Cell type-specificity of mosaic chromosome 1q gain resolved by single-nucleus RNA-seq in a case of pharmacoresistant epilepsy associated with hyaline protoplasmic astrocytopathy
Ontology highlight
ABSTRACT: Introduction: Mosaic gain of chromosome 1q (chr1q) is a recently described driver of malformation of cortical development (MCD) and pharmacoresistant epilepsy. Hyaline protoplasmic astrocytopathy of the neocortex is a rare neuropathological finding seen in cases of pharmacoresistant epilepsy with focal cortical dysplasia or other MCD. The cell-type specificity of mosaic chr1q gain in the brain is unknown, and no data exists on the molecular signatures of hyaline protoplasmic astrocytopathy. Methods: We present a child with pharmacoresistant epilepsy who underwent epileptic focus resections at age 3 and 5 years and was found to have mosaic chr1q gain and hyaline protoplasmic astrocytopathy with partial reduction in seizure burden after the second surgery. We performed single-nucleus RNA-sequencing (snRNA-seq) of brain tissue from the second resection. Results: Remarkably, snRNA-seq showed increased expression of chr1q genes only in select populations of neurons and astrocytes. Differentially expressed genes correlating with inferred chr1q gain included AKT3 and genes associated with cell adhesion or migration. A subcluster of astrocytes demonstrated enrichment for synapse-associated transcripts, possibly linked to the astrocytic inclusions observed in hyaline protoplasmic astrocytopathy. Discussion: snRNA-seq may be used to infer the cell type-specificity of mosaic chromosomal copy number variations and identify associated gene expression alterations, which in the case of chr1q gain may involve aberrations in cell migration. Future studies using spatial profiling could yield further insights on the molecular signatures of hyaline protoplasmic astrocytopathy.
Project description:Somatic mosaicism is a known cause of neurological disorders, including developmental brain malformations and epilepsy. Brain mosaic copy number gain of chromosome 1q is associated with cortical malformations, early-onset epilepsy, and developmental delay. Pathogenic brain mosaicism is traditionally attributed to post-zygotic genetic alterations arising in a neural progenitor cell during fetal development. However, in our cohort, in at least five of six patients with brain mosaic copy number gain of chromosome 1q, the alteration occurred pre-conception. We observed a third non-constitutive, but parentally-derived, haplotype for chromosome 1q in patient brain tissue, demonstrating that the copy number alteration occurred in a gamete pre-conception. The altered cells had no representation in parental buccal or proband blood or buccal samples, but were prominently observed in proband brain tissue, suggesting the copy number gain was lost in most cell lineages during embryonic development. Single-nuclei genotyping coupled with gene expression profiling revealed a strong enrichment of the chromosome 1q gain in astrocytes, which correlated with the unusual finding of hyaline astrocytic inclusions in all six cases.
Project description:Malformations of cortical development (MCD) are present in up to 40% of children with pharmacoresistant epilepsy. Although epilepsy surgery can be successful in a subset of children, not all forms of MCD are operable. Understanding the genetic and neurobiological mechanisms underlying MCD and MCD-related epilepsy are necessary for the development of novel anti-epilepsy drugs. The tish (telencephalic internal structural heterotopia) rat is a unique model of MCD and spontaneous seizures, but the underlying genetic mutation has been, heretofore, unknown. DNA and RNA-sequencing revealed that a deletion encompassing a previously unannotated exon markedly diminished EML1 transcript and protein abