to the number of neurons present in each culture at the time of Pen1- CRADD treatment and are reported as means 5 SEM. Immunofluorescence Microscopy Cells cultured on uncoated German glass coverslips transfected with pCAG/FLAG-CRADD WT or CRADD-TLIS variant cDNAs 1120 The American Journal of Human Genetics 99, 1117–1129, Nove were rinsed with PBS at 37�C and fixed with 4% paraformaldehyde at 21�C for 15 min. Cells were then prepared for immunofluores- cence microscopy and documented as described previously.44 The primary and secondary antibodies used were anti-FLAG M2 (Sigma, 1:1,000) and Alexa Fluor 488-goat anti-mouse IgG1 (ThermoFisher, 1:400), respectively. Nuclei were stained with 1 mg/mL DAPI (Santa Cruz Biotechnology), and coverslips were mounted with Fluoromount-G (Electron Microscopy Sciences). CRADD and PIDD Co-immunoprecipitation Co-immunoprecipitation was performed as described previ- ously.44,45 In brief, pCAG/FLAG-CRADD WT and CRADD- TLIS variant cDNAs or empty vector were co-transfected with pcDNA3.1/V5-PIDD1-DD cDNA in Neuro2a and HEK293T cells for 40–48 hr, after which whole-cell lysates were clarified by centri- fugation at 14,000 rpm for 15 min and incubated with anti-FLAG M2 affinity resin (Sigma) for 2 hr with end-over-end mixing at 4�C. Resin beads were pelleted and washed 33 in Tris-buffered saline (pH 7.2) with protease inhibitor cocktail (Roche), phospha- tase inhibitor (PhosSTOP, Roche), 1 mM PMSF, and 0.5%–1% Tween-20, before elution with 150 ng/mL 3x-FLAG peptide (Sigma). Input lysates and eluates were denatured in sample buffer at 100�C for 10 min and then immunoblotted with anti-FLAG (Sigma, 1:1,000) and anti-V5 (ThermoFisher, 1:5,000) antibodies. Caspase-2 and CRADD Co-immunoprecipitation Mouse IgG or rabbit IgG magnetic beads (Invitrogen) were pre- coated with affinity-purified anti-caspase-2 antibody42 for 2 hr at 4�C on a rotator. In total, 10 mL of anti-caspase-2 antibody was used per 30 mL of beads. PC12 cells were transduced with Pen1- CRADD for 5 hr. Cell lysates were prepared using CHAPS lysis buffer (150 mM KCl, 50 mM HEPES, 0.1% CHAPS, and protease in- hibitor cocktail [pH 7.4]).22 Lysates (70–120 mg) were loaded onto anti-caspase-2 antibody pre-coated beads and incubated overnight at 4�C on a rotator. After overnight incubation, the captured pro- teins were boiled off the beads at 100�C for 5 min. The immuno- precipitated samples, along with inputs, were then subjected to western blot analysis using an anti-CRADD antibody (Abnova), an anti-caspase-2 antibody (Alexis), or an anti-ERK antibody (Santa Cruz Biotechnology). Proteins were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences). Caspase-2 Activity Assay We adapted an unbiased caspase-2 activity measurement from Jean et al.43 bVAD-fmk (biotin-Val-Ala-DL-Asp-fluoromethylke- tone; 50 mM, MP Biomedicals), a biotinylated pan-caspase inhibi- tor that traps active caspases, was added to PC12 cultures 1 hr prior to treatment with Pen1-CRADD. Cells were lysed in CHAPS buffer. Active caspase-bVAD-fmk complexes were pulled out with strepta- vidin-coated beads (Invitrogen). Active caspase-2 was detected by western blotting using affinity-purified polyclonal anti-caspase-2 antibody.42 Results Clinical Presentation and Molecular Genetic Analysis of CRADD-Associated Lissencephaly Review of brain neuroimaging from more than 1,400 subjects with lissencephaly revealed a relatively mild lissencephaly variant characterized by megalencephaly, mber 3, 2016 Figure 1. Brain Imaging of TLIS-Affected Individuals with CRADD Mutations (A–D) MRI in subject LR04-101a1 at 5 years shows normal brainstem and cerebellum (A, B), frontal predominant symmetric pa- chygyria with reduced number of gyri, shallow sulci, and mildly thickened cortex (double arrowheads in B, C, and D with ar- rowheads located only in the left hemi- sphere). (E–H) MRI in subject LR05-279a2 at 7 years demonstrates the same pattern as in the top row (double arrowheads in F, G, and H). The enlargement in the inferior cere- bellum is a mild Chiari malformation type I (asterisk in E). (I–L) Age-matched normal control images. The cortex in TLIS-affected individuals is mildly thick, measuring 5–7 mm in the frontal lobes (see C and G) compared to normal cortex, measuring 3–4 mm (inset in K). MRI image section planes: A, B, E, F, I, J, sagittal T1-weighted images; C, G, K, axial T1-weighted images; D, H, L, coronal T2- weighted images. anterior-predominant pachygyria with shallow and un- usually wide sulci, mildly thick cortex (5–7 mm) especially in the frontal regions (TLIS), and no non-cortical malfor- mations in 13 families (Figures 1, S1, and S2). Pedigree analysis suggested autosomal-recessive inheritance. Trio- based whole-exome sequencing (WES) in two families with TLIS detected a homozygous mutation of CRADD (GenBank: NM_003805.3; c.382G>C [p.Gly128Arg]) in two siblings from a Mennonite family that segregated in the parents. To identify additional mutations, we performed Sanger sequencing of the two coding exons of CRADD in another 18 subjects with TLIS and detected homozygous missense mutations in two affected siblings born to consanguineous Turkish parents (c.508C>T [p.Arg170Cys]) and a pro- band with Finnish ancestry (c.509G>A [p.Arg170His]), as well as a single variant (c.491T>G [p.Phe164Cys]) in an American boy of Western European background (Table 1). Notably, we previously detected a 3.07 Mb deletion of chromosome 12q22 (chr12: 92,443,579– 95,515,465; hg38) in this boy that includes the remaining CRADD allele.46 Targeted panel sequencing in another 148 LIS-affected subjects for whom molecular genetic testing did not detect mutations in any of the known LIS-associ- ated genes did not reveal additional CRADD mutations. The four CRADD mutations identified in six individ- uals from four unrelated families with different ethnic backgrounds are all rare variants (ExAC allele frequency < 0.0005) with high pathogenicity scores (Table 1) that alter highly conserved residues within the CRADD death domain (Figure 2A). The first CRADD mutation (c.382G>C [p.Gly128Arg]) that we identified was previ- The American Jou ously reported in another large family with multiple members presenting with mild to moderate ID;44 both families belong to the Mennonite (Plain) population of Pennsylvania. The prior family was added to the study, and brain MRI performed in three of seven affected indi- viduals revealed TLIS with normal cerebellum, in line with the MRI features in all other CRADD-TLIS-affected subjects. Brain imaging and additional clinical data for all affected individuals are shown in Figures 1 and S2 and summarized in Table S1 and Figure S1. Case reports are provided in the Supplemental Data. Of 13 CRADD-TLIS- affected subjects, 9 (69%) had mild intellectual disability (ID), while three had mild to moderate ID and one had borderline ID (IQ ¼ 70–75). Seizures (3 of 13) and addi- tional abnormalities such as hydrocephalus due to Chiari malformation (MIM: 118420) were associated with lower cognitive function. None of the CRADD-TLIS-affected subjects had congenital anomalies other than TLIS, and all were in good general health. Clinical course of the TLIS-affected subjects without CRADD mutations was similar to the CRADD-associated group. However, all TLIS-affected individuals without CRADD mutations had either normal or small head size. In summary, indi- viduals with recessive CRADD mutations (n ¼ 13) pre- sented with TLIS, mild to moderate ID, epilepsy (3 of 13), and either enlarged head size with occipitofrontal circumference (OFC) 2–3 SD (mild megalencephaly in 9 of 13) or true megalencephaly with OFC > 3 SD (2 of 13). One individual had relative megalencephaly, and 1 of the 13 TLIS-affected individuals studied did not have megalencephaly (Table S1). rnal of Human Genetics 99, 1117–1129, November 3, 2016 1121 T a b le 1 . G e n o ty p e s o f In d iv id u a ls w it h C R A D D -A ss o c ia te d L is se n c e p h a ly T L IS ID S e x E th n ic O ri g in G e n o m ic P o si ti o n (h g 1 9 ) A ff e c te d T ra n sc ri p t H G V S F o rm a t (N M _ 0 0 3 8 0 5 .3 ) A ff e c te d P ro te in H G V S F o rm a t E x A C F re q u e n c y a F re q u e n c y in th e P o p u la ti o n o f O ri g in a P o ly P h e n -2 S c o re S IF T M u ta ti o n T a st e r C A D D P h y lo P d b S N P L R 0 4 -1 0 1 2 x F M e n n o n it e c h r1 2 : g .