Smith et al., Supplemental Materials 1

1 Folding and lamination of the human neocortex depend on the sodium 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 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 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 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 to the McCarroll lab in ice-cold

132 Hibernate-E medium (ThermoFisher). The tissue was transferred to a dissection dish and submerged in

133 ice-cold L-15 Medium (ThermoFisher) supplemented with 10% FBS (ThermoFisher). The medium was

134 kept refrigerated throughout the dissection. Upon removal of the meninges, the hemisphere was divided

135 along the rostro-caudal axis into 6 serial coronal slabs, each ~5cm thick, spanning the rostro-caudal

136 axis. Regions of interest (Fig S2, Fig 4) were microdissected under visual guidance of a stereoscope

137 (Leica MZ10), and identified using a fetal human atlas as reference.(Bayer et al., 2005) Single cell

138 suspensions were produced using the MACS Neural Tissue Dissociation kit [papain (P), Miltenyi

139 Biotec)] as described (Florio et al., 2015) with modifications. Briefly, tissue fragments containing

140 regions of interest were transferred into 15 mL falcon tubes containing 2 mL of “ mix 1”

141 (containing papain) and incubated at 37 C for 20’ in a benchtop incubator without agitation. Tubes

142 containing digested tissue were then transferred onto ice and 30 µL of “Enzyme mix 2” (containing Smith et al., Supplemental Materials 6

143 papain inhibitor) were added to each sample. Tissue chunks were carefully titrated with a P1000

144 pipette, the triturated cells were centrifuged at 300 g for 5’, and the cell pellets were resuspended in 1x

145 PBS containing 0.01% BSA. Cell suspensions were then passed through a pre-wet 40 µm filter into a

146 new tube on ice, and diluted to a final concentration of 220 cells/µl for Drop-Seq.

147

148 Single-nucleus suspensions for Drop-Seq

149 Nuclei were isolated from frozen cortical tissue (240-days-old donor), from 4 anatomically distinct

150 cortical regions (BA10, BA41/42, BA17, BA40, Fig 5), dissected by NIH BioBank and stored in

151 individual microcentrifuge tubes at -80°C. Single-nucleus suspensions and processed for DropSeq as

152 described in detail at: https://protocols.io/view/extraction-of-nuclei-from-brain-tissue-2srged6, with

153 modifications detailed (Krienen et al., 2019).

154

155 Drop-seq sequencing

156 Single-cell cDNA libraries were processed and sequenced using Illumina short read sequencing on a

157 Nova-seq platform at the Broad Institute. Libraries were sequenced at an average depth of >8 reads per

158 UMI. Sequencing reads were aligned to the hg19 human reference genome. For the fetal dataset, only

159 reads mapping to exons were used; for the postnatal dataset, reads mapping to either exons and introns

160 were used. Software and core computational analysis for alignment and downstream processing of

161 Drop-seq sequencing reads are freely available at https://github.com/broadinstitute/Drop-seq/releases.

162

163 Independent component analysis and clustering

164 Cell selection was performed as described (Saunders et al., 2018) for each sequencing pool

165 independently and the resulting digital (DGE) matrices were then merged into unique Smith et al., Supplemental Materials 7

166 “dataset DGEs” (i.e. fetal and postnatal) which were analyzed separately. Independent component

167 analysis (ICA) was performed using the R’ fastICA package on each dataset’s DGE, after

168 normalization and variable gene selection, as described in Saunders 2015 and Krienen 2019, with

169 modifications. Single-cell/nucleus libraries containing fewer than 500 genes were excluded from the

170 analysis, and genes encoding Mitochondrial RNAs ('^RPS'), Ribosomal ('^RPS', '^RPL'), and

171 Histones ('^HIST) were filtered out from the DGEs prior to ICA. The number of significant

172 components used for clustering was determined using the JackStraw function from Seurat

173 (https://www.rdocumentation.org/packages/Seurat/versions/3.1.4). Cells identified as doublets or

174 outliers were iteratively removed through multiple rounds of clustering. Clusters were identified using

175 canonical cell type markers (Fig S2, S4), and only neural clusters (i.e. excitatory neurons, inhibitory

176 neurons, glia, and neural progenitor cells) were selected in the analysis. A final round of clustering was

177 then run on the filtered DGEs, and the optimal clustering resolutions were chosen based on the salience

178 of unique markers within each cluster, as described (Saunders et al., 2018).

179 Statistical analysis

180 Data analysis was performed using R studio. Source code available by request. Gene expression

181 normalization and scaling methods for each analysis are specified in figure legends. Clustering

182 Heatmaps were generated using R’s pheatmap package, using the ‘ward.D2’ clustering method and

183 euclidean clustering distance. Pairwise gene correlation coefficients were calculated upon relative-

184 count normalization and scaling using R’s cor function. tSNE embeddings were generated as described

185 in Saunders 2015. UMAP embeddings were generated using R’s uwot package using as input a matrix

186 wherein each row is a cell in the DGE and each column is the aggregated expression, for that given cell,

187 of the genes positively loading onto each individual component used in ICA. The resulting matrixes

188 was then scaled by cell using R’scale function. The UMAP algorithm was run with default parameters, Smith et al., Supplemental Materials 8

189 with a n_neighbors equal to the square root of number of cells in the dataset. Pseudotime analysis was

190 performed using Monocle 3 (https://cole-trapnell-lab.github.io/monocle3/). The analysis was run using

191 default parameters and using as input a subset of the fetal dataset’s DGE containing only cells assigned

192 to EN clusters.

