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All research involving humans and animals have been approved by the Institutional Review Boards of The University of Chicago, The University of Toronto, and The University of California, Los Angeles. All families provided written informed consent for the collection of samples and subsequent analysis.
Genomic DNA from autism and control subjects were obtained from various sources as described below. Ethnic breakdown for all autism and control groups are provided in Table S5. We obtained autism samples from several DNA repositories including the Autism Genetics Resource Exchange (AGRE) (n = 793) and the National Institutes of Mental Health (NIMH) (n = 313). For the AGRE sample set, the Autism Diagnostic Interview–Revised (ADI-R), Autism Diagnostic Observation Schedule (ADOS), Raven and Handedness, Peabody and Vineland assessments were performed. Medical histories and physical neurological exams were also collected. Additional phenotypic data on the AGRE sample set are available on the AGRE website (http://www.agre.org/). Genomic DNA from control subjects were obtained from the NIMH Genetics Initiative Control sample set (n = 1161); these subjects were screened for any Axis I mental health disorders and none had a diagnosis of autism. Genomic DNA was also obtained from several Canadian institutions including The Hospital for Sick Children in Toronto and in child diagnostic centers in Hamilton, Ontario, and in St. John's, Newfoundland (n = 529). For the Canadian autism cohort, all subjects met ADI-R and ADOS criteria conclusively or on a clinical best estimate. Most index patients (∼75%) were screened for fragile×mutations and were karyotyped. Wherever possible, experiments were performed on blood-derived genomic DNA (80%); otherwise, DNA from cell lines was used. Control DNA was isolated from cell lines from the Ontario Population Genomics Platform (n = 570). Subjects living in Ontario, Canada were recruited by telephone from a list of randomly selected residential telephone numbers for Ontario and from population-based Tax Assessment Rolls of the Ontario Ministry of Finance. Health and Ancestry of these subjects is self reported in an extensive questionnaire.
Association analyses were performed on existing data generated on the following three SNP genotyping platforms: 1) Affymetrix 5.0 data available on 777 AGRE families by the Autism Consortium [21]; 2) IlluminaHap550 data available on 943 AGRE families by the microarray facility at Children's Hospital of Philadelphia (www.agre.org/) (Bucan and Hakonarson, unpublished); and 3) Affymetrix 500 K platform data on 60 families [18]. PLINK v1.03 was used for the analysis [45]. Two different types of analyses were performed. First, we performed the transmission disequilibrium test (TDT) [24] with permutation for families with 2 genotyped parents and 1 or more affected offspring. The permutation procedure flips transmitted/untransmitted status constantly for all SNPs for a given family, thereby preserving the linkage disequilibrium and linkage information between markers and siblings. Second, we used DFAM for all individuals. DFAM within PLINK implements the sib-TDT [25] and also allows for unrelated individuals to be included (via a clustered-analysis using the Cochran-Mantel-Haesnzel) and can be used to combine discordant sibship data, parent-offspring trio data and unrelated case/control data in a single analysis. Region-wide significance for both tests was estimated using the mperm option in PLINK which uses permutation to correct for multiple testing of all the markers within the region while taking linkage disequilibrium into account.
Genes (accession numbers) examined in this study include: ALDOA (NM_000034.2), DOC2A (NM_003586.2), HIRIP3 (NM_003609.2), MAPK3 (NM_002746.2), MAZ (NM_002383.2), PPP4C (NM_002720.1), SEZ6L2 (NM_201575.2), and TAOK2 (NM_004783.2). PCR-amplification primers were designed using Primer3 (http://frodo.wi.mit.edu/) with M13 Forward and Reverse Tails added to each primer to facilitate high-throughput DNA sequencing (Table S8). DNA was amplified in a reaction comprised of: 20 ng genomic DNA, 1× buffer I (1.5 mM MgCl2, Applied Biosystems, Foster City, CA), 1 mM dNTPs (Applied Biosystems), 0.4 µM primer (each of forward and reverse, IDT, Coralville, IA), and 0.25 units AmpliTaq Gold (Applied Biosystems) in a total volume of 10 µl. Thermocycling conditions were as follows: 94°C for 10 min; 35 cycles of 94°C for 30 sec, annealing temperature (53–60°C) for 30 sec, and 72°C for 30 sec; and final extension of 72°C for 10 min. Variations in reaction composition and cycling conditions were required for a small number of amplicons. PCR products were purified in a 10 ul reaction comprised of 6.6 units Exonuclease I and 0.66 units shrimp alkaline phosphatase that were incubated at 37°C for 30 min followed by 80°C for 15 minutes. Sequencing reactions were performed using Big Dye terminators on an ABI 3730XL 96-capillary automated 3730XL DNA sequencer (Applied Biosystems) at The University of Chicago DNA Sequencing and Genotyping Core Facility. Sequence data were imported as AB1 files into Mutation Surveyor v3.10 (SoftGenetics, State College, PA). Sequence contigs were assembled by aligning the AB1 files against GenBank reference sequence files that were obtained from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/Genbank/). Reference sequences included the complete 5′ untranslated region (UTR), coding sequence and associated splice-sites, intronic sequence, and complete 3′ UTR. The imported GenBank files provide annotated features for each gene that include base count, intron/exon boundaries, amino acid sequence, and previously reported mutations and single nucleotide polymorphisms (SNPs) from the SNP database (dbSNP) (http://www.ncbi.nlm.nih.gov/projects/SNP/). To screen for putative mutations, the entire length of the sample trace was manually inspected for quality and variation from the reference trace. All detected variants were visually reviewed by two trained individuals and were confirmed using bi-directional sequencing.
Human and mouse SEZ6L2 expression studies: Mouse in situ hybridization experiments were performed as previously described [46] on wildtype CD-1 whole embryos using DIG-labeled RNA probe for Sez6l2 (IMAGE clone 6467632, Invitrogen). Human in situ hybridization experiments were performed on fresh frozen post-fixed tissues as previously described. [47]. The SEZ6L2-specific sequence (MHS1011-59266) was obtained from OpenBiosystem (Huntsville, AL), sequenced for verification, and checked for specificity with BLAST against the human genome. In vitro transcription was then performed to generate S35-labeled cRNA. Labeled cRNA was hybridized on 20 µm thick cryostat frozen tissue sections, sectioned into either coronal or sagittal plane and opposed to autoradiography films for two to five days. Slides were then coated with NTB2 autoradiography emulsion (Kodak, New Haven, CT), exposed for four weeks, and developed. Following staining with cresyl violet, emulsion dipped slides were cover-slipped and imaged using Nikon Eclipse E600 microscope with a Digital Capture System built around spot cooled CCD camera. Corresponding sense probes were used on sections adjacent to those used for antisense probes.
Gene selection was performed using the UCSC Genome browser (http://genome.ucsc.edu/) and literature review of articles published in PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez). PolyPhen was used to predict whether amino acid substitutions affect protein function. The differences in frequency of any variant between cases and controls were assessed using the Fishers Exact test.
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