Comprehensive multi-omic profiling of somatic mutations in malformations of cortical development

Malformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fraction in brain tissue resected to treat epilepsy. Here, we report genetic atlas from 283 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. Moreover, the unique spatiotemporal expression patterns deconvolved from single-nuclear transcriptional sequences of mutated genes in control and patient brains suggest critical roles driving excitatory neurogenic pools during brain development, and in establishing neuronal excitation after birth. A fresh-frozen brain tissue (~50 mg) was placed into a glass dounce homogenizer containing 1 ml cold lysis buffer (0.05% (v/v) NP-40, 10 mM Tris (pH 7.4), 3 mM MgCl2, 10 mM NaCl) and dounce 10 times with a loose pestle and following 10 times with a tight pestle. The homogenate was incubated for 10 min in RT. 9 ml of wash buffer (1% BSA in 1X PBS) was added to the homogenate and filtered by a 30 um cell strainer. The strained homogenate was spun down in 500 g to remove the supernatant. The pellet was resuspended with 5 ml of wash buffer. Straining and spinning down steps were performed once more, and the pellet was resuspended into 500 ul of wash buffer. 10 ul of nuclei resuspension was mixed with counting solution (0.02% Tween 20, 0.1ug/ml DAPI, 1% BSA in 1X PBS) and nuclei density was measured by manual nuclei counting using DAPI signal. The resuspension was diluted by wash buffer to make the desired concentration (800~1000 nuclei/ul). Maximum 2 samples were pooled together targeting 10000 nuclei per reaction. Gel beads emulsion (GEM) generation, cDNA, and sequencing library constructions were performed in accordance with instructions in the Chromium Single Cell 3' Reagent Kits User Guide (v3.1). A library pool was sequenced with 800 million read pairs using NovaSeq 6000.