[ 9 4 2 4 3 8 2 9 G > C ] c .[ 3 8 2 G > C ]; [3 8 2 G > C ] p .[ G ly 1 2 8 A rg ]; [G ly 1 2 8 A rg ] 0 .0 0 0 0 0 8 0 .0 1 7 2 1 .0 to le ra te d d is e a se c a u si n g 2 8 .3 8 ,4 6 6 rs 3 8 7 9 0 6 8 6 1 L R 0 5 -2 7 9 F, M T u rk is h c h r1 2 : g .[ 9 4 2 4 3 9 5 5 C > T ] c .[ 5 0 8 C > T ]; [5 0 8 C > T ] p .[ A rg 1 7 0 C y s] ;[ A rg 1 7 0 C y s] 0 .0 0 0 0 0 8 3 n .e . 0 .0 3 1 d a m a g in g d is e a se c a u si n g 2 4 .8 1 ,6 5 4 N A L R 0 0 -1 5 0 F F in n is h c h r1 2 : g .[ 9 4 2 4 3 9 5 6 G > A ] c .[ 5 0 9 G > A ]; [5 0 9 G > A ] p .[ A rg 1 7 0 H is ]; [A rg 1 7 0 H is ] 0 .0 0 0 5 2 7 7 0 .0 0 6 6 6 5 0 .9 9 5 d a m a g in g d is e a se c a u si n g 2 6 .2 2 ,1 7 9 rs 1 4 1 1 7 9 7 7 4 L R 0 2 -0 0 6 M W e st e rn E u ro p e a n c h r1 2 : g .[ 9 4 2 4 3 9 3 8 T > G ] c .[ 4 9 1 T > G ] p .P h e 1 6 4 C y s 0 .0 0 0 0 2 4 9 6 n .e . 1 .0 d a m a g in g d is e a se c a u si n g 2 3 .2 6 ,9 1 0 rs 3 7 0 9 1 6 9 6 8 a rr [h g 1 9 ]1 2 q 2 2 (9 2 ,8 3 7 , 3 5 5 – 9 5 ,9 0 9 ,2 4 1 x 1 ) N A N A N A N A N A N A N A N A N A N A A b b re v ia ti o n s a re a s fo ll o w s: T L IS , ‘‘ th in ’’ li ss e n c e p h a ly ; h g 1 9 , G e n o m e R e fe re n c e C o n so rt iu m H u m a n B u il d 3 7 (G R C h 3 7 ); H G V S , H u m a n G e n o m e V a ri a ti o n S o c ie ty ; E x A C , E x o m e A g g re g a ti o n C o n so rt iu m ; C A D D , C o m b in e d A n n o ta ti o n D e p e n d e n t D e p le ti o n ; F , fe m a le ; M , m a le ; N A , n o t a p p li c a b le ; n .e ., n o t e st im a te d . a N o n e o f th e v a ri a n ts w e re o b se rv e d h o m o zy g o u sl y . 1122 The American Journal of Human Genetics 99, 1117–1129, Nove TLIS Variants Cluster in the CRADD Death Domain In response to intrinsic death signals (e.g., genotoxic stress or neurotrophic factor withdrawal), the C-terminal CRADD-DD interacts with the PIDD-DD to form the PIDDosome enabling the N-terminal CRADD CARD domain to interact with and activate caspase-2.17,18 TLIS-associated CRADD variants (p.Gly128Arg, p.Phe164Cys, p.Arg170Cys, and p.Arg170His) cluster in the DD (Figure 2A), suggesting that they may disrupt interaction with the PIDD-DD. TLIS CRADD Variants Do Not Disrupt Binding with PIDD or Caspase-2 To determine the functional impact of the TLIS-CRADD death domain mutations, we first investigated binding of the CRADD variants to PIDD and caspase-2 and then assessed their ability to induce apoptosis by activating caspase-2. Ultimately, these experiments showed that TLIS-CRADD variants fail to drive caspase-2-initiated apoptosis without significant loss of binding to either PIDD or caspase-2. In human cells, exogenously expressed p.Gly128Arg CRADD is present at very low abundance, indicating decreased protein stability relative to wild-type (WT) CRADD (Figure 2B). Additionally, it forms abnormal cytosolic clusters (Figure S3) and fails to interact with PIDD in vitro18,44 (Figure 2D). Dermal fibroblasts from p.Gly128Arg CRADD TLIS-affected subjects are essentially null for CRADD abundance (Figure 2C) relative to hetero- zygous parent and normal human dermal fibroblasts. Fibroblasts were not available from other CRADD TLIS- affected individuals. However, recombinant p.Phe164Cys, p.Arg170Cys, and p.Arg170His CRADD overexpressed in human cells (ARPE-19, HEK293T) and mouse neuroblasts (Neuro2a) displayed protein abundance and cellular localization comparable to that observed for WT CRADD (Figures 2B and S3), suggesting that the reduced protein abundance observed for p.Gly128Arg CRADD is not a primary pathogenic mechanism in TLIS. Because the TLIS-CRADD mutations cluster in the DD (Figure 2A) where CRADD and PIDD interact,18 we investi- gated whether the CRADD variants disrupted interaction with PIDD (PIDD-DD) as we observed for p.Gly128Arg CRADD.44 Immunoprecipitation of recombinant FLAG- CRADD overexpressed in Neuro2a (data not shown) and HEK293T cells co-precipitated V5-PIDD-DD for each of the CRADD-TLIS variants except p.Gly128Arg (Figure 2D). These data suggest a pathophysiological mech- anism other than disrupted PIDD interaction, consistent with our protein structural modeling (see Figure S4 and Discussion) and with the earlier finding that neuronal caspase-2 activation requires CRADD expression but can occur independent of PIDD expression.22 Upon activation, the N-terminal caspase-recruitment domain (CARD) of CRADD (Figure 2A) binds with cas- pase-2 to induce caspase-2 activity.15,16 To determine whether CRADD-TLIS variants can activate caspase-2, we first determined whether they could bind to caspase-2. To mber 3, 2016 A B C D F E Figure 2. TLIS CRADD Variants Do Not Disrupt Binding with PIDD or Caspase-2 (A) Functional domain structure of CRADD showing TLIS substitutions clustered in the C-terminal death domain (DD, amino acids 116–188). CARD, N-terminal cas- pase-recruitment domain (amino acids 1–91). CRADD substitutions p.Gly128Arg, p.Arg170Cys, and p.Arg170His are ho- mozygous in individuals with TLIS. The p.Phe164Cys encoding CRADD allele (c.491T>G) is in trans with a 3.07 Mb dele- tion of chromosome 12q22 (denoted by the two red Xs) in one TLIS-affected subject (see Table 1). (B) Immunoblot (IB) of recombinant FLAG-CRADD TLIS variants overexpressed in HEK293T cells (n ¼ 6). NTC, non-trans- fected control. Receptor-interacting serine/ threonine-protein kinase 1 (RIPK1 [MIM: 603453]; 78 kDa) immunoblotting was used as a loading control. (C) Top: western blot of CRADD (23 kDa) from dermal fibroblasts of a TLIS-affected subject homozygous for p.Gly128Arg CRADD (p.Gly128Arg/p.Gly128Arg) versus heterozygous parent (WT/p.Gly128Arg) and normal human dermal fibroblasts (WT/WT) (n ¼ 4). Anti-b-actin (42 kDa) immunoblotting was used as a loading control. Bottom: Reverse transcriptase- PCR of the full-length human CRADD cod- ing sequence (GenBank: NM_003805.3; 658 bp) from CRADD p.Gly128Arg (c.382G>C) TLIS-affected subject dermal fibroblasts (p.Gly128Arg/p.Gly128Arg) versus heterozygous parent (WT/ p.Gly128Arg) and normal fibroblasts (WT/ WT) demonstrating stability of the mutant transcript. Note: the WT/WT lane of the RT-PCR gel was cropped from the same gel usedforthesubjectandheterozygousparent samples. Cropping is denoted by the white bar separating lanes 2 and 3. (D) FLAG-CRADD WT and TLIS variants were co-overexpressed with V5-PIDD-DD in HEK293T cells (as indicated) for 40 hr after which FLAG-CRADD was immunopre- cipitated (IP) from whole cell lysates (input) with anti-FLAG M2affinity resin. Co-precip- itated complexes were first immunoblotted (IB) with anti-V5 antibody (PIDD-DD). Blots were then stripped and reprobed with anti-FLAG antibody (CRADD). CRADD WT and each of the TLIS CRADD variants except for p.Gly128Arg CRADD co-precipitated PIDD-DD (n ¼ 3). (E and F) Immunoprecipitation (IP) of caspase-2 from PC12 cells 5 hr after the cells were transduced with 27 nM Pen1-CRADD proteins as indicated (n ¼ 2–3). Caspase-2 co-precipitated Pen1-CRADD WT and TLIS variants in similar abundances when corrected for Pen1- CRADD abundance in whole cell lysates (input) as shown in (F). Blots were stripped and reprobed with anti-caspase-2. Error bars repre- sent SEM. NS, no significant difference; control, non-transduced cells. ERK immunoblotting was used as an input lysate loading control. CRADD p.Gly128Arg abundance is always lower in transduced PC12 cells, in agreement with our previous findings.44 efficiently deliver CRADD-TLIS variant proteins into cells without the cytotoxic side effects of transient transfection, CRADD-TLIS protein variants were directly linked to Pene- tratin-1 (Pen1) by a disulfide bond. Pen1 facilitates