Smith et al., Supplemental Materials 1 1 Folding and lamination of the human neocortex depend on the sodium potassium pump 2 alpha 3 (ATP1A3) subunit 3 4 5 Richard S. Smith1, Marta Florio2,3, Shyam K. Akula1,4, Jennifer E. Neil1, Yidi Wang1, R. Sean 1 2,3 2,3 2,3 5 6 Hill , Melissa Goldman , Christopher D. Mullally , Nora Reed , Luis Bello-Espinosa , Laura 7 Flores-Sarnat6, Fabiola Paoli Monteiro7, Casella B. Erasmo8, Filippo Pinto e Vairo9,10, Eva 8 Morava10, A. James Barkovich11, Joseph Gonzalez-Heydrich12, Catherine A. Brownstein1, 9 Steven A. McCarroll2,3, Christopher A. Walsh1 10 11 1Division of Genetics and Genomics, Howard Hughes Medical Institute, Broad Institute of MIT and Harvard, 12 Manton Center for Orphan Disease Research, Boston Children’s Hospital, Harvard Medical School, Boston, MA 13 02115, USA 14 15 2Department of Genetics, Harvard Medical School, Boston, MA 02115, USA 16 17 3Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA 18 4Harvard-MIT MD/PhD Program; Program in Neuroscience; Harvard Medical School, Boston, MA, 02115, USA 19 20 5Arnold Palmer Hospital for Children, Orlando, Fl, 32806, USA 21 6University of Calgary and Alberta Children´s Hospital Research Institute (Owerko Centre), Dept of Paediatrics 22 and Clinical Neurosciences, Calgary, Alberta, Canada 23 24 7Mendelics Genomic Analysis, CEP 04013-000, São Paulo, SP, Brazil 25 26 8Children's Institute, Hospital das Clinicas, São Paulo, SP, Brazil 27 28 9Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA 29 30 10Department of Clinical Genomics, Mayo Clinic, Rochester, MN, 55905, USA 31 32 11Benioff Children's Hospital, Departments of Radiology, Pediatrics, Neurology, and Neurological Surgery, 33 University of California San Francisco, San Francisco, CA, 94117, USA 34 35 12Department of Psychiatry, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA 36 37 38 *Correspondence should be addressed to: [email protected] and 39 [email protected] 40 41 42 43 44 45 46 Smith et al., Supplemental Materials 2 47 EXPERIMENTAL DETAILS 48 Human subjects and samples 49 Individuals presented herein were identified and evaluated in a clinical setting, and biological samples 50 collected after obtaining written informed clinical and/or research consent. Human subject research was 51 conducted according to protocols approved by the institutional review boards of Boston Children’s 52 Hospital, Beth Israel Deaconess Medical Center and the Mayo Clinic. Fetal brain tissue was received 53 after release from clinical pathology, with a maximum post-mortem interval of 4 h. Cases with known 54 anomalies were excluded. Tissue was transported in Hibernate-E medium (Thermo Fisher) on ice to 55 either the Walsh laboratory or McCarroll laboratory for downstream processing. The neonatal brain 56 sample was obtained from the University of Maryland Brain and Tissue Bank of the NIH 57 NeuroBioBank (sample number UMBN 5817) and stored at -80C until further processing. 58 59 Human genetics sequencing and analysis 60 For Case A and B, whole exome sequencing and data processing were performed by the Genomics 61 Platform at the Broad Institute of MIT and Harvard. Libraries from DNA samples (>250ng of DNA, at 62 >2 ng/ul) the Human Core Exome Kit from Twist Biosciences was used to capture target regions (~38 63 Mb target), and sequencing was performed on the Illumina NovaSeq 6000 (150 bp paired reads) to 64 cover >96% of targets at 20x and a mean target coverage of >100x. Sample identity quality assurance 65 checks were performed on each sample. The exome sequencing data was de-multiplexed and each 66 sample's sequence data were aggregated into a single Picard BAM file. Exome sequencing data was 67 processed through a pipeline based on Picard, using base quality score recalibration and local 68 realignment at known indels. The BWA aligner was used for mapping reads to the human genome build 69 38. Single nucleotide variants (SNVs) and insertions/deletions (indels) were jointly called across all 70 samples using Genome Analysis Toolkit (GATK) HaplotypeCaller package version 4. Default filters Smith et al., Supplemental Materials 3 71 were applied to SNV and indel calls using the GATK Variant Quality Score Recalibration (VQSR) 72 approach. Annotation was performed using Variant Effect Predictor (VEP). The variant call sets were 73 uploaded to seqr (https://seqr.broadinstitute.org/) for collaborative analysis between the Center for 74 Mendelian Genomics (CMG) and investigator. Identified variants were validated with Sanger 75 sequencing run in all available members of the family. For Case C, WES was performed in Mendelics 76 Genomic Analysis facilities using Illumina NovaSeq 6000. Sequencing library was built with Illumina 77 Nextera Flex and for capture of target regions Customized Exome Kit from Twist Biosciences was 78 used. Sequencing of sample resulted in a total of 27.173.797 paired 101 bp sequences mapped to 79 b37/hg19 human genome reference. 