193 Graphics

194 Graphics prepared in Adobe Illustrator and using BioRender software. Smith et al., Supplemental Materials 9

195 SUPPLEMENTAL RESULTS

196 Supplemental Text 197 Clinical descriptions of Case A-D; Related to Figure 1 198 199 Table S1 200 PMG-associated ATP1A3 alleles 201 202 Figure S1 203 Sequencing and conservation of Cases A-D with ATP1A3 pathogenic variants; Related to 204 Figure 1 205 206 Figure S2 207 Anatomical origin and gene-expression markers for cortical fetal samples used in 208 DropSeq experiments; Related to Figure 4 209 210 Figure S3 211 Cell-type specific expression and pair-wise correlation of ATP1A1-3 in the fetal human 212 neocortex; Related to Figure 4 213 214 Figure S4 215 Clustering analysis of ATP1A3 expression in the infant human cortex, Related to Figure 5 216 217 Figure S5 218 Pair-wise correlation of ATP1A1-3 in the infant human neocortex; Related to Figure 5 219 220 Table S2. 221 analysis of the top-100 genes correlating with ATP1A3 in the EN cluster in 222 the postnatal cortex, Related to Figure 5 223 224 Figure S6 225 Comparison of ATP1A3 expression within cortical interneurons isolated from mice, 226 macaque, and human; Related to Figure 5 227 228 Figure S7 229 Schematic of ATP1A3 variants with respect to disease severity 230 231 Table S3 232 Summary of published ATP1A3 variants associated with disease with original references 233 and phenotypic features 234 235 236 237 238 239 CLINICAL DESCRIPTIONS 240 Case A: Bilateral Frontoparietal PMG (p.Arg901Met) Smith et al., Supplemental Materials 10

241 Case A, a female child of nonconsanguineous parents from the Azores of Portugal, was born at 242 full term by spontaneous vaginal delivery following an unremarkable pregnancy. She required 243 oxygen for respiratory distress at birth, exhibited neonatal jaundice and was discharged from 244 intensive care after 10 days. Her developmental delays were noted at an early age, as she could not 245 hold a pacifier. At 2 and 3 years of age, she had surgical corrections for strabismus. Drooling was a 246 significant concern by 6 years of age. She had Botox injections in childhood for her spastic 247 quadriparesis. Episodes suspicious for seizure activity occurred around 8.5 years and at about 9 248 years she developed focal impaired awareness seizures, which was treated with oxcarbazepine. 249 Evaluation at 9 years and 2 months of age revealed a head circumference of 51 cm (-0.73 standard 250 deviations, SD), length of 110.6 cm (-3.82 SD) and weight of 17.7 kg (-2.92 SD). She was 251 developmentally and cognitively delayed, able to sit alone and walk using a walker with assistance, 252 point and use some sign language but had no speech, and could hold a pencil but not write. Drooling 253 was still an issue and she did not chew. A prior barium swallow study was negative for aspiration. 254 She attended a public school in a contained class and received physical, occupational and speech 255 therapies. Asymmetric spastic quadriparesis (left more significant than right) with hypertonia and 256 athetoid movements of the hands and fingers were noted on examination, as were a high palate, thin 257 upper lip and smooth philtrum. 258 Investigations included an EEG at 8 years 8 months of age that was abnormal and showed no 259 clear anterior to posterior gradient. Alpha activity was more prominent over the frontal area with 260 some attenuation of the amplitude and slightly faster frequencies over the posterior regions. A clear 261 beta activity could not be identified over the anterior or central areas in a consistent manner. 262 Throughout the recording, spikes and high amplitude sharp and slow waves were noted bilaterally 263 over the frontotemporal areas, with the right being greater than left. Brain MRI performed at 5 264 months showed polymicrogyria (PMG) in the perisylvian cortex bilaterally, as well as extensive 265 thinning of the spinal cord from T1-T12. MRI at 9 years and 3 months of age revealed PMG in the 266 perisylvian cortex, which was thicker than at 5 months, as well as in the suprasylvian cortex, 267 predominantly frontally but extending much farther rostrally. The parietal lobes were very abnormal 268 and pachygyric in appearance. The posterior part of frontal and temporal cortex was abnormally 269 thick, with somewhat shallow sulci, giving a pachygyric appearance. These abnormalities were 270 bilateral but asymmetric, with the left more affected than the right. The most anterior aspects of the 271 frontal and parietal lobes, as well as the medial and inferior cerebral cortex, were spared. However, it 272 is worth noting the pachygyric appearance may be the result of averaging of a large segment of the Smith et al., Supplemental Materials 11