high- efficiency cellular uptake of linked proteins, and the reducing environment of the cytosol then cleaves the disulfide linkage releasing the transported protein.40,41 PC12 cells were transduced with Pen1-linked constructs of each of the CRADD-TLIS variants and caspase-2 was immunoprecipitated from whole-cell lysates 5 hr The American Jou after transduction. Immunoprecipitated caspase-2 pulled down each of the CRADD-TLIS variants, suggesting that all of the CRADD-TLIS variants can interact with caspase-2 when the blots are normalized for CRADD abundance (Figures 2E and 2F). TLIS Variants Disrupt CRADD’s Ability to Drive Neuronal Apoptosis One parsimonious explanation for TLIS could be that loss of CRADD function results in reduction of rnal of Human Genetics 99, 1117–1129, November 3, 2016 1123 Figure 3. TLIS-CRADD Variants Fail to Activate Caspase-2-Initiated Apoptosis (A and B) Pen1-CRADD WT (27 nM) drives apoptosis 1 day after transduction in PC12 cells (A) and in primary sympathetic neurons from Cradd�/� mice (B) whereas Pen1-TLIS-CRADD variants (27 nM) do not (n ¼ 4 for each cell type). Control indicates non-transduced cells. (C) TLIS-CRADD variants fail to activate caspase-2. PC12 cells were treated with 50 mM bVAD-fmk for 1 hr followed by transduction with 27 nM Pen1-CRADD as indicated for 2 hr. Caspase-bVAD-fmk complexes in whole-cell lysates were precipitated with streptavidin and immu- noblotted for caspase-2. Precipitated cas- pase-2 band optical density was used as a measure of relative caspase-2 activity42,43 (n ¼ 3). Error bars represent SEM. Control indicates non-transduced cells. caspase-2-mediated apoptosis, resulting in megalence- phaly and TLIS from failure to prune inappropriate neu- rons and/or neuropil. Overexpression of WT CRADD in PC12 and other mammalian cells (e.g., HEK293) is suffi- cient to activate caspase-2 and promote apoptosis.15,47 To examine whether CRADD-TLIS variants fail to induce apoptosis, PC12 cells were transduced with Pen1-linked human WT and CRADD-TLIS variants and cell survival was measured 1 day after treatment. Only WT CRADD import induced apoptosis in PC12 cells. Transduc- tion of PC12 cells with Pen1-linked CRADD-TLIS vari- ants (p.Gly128Arg, p.Phe164Cys, p.Arg170Cys, and p.Arg170His) failed to induce cell death (Figure 3A). To confirm this finding in neurons lacking endogenous CRADD, primary sympathetic neurons from Cradd knockout (Cradd�/�) mice were transduced with Pen1- linked human WT and p.Gly128Arg CRADD. CRADD WT resulted in significant cell death of primary sympathetic neurons from Cradd�/� mice 1 day after transduction, whereas cell survival after transduction of p.Gly128Arg CRADD was indistinguishable from that of non-trans- duced cells (Figure 3B). CRADD TLIS Variants Do Not Activate Caspase-2 All CRADD-TLIS variants bind to caspase-2 but only WT CRADD induces cell death, so we next determined whether CRADD-TLIS variants can induce caspase-2 activation. To measure caspase-2 activity, we used the caspase-affinity 1124 The American Journal of Human Genetics 99, 1117–1129, November 3, 2016 ligand biotin-Val-Ala-DL-Asp-fluoro- methylketone (bVAD-fmk), a pan-cas- pase substrate/inhibitor that binds to the first active caspase, and which we previously showed is effective in measuring caspase-2 activity in PC12 cells.42,43 PC12 cells were treated with bVAD-fmk for 1 hr prior to trans- duction with Pen1-CRADD WT and TLIS variants. Two hours after transduction, cells were har- vested for streptavidin pulldown of bVAD-fmk:caspase complexes followed by western blotting for caspase-2 (Figure 3C). We found that only WT CRADD induces cas- pase-2 activity; the CRADD-TLIS variants do not. This sug- gests that although the CRADD-TLIS variants can bind caspase-2, they are unable to activate caspase-2 and induce apoptosis. In sum, in vitro experiments demonstrated functional defects in caspase-2 activation despite no consistent defects in protein interactions with PIDD or caspase-2. Cradd Knockout Mice Display Megalencephaly and Seizures Consistent with TLIS To determine whether loss of CRADD function results in a TLIS-like phenotype in mice, we analyzed cortical morphology and head size in Cradd�/� mice. The Cradd�/� mice used were previously reported to have normal develop- ment, although brain morphology and head size were not studied.17 Volumetric micro-CT skull analysis of Cradd�/� mice relative to heterozygous (Craddþ/�) and WT controls demonstrated that all mutant mice had a statistically signif- icant increase in endocast volume that corresponds to increased brain volume (Figure 4). Morphological analysis of Cradd�/� cortex revealed normal layering indistinguish- able from WT mice (Figure S5). Notably, 8 of 31 (26%) Cradd�/� mice displayed handling-induced seizures; no WT or Craddþ/� littermates (0 of 60) had seizures. Figure 4. Megalencephaly in Cradd�/� Knockout Mice (A) Boxplot showing the ratio of endocranial volume to body weight in Cradd control (heterozygous and wild-type, Het-WT) (n ¼ 9) and Cradd�/� knockout (Hom) (n ¼ 8) mice. (B) Boxplot demonstrating the ratio of endocranial volume to hu- merus surface area in Cradd control (Het-WT) (n ¼ 5) and Cradd�/� (Hom) (n ¼ 3) mice. Wild-type and heterozygous mice are shown with closed and open circles, respectively. Megalencephaly with normal cortical morphology in Cradd�/� mice coupled with loss of function in driving apoptosis through caspase-2 activation in the CRADD- TLIS variants in vitro suggest that the cortical malforma- tion in CRADD-TLIS more likely results from reduced caspase-2-mediated apoptosis during cortical development than a neuronal migration disorder. Discussion CRADD/Caspase-2-Driven Apoptosis Is Required for Normal Development of Neocortex Here, we describe a mild ‘‘thin’’ lissencephaly (TLIS) result- ing from loss of CRADD function and reduced caspase-2- initiated neuronal apoptosis. Recent reviews have postu- lated that decreased apoptosis could underlie some human brain overgrowth disorders (e.g., megalencephaly).48 How- ever, to date no human cortical malformations associated with decreased apoptosis have been reported. The poten- tial impact of reduced apoptosis during mammalian brain development was previously demonstrated by brain mal- formations in caspase-3-,49 caspase-9-,50,51 and Apaf1-52 deficient mouse embryos and by increased forebrain size in EphA7�/� mice53 resulting from decreased apoptosis of cortical progenitor cells. In contrast to the pachygyria and mildly thick cortex associated with TLIS in humans, caspase-3 (Casp3), caspase-9 (Casp9), and Apaf1 (Apaf1) knockout mice displayed neural tube closure defects, fore- brain protrusions of ectopic masses of cortical tissue that deformed the anterior skull, and widespread increased cell density from decreased apoptosis of progenitor cells throughout the brain including the cortex, cerebellum, and brainstem. Overgrowth of progenitors in these mice is thought to result largely from loss of apoptosis of morphogen-producing cells early in development (e.g., FGF8), altering the spatiotemporal morphogen gradients that regulate progenitor expansion and ensure proper The American Jou forebrain development.54 During zebrafish development, excessive activation of CRADD/caspase-2 signaling through persistent phosphomimetic activation of Pidd (pidd1) resulted in a severe microcephaly phenotype.19 TLIS CRADD Variants Fail to Activate Caspase-2- Initiated Apoptosis without Disrupting Interaction between CRADD and PIDD The recent published structure of the PIDDosome in solution suggests that the PIDDosome core is composed of a CRADD-DD:PIDD-DD stoichiometry of 5:5 with 1–2 additional loosely associated CRADD-DDs.55 The solu- tion structure suggests that the amino acids altered by TLIS CRADD mutations do not participate in intra- or inter-molecular interactions that are critical to the core structure of the PIDDosome, and as such should not alter caspase-2 activation through disrupted CRADD- DD:PIDD-DD interaction. To