96.1 % of the Nextera exome reference was sampled at 10X or 80 more. Exome reads were mapped to b37/hg19 reference using BWA MEM software (http://bio- 81 bwa.sourceforge.net/). Resulting BAM files were genotyped using Broad Institute best practices with 82 GATK (https://software.broadinstitute.org/gatk/). Resulting VCF files were processed using Mendelics 83 in-house pipeline for annotation and filtering. “In silico” pathogenicity evaluation was performed using 84 VSA, a Mendelics proprietary machine-learning based software. Aligned BAM files were also 85 processed by ExomeDepth in order to identify CNVs. The heterozygous c.2771T>C; p.Leu24Pro 86 variant in ATP1A3 was validated in the proband by Sanger sequencing. The proband’s parents were 87 also investigated for the ATP1A3 variant using Sanger sequencing, which was found to be absent in 88 both, in peripheral blood samples. For Case D, WES was performed at GeneDx. Genomic DNA from 89 the submitted specimen was enriched for the complete coding regions and splice site junctions for most 90 genes of the human genome using a proprietary capture system developed by GeneDx for next- 91 generation sequencing with CNV calling (NGS-CNV). The enriched targets were simultaneously 92 sequenced with paired-end reads on an Illumina platform. Bi-directional sequence reads were 93 assembled and aligned to reference sequences based on NCBI RefSeq transcripts and human genome 94 build GRCh37/UCSC hg19. Using a custom-developed analysis tool (XomeAnalyzer), data were Smith et al., Supplemental Materials 4 95 filtered and analyzed to identify sequence variants and most deletions and duplications involving three 96 or more coding exons (Retterer et al., 2016). 97 98 Phenotypic assessment 99 All affected individuals and clinical data were examined by neurologists and/or geneticists, and 100 radiologists, and polymicrogyria was diagnosed using criteria described previously (Jansen et al., 2015; 101 Smith and Walsh, 2020). Supplemental text below summarizes their phenotypes and detailed clinical 102 and radiographic evaluations. 103 104 Human brain tissue preparation and mRNA in situ hybridization 105 Performed as previously described Smith et al3. Briefly, following fixation (4% PFA) and 106 cryoprotection (30% sucrose), brains were frozen using Isopentane on dry ice. Samples were sectioned 107 at 20 – 30 µm thickness (Leica Cryostat), mounted immediately onto warm charged SuperFrost Plus 108 slides (Fisher), and stored at –80°C. We followed manufacturer’s standard protocol for multiplex 109 fluorescent in situ hybridization (Multiplex Version 2 kit, Advanced Cell Diagnostics). In situ probe 110 ACD catalog numbers are as follows: ATP1A3: 503941, Vimentin: 479411, Eomes: 429691. Whole 111 tissue mRNA in situ imaging was performed on a Zeiss Axio Observer with automated image. (Figure 112 3B) Bright-field images were background corrected by Zen Blue Software for center intensity 113 illumination and stitched together (Figure 3B). 114 115 Bulk human cortex gene expression analysis 116 The Allen Human Brain Atlas (ABA) publishes a rich dataset of cortical genetic expression across 117 cortical brain regions, from age 8 weeks post conception to adult ages.(Jones et al., 2009) BrainSpan 118 data analysis of ATP1A1 (chr1:116,915,289-116,952,883, GRCh37/hg19), ATP1A2 (chr1:160,085,548- Smith et al., Supplemental Materials 5 119 160,113,381, GRCh37/hg19), and ATP1A3 (chr19:42,470,733-42,498,384, GRCh37/hg19) was 120 performed. ATP1A4 is not expressed in the CNS and was not included as the read counts were near 121 zero. RNA-seq expression measured in RPKM (reads per kilobase exon per million mapped reads) was 122 obtained from the BrainSpan project data and summarized to Gencode v10 exons for all annotated 123 neocortical tissues aged 12 weeks post conception to 36 years. To obtain a moving average across ages, 124 we fit a polynomial to the data using Igor pro software. Brain regions for Figure 3B include: 125 dorsolateral prefrontal cortex; ventrolateral prefrontal cortex; anterior (rostral) cingulate (medial 126 prefrontal) cortex; orbital frontal cortex; primary motor-sensory cortex; parietal neocortex; posterior 127 (caudal) superior temporal cortex (area 22c), inferolateral temporal cortex (area 20); occipital 128 neocortex, thalamic regions and hippocampus, among others. 129 130 Single-cell suspensions for Drop-Seq 131 The intact left brain hemisphere of a 21 wpc specimen was transported