273 cortex as a result of thick sections on a 20 year old MRI. The corpus callosum, basal ganglia and 274 midline structures appeared normal. The cerebral white matter volume was significantly reduced 275 compared to the 5-month study with otherwise normal myelination. Lateral ventricles were mildly 276 enlarged, and the cerebellum was disproportionately large when compared to the cerebrum. The 277 spinal cord was not included in the MRI at 9 years. Unremarkable genetic testing included a 278 karyotype (46, XX), array CGH, and Fragile X, as well as ARX gene sequencing. 279 280 Case B: Extensive Bilateral PMG (c.2921+1G>A) 281 Case B, a male child of nonconsanguineous parents from the Philippines, was born at 38 weeks’ 282 gestation by Cesarean section due to decreased fetal heart rate following a pregnancy complicated by 283 maternal gestational diabetes. At birth, his head circumference was 34 cm (-0.83 SD) and weight 284 was 2.995 kg (-0.91 SD), and he developed seizures 12 hours after delivery. Extensive bilateral 285 polymicrogyria was identified by brain MRI at 6 weeks of age and video EEGs confirmed a severe 286 epileptic encephalopathy that proved resistant to essentially all antiepileptic drugs. EEGs at 2 287 months of age showed abundant electroclinical and electrographic seizures beginning predominantly 288 from the right parasagittal region but also from the left and bilateral parasagittal regions. Some 289 seizures had diffuse onset associated with head jerks, and interictally, there were independent 290 epileptogenic discharges from the right and left parasagittal regions activated during sleep. Visual 291 and auditory evoked potential studies were normal at 4 and 5 months of age respectively. He 292 required nasogastric tube followed later by a G-tube for feeding. 293 Evaluation at 14 months revealed microcephaly, with a head circumference of 41.5cm (-4.63 294 SD); his weight was 10.4 kg (-1.61 SD) and height was 73cm (-0.38 SD). He exhibited severe global 295 developmental delay (never rolled over, sat up or talked), intermittent nystagmus, significant axial 296 and appendicular hypotonia, and hyporeflexia. His severe epileptic encephalopathy at 14 months was 297 described by 24-hour video-EEG study as having a poorly organized and slow background with 298 excessively frequent multifocal independent epileptiform discharges throughout almost all brain 299 regions, though no electrographic seizures were noted on that study. Brain MRI at 14 months noted 300 widespread bilateral polymicrogyria with some calcification at the cortical-white matter junction. 301 While myelination was slightly delayed at 2 months, it was markedly delayed at 14 months with 302 progressive volume loss, and the corpus callosum was normally formed but thin. The basal ganglia 303 appeared atrophic compared to the initial scan. Initial MRI studies revealed a small, cyst-like 304 (perhaps cavitation) areas observed within the hippocampal heads and bodies (left side slightly Smith et al., Supplemental Materials 12

305 larger). Follow-up studies the following year demonstrate hippocampi are much smaller with what 306 appears to be central atrophy (possibly shrinkage after cavitation) of the hippocampal head and 307 ventral body. Adjacent white matter also shrinking but, most likely, gray matter (hippocampi) 308 shrunk first. Other examinations included a karyotype and 22q11 FISH assay, chromosomal 309 microarray, ADGRG1 (GPR56) gene sequencing, urine organic and plasma amino acid testing, 310 acylcarnitine profile and very long chain fatty acid testing, which were all unremarkable. 311

312 Case C: Unilateral PMG (p.Leu924Pro) 313 Case C is a male born to nonconsanguineous parents from Brazil. His 32-year-old mother has a 314 personal history of hypothyroidism. After an uneventful pregnancy, he was born at 38 weeks of 315 gestation by Cesarean section for acute fetal distress, weighed 3.66 kg (0.2 SD) and had Apgar 316 scores of 6 and 8 at 1 and 5 minutes, respectively. Hypotonia and a left clubfoot (for which casting 317 was indicated) were noted at birth. Episodes of upward rolling of the eyes on the first day of life 318 prompted admission to the NICU. An EEG confirmed the presence of electroclinical seizures and 319 phenobarbital was initiated. On day 3, he exhibited apnea and hypertonia, however 24-hour video- 320 EEG study failed to detect any significant abnormalities. Neurological exam revealed episodic 321 dystonic posturing of the upper left extremity, alternating with excessive movements of closing his 322 left hand with thumb adduction. Three electroencephalographic focal seizures and a clinical 323 epileptic episode characterized by eye blinking and apnea occurred on day 4, and an EEG on day 5 324 described the epileptic seizures as periodic discharges with right posterior projection. Brain MRI, 325 also on day 5, showed extensive right hemispheric polymicrogyria involving the right frontal, 326 parietal, and temporal lobes, and the insula, as well as a small foci of signal abnormality in the 327 periventricular white matter, compatible with neonatal ischemic injury. He was discharged at 13 328 days of age with a phenobarbital and levetiracetam regime. 329 His subsequent neuropsychomotor development was delayed and he required hospital admission 330 for epileptic episodes at 2 and 4 months of age. Two EEGs around 4 months showed: (1) moderate 331 periodic epileptic activity localized to the left temporal region and more rarely bilateral central 332 projection, and low delta wave surges with left hemisphere projection (occasionally rhythmically 333 occurring) suggestive of a localized rhythmic delta activity pattern; and (2) infrequent slow wave 334 surges bilaterally in the anterior regions, and epileptic paroxysms with sharp wave morphology in 335 the left temporal region and rare occurrences in the posterior regions of the left hemisphere and 336 midline region. He was admitted again at 5 months due to bradycardia, without associated seizures, Smith et al., Supplemental Materials 13