explore this further, we generated three protein models including the CRADD- TLIS substitutions using SWISS-MODEL, IRECS, and PyMOL (Figure S4). Based on our models and the published data, CRADD Arg170 forms a hydrogen bond with Glu188 on another CRADD-DD, while the adjacent amino acid Ala169 interacts either with Val189 on another CRADD- DD or Arg862 and Arg864 on a PIDD-DD (Figure S4). The substitution of CRADD Arg170 to Cys or His should inhibit hydrogen bond formation with Glu188. How- ever, others recently determined that the 5:5 CRADD- DD:PIDD-DD PIDDosome core is not disrupted by substi- tution of CRADD Glu188 with Lys and that Glu188 is solvent accessible in the core PIDDosome molecule in solution. This suggests that Arg170 interaction with Glu188 is not fundamentally important to formation of the PIDDosome.55 Our models also predicted that Gly128 and Phe164 do not participate in an interaction interface and should therefore have no direct contact with any residues of the PIDD-DD (Figure S4). Our co-immunoprecipitation data showing that all FLAG-CRADD TLIS variants except for p.Gly128Arg CRADD co-precipitate V5-PIDD-DD at abundances similar to FLAG-CRADD WT support the modeling data which suggest that TLIS-associated pathophysiology does not result from impaired CRADD-DD:PIDD-DD interac- tions. Previous in vitro mutational analysis suggested that p.Arg170Ala-substituted CRADD exhibited a domi- nant-negative effect interfering with formation of the PIDDosome core by disrupting the interaction be- tween CRADD and PIDD death domains.56 However, p.Arg170His and p.Arg170Cys CRADD co-precipitated PIDD-DD at abundances comparable to those co-precipi- tated by wild-type CRADD in our experiments (Figure 2D). Further, adult CRADD p.Arg170His and p.Arg170Cys het- erozygotes had no symptoms suggestive of impaired CRADD function, including no unusual sensitivity to gen- otoxic stress. This suggests that the recent resolution of the structure of the PIDDosome in solution55 provides a more physiologically relevant model consistent with our data. rnal of Human Genetics 99, 1117–1129, November 3, 2016 1125 The lack of significant phenotypic differences between TLIS-affected individuals homozygous for p.Gly128Arg CRADD that disrupts interaction with the PIDD-DD versus TLIS-affected individuals with CRADD variants that do not disrupt PIDD-DD binding suggests that activation of caspase-2 may be the only function of the PIDDosome dur- ing brain development. Alternatively, formation of the PIDDosome may not be required for neuronal apoptosis during brain development. Amyloid-b-induced, CRADD/ caspase-2-initiated neuronal apoptosis persists in rat hip- pocampal neurons depleted of PIDD and NGF withdrawal induces CRADD/caspase-2 driven death of PIDD-null (Pidd1�/�) mouse sympathetic neurons.22 The TLIS Phenotype Is Present but Less Pronounced in Cradd Knockout Mice Complete loss of CRADD expression during brain devel- opment in Cradd�/� mice resulted in megalencephaly (Figure 4) and seizures without obvious changes in cortical thickness or lamination (Figure S5). This is in distinct contrast to the severe malformations of brain development described above for caspase-3-, caspase-9-, and Apaf1-null mice. Previous work demonstrated that Casp2�/� mice have significantly reduced body weight and bone volume.57 We also observed reduced body weight in Cradd�/� mice. Further study showed significantly decreased limb size, indicating that the low weight correlated with small body size (Table S2, Figure S6). The decreased body weight and bone volume in Casp2�/� and Cradd�/� mice corre- lates with our functional results, which demonstrate loss of caspase-2 activation in the absence of active CRADD. The head size of Casp2�/� mice has not yet been assessed. CRADD activation of caspase-2 in neurons is required for neurotrophic factor withdrawal-induced apoptosis and for dendritic spine collapse and neuronal apoptosis in response to amyloid-b aggregation,22,58 suggesting a role for CRADD/caspase-2 signaling in synaptic plasticity and cortical architecture during mammalian brain develop- ment. The relative lack of a significant CNS phenotype in early studies of Casp2�/� mice and the mild megalence- phaly described in this study for Cradd�/� mice, in contrast to the lissencephaly variant and ID observed in CRADD- TLIS-affected subjects, suggests that caspase-2 activity may be less important in development of the mouse brain than it is in the human brain. Other Disorders Caused by Decreased Caspase- Mediated Apoptosis Caspase-dependent apoptosis is characterized by ordered disruption of the cell without induction of inflamma- tion.59 Caspase activation can be triggered by extracellular signals (extrinsic pathway regulated by multiple death re- ceptors) or intrinsic stress signals such as DNA damage (intrinsic or mitochondrial pathway).60 Caspase-depen- dent apoptosis is involved in several different human dis- 1126 The American Journal of Human Genetics 99, 1117–1129, Nove eases including neurodegenerative, cardiovascular, and autoimmune disorders and cancer (reviewed by Favaloro et al.59). Autoimmune lymphoproliferative syndrome is a Mendelian disorder caused by impaired caspase-medi- ated apoptosis (MIM: 601859, 603909, and 607271)61 associated with mutations in FAS,62 FASLG,63 CASP10,64 and CASP865 (MIM: 134637, 134638, 601762, and 601763, respectively). Individuals with this syndrome develop chronic lymphoproliferation with lymphadenop- athy, hepatosplenomegaly, autoimmunity, and increased rates of malignancy61 but have normal development and intellectual function. Conclusions Our data suggest that CRADD/caspase-2 signaling is required for normal development of the human neocortex and normal cognitive function and that decreased caspase- 2-mediated apoptosis during human development can result in cortical malformation. Further investigation is required to delineate the role of CRADD/caspase-2 signaling in homeostatic regulation of cortical devel- opment. It is not clear whether the megalencephaly, un- dulating pachygyria, increased cortical thickness, and intellectual disability characteristic of CRADD-TLIS result from reduced pruning of synaptic connections, decreased programmed cell death of inappropriate neurons, or a combination of the two. Supplemental Data Supplemental Data include seven figures and two tables and can be found with this article online at http://dx.doi.org/10.1016/j. ajhg.2016.09.010. Acknowledgments The authors are grateful to the TLIS-affected individuals and their families for their contributions to this study. We thank Prof. Tak Mak (University of Toronto) for the Cradd�/� mice; the Coriell Institute NIGMS Cell Repository for generation of cell lines; R. Wagner, L. Basom, and K. Beutel for animal care; and K. Bakalov, Q. Nguyen, and K. Jacob for technical assistance. Research reported in this publication was supported by the Deut- sche Forschungsgemeinschaft (DI 2079/2-1 and DI 2170/2-2 to N.D.D.), HHMI Undergraduate Science Education Awards (52006294 and 52007538 to Franklin & Marshall College, R.N.J., Principal Investigator), the Center for Research on Women and Newborn Health and ConnectCare3 (to R.N.J.), the National Eye Institute (NEI) of the NIH (Vision Training Grant T32 EY013933 to M.I.A.), and the National Institute of Neurological Disorders and Stroke (NINDS) of the NIH (K08NS092898 to G.M.M., R01NS081333 to C.M.T., R01NS080390 to K.J.M., and R01NS092772 to W.B.D.). The Clinic for Special Children is funded by charitable contributions from private donors and the communities it serves. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or other organizations listed above. The funding sour- ces had no role in the design and conduct of the study, collec- tion, management, analysis, and interpretation of the data, mber 3, 2016 http://dx.doi.org/10.1016/j.ajhg.2016.09.010 http://dx.doi.org/10.1016/j.ajhg.2016.09.010 preparation, review, or approval of the manuscript, or decision to submit the manuscript for publication. All authors discussed and commented on the manuscript. Received: August 5, 2016 Accepted: September 19, 2016 Published: October 20, 2016 Web Resources 3D Slicer, https://www.slicer.org/ 1000 Genomes, http://www.internationalgenome.org/ CADD, http://cadd.gs.washington.edu/ dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/ Developmental Brain ADC Atlas Creation from Clinical Images, http://www.nmr.mgh.harvard.edu/~you2/publications/ BabyAtlasing_OHBM14.pdf DRAMMS Image Registration Software, https://www.cbica.upenn. edu/sbia/software/dramms/ ExAC Browser, http://exac.broadinstitute.org/ GenBank, http://www.ncbi.nlm.nih.gov/genbank/ IRECS, http://irecs.bioinf.mpi-inf.mpg.de/ MutationTaster, http://www.mutationtaster.org/ NHLBI Exome Sequencing Project (ESP) Exome Variant Server, http://evs.gs.washington.edu/EVS/ OMIM, http://www.omim.org/ PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/ PyMOL, http://www.pymol.org RCSB Protein Data Bank, http://www.rcsb.org/pdb/home/ home.do RefSeq, http://www.ncbi.nlm.nih.gov/RefSeq SIFT, http://sift.bii.a-star.edu.sg/ SWISS-MODEL, http://swissmodel.expasy.org/ UCSC Genome Browser, http://genome.ucsc.edu UCSC Genome Browser: Downloads, http://hgdownload.cse.ucsc. edu/downloads.html References 1. Bystron, I., Blakemore, C., and Rakic, P. (2008). Development of the human cerebral cortex: Boulder Committee revisited. Nat. Rev. Neurosci. 9, 110–122. 2. Dekkers, M.P., Nikoletopoulou, V., and Barde, Y.A. (2013). Cell biology in neuroscience: Death of developing neurons: new insights and implications for connectivity. J. Cell Biol. 203, 385–393. 3. Hu, W.F., Chahrour, M.H., and Walsh, C.A. (2014). The diverse genetic landscape of neurodevelopmental disorders. Annu. Rev. Genomics Hum. Genet. 15, 195–213. 4. McIlwain, D.R., Berger, T., and Mak, T.W. (2013). Caspase functions in cell death and disease. Cold Spring Harb. Per- spect. Biol. 5, a008656. 5. Rakic, P. (2009). Evolution of the neocortex: a perspective from developmental biology. Nat. Rev. Neurosci. 10, 724–735. 6. Fischl, B., and Dale, A.M. (2000). Measuring the thickness of the human cerebral cortex from magnetic resonance images. Proc. Natl. Acad. Sci. USA 97, 11050–11055. 7. Dobyns, W.B., Reiner, O., Carrozzo, R., and Ledbetter, D.H. (1993). Lissencephaly. A human brain malformation associ- ated with deletion of the LIS1 gene located at chromosome 17p13. JAMA 270, 2838–2842. The American Jou 8. Kato, M., and Dobyns, W.B. (2003). Lissencephaly and the molecular basis of neuronal migration. Hum. Mol. Genet. 12, R89–R96. 9. Moon, H.M., and Wynshaw-Boris, A. (2013). Cytoskeleton in action: lissencephaly, a neuronal migration disorder. Wiley Interdiscip. Rev. Dev. Biol. 2, 229–245. 10. Jamuar, S.S., and Walsh, C.A. (2015). Genomic variants and variations in malformations of cortical development. Pediatr. Clin. North Am. 62, 571–585. 11. Avela, K., Toiviainen-Salo, S., Karttunen-Lewandowski, P., Kauria, L., Valanne, L., and Salonen-Kajander, R. (2012). Fron- totemporal pachygyria-two new patients. Eur. J. Med. Genet. 55, 753–757. 12. Guzel, A.,Tatli,M., Bilguvar, K., Diluna,M.L., Bakkaloglu,B., Oz- turk, A.K., Bayrakli, F., and Gunel, M. (2007). Apparently novel genetic syndrome of pachygyria, mental retardation, seizure, and arachnoid cysts. Am. J. Med. Genet. A. 143A, 672–677. 13. Phadke, S.R., Girisha, K.M., and Phadke, R.V. (2007). A new autosomal recessive disorder of bilateral frontotemporal pa- chygyria without microcephaly: report of a case and review of literature. Neurol. India 55, 57–60. 14. Ramirez, D., Lammer, E.J., Johnson, C.B., and Peterson, C.D. (2004). Autosomal recessive frontotemporal pachygyria. Am. J. Med. Genet. A. 124A, 231–238. 15. Ahmad, M., Srinivasula, S.M., Wang, L., Talanian, R.V., Lit- wack, G., Fernandes-Alnemri, T., and Alnemri, E.S. (1997). CRADD, a novel human apoptotic adaptor molecule for cas- pase-2, and FasL/tumor necrosis factor receptor-interacting protein RIP. Cancer Res. 57, 615–619. 16. Duan, H., and Dixit, V.M. (1997). RAIDD is a new ‘death’ adaptor molecule. Nature 385, 86–89. 17. Berube, C., Boucher, L.M., Ma, W., Wakeham, A., Salmena, L., Hakem, R., Yeh, W.C., Mak, T.W., and Benchimol, S. (2005). Apoptosis caused by p53-induced protein with death domain (PIDD) depends on the death adapter protein RAIDD. Proc. Natl. Acad. Sci. USA 102, 14314–14320. 18. Tinel, A., and Tschopp, J. (2004). The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science 304, 843–846. 19. Ando, K., Kernan, J.L., Liu, P.H., Sanda, T., Logette, E., Tschopp, J., Look, A.T., Wang, J., Bouchier-Hayes, L., and Sidi, S. (2012). PIDD death-domain phosphorylation by ATM controls pro- death versus prosurvival PIDDosome signaling. Mol. Cell 47, 681–693. 20. Shearwin-Whyatt, L.M., Harvey, N.L., and Kumar, S. (2000). Subcellular localization and CARD-dependent oligomerization of the death adaptor RAIDD. Cell Death Differ. 7, 155–165. 21. Jabado, O., Wang, Q., Rideout, H.J., Yeasmin, M., Guo, K.X., Vekrellis, K., Papantonis, S., Angelastro, J.M., Troy, C.M., and Stefanis, L. (2004). RAIDD aggregation facilitates apoptotic death of PC12 cells and sympathetic neurons. Cell Death Differ. 11, 618–630. 22. Ribe, E.M., Jean, Y.Y., Goldstein, R.L., Manzl, C., Stefanis, L., Villunger, A., and Troy, C.M. (2012). Neuronal caspase 2 activ- ity and function requires RAIDD, but not PIDD. Biochem. J. 444, 591–599. 23. D’Amelio, M., Cavallucci, V., and Cecconi, F. (2010). Neuronal caspase-3 signaling: not only cell death. Cell Death Differ. 17, 1104–1114. 24. D’Amelio, M., Sheng, M., and Cecconi, F. (2012). Caspase-3 in the central nervous system: beyond apoptosis. Trends Neurosci. 35, 700–709. rnal of Human Genetics 99, 1117–1129, November 3, 2016 1127 https://www.slicer.org/ http://www.internationalgenome.org/ http://cadd.gs.washington.edu/ http://www.ncbi.nlm.nih.gov/projects/SNP/ http://www.nmr.mgh.harvard.edu/%7Eyou2/publications/BabyAtlasing_OHBM14.pdf http://www.nmr.mgh.harvard.edu/%7Eyou2/publications/BabyAtlasing_OHBM14.pdf https://www.cbica.upenn.edu/sbia/software/dramms/ https://www.cbica.upenn.edu/sbia/software/dramms/ http://exac.broadinstitute.org/ http://www.ncbi.nlm.nih.gov/genbank/ http://irecs.bioinf.mpi-inf.mpg.de/ http://www.mutationtaster.org/ http://evs.gs.washington.edu/EVS/ http://www.omim.org/ http://genetics.bwh.harvard.edu/pph2/ http://www.pymol.org http://www.rcsb.org/pdb/home/home.do http://www.rcsb.org/pdb/home/home.do http://www.ncbi.nlm.nih.gov/RefSeq http://sift.bii.a-star.edu.sg/ http://swissmodel.expasy.org/ http://genome.ucsc.edu http://hgdownload.cse.ucsc.edu/downloads.html http://hgdownload.cse.ucsc.edu/downloads.html http://refhub.elsevier.com/S0002-9297(16)30390-1/sref1 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref1 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref1 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref2 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref2 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref2 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref2 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref3 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref3 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref3 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref4 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref4 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref4 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref5 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref5 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref6 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref6 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref6 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref7 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref7 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref7 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref7 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref8 