Conserved transcriptional regulation by BRN1 and BRN2 in neocortical progenitors drives mammalian neural specification and neocortical expansion.

The neocortex varies in size and complexity among mammals due to the tremendous variability in the number and diversity of neuronal subtypes across species. The increased cellular diversity is paralleled by the expansion of the pool of neocortical progenitors and the emergence of indirect neurogenesis during brain evolution. The molecular pathways that control these biological processes and are disrupted in neurological disorders remain largely unknown. Here we show that the transcription factors BRN1 and BRN2 have an evolutionary conserved function in neocortical progenitors to control their proliferative capacity and the switch from direct to indirect neurogenesis. Functional studies in mice and ferrets show that BRN1/2 act in concert with NOTCH and primary microcephaly genes to regulate progenitor behavior. Analysis of transcriptomics data from genetically modified macaques provides evidence that these molecular pathways are conserved in non-human primates. Our findings thus demonstrate that BRN1/2 are central regulators of gene expression programs in neocortical progenitors critical to determine brain size during evolution.

Analysis of DNA from brain tissue on stereo-EEG electrodes reveals mosaic epilepsy-related variants.

Somatic mosaic variants contribute to focal epilepsy, but genetic analysis has been limited to patients with drug-resistant epilepsy (DRE) who undergo surgical resection, as the variants are mainly brain-limited. Stereoelectroencephalography (sEEG) has become part of the evaluation for many patients with focal DRE, and sEEG electrodes provide a potential source of small amounts of brain-derived DNA. We aimed to identify, validate, and assess the distribution of potentially clinically relevant mosaic variants in DNA extracted from trace brain tissue on individual sEEG electrodes. We enrolled a prospective cohort of eleven pediatric patients with DRE who had sEEG electrodes implanted for invasive monitoring, one of whom was previously reported. We extracted unamplified DNA from the trace brain tissue on each sEEG electrode and also performed whole-genome amplification for each sample. We extracted DNA from resected brain tissue and blood/saliva samples where available. We performed deep panel and exome sequencing on a subset of samples from each case and analysis for potentially clinically relevant candidate germline and mosaic variants. We validated candidate mosaic variants using amplicon sequencing and assessed the variant allele fraction (VAF) in amplified and unamplified electrode-derived DNA and across electrodes. We extracted DNA from >150 individual electrodes from 11 individuals and obtained higher concentrations of whole-genome amplified vs unamplified DNA. Immunohistochemistry confirmed the presence of neurons in the brain tissue on electrodes. Deep sequencing and analysis demonstrated similar depth of coverage between amplified and unamplified samples but significantly more called mosaic variants in amplified samples. In addition to the mosaic PIK3CA variant detected in a previously reported case from our group, we identified and validated four potentially clinically relevant mosaic variants in electrode-derived DNA in three patients who underwent laser ablation and did not have resected brain tissue samples available. The variants were detected in both amplified and unamplified electrode-derived DNA, with higher VAFs observed in DNA from electrodes in closest proximity to the electrical seizure focus in some cases. This study demonstrates that mosaic variants can be identified and validated from DNA extracted from trace brain tissue on individual sEEG electrodes in patients with drug-resistant focal epilepsy and in both amplified and unamplified electrode-derived DNA samples. Our findings support a relationship between the extent of regional genetic abnormality and electrophysiology, and suggest that with further optimization, this minimally invasive diagnostic approach holds promise for advancing precision medicine for patients with DRE as part of the surgical evaluation.

Rare variation in non-coding regions with evolutionary signatures contributes to autism spectrum disorder risk.

Little is known about the role of non-coding regions in the etiology of autism spectrum disorder (ASD). We examined three classes of non-coding regions: human accelerated regions (HARs), which show signatures of positive selection in humans; experimentally validated neural VISTA enhancers (VEs); and conserved regions predicted to act as neural enhancers (CNEs). Targeted and whole-genome analysis of >16,600 samples and >4,900 ASD probands revealed that likely recessive, rare, inherited variants in HARs, VEs, and CNEs substantially contribute to ASD risk in probands whose parents share ancestry, which enriches for recessive contributions, but modestly contribute, if at all, in simplex family structures. We identified multiple patient variants in HARs near IL1RAPL1 and in VEs near OTX1 and SIM1 and showed that they change enhancer activity. Our results implicate both human-evolved and evolutionarily conserved non-coding regions in ASD risk and suggest potential mechanisms of how regulatory changes can modulate social behavior.

Neuropathologically directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, < 1% were transmitted by misfolded PrP, ~ 15% are inherited, and ~ 85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate localized initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of > 5000× across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a localized presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Chromosomal structural rearrangements implicate long non-coding RNAs in rare germline disorders.

In recent years, there has been increased focus on exploring the role the non-protein-coding genome plays in Mendelian disorders. One class of particular interest is long non-coding RNAs (lncRNAs), which has recently been implicated in the regulation of diverse molecular processes. However, because lncRNAs do not encode protein, there is uncertainty regarding what constitutes a pathogenic lncRNA variant, and thus annotating such elements is challenging. The Developmental Genome Anatomy Project (DGAP) and similar projects recruit individuals with apparently balanced chromosomal abnormalities (BCAs) that disrupt or dysregulate genes in order to annotate the human genome. We hypothesized that rearrangements disrupting lncRNAs could be the underlying genetic etiology for the phenotypes of a subset of these individuals. Thus, we assessed 279 cases with BCAs and selected 191 cases with simple BCAs (breakpoints at only two genomic locations) for further analysis of lncRNA disruptions. From these, we identified 66 cases in which the chromosomal rearrangements directly disrupt lncRNAs. In 30 cases, no genes of any other class aside from lncRNAs are directly disrupted, consistent with the hypothesis that lncRNA disruptions could underly the phenotypes of these individuals. Strikingly, the lncRNAs MEF2C-AS1 and ENSG00000257522 are each disrupted in two unrelated cases. Furthermore, we experimentally tested the lncRNAs TBX2-AS1 and MEF2C-AS1 and found that knockdown of these lncRNAs resulted in decreased expression of the neighboring transcription factors TBX2 and MEF2C, respectively. To showcase the power of this genomic approach for annotating lncRNAs, here we focus on clinical reports and genetic analysis of seven individuals with likely developmental etiologies due to lncRNA disruptions.

Contributions of Germline and Somatic Mosaic Genetics to Thoracic Aortic Aneurysms in Nonsyndromic Individuals.

BACKGROUND: Thoracic aortic aneurysm (TAA) is associated with significant morbidity and mortality. Although individuals with family histories of TAA often undergo clinical molecular genetic testing, adults with nonsyndromic TAA are not typically evaluated for genetic causes. We sought to understand the genetic contribution of both germline and somatic mosaic variants in a cohort of adult individuals with nonsyndromic TAA at a single center. METHODS AND RESULTS: One hundred eighty-one consecutive patients <60 years who presented with nonsyndromic TAA at the Massachusetts General Hospital underwent deep (>500×) targeted sequencing across 114 candidate genes associated with TAA and its related functional pathways. Samples from 354 age- and sex-matched individuals without TAA were also sequenced, with a 2:1 matching. We found significant enrichments for germline (odds ratio [OR], 2.44, P=4.6×10-6 [95% CI, 1.67-3.58]) and also somatic mosaic variants (OR, 4.71, P=0.026 [95% CI, 1.20-18.43]) between individuals with and without TAA. Likely genetic causes were present in 24% with nonsyndromic TAA, of which 21% arose from germline variants and 3% from somatic mosaic alleles. The 3 most frequently mutated genes in our cohort were FLNA (encoding Filamin A), NOTCH3 (encoding Notch receptor 3), and FBN1 (encoding Fibrillin-1). There was increased frequency of both missense and loss of function variants in TAA individuals. CONCLUSIONS: Likely contributory dominant acting genetic variants were found in almost one quarter of nonsyndromic adults with TAA. Our findings suggest a more extensive genetic architecture to TAA than expected and that genetic testing may improve the care and clinical management of adults with nonsyndromic TAA.

Rare germline disorders implicate long non-coding RNAs disrupted by chromosomal structural rearrangements.

In recent years, there has been increased focus on exploring the role the non-protein-coding genome plays in Mendelian disorders. One class of particular interest is long non-coding RNAs (lncRNAs), which has recently been implicated in the regulation of diverse molecular processes. However, because lncRNAs do not encode protein, there is uncertainty regarding what constitutes a pathogenic lncRNA variant, and thus annotating such elements is challenging. The Developmental Genome Anatomy Project (DGAP) and similar projects recruit individuals with apparently balanced chromosomal abnormalities (BCAs) that disrupt or dysregulate genes in order to annotate the human genome. We hypothesized that rearrangements disrupting lncRNAs could be the underlying genetic etiology for the phenotypes of a subset of these individuals. Thus, we assessed 279 cases with BCAs and selected 191 cases with simple BCAs (breakpoints at only two genomic locations) for further analysis of lncRNA disruptions. From these, we identified 66 cases in which the chromosomal rearrangements directly disrupt lncRNAs. Strikingly, the lncRNAs MEF2C-AS1 and ENSG00000257522 are each disrupted in two unrelated cases. Furthermore, in 30 cases, no genes of any other class aside from lncRNAs are directly disrupted, consistent with the hypothesis that lncRNA disruptions could underly the phenotypes of these individuals. To showcase the power of this genomic approach for annotating lncRNAs, here we focus on clinical reports and genetic analysis of two individuals with BCAs and additionally highlight six individuals with likely developmental etiologies due to lncRNA disruptions.

Cell-type-specific effects of autism-associated 15q duplication syndrome in the human brain.

Recurrent copy-number variation represents one of the most well-established genetic drivers in neurodevelopmental disorders, including autism spectrum disorder. Duplication of 15q11-q13 (dup15q) is a well-described neurodevelopmental syndrome that increases the risk of autism more than 40-fold. However, the effects of this duplication on gene expression and chromatin accessibility in specific cell types in the human brain remain unknown. To identify the cell-type-specific transcriptional and epigenetic effects of dup15q in the human frontal cortex, we conducted single-nucleus RNA sequencing and multi-omic sequencing on dup15q-affected individuals (n = 6) as well as individuals with non-dup15q autism (n = 7) and neurotypical control individuals (n = 7). Cell-type-specific differential expression analysis identified significantly regulated genes, critical biological pathways, and differentially accessible genomic regions. Although there was overall increased gene expression across the duplicated genomic region, cellular identity represented an important factor mediating gene-expression changes. As compared to other cell types, neuronal subtypes showed greater upregulation of gene expression across a critical region within the duplication. Genes that fell within the duplicated region and had high baseline expression in control individuals showed only modest changes in dup15q, regardless of cell type. Of note, dup15q and autism had largely distinct signatures of chromatin accessibility but shared the majority of transcriptional regulatory motifs, suggesting convergent biological pathways. However, the transcriptional binding-factor motifs implicated in each condition implicated distinct biological mechanisms: neuronal JUN and FOS networks in autism vs. an inflammatory transcriptional network in dup15q microglia. This work provides a cell-type-specific analysis of how dup15q changes gene expression and chromatin accessibility in the human brain, and it finds evidence of marked cell-type-specific effects of this genetic driver. These findings have implications for guiding therapeutic development in dup15q syndrome, as well as understanding the functional effects of copy-number variants more broadly in neurodevelopmental disorders.

Neuropathologically-directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, ~1% were transmitted by misfolded PrP, ~15% are inherited, and ~85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate focal initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of >5,000X across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a focal presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

ARX regulates cortical interneuron differentiation and migration.

Mutations in aristaless-related homeobox ( ARX ) are associated with neurodevelopmental disorders including developmental epilepsies, intellectual disabilities, and autism spectrum disorders, with or without brain malformations. Aspects of these disorders have been linked to abnormal cortical interneuron (cIN) development and function. To further understand ARX's role in cIN development, multiple Arx mutant mouse lines were interrogated. We found that ARX is critical for controlling cIN numbers and distribution, especially, in the developing marginal zone (MZ). Single cell transcriptomics and ChIP-seq, combined with functional studies, revealed ARX directly or indirectly regulates genes involved in proliferation and the cell cycle (e.g., Bub3 , Cspr3 ), fate specification (e.g., Nkx2.1 , Maf , Mef2c ), and migration (e.g., Nkx2.1 , Lmo1 , Cxcr4 , Nrg1 , ErbB4 ). Our data suggest that the MZ stream defects primarily result from disordered cell-cell communication. Together our findings provide new insights into the mechanisms underlying cIN development and migration and how they are disrupted in several disorders.

Cell-type-specific effects of autism-associated chromosome 15q11.2-13.1 duplications in human brain.

Recurrent copy number variation represents one of the most well-established genetic drivers in neurodevelopmental disorders, including autism spectrum disorder (ASD). Duplication of 15q11.2-13.1 (dup15q) is a well-described neurodevelopmental syndrome that increases the risk of ASD by over 40-fold. However, the effects of this duplication on gene expression and chromatin accessibility in specific cell types in the human brain remain unknown. To identify the cell-type-specific transcriptional and epigenetic effects of dup15q in the human frontal cortex we conducted single-nucleus RNA-sequencing and multi-omic sequencing on dup15q cases (n=6) as well as non-dup15q ASD (n=7) and neurotypical controls (n=7). Cell-type-specific differential expression analysis identified significantly regulated genes, critical biological pathways, and differentially accessible genomic regions. Although there was overall increased gene expression across the duplicated genomic region, cellular identity represented an important factor mediating gene expression changes. Neuronal subtypes, showed greater upregulation of gene expression across a critical region within the duplication as compared to other cell types. Genes within the duplicated region that had high baseline expression in control individuals showed only modest changes in dup15q, regardless of cell type. Of note, dup15q and ASD had largely distinct signatures of chromatin accessibility, but shared the majority of transcriptional regulatory motifs, suggesting convergent biological pathways. However, the transcriptional binding factor motifs implicated in each condition implicated distinct biological mechanisms; neuronal JUN/FOS networks in ASD vs. an inflammatory transcriptional network in dup15q microglia. This work provides a cell-type-specific analysis of how dup15q changes gene expression and chromatin accessibility in the human brain and finds evidence of marked cell-type-specific effects of this genetic driver. These findings have implications for guiding therapeutic development in dup15q syndrome, as well as understanding the functional effects CNVs more broadly in neurodevelopmental disorders.

Spatial Single-cell Analysis Decodes Cortical Layer and Area Specification.

The human cerebral cortex, pivotal for advanced cognitive functions, is composed of six distinct layers and dozens of functionally specialized areas1,2. The layers and areas are distinguished both molecularly, by diverse neuronal and glial cell subtypes, and structurally, through intricate spatial organization3,4. While single-cell transcriptomics studies have advanced molecular characterization of human cortical development, a critical gap exists due to the loss of spatial context during cell dissociation5,6,7,8. Here, we utilized multiplexed error-robust fluorescence in situ hybridization (MERFISH)9, augmented with deep-learning-based cell segmentation, to examine the molecular, cellular, and cytoarchitectural development of human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing 16 million single cells, spans eight cortical areas across four time points in the second and third trimesters. We uncovered an early establishment of the six-layer structure, identifiable in the laminar distribution of excitatory neuronal subtypes by mid-gestation, long before the emergence of cytoarchitectural layers. Notably, while anterior-posterior gradients of neuronal subtypes were generally observed in most cortical areas, a striking exception was the sharp molecular border between primary (V1) and secondary visual cortices (V2) at gestational week 20. Here we discovered an abrupt binary shift in neuronal subtype specification at the earliest stages, challenging the notion that continuous morphogen gradients dictate mid-gestation cortical arealization6,10. Moreover, integrating single-nuclei RNA-sequencing and in situ whole transcriptomics revealed an early upregulation of synaptogenesis in V1-specific Layer 4 neurons, suggesting a role of synaptogenesis in this discrete border formation. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This work not only provides a valuable resource for the field, but also establishes a spatially resolved single-cell analysis paradigm that paves the way for a comprehensive developmental atlas of the human brain.

High-resolution detection of copy number alterations in single cells with HiScanner.

Improvements in single-cell whole-genome sequencing (scWGS) assays have enabled detailed characterization of somatic copy number alterations (CNAs) at the single-cell level. Yet, current computational methods are mostly designed for detecting chromosome-scale changes in cancer samples with low sequencing coverage. Here, we introduce HiScanner (High-resolution Single-Cell Allelic copy Number callER), which combines read depth, B-allele frequency, and haplotype phasing to identify CNAs with high resolution. In simulated data, HiScanner consistently outperforms state-of-the-art methods across various CNA types and sizes. When applied to high-coverage scWGS data from human brain cells, HiScanner shows a superior ability to detect smaller CNAs, uncovering distinct CNA patterns between neurons and oligodendrocytes. For 179 cells we sequenced from longitudinal meningioma samples, integration of CNAs with point mutations revealed evolutionary trajectories of tumor cells. These findings show that HiScanner enables accurate characterization of frequency, clonality, and distribution of CNAs at the single-cell level in both non-neoplastic and neoplastic cells.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Neurodevelopmental disorders associated variants in ADAT3 disrupt the activity of the ADAT2/ADAT3 tRNA deaminase complex and impair neuronal migration.

The ADAT2/ADAT3 complex catalyzes the adenosine to inosine modification at the wobble position of eukaryotic tRNAs. Mutations in ADAT3 , the catalytically inactive subunit of the ADAT2/ADAT3 complex, have been identified in patients presenting with severe neurodevelopmental disorders (NDDs). Yet, the physiological function of ADAT2/ADAT3 complex during brain development remains totally unknown. Here we showed that maintaining a proper level of ADAT2/ADAT3 catalytic activity is required for correct radial migration of projection neurons in the developing mouse cortex. In addition, we not only reported 7 new NDD patients carrying biallelic variants in ADAT3 but also deeply characterize the impact of those variants on ADAT2/ADAT3 structure, biochemical properties, enzymatic activity and tRNAs editing and abundance. We demonstrated that all the identified variants alter both the expression and the activity of the complex leading to a significant decrease of I 34 with direct consequence on their steady-state. Using in vivo complementation assays, we correlated the severity of the migration phenotype with the degree of the loss of function caused by the variants. Altogether, our results indicate a critical role of ADAT2/ADAT3 during cortical development and provide cellular and molecular insights into the pathogenicity of ADAT3-related neurodevelopmental disorder.

Contrasting somatic mutation patterns in aging human neurons and oligodendrocytes.

Characterizing somatic mutations in the brain is important for disentangling the complex mechanisms of aging, yet little is known about mutational patterns in different brain cell types. Here, we performed whole-genome sequencing (WGS) of 86 single oligodendrocytes, 20 mixed glia, and 56 single neurons from neurotypical individuals spanning 0.4-104 years of age and identified >92,000 somatic single-nucleotide variants (sSNVs) and small insertions/deletions (indels). Although both cell types accumulate somatic mutations linearly with age, oligodendrocytes accumulated sSNVs 81% faster than neurons and indels 28% slower than neurons. Correlation of mutations with single-nucleus RNA profiles and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed across the genome similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These stark differences suggest an assortment of active mutagenic processes in oligodendrocytes and neurons.

Somatic cancer driver mutations are enriched and associated with inflammatory states in Alzheimer's disease microglia.

Alzheimer's disease (AD) is an age-associated neurodegenerative disorder characterized by progressive neuronal loss and pathological accumulation of the misfolded proteins amyloid-ß and tau1,2. Neuroinflammation mediated by microglia and brain-resident macrophages plays a crucial role in AD pathogenesis1-5, though the mechanisms by which age, genes, and other risk factors interact remain largely unknown. Somatic mutations accumulate with age and lead to clonal expansion of many cell types, contributing to cancer and many non-cancer diseases6,7. Here we studied somatic mutation in normal aged and AD brains by three orthogonal methods and in three independent AD cohorts. Analysis of bulk RNA sequencing data from 866 samples from different brain regions revealed significantly higher (~two-fold) overall burdens of somatic single-nucleotide variants (sSNVs) in AD brains compared to age-matched controls. Molecular-barcoded deep (>1000X) gene panel sequencing of 311 prefrontal cortex samples showed enrichment of sSNVs and somatic insertions and deletions (sIndels) in cancer driver genes in AD brain compared to control, with recurrent, and often multiple, mutations in genes implicated in clonal hematopoiesis (CH)8,9. Pathogenic sSNVs were enriched in CSF1R+ microglia of AD brains, and the high proportion of microglia (up to 40%) carrying some sSNVs in cancer driver genes suggests mutation-driven microglial clonal expansion (MiCE). Analysis of single-nucleus RNA sequencing (snRNAseq) from temporal neocortex of 62 additional AD cases and controls exhibited nominally increased mosaic chromosomal alterations (mCAs) associated with CH10,11. Microglia carrying mCA showed upregulated pro-inflammatory genes, resembling the transcriptomic features of disease-associated microglia (DAM) in AD. Our results suggest that somatic driver mutations in microglia are common with normal aging but further enriched in AD brain, driving MiCE with inflammatory and DAM signatures. Our findings provide the first insights into microglial clonal dynamics in AD and identify potential new approaches to AD diagnosis and therapy.

An evolutionary perspective on complex neuropsychiatric disease.

The forces of evolution-mutation, selection, migration, and genetic drift-shape the genetic architecture of human traits, including the genetic architecture of complex neuropsychiatric illnesses. Studying these illnesses in populations that are diverse in genetic ancestry, historical demography, and cultural history can reveal how evolutionary forces have guided adaptation over time and place. A fundamental truth of shared human biology is that an allele responsible for a disease in anyone, anywhere, reveals a gene critical to the normal biology underlying that condition in everyone, everywhere. Understanding the genetic causes of neuropsychiatric disease in the widest possible range of human populations thus yields the greatest possible range of insight into genes critical to human brain development. In this perspective, we explore some of the relationships between genes, adaptation, and history that can be illuminated by an evolutionary perspective on studies of complex neuropsychiatric disease in diverse populations.

Somatic Mosaicism in PIK3CA Variant Correlates With Stereoelectroencephalography-Derived Electrophysiology.

Objectives: Brain-limited pathogenic somatic variants are associated with focal pediatric epilepsy, but reliance on resected brain tissue samples has limited our ability to correlate epileptiform activity with abnormal molecular pathology. We aimed to identify the pathogenic variant and map variant allele fractions (VAFs) across an abnormal region of epileptogenic brain in a patient who underwent stereoelectroencephalography (sEEG) and subsequent motor-sparing left frontal disconnection. Methods: We extracted genomic DNA from peripheral blood, brain tissue resected from peri-sEEG electrode regions, and microbulk brain tissue adherent to sEEG electrodes. Samples were mapped based on an anatomic relationship with the presumed seizure onset zone (SOZ). We performed deep panel sequencing of amplified and unamplified DNA to identify pathogenic variants with subsequent orthogonal validation. Results: We detect a pathogenic somatic PIK3CA variant, c.1624G>A (p.E542K), in the brain tissue samples, with VAF inversely correlated with distance from the SOZ. In addition, we identify this variant in amplified electrode-derived samples, albeit with lower VAFs. Discussion: We demonstrate regional mosaicism across epileptogenic tissue, suggesting a correlation between variant burden and SOZ. We also validate a pathogenic variant from individual amplified sEEG electrode-derived brain specimens, although further optimization of techniques is required.

Somatic Mosaicism in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Reveals Widespread Degeneration from Focal Mutations.

Although mutations in dozens of genes have been implicated in familial forms of amyotrophic lateral sclerosis (fALS) and frontotemporal degeneration (fFTD), most cases of these conditions are sporadic (sALS and sFTD), with no family history, and their etiology remains obscure. We tested the hypothesis that somatic mosaic mutations, present in some but not all cells, might contribute in these cases, by performing ultra-deep, targeted sequencing of 88 genes associated with neurodegenerative diseases in postmortem brain and spinal cord samples from 404 individuals with sALS or sFTD and 144 controls. Known pathogenic germline mutations were found in 20.6% of ALS, and 26.5% of FTD cases. Predicted pathogenic somatic mutations in ALS/FTD genes were observed in 2.7% of sALS and sFTD cases that did not carry known pathogenic or novel germline mutations. Somatic mutations showed low variant allele fraction (typically <2%) and were often restricted to the region of initial discovery, preventing detection through genetic screening in peripheral tissues. Damaging somatic mutations were preferentially enriched in primary motor cortex of sALS and prefrontal cortex of sFTD, mirroring regions most severely affected in each disease. Somatic mutation analysis of bulk RNA-seq data from brain and spinal cord from an additional 143 sALS cases and 23 controls confirmed an overall enrichment of somatic mutations in sALS. Two adult sALS cases were identified bearing pathogenic somatic mutations in DYNC1H1 and LMNA, two genes associated with pediatric motor neuron degeneration. Our study suggests that somatic mutations in fALS/fFTD genes, and in genes associated with more severe diseases in the germline state, contribute to sALS and sFTD, and that mosaic mutations in a small fraction of cells in focal regions of the nervous system can ultimately result in widespread degeneration.