337 ultimately went into cardiogenic shock due to acute viral myocarditis and was discharged after 338 hemodynamic recovery. Upon evaluation at 8 months of age, he was on a ketogenic diet and in use 339 of Cannabidiol 4.4 mg/kg/day, Topiramate 50 50 16.5 mg/kg/day, phenobarbital 7.25 mg/kg/day. 340 His examination revealed a head circumference of 42.5 cm (-1.75 SD), global hypotonia with present 341 reflexes, very poor eye contact, babbling but no head control, rolling or gripping of objects. 342 343 Case D: Extensive Multifocal Bilateral PMG (p.Gln851Arg) 344 Case D is a male born to nonconsanguineous parents of European decent by emergency Cesarean 345 section at 41 weeks gestation following a pregnancy complicated by fetal hydronephrosis. He had 346 Apgar scores of 1 and 8 at 1 and 5 minutes respectively, and a birth weight of 3.8 kg (0.49 SD). He 347 required a 30-day neonatal hospital stay, as his early course was complicated by seizures and he was 348 diagnosed with a neonatal encephalopathy requiring cooling. Brain MRI soon after birth revealed 349 bilateral polymicrogyria involving the right cerebral hemisphere more extensively than the left, most 350 severe in the right fronto-temporo-parietal lobes and insula with relative sparing of the right occipital 351 lobe. There was progression of diffuse white matter volume loss in the right cerebral hemisphere, 352 right basal ganglia and corresponding progressive volume loss in the right cerebral peduncle, right 353 aspect of the pons and upper medulla. Progression of ex vacuo dilatation of the right lateral ventricle 354 was also noted, as was a small corpus callosum, especially the splenium. There was mild scattered 355 ethmoid sinus mucosal thickening, and no evidence of an intracranial mass, acute intracranial 356 hemorrhage or infarct, or extra axial fluid collection. 357 At 4 months of age, he underwent surgery for congenital hip dysplasia. At that time, he was 358 noted to have frequent breath-holding spells, which worsened after the surgery. A video EEG then 359 showed slowing during these spells and they were initially not considered to be epileptiform. His 360 seizures are characterized by back and forth eye movements and sometimes involve shaking of his 361 hands. He is treated with Phenobarbital, Keppra and Trileptal, is reported to have persistent 362 intermittent abnormal eye movements and has spastic left hemiparesis, mainly involving his left arm 363 but also his face. 364 At 15 and 30 months of age, head circumference was 42.7 cm (-3.5 SD) and 44.2 cm (-3.3 SD), 365 respectively. On examination at 18 months of age, he exhibited very low tone with poor head 366 control. Developmentally, he babbled, could not roll over, sat with support and tended to play with 367 his right hand. He had some persistent feeding problems, was not yet receiving any solid food and Smith et al., Supplemental Materials 14

368 was reported to have constant constipation. He was described to sleep like a newborn, waking up 369 during the night and asking to be fed. He receives speech, physical and occupational therapies. 370 371

372

373 Smith et al., Supplemental Materials 15

374 Table S1, PMG-associated ATP1A3 alleles

Case A Case B Case C Case D Genomic g.41968902C>A g.41967661C>T g.41968833A>G g.41969571T>C coordinate (GRCh38) cDNA notation c.2702G>T c.2921+1G>A c.2771T>C c.2552A>G (NM_152296.4) notation p.Arg901Met NA p.Leu924Pro p.Gln851Arg PhyloP 7.76 [-20.0;10.0] NA 9.29 [-20.0;10.0] 9.29 [-20.0;10.0] SIFT (v6.2.0) Deleterious NA Deleterious Deleterious (score: 0, (score: 0.01, (score: 0.01, median: 3.46) median: 3.46) median: 3.45) Mutation Taster Disease causing NA Disease causing Disease causing (v2013) (prob: 1) (prob: 1) (prob: 1) PolyPhen-2 Probably NA Probably Probably damaging (1.000) damaging (1.000) damaging (1.000) Splice predictions NA Predicted change NA NA at nearest natural at donor site 1 splice junction bps upstream: - 100.0%MaxEnt: -100.0% NNSPLICE: - 100.0% SSF: -100.0% gnomAD v.2.1.1 0 0 0 0 (MAF) 375

376

377 378 Smith et al., Supplemental Materials 16

Figure S1. Sequencing and conservation of Cases A-D with ATP1A3 pathogenic variants; Related to Figure 1

379 380 Figure S1. Sequencing and conservation of Cases A-D with ATP1A3 pathogenic variants; 381 Related to Figure 1 382 (A) Left, Representative ATP1A3 Sanger chromatograms from unaffected (black text) parents and

383 affected individual A, possessing a de novo heterozygous G>T substitution. . Center, Case B Sanger

384 chromatograms from unaffected (black text) parents and affected de novo individual B, possessing a

385 de novo heterozygous G>A substitution. Right, Case C Sanger chromatograms from unaffected

386 (black text) parents and affected de novo individual C, possessing a de novo heterozygous T>C

387 substitution. (B) ATP1A3 sequence alignments of the amino acids surrounding the PMG associated

388 mutations from Na,K-ATPase alpha-3 isoform ortholog across species, showing high degree of

389 conservation. Smith et al., Supplemental Materials 17

Figure S2, Anatomical origin and gene-expression markers for cortical fetal samples used in DropSeq experiments; Related to Figure 4 390 Smith et al., Supplemental Materials 18

391 Figure S2, Anatomical origin and gene-expression markers for cortical fetal samples used 392 in DropSeq experiments; Related to Figure 4 393 (A) Left, Photograph of the fetal neocortical specimen (21 wpc) used for DropSeq, prior to 394 microdissection. Anatomical axes are indicated (R: rostral, C: caudal, D: dorsal, M: medial). 395 Horizontal white lines indicate approximate location of dissection boundaries between cortical 396 slabs (I-VI). Right, Representative Nissl-stained coronal section images of a 21wpc neocortex 397 depicting anatomical detail and location of samples [source: Atlas of the Developing Human 398 Brain, BrainSpan (www.brainspan.org). Fr: Frontal; Par: Parietal; Temp: Temporal; Occ: 399 Occipital; Orb: Orbital Frontal Cortex; Ins: Insula; Cing: Cingulate cortex; dV1 and mV1: 400 dorsal and medial Primary Visual Cortex, respectively; Str: Striatum; Hp: Hippocampus; CGE 401 and MGE: caudal and medial Ganglionic Eminence, respectively. (B) Bar graphs showing 402 relative expression of regional marker genes (y-axis) across samples (x-axis, samples 1-11, 403 ordered from caudal to rostral). Note the enrichment of LMO3, VSTM2L, CBLN2, CRYM, NTS, 404 GRP in frontal and prefrontal cortex samples (samples 9-11); NR2F1, LPL, TENM2, MET in 405 occipital cortex samples; ANGPTL1, HGF in medial occipital cortex samples including portion 406 of hippocampus; NR2F2 in temporo-parietal cortex samples; NKX2-1 in medial temporal cortex 407 samples including MGE; CRABP1 in medial cortex samples including CGE; and SYNPR in 408 insular cortex samples including striatum. Expression of each gene was sum-aggregated by 409 sample, then gene expression was relative-count normalized (divided by total counts/sample x 410 100k). (C) Heatmap showing hierarchical clustering and expression specificity of selected cell- 411 type markers (columns) in the different clusters (rows). Note the highest relative expression of 412 ATP1A3 is the EN.4 SP cluster, marked by deep-layer EN marker NEFL and SP marker LPL. 413 Expression of each gene was sum-aggregated by cluster, relative-count normalized, then 414 rescaled from 0 to 1. (D) Dot-plot showing specific enrichment of subplate markers in the EN.4 415 SP cluster. Color scale codes for mean gene expression by cluster; size of the dots codes for 416 percentage of cells expressing a given gene in each cluster. RG: radial glia IP: intermediate 417 progenitors (div: dividing, diff: differentiating, cx: cortex, str: striatum); OPC: oligodendrocyte 418 progenitor cell; IN: inhibitory neurons, EN: excitatory neurons (CP: cortical plate, IZ:

419 intermediate zone); astro: astrocytes; OPC: oligodendrocyte progenitor cells. Smith et al., Supplemental Materials 19

Figure S3 Cell-type specific expression and pair-wise correlation of ATP1A1-3 in the fetal human neocortex; Related to Figure 4

420 Smith et al., Supplemental Materials 20

421 Figure S3 Cell-type specific expression and pair-wise correlation of ATP1A1-3 in the fetal 422 human neocortex; Related to Figure 4 423 424 (A) Bar graphs showing relative expression of ATP1A1, ATP1A2 and ATP1A3 (y-axis) across

425 clusters. Gene expression (y-axis) was normalized by cell (divided by total UMIs/cell x 100k),

426 then the mean value for each gene was aggregated by cluster and rescaled from 0 to 1. Note the

427 enrichment of ATP1A1 and ATP1A3 in the EN4.SP cluster, and the enrichment of ATP1A2 in

428 glial clusters (anti-correlating with ATP1A3). (B) Dot plots showing pairwise correlation

429 coefficients between ATP1A1, ATP1A2 and ATP1A3 expression across clusters. Dots represent

430 Spearmann’s correlation coefficients (r), color coded by association strength. Note the positive

431 correlation between expression of ATP1A3 and ATP1A1, but not ATP1A2, in the EN SP cluster.

432 (C) Scatter plots showing pairwise comparisons of ATP1A1, ATP1A2 and ATP1A3 expression

433 in single cells. Each dot represents a cell. Gene expression was normalized by cell. Pearson

434 correlation coefficients (R) for each pair are indicated above each plot.

435 436 437 438 Smith et al., Supplemental Materials 21

439 440 441

Figure S4. Clustering analysis of ATP1A3 expression in the postnatal cortex, Related to Figure 5

Smith et al., Supplemental Materials 22

442 443 Figure S4. Clustering analysis of ATP1A3 expression in the postnatal cortex, Related to

444 Figure 5

445 8-month old neocortex of 51,878 single nuclei profiled by DropSeq. EN: excitatory neurons; IN:

446 inhibitory neurons. Heatmap showing hierarchical clustering and expression specificity of

447 selected cell-type markers (columns) in the different clusters (rows). Mean gene expression was

448 aggregated by cluster, then rescaled from 0 to 1. Representative cortical marker genes including

449 EN layer 2-3 marker CUX2, EN layer-5 marker FEZF2, EN layer-4 marker RORB, MGE-

450 derived IN markers SST+, VIP+, CGE-derived IN maker LAMP5.

451 452 453 454 455 456 457 Smith et al., Supplemental Materials 23

458

Table S2. Gene ontology analysis of the top-100 genes correlating with ATP1A3 in the EN cluster in the infant cortex, Related to Figure 5

459 460 461 462 463 464 465 466 Smith et al., Supplemental Materials 24

467

Figure S5. Pair-wise correlation of ATP1A1-3 in the infant human neocortex; Related to Figure 5

468 469 470 Figure S5. Pair-wise correlation of ATP1A1-3 beta subunits in the infant human 471 neocortex; Related to Figure 5 472 Left, Bar graphs showing relative expression of ATP1A1, ATP1A2 and ATP1A3 (y-axis) across

473 clusters. Gene expression (y-axis) was normalized by cell (divided by total UMIs/cell x 100k),

474 then the mean value for each gene was aggregated by cluster and rescaled from 0 to 1. Right,

475 Dot plots showing pairwise correlation coefficients between ATP1A1, ATP1A2 and ATP1A3 Smith et al., Supplemental Materials 25

476 expression across clusters. Dots represent Spearmann’s correlation coefficients (r), color coded

477 by association strength. Of note, within ENs, the only cluster in which ATP1A3 is coexpressed

478 with ATP1B1 significantly is layer 6 (SP_NR42A), labeled with green box. Also, note the

479 positive correlation between expression of ATP1A3 and ATP1A1, within the MGE derived

480 cluster, most notably parvalbumin (PVALB), labeled with red box. EN, excitatory neuron; IN,

481 inhibitory neuron.

482 483 Smith et al., Supplemental Materials 26

Figure S6, Comparison of ATP1A3 expression within cortical interneurons isolated from mice, macaque, and human; Related to Figure 5

484 485 Smith et al., Supplemental Materials 27

486 Figure S6, Comparison of ATP1A3 expression within cortical interneurons isolated from mice,

487 macaque, and human; Related to Figure 5

488 (A) Human vs. mouse, marmoset, and macaque comparing the four major neocortical interneuron

489 classes: somatostatin (SST+), parvalbumin (PVALB+), Vasoactive intestinal peptide (VIP+), or

490 differentiation marker clade (ID2+). (B) Striatal interneurons colored by in one of the four major

491 neocortical classes: cholecystokinin (CCK+), choline acetyltransferase (CHAT+), SST+, tyrosine

492 hydroxylase (TH+), PVALB+, neuropeptide TAC+. Scaled expression levels (number of transcripts

493 per 100k) for ATP1A3 and ATP1A1. Data from recently published collaborative study

494 (Krienen et al, 2020). (C) ATP1A3 in situ demonstrates ubiquitous expression in the human adult

495 cerebral cortex. (C) Image downloaded from the Allen Brain Atlas.

496 497 498 499 500 501 502 503 504 505 506 507 508 509 Smith et al., Supplemental Materials 28

Figure S7 Schematic of ATP1A3 variants with respect to disease severity

510 Smith et al., Supplemental Materials 29

511 Figure S7 Schematic of ATP1A3 variants with respect to disease severity. 512 513 (A) Phenotypes associated with ATP1A3 are complex and overlapping, one way to organize

514 them is by “severity” (i.e. age of onset), as done by Sweadner et al. 2019. Red corresponds to

515 “severe”, blue to “mild”, and purple to “mixed” (e.g. different presentations with same

516 mutation). Diseases like AHC and PMG have clear presentation in pre and early postnatal

517 period, whereas patients of RDP may be asymptomatic until adulthood. Grouping mutations by

518 the onset of associated symptoms in those patients suggests that mutations in the TM domains,

519 and particularly in TMs 5-8 are more likely to lead to severe/early onset disease, and mutations

520 in non-TM domains are more likely to lead to later onset ATP1A3 related disease such as RDP.

521 See Supplemental Table S3 for sources of each allele. See Figure 2C for disease specific

522 mutations.

523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 Smith et al., Supplemental Materials 30

549 Table S3: Summary of published ATP1A3 variants associated with disease with original references 550 and phenotypic features, See Figure 2C.