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref8 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref8 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref9 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref9 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref9 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref10 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref10 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref10 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref11 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref11 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref11 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref11 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref12 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref12 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref12 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref12 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref13 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref13 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref13 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref13 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref14 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref14 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref14 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref15 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref15 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref15 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref15 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref15 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref16 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref16 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref17 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref17 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref17 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref17 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref17 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref18 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref18 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref18 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref19 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref19 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref19 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref19 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref19 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref20 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref20 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref20 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref21 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref21 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref21 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref21 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref21 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref22 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref22 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref22 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref22 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref23 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref23 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref23 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref24 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref24 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref24 25. Bergeron, L., Perez, G.I., Macdonald, G., Shi, L., Sun, Y., Juris- icova, A., Varmuza, S., Latham, K.E., Flaws, J.A., Salter, J.C., et al. (1998). Defects in regulation of apoptosis in caspase-2- deficient mice. Genes Dev. 12, 1304–1314. 26. Kumar, S., Kinoshita, M., Noda, M., Copeland, N.G., and Jen- kins, N.A. (1994). Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mamma- lian IL-1 beta-converting enzyme. Genes Dev. 8, 1613–1626. 27. Wang, L., Miura, M., Bergeron, L., Zhu, H., and Yuan, J. (1994). Ich-1, an Ice/ced-3-related gene, encodes both positive and negative regulators of programmed cell death. Cell 78, 739–750. 28. Carlsson, Y., Schwendimann, L., Vontell, R., Rousset, C.I., Wang, X., Lebon, S., Charriaut-Marlangue, C., Supramaniam, V., Hagberg, H., Gressens, P., and Jacotot, E. (2011). Genetic in- hibition of caspase-2 reduces hypoxic-ischemic and excito- toxic neonatal brain injury. Ann. Neurol. 70, 781–789. 29. Gripp, K.W., Hopkins, E., Doyle, D., and Dobyns, W.B. (2010). High incidence of progressive postnatal cerebellar enlarge- ment in Costello syndrome: brain overgrowth associated with HRAS mutations as the likely cause of structural brain and spinal cord abnormalities. Am. J. Med. Genet. A. 152A, 1161–1168. 30. Boyle, E.A., O’Roak, B.J., Martin, B.K., Kumar, A., and Shen- dure, J. (2014). MIPgen: optimized modeling and design of molecular inversion probes for targeted resequencing. Bioin- formatics 30, 2670–2672. 31. Hiatt, J.B., Pritchard, C.C., Salipante, S.J., O’Roak, B.J., and Shendure, J. (2013). Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation. Genome Res. 23, 843–854. 32. Gage, G.J., Kipke, D.R., and Shain, W. (2012). Whole animal perfusion fixation for rodents. J. Vis. Exp. 65, 3564. 33. Fedorov, A., Beichel, R., Kalpathy-Cramer, J., Finet, J., Fillion- Robin, J.C., Pujol, S., Bauer, C., Jennings, D., Fennessy, F., Sonka, M., et al. (2012). 3D Slicer as an image computing plat- form for the Quantitative Imaging Network. Magn. Reson. Imaging 30, 1323–1341. 34. Dawant, B.M., Christensen, G.E., Fitzpatrick, J.M., and Rueck- ert, D., eds. (2012). Biomedical Image Registration, 5th Inter- national Workshop, WBIR 2012, Nashville, Tennessee, USA, July 7–8, 2012, Proceedings. In Lecture Notes in Computer Science (New York: Springer). 35. Ou, Y., Sotiras, A., Paragios, N., and Davatzikos, C. (2011). DRAMMS: Deformable registration via attribute matching and mutual-saliency weighting. Med. Image Anal. 15, 622–639. 36. Doshi, J.J., Erus, G., Ou, Y., and Davatzikos, C. (2013). Ensemble-based medical image labeling via sampling morpho- logical appearance manifolds. Proceedings of MICCAI Chal- lenge Workshop on Segmentation: Algorithms, Theory and Applications, 148–151. 37. Guimond, A., Meunier, J., and Thirion, J.-P. (2000). Average brain models: A convergence study. Comput. Vis. Image Underst. 77, 192–210. 38. Denault, J.B., and Salvesen, G.S. (2008). Apoptotic caspase activation and activity. Methods Mol. Biol. 414, 191–220. 39. Akpan, N., Serrano-Saiz, E., Zacharia, B.E., Otten, M.L., Du- cruet, A.F., Snipas, S.J., Liu, W., Velloza, J., Cohen, G., Sosunov, S.A., et al. (2011). Intranasal delivery of caspase-9 inhibitor re- duces caspase-6-dependent axon/neuron loss and improves neurological function after stroke. J. Neurosci. 31, 8894–8904. 1128 The American Journal of Human Genetics 99, 1117–1129, Nove 40. Davidson, T.J., Harel, S., Arboleda, V.A., Prunell, G.F., Shelan- ski, M.L., Greene, L.A., and Troy, C.M. (2004). Highly efficient small interfering RNA delivery to primary mammalian neu- rons induces microRNA-like effects before mRNA degradation. J. Neurosci. 24, 10040–10046. 41. Troy, C.M., Stefanis, L., Prochiantz, A., Greene, L.A., and She- lanski, M.L. (1996). The contrasting roles of ICE family prote- ases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation. Proc. Natl. Acad. Sci. USA 93, 5635–5640. 42. Troy, C.M., Stefanis, L., Greene, L.A., and Shelanski, M.L. (1997). Nedd2 is required for apoptosis after trophic factor withdrawal, but not superoxide dismutase (SOD1) downregu- lation, in sympathetic neurons and PC12 cells. J. Neurosci. 17, 1911–1918. 43. Jean, Y.Y., Ribe, E.M., Pero, M.E., Moskalenko, M., Iqbal, Z., Marks, L.J., Greene, L.A., and Troy, C.M. (2013). Caspase-2 is essential for c-Jun transcriptional activation and Bim induc- tion in neuron death. Biochem. J. 455, 15–25. 44. Puffenberger, E.G., Jinks, R.N., Sougnez, C., Cibulskis, K., Wil- lert, R.A., Achilly, N.P., Cassidy, R.P., Fiorentini, C.J., Heiken, K.F., Lawrence, J.J., et al. (2012). Genetic mapping and exome sequencing identify variants associated with five novel dis- eases. PLoS ONE 7, e28936. 45. Jinks, R.N., Puffenberger, E.G., Baple, E., Harding, B., Crino, P., Fogo, A.B., Wenger, O., Xin, B., Koehler, A.E., McGlincy, M.H., et al. (2015). Recessive nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum is caused by homozy- gous protein-truncating mutations of WDR73. Brain 138, 2173–2190. 46. Sajan,S.A.,Fernandez,L., Nieh, S.E.,Rider, E., Bukshpun, P.,Wa- kahiro, M., Christian, S.L., Rivière, J.B., Sullivan, C.T., Sudi, J., et al. (2013). Both rare and de novo copy number variants are prevalent in agenesis of the corpus callosum but not in cere- bellar hypoplasia or polymicrogyria. PLoS Genet. 9, e1003823. 47. Wang, Q., Maniati, M., Jabado, O., Pavlaki, M., Troy, C.M., Greene, L.A., and Stefanis, L. (2006). RAIDD is required for apoptosis of PC12 cells and sympathetic neurons induced by trophic factor withdrawal. Cell Death Differ. 13, 75–83. 48. Mirzaa, G.M., and Poduri, A. (2014). Megalencephaly and hemimegalencephaly: breakthroughs in molecular etiology. Am. J. Med. Genet. C. Semin. Med. Genet. 166C, 156–172. 49. Kuida, K., Zheng, T.S., Na, S., Kuan, C., Yang, D., Karasuyama, H., Rakic, P., and Flavell, R.A. (1996). Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384, 368–372. 50. Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M., Soengas, M.S., Elia, A., de la Pompa, J.L., Kagi, D., Khoo, W., et al. (1998). Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339–352. 51. Kuida, K., Haydar, T.F., Kuan, C.Y., Gu, Y., Taya, C., Kara- suyama, H., Su, M.S., Rakic, P., and Flavell, R.A. (1998). Reduced apoptosis and cytochrome c-mediated caspase acti- vation in mice lacking caspase 9. Cell 94, 325–337. 52. Yoshida, H., Kong, Y.Y., Yoshida, R., Elia, A.J., Hakem, A., Hakem, R., Penninger, J.M., and Mak, T.W. (1998). Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739–750. 53. Depaepe, V., Suarez-Gonzalez, N., Dufour, A., Passante, L., Gorski, J.A., Jones, K.R., Ledent, C., and Vanderhaeghen, P. (2005). Ephrin signalling controls brain size by regulating apoptosis of neural progenitors. Nature 435, 1244–1250. mber 3, 2016 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref25 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref25 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref25 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref25 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref26 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref26 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref26 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref26 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref26 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref27 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref27 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref27 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref27 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref28 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref28 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref28 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref28 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref28 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref29 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref29 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref29 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref29 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref29 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref29 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref30 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref30 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref30 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref30 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref31 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref31 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref31 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref31 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref32 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref32 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref33 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref33 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref33 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref33 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref33 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref35 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref35 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref35 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref37 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref37 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref37 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref38 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref38 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref39 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref39 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref39 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref39 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref39 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref40 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref40 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref40 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref40 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref40 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref41 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref41 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref41 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref41 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref41 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref42 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref42 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref42 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref42 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref42 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref43 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref43 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref43 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref43 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref44 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref44 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref44 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref44 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref44 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref45 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref45 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref45 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref45 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref45 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref45 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref46 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref46 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref46 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref46 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref46 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref47 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref47 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref47 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref47 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref48 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref48 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref48 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref49 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref49 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref49 