Publications

Importance Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy subtype and is often refractory to anti-seizure medications. While most MTLE patients do not have pathogenic germline genetic variants, the contribution of post-zygotic (i.e., somatic) variants in the brain is unknown.

Objective To test the association between pathogenic somatic variants in the hippocampus and MTLE.

Design This case-control genetic association study analyzed the DNA derived from hippocampal tissue of neurosurgically-treated patients with MTLE and age- and sex-matched neurotypical controls. Participants were enrolled from 1988 through 2019 and clinical data was collected retrospectively. Whole-exome and gene-panel sequencing (depth>500X) were used to identify candidate pathogenic somatic variants. A subset of novel variants were functionally evaluated using cellular and molecular assays.

Setting Level 4 epilepsy centers, multi-center study.

Participants Non-lesional and lesional (mesial temporal sclerosis, focal cortical dysplasia, and low-grade epilepsy-associated tumors) drug-resistant MTLE patients who underwent anterior medial temporal lobectomy. All patients with available frozen tissue and appropriate consents were included. Control brain tissue was obtained from neurotypical donors at brain banks.

Exposures Drug-resistant MTLE.

Main Outcomes and Measures Presence and abundance of pathogenic somatic variants in the hippocampus versus the unaffected temporal neocortex.

Results Samples were obtained from 105 MTLE patients (52 male, 53 female; age: MED [IQR], 32 [26-44]) and 30 neurotypical controls (19 male, 11 female; age: MED [IQR], 37 [18-53]). Eleven pathogenic somatic variants, enriched in the hippocampus relative to the unaffected temporal neocortex (MED [IQR], 1.92 [1.5-2.7] vs 0.3 [0-0.9], p<0.05), were detected in MTLE patients but not in the controls. Ten of these variants were in PTPN11, SOS1, KRAS, BRAF, and NF1, all predicted to constitutively activate Ras/Raf/MAPK signaling. Immunohistochemical studies of variant-positive hippocampal tissue demonstrated increased Erk1/2 phosphorylation, indicative of Ras/Raf/MAPK activation, predominantly in glial cells. Molecular assays showed abnormal liquid-liquid phase separation for the PTPN11 variants as a possible dominant gain-of-function mechanism.

Conclusions and Relevance Hippocampal somatic variants, particularly those activating Ras/Raf/MAPK signaling, may contribute to the pathogenesis of sporadic, drug-resistant MTLE. These findings may provide a novel genetic mechanism and highlight new therapeutic targets for this common indication for epilepsy surgery.

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 due to 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 to 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; thirty-one percent 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 approximately 100 de novo enhancers in genes, such as in MYO1F and AGAP2. Together, our results underscore the effect of KMT2D haploinsufficiency on (dys)regulation of enhancer states and gene transcription and provide a framework for the identification of therapeutic targets and biomarkers in preparation for clinical trial readiness.

While inherited and de novo copy number variants (CNV) have been implicated in the genetic architecture of schizophrenia (SCZ), the contribution of somatic CNVs (sCNVs), present in some but not all cells of the body, remains unknown. Here we explore the role of sCNVs in SCZ by analyzing blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls. sCNVs were more common in cases (0.91%) than in controls (0.51%, p = 2.68e-4). We observed recurrent somatic deletions of exons 1-5 of the NRXN1 gene in 5 SCZ cases. Allele-specific Hi-C maps revealed ectopic, allele-specific loops forming between a potential novel cryptic promoter and non-coding cis regulatory elements upon deletions in the 5’ region of NRXN1. We also observed recurrent intragenic deletions of ABCB11, a gene associated with anti-psychotic response, in 5 treatment-resistant SCZ cases. Taken together our results indicate an important role of sCNVs to SCZ risk and treatment-responsiveness.

Dementia in Alzheimer’s disease progresses alongside neurodegeneration, but the specific events that cause neuronal dysfunction and death remain poorly understood. During normal ageing, neurons progressively accumulate somatic mutations at rates similar to those of dividing cells which suggests that genetic factors, environmental exposures or disease states might influence this accumulation. Here we analysed single-cell whole-genome sequencing data from 319 neurons from the prefrontal cortex and hippocampus of individuals with Alzheimer’s disease and neurotypical control individuals. We found that somatic DNA alterations increase in individuals with Alzheimer’s disease, with distinct molecular patterns. Normal neurons accumulate mutations primarily in an age-related pattern (signature A), which closely resembles ‘clock-like’ mutational signatures that have been previously described in healthy and cancerous cells. In neurons affected by Alzheimer’s disease, additional DNA alterations are driven by distinct processes (signature C) that highlight C>A and other specific nucleotide changes. These changes potentially implicate nucleotide oxidation, which we show is increased in Alzheimer’s-disease-affected neurons in situ. Expressed genes exhibit signature-specific damage, and mutations show a transcriptional strand bias, which suggests that transcription-coupled nucleotide excision repair has a role in the generation of mutations. The alterations in Alzheimer’s disease affect coding exons and are predicted to create dysfunctional genetic knockout cells and proteostatic stress. Our results suggest that known pathogenic mechanisms in Alzheimer’s disease may lead to genomic damage to neurons that can progressively impair function. The aberrant accumulation of DNA alterations in neurodegeneration provides insight into the cascade of molecular and cellular events that occurs in the development of Alzheimer’s disease.

Replication errors and various genotoxins cause DNA double-strand breaks (DSBs) where error-prone repair creates genomic mutations, most frequently focal deletions, and defective repair may lead to neurodegeneration. Despite its pathophysiological importance, the extent to which faulty DSB repair alters the genome, and the mechanisms by which mutations arise, have not been systematically examined reflecting ineffective methods. Here, we develop PhaseDel, a computational method to detect focal deletions and characterize underlying mechanisms in single-cell whole genome sequences (scWGS). We analyzed high-coverage scWGS of 107 single neurons from 18 neurotypical individuals of various ages, and found that somatic deletions increased with age and in highly expressed genes in human brain. Our analysis of 50 single neurons from DNA repair-deficient diseases with progressive neurodegeneration (Cockayne syndrome, Xeroderma pigmentosum, and Ataxia telangiectasia) reveals elevated somatic deletions compared to age-matched controls. Distinctive mechanistic signatures and transcriptional associations suggest roles for somatic deletions in neurodegeneration.

The accumulation of somatic DNA mutations over time is a hallmark of aging in many dividing and nondividing cells but has not been studied in postmitotic human cardiomyocytes. Using single-cell whole-genome sequencing, we identified and characterized the landscape of somatic single-nucleotide variants (sSNVs) in 56 single cardiomyocytes from 12 individuals (aged from 0.4 to 82 years). Cardiomyocyte sSNVs accumulate with age at rates that are faster than in many dividing cell types and nondividing neurons. Cardiomyocyte sSNVs show distinctive mutational signatures that implicate failed nucleotide excision repair and base excision repair of oxidative DNA damage, and defective mismatch repair. Since age-accumulated sSNVs create many damaging mutations that disrupt gene functions, polyploidization in cardiomyocytes may provide a mechanism of genetic compensation to minimize the complete knockout of essential genes during aging. Age-related accumulation of cardiac mutations provides a paradigm to understand the influence of aging on cardiac dysfunction.

The autosomal recessive genome instability disorder Ataxia-telangiectasia, caused by mutations in ATM kinase, is characterized by the progressive loss of cerebellar neurons. We find that DNA damage associated with ATM loss results in dysfunctional behaviour of human microglia, immune cells of the central nervous system. Microglial dysfunction is mediated by the pro-inflammatory RELB/p52 non-canonical NF-κB transcriptional pathway and leads to excessive phagocytic clearance of neuronal material. Activation of the RELB/p52 pathway in ATM-deficient microglia is driven by persistent DNA damage and is dependent on the NIK kinase. Activation of non-canonical NF-κB signalling is also observed in cerebellar microglia of individuals with Ataxia-telangiectasia. These results provide insights into the underlying mechanisms of aberrant microglial behaviour in ATM deficiency, potentially contributing to neurodegeneration in Ataxia-telangiectasia.

Pathogenic variants in the SRCAP (SNF2-related CREBBP activator protein) gene, which encodes a chromatin-remodeling ATPase, cause neurodevelopmental disorders including Floating Harbor syndrome (FLHS). Here, we report the discovery of a de novo transposon insertion in SRCAP exon 13 from trio genome sequencing in a 28-year-old female with failure to thrive, developmental delay, mood disorder and seizure disorder. The insertion was a full-length (~2.8 kb), antisense-oriented SVA insertion relative to the SRCAP transcript, bearing a 5’ transduction and hallmarks of target-primed reverse transcription. The 20-bp 5’ transduction allowed us to trace the source SVA element to an intron of a long non-coding RNA on chromosome 12, which is highly expressed in testis. RNA sequencing and qRT-PCR confirmed significant depletion of SRCAP expression and low-level exon skipping in the proband. This case highlights a novel disease-causing structural variant and the importance of transposon analysis in a clinical diagnostic setting.

Somatic mutations are DNA variants that occur after the fertilization of zygotes and accumulate during the developmental and aging processes in the human lifespan. Somatic mutations have long been known to cause cancer, and more recently have been implicated in a variety of non-cancer diseases. The patterns of somatic mutations, or mutational signatures, also shed light on the underlying mechanisms of the mutational process. Advances in next-generation sequencing over the decades have enabled genome-wide profiling of DNA variants in a high-throughput manner; however, unlike germline mutations, somatic mutations are carried only by a subset of the cell population. Thus, sensitive bioinformatic methods are required to distinguish mutant alleles from sequencing and base calling errors in bulk tissue samples. An alternative way to study somatic mutations, especially those present in an extremely small number of cells or even in a single cell, is to sequence single-cell genomes after whole-genome amplification (WGA); however, it is critical and technically challenging to exclude numerous technical artifacts arising during error-prone and uneven genome amplification in current WGA methods. To address these challenges, multiple bioinformatic tools have been developed. In this review, we summarize the latest progress in methods for identification of somatic mutations and the challenges that remain to be addressed in the future.

While oncogenic mutations have been found in non-diseased, proliferative non-neural tissues, their prevalence in the human brain is unknown. Targeted sequencing of genes implicated in brain tumors in 418 samples derived from 110 individuals of varying ages, without tumor diagnoses, detected oncogenic somatic single-nucleotide variants (sSNVs) in 5.4% of the brains, including IDH1 R132H. These mutations were largely present in subcortical white matter and enriched in glial cells, and surprisingly, were less common in older individuals. A depletion of high-allele frequency sSNVs representing macroscopic clones with age was replicated by analysis of bulk RNAseq data from 1,816 non-diseased brain samples ranging from fetal to old age. We also describe large clonal copy number variants, and that sSNVs show mutational signatures resembling those found in gliomas, suggesting that mutational processes of the normal brain drive early glial oncogenesis. This study helps understand the origin and early evolution of brain tumors.