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.

A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer's disease.

Many aging individuals accumulate the pathology of Alzheimer's disease (AD) without evidence of cognitive decline. Here we describe an integrated neurodegeneration checkpoint response to early pathological changes that restricts further disease progression and preserves cognitive function. Checkpoint activation is mediated by the REST transcriptional repressor, which is induced in cognitively-intact aging humans and AD mouse models at the onset of amyloid ß-protein (Aß) deposition and tau accumulation. REST induction is mediated by the unfolded protein response together with ß-catenin signaling. A consequence of this response is the targeting of REST to genes involved in key pathogenic pathways, resulting in downregulation of gamma secretase, tau kinases, and pro-apoptotic proteins. Deletion of REST in the 3xTg and J20 AD mouse models accelerates Aß deposition and the accumulation of misfolded and phosphorylated tau, leading to neurodegeneration and cognitive decline. Conversely, viral-mediated overexpression of REST in the hippocampus suppresses Aß and tau pathology. Thus, REST mediates a neurodegeneration checkpoint response with multiple molecular targets that may protect against the onset of AD.

Schizophrenia-associated somatic copy-number variants from 12,834 cases reveal recurrent NRXN1 and ABCB11 disruptions.

While germline copy-number variants (CNVs) contribute to schizophrenia (SCZ) risk, the contribution of somatic CNVs (sCNVs)-present in some but not all cells-remains unknown. We identified sCNVs using blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls, filtering sCNVs at loci recurrently mutated in clonal blood disorders. Likely early-developmental sCNVs were more common in cases (0.91%) than controls (0.51%, p = 2.68e-4), with recurrent somatic deletions of exons 1-5 of the NRXN1 gene in five SCZ cases. Hi-C maps revealed ectopic, allele-specific loops forming between a potential cryptic promoter and non-coding cis-regulatory elements upon 5' deletions in NRXN1. We also observed recurrent intragenic deletions of ABCB11, encoding a transporter implicated in anti-psychotic response, in five treatment-resistant SCZ cases and showed that ABCB11 is specifically enriched in neurons forming mesocortical and mesolimbic dopaminergic projections. Our results indicate potential roles of sCNVs in SCZ risk.

Characterizing the molecular impact of KMT2D variants on the epigenetic and transcriptional landscapes in Kabuki syndrome.

Kabuki syndrome (KS) is a rare, multisystem disorder with a variable clinical phenotype. The majority of KS is caused by dominant loss-of-function mutations in KMT2D (lysine methyltransferase 2D). KMT2D mediates chromatin accessibility by adding methyl groups to lysine residue 4 of histone 3, which plays a critical role in cell differentiation and homeostasis. The molecular underpinnings of KS remain elusive partly because of a lack of histone modification data from human samples. Consequently, we profiled and characterized alterations in histone modification and gene transcription in peripheral blood mononuclear cells (PBMCs) from 33 patients with KMT2D mutations and 36 unaffected healthy controls. Our analysis identified unique enhancer signatures in H3K4me1 and H3K4me2 in KS compared with controls. Reduced enhancer signals were present for promoter-distal sites of immune-related genes for which co-binding of PBMC-specific transcription factors was predicted; 31% of super-enhancers of normal blood cells overlapped with disrupted enhancers in KS, supporting an association of reduced enhancer activity of immune-related genes with immune deficiency phenotypes. In contrast, increased enhancer signals were observed for promoter-proximal regions of metabolic genes enriched with EGR1 and E2F2 motifs, whose transcriptional levels were significantly increased in KS. Additionally, we identified ~100 de novo enhancers in genes, such as in MYO1F and AGAP2. Together, our results underscore the effect of KMT2D haploinsufficiency on dysregulation of enhancer states and gene transcription and provide a framework for the identification of therapeutic targets and biomarkers in preparation for clinical trial readiness.

Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).

A Pan-Cancer Compendium of Genes Deregulated by Somatic Genomic Rearrangement across More Than 1,400 Cases.

A systematic cataloging of genes affected by genomic rearrangement, using multiple patient cohorts and cancer types, can provide insight into cancer-relevant alterations outside of exomes. By integrative analysis of whole-genome sequencing (predominantly low pass) and gene expression data from 1,448 cancers involving 18 histopathological types in The Cancer Genome Atlas, we identified hundreds of genes for which the nearby presence (within 100 kb) of a somatic structural variant (SV) breakpoint is associated with altered expression. While genomic rearrangements are associated with widespread copy-number alteration (CNA) patterns, approximately 1,100 genes-including overexpressed cancer driver genes (e.g., TERT, ERBB2, CDK12, CDK4) and underexpressed tumor suppressors (e.g., TP53, RB1, PTEN, STK11)-show SV-associated deregulation independent of CNA. SVs associated with the disruption of topologically associated domains, enhancer hijacking, or fusion transcripts are implicated in gene upregulation. For cancer-relevant pathways, SVs considerably expand our understanding of how genes are affected beyond point mutation or CNA.

NGSCheckMate: software for validating sample identity in next-generation sequencing studies within and across data types.

In many next-generation sequencing (NGS) studies, multiple samples or data types are profiled for each individual. An important quality control (QC) step in these studies is to ensure that datasets from the same subject are properly paired. Given the heterogeneity of data types, file types and sequencing depths in a multi-dimensional study, a robust program that provides a standardized metric for genotype comparisons would be useful. Here, we describe NGSCheckMate, a user-friendly software package for verifying sample identities from FASTQ, BAM or VCF files. This tool uses a model-based method to compare allele read fractions at known single-nucleotide polymorphisms, considering depth-dependent behavior of similarity metrics for identical and unrelated samples. Our evaluation shows that NGSCheckMate is effective for a variety of data types, including exome sequencing, whole-genome sequencing, RNA-seq, ChIP-seq, targeted sequencing and single-cell whole-genome sequencing, with a minimal requirement for sequencing depth (>0.5X). An alignment-free module can be run directly on FASTQ files for a quick initial check. We recommend using this software as a QC step in NGS studies. AVAILABILITY: https://github.com/parklab/NGSCheckMate.