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref49 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref50 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref50 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref50 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref50 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref51 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref51 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref51 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref51 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref52 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref52 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref52 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref52 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref53 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref53 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref53 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref53 54. Nonomura, K., Yamaguchi, Y., Hamachi, M., Koike, M., Uchiyama, Y., Nakazato, K., Mochizuki, A., Sakaue-Sawano, A., Miyawaki, A., Yoshida, H., et al. (2013). Local apoptosis modulates early mammalian brain development through the elimination of morphogen-producing cells. Dev. Cell 27, 621–634. 55. Nematollahi, L.A., Garza-Garcia, A., Bechara, C., Esposito, D., Morgner, N., Robinson, C.V., and Driscoll, P.C. (2015). Flexible stoichiometry and asymmetry of the PIDDosome core com- plex by heteronuclear NMR spectroscopy and mass spectrom- etry. J. Mol. Biol. 427, 737–752. 56. Jang, T.H., Seo, E.K., and Park, H.H. (2013). Analysis of muta- tion effects on PIDDosome core complex. Appl. Biochem. Bio- technol. 170, 210–218. 57. Shalini, S., Dorstyn, L., Wilson, C., Puccini, J., Ho, L., and Ku- mar, S. (2012). Impaired antioxidant defence and accumula- tion of oxidative stress in caspase-2-deficient mice. Cell Death Differ. 19, 1370–1380. 58. Pozueta, J., Lefort, R., Ribe, E.M., Troy, C.M., Arancio, O., and Shelanski, M. (2013). Caspase-2 is required for dendritic spine and behavioural alterations in J20 APP transgenic mice. Nat. Commun. 4, 1939. 59. Favaloro, B., Allocati, N., Graziano, V., Di Ilio, C., and De Lau- renzi, V. (2012). Role of apoptosis in disease. Aging (Albany, N.Y.) 4, 330–349. The American Jou 60. Troy, C.M., and Jean, Y.Y. (2015). Caspases: therapeutic targets in neurologic disease. Neurotherapeutics 12, 42–48. 61. Teachey, D.T., Seif, A.E., and Grupp, S.A. (2010). Advances in the management and understanding of autoimmune lymphoproliferative syndrome (ALPS). Br. J. Haematol. 148, 205–216. 62. Fisher, G.H., Rosenberg, F.J., Straus, S.E., Dale, J.K., Middleton, L.A., Lin, A.Y., Strober, W., Lenardo, M.J., and Puck, J.M. (1995). Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syn- drome. Cell 81, 935–946. 63. Wu, J., Wilson, J., He, J., Xiang, L., Schur, P.H., and Mountz, J.D. (1996). Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease. J. Clin. Invest. 98, 1107–1113. 64. Wang, J., Zheng, L., Lobito, A., Chan, F.K., Dale, J., Sneller, M., Yao, X., Puck, J.M., Straus, S.E., and Lenardo, M.J. (1999). In- herited human Caspase 10 mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lym- phoproliferative syndrome type II. Cell 98, 47–58. 65. Chun, H.J., Zheng, L., Ahmad, M., Wang, J., Speirs, C.K., Sie- gel, R.M., Dale, J.K., Puck, J., Davis, J., Hall, C.G., et al. (2002). Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency. Nature 419, 395–399. rnal of Human Genetics 99, 1117–1129, November 3, 2016 1129 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref54 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref54 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref54 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref54 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref54 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref54 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref55 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref55 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref55 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref55 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref55 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref56 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref56 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref56 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref57 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref57 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref57 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref57 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref58 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref58 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref58 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref58 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref59 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref59 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref59 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref60 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref60 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref61 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref61 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref61 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref61 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref62 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref62 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref62 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref62 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref62 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref63 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref63 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref63 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref63 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref64 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref64 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref64 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref64 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref64 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref65 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref65 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref65 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref65 http://refhub.elsevier.com/S0002-9297(16)30390-1/sref65 Mutations in CRADD Result in Reduced Caspase-2-Mediated Neuronal Apoptosis and Cause Megalencephaly with a Rare Lissencepha ... Introduction Subjects and Methods Research Subjects Whole-Exome Sequencing Targeted Panel Sequencing with Single-Molecule Molecular Inversion Probe Cradd Knockout Mice Whole-Animal Perfusion Fixation Cortical/Brain Histology Volumetric Mouse Skull CT cDNA Cloning and Mutagenesis Transduction of Pen1-CRADD Cell Lines and Transfection PC12 Cell Culture and Viability Primary Sympathetic Neuron Culture and Viability Immunofluorescence Microscopy CRADD and PIDD Co-immunoprecipitation Caspase-2 and CRADD Co-immunoprecipitation Caspase-2 Activity Assay Results Clinical Presentation and Molecular Genetic Analysis of CRADD-Associated Lissencephaly TLIS Variants Cluster in the CRADD Death Domain TLIS CRADD Variants Do Not Disrupt Binding with PIDD or Caspase-2 TLIS Variants Disrupt CRADD’s Ability to Drive Neuronal Apoptosis CRADD TLIS Variants Do Not Activate Caspase-2 Cradd Knockout Mice Display Megalencephaly and Seizures Consistent with TLIS Discussion CRADD/Caspase-2-Driven Apoptosis Is Required for Normal Development of Neocortex TLIS CRADD Variants Fail to Activate Caspase-2-Initiated Apoptosis without Disrupting Interaction between CRADD and PIDD The TLIS Phenotype Is Present but Less Pronounced in Cradd Knockout Mice Other Disorders Caused by Decreased Caspase-Mediated Apoptosis Conclusions Supplemental Data Acknowledgments Web Resources References