Sequence Variant Protein First Report Severity Documented Phenotype(s) (NM_152296.5) Variant 55G>A R19C* Allocco 2019 S CH/Schizencephaly 266delG 89fs Holze 2018 S HT/DD 367G>C G123R Sampson 2016 M RDP 385G>A V129M Smedemark-Margulies 2016 M COS/DD 410C>A S137Y Heinzen 2012 S AHC 410C>T S137F Heinzen 2012 S AHC 409-411delGAG S137del Wilcox 2015 M RDP/PXD 419A>T Q140L Heinzen 2012 S AHC 420G>C or T Q140H Prange 2017 S RDP/EIEE 821T>A I274N Rosewich 2012, Heinzen 2012 S AHC 821T>C I274T de Carvalho Aguiar 2004 M RDP 829G>A E277K de Carvalho Aguiar 2004 M AHC, RDP 946G>A G316S Sweadner 2016 M RDP/CA 958G>A A320T Trump 2016 S EIEE/PXD 958G>C A320P ClinVar 965T>A V322D Rosewich 2012 S AHC 967C>T P323S Masoud 2017 S AHC 968C>T P323L ClinVar** 970G>C E324Q Panagiotakaki 2015, Viollet 2015 S AHC 971A>G E324G Prange 2017 S AHC 972G>C E324D Viollet 2015 S AHC 974G>A G325D Lee 2014 S DD/CA 977T>G L326R Panagiotakaki 2015, Viollet 2015 S AHC 976-978delCTG L327del Kamm 2008 M RDP 983C>A A328D Sweadner 2019 S 998G>T C333F Heinzen 2012 S AHC 1003A>C T335P Rosewich 2014a S AHC 1004C>T T335M ClinVar 1004C>A T335K ClinVar 1012G>C A338P Gurrieri 2016 S AHC 1036T>C C346R Marzin 2018 S HT/EIEE/DD 1072G>T G358C Sasaki 2014 S EIEE/AHC 1072G>A G358S Panagiotakaki 2015 S AHC 1073G>T G358V Paciorkowski 2015 S EIEE 1073G>A G358D Pereira 2015 M AHC/RDP Smith et al., Supplemental Materials 31

1079C>G T360R Prange 2017 S EIEE/CA/DYT 1088T>A I363N Paciorkowski 2015 S EIEE/PXD/MC 1088T>C I363T Kim 2020 S AHC/EIEE 1096G>C D366H Sweadner 2019 M RDP 1108G>A T370A Sweadner 2019 M 1109C>A T370N Yang 2014, Rosewich 2014a M AHC, RDP 1112T>C L371P Rosewich 2012 S AHC 1123C>T R375C ClinVar 1124G>A R375H ClinVar 1144T>C W382R Rosewich 2012 M AHC/RDP 1244C>A A415D Sweadner 2019 M 1250T>C L417P Rosewich 2014a M RDP 1387G>A R463C* Allocco 2019; Aryastarkhova 2019 S CH/Schizencephaly, RDP** 1747G>T D583Y Nicita 2016(Nicita et al., 2016)35 M PXD/RDP 1765G>T V589F Richards 2018; Tran 2020 S EIEE/AHC/RDP 1786T>C C596R Viollet 2015 S AHC 1790G>C R597P Wenzel 2017 M RDP 1795G>A A599T ClinVar 1825G>T D609Y Marzin 2018 S HT/DD/EIEE 1838C>T T613M de Carvalho Aguiar 2004; Balint 2019 M RDP, PXD 2041G>A A681T Torres 2018 M DD/ASDa 2051C>T S684F Svetel 2010 M RDP 2116G>A G706R Yang 2014 S AHC 2140G>A A714T Meijer 2016 M RDP 2144T>C L715P Panagiotakaki 2015 S AHC 2224G>T D742Y Marzin 2018 S EIEE/PXD 2227G>C D743H Meijer 2016 M RDP 2266-2268delGAC D743del Schirzini 2018 S EIEE/AHC/HT 2263G>A G755S Heinzen 2012 S AHC 2263G>T G755C Rosewich 2012 S AHC 2264G>T G755V Viollet 2015 S AHC 2264G>C G755A Sasaki 2014 S AHC 2266C>T R756C Dard 2015 M RECA 2267G>A R756H Brashear 2012 M RECA 2267G>T R756L Yano 2017 M RECA 2270T>C L757P Rosewich 2014a S AHC 2272A>T I758F Sweadner 2019 M 2273T>G I758S de Carvalho Aguiar 2004 M RDP 2281A>C N761H Viollet 2015 S AHC 2302T>C Y768H Viollet 2015 S AHC Smith et al., Supplemental Materials 32

2303T>C Y768C Viollet 2015 S AHC 2305A>C T769P Viollet 2015 S AHC 2309T>G L770R Yang 2014 S AHC 2312C>A T771N Sasaki 2014 S AHC 2312C>T T771I Yang 2014 S AHC 2314A>C S772R Panagiotakaki 2015, Viollet 2015 S AHC 2316C>A S772R Rosewich 2012 S AHC 2316C>G S772R Yang 2014 S AHC 2317A>C N773H Viollet 2015 S AHC 2318A>G N773S Heinzen 2012 S AHC 2318A>T N773I Rosewich 2012 S AHC 2318A>C N773T Yang 2015 S AHC 2323C>A P775L ClinVar (2) 2324C>T P775T ClinVar 2332A>C T778P Gasser 2020 S RDP/EP 2338T>C F780L de Carvalho Aguiar 2004 M RDP 2401G>A D801N Heinzen 2012 (>300) S AHC, AHC/RDP 2401G>T D801Y de Carvalho Aguiar 2004 M-S AHC, RDP 2401G>C D801H ClinVar 2402A>T D801V Panagiotakaki 2015 S AHC 2403T>A D801E Hoei-Hansen 2014 S AHC 2405T>G L802R Zúñiga-Ramírez 2019 S PXD 2405T>C L802P Yang 2014 S AHC 2408G>A G803D Gall 2017 S CA/DYT/DD/MC/HT 2411C>T T804I Ulate-Campos 2014, Rosewich 2014b S AHC 2413G>A D805N Viollet 2015 S AHC 2413G>C D805H Yang 2014 S AHC 2415C>G D805E Rosewich 2014 S AHC 2417T>G M806R Heinzen 2012 S AHC 2417T>A M806K Yang 2014 S AHC 2423C>T P808L Yang 2014 S AHC 2428A>T I810F Rosewich 2014 S AHC/RDP 2429T>G I810S Heinzen 2012 S AHC 2429T>A I810N Yang 2014 S AHC 2431T>C S811P Heinzen 2012 S AHC 2438C>T A813V Kubota 2017 M COS/ASD 2443G>A E815K Heinzen 2012 ( >200) S AHC, AHC/COS 2452G>A E818K Demos 2014 M CAPOS, AHC/CAPOS 2479A>T R827W Gasser 2020 S AHC 2501T>C L834S Yang 2015 S AHC Smith et al., Supplemental Materials 33

2516T>C L839P Yang 2014 S AHC 2542+1G>A SPLICE Viollet 2015 S AHC 2542+2T>C SPLICE Viollet 2015 S AHC 2552A>G Q851R Masoud 2017, This study S AHC, PMG 2552A>C Q851P Yang 2015 S AHC/EIEE 2558T>G L853R Rodriguez-Quiroga 2016 M AHC, RDP, AHC/RDP 2600G>A G867D Rosewich 2014b M AHC/RDP 2600G>A G867N Sweney 2015*** S AHC, RDP 2663T>C L888P Panagiotakaki 2015, This study S AHC, PMG 2677G>A G893R Yang 2014 S AHC 2702G>C R901T Viollet 2015 S AHC 2702G>T R901M This study S PMG 2736-2738delCTT F913del Ishihara 2019 S AHC/HT/EIEE 2755- 2757delGTC V919del Heinzen 2012 S AHC 2767G>T D923Y Rosewich 2012 S AHC 2767G>A D923N Zanotti 2008 M AHC/RDP 2767G>T D923T Sweney 2015*** S AHC 2810T>C L924P Aryastarkhova 2019, This study S EIEE/MC, PMG 2780G>A C927Y Ishii 2013 S AHC 2780G>T C927F Sasaki 2014 S AHC 2781C>G C927W Ulate-Campos 2014 S AHC 2788C>T R930W Meijer 2016 M RDP 2839G>A G947R Heinzen 2012 (>30) S AHC, EIEE, HT/AHC 2839G>C G947R Heinzen 2012 (>50) S AHC, EIEE, HT/AHC 2851G>A E951K Panagiotakaki 2015, Viollet 2015 S AHC, AHC/RDP 2864C>A A955D Heinzen 2012 S AHC 2960+1G>A SPLICE This study S PMG 2974G>C D992H Sweadner 2019 S AHC 2974G>T D992Y Heinzen 2012 S AHC 3191-3193dupTAC Y1013YY Blanco-Arias 2009 M RDP 551 552 *Seen in recessive case 553 **Atypical presentation/responsive to L-DOPA treatment 554 ***Could not locate original report, earliest found reference 555 556 Methods: Based on methods and criteria used by Sweadner et al. 201917; see supplementary table e-4. 557 Severity was defined by age of onset of symptoms (S = severe/neonatal onset, M = milder/later onset). 558 Phenotypes were annotated based on review of published clinical information in the original report of each 559 allele, classified with descriptions/keywords. Some mutations were originally reported on a different 560 transcript, but were adjusted to reflect NM_152296.5, the canonical ATP1A3 transcript, as done by 561 Sweadner et al. 2019. Annotations on Protter (http://wlab.ethz.ch/protter/) on Figure 2C and Figure S6.

Phenotype Smith et al., Supplemental Materials 34

HT Hypotonia (diffuse) MC Microcephaly DD Developmental Delay EIEE Early Infantile Epileptic Encephalopathy AHC Alternating Hemiplegia of Childhood RDP Rapid Onset -Parkinsonism RECA Recurrent Episodes of Cerebellar Ataxia (Fever Induced) CAPOS Cerebellar Ataxia, Areflexia, Pes Cavus, Optic Atrophy, and Sensorineural Hearing Loss PXD Paroxysmal Dystonia CH Congenital Hydrocephalus PMG Polymicrogryria COS Childhood Onset CA Cerebellar Ataxia (or confirmed cerebellar atrophy on MRI) DYT Dystonia/Generalized 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 Smith et al., Supplemental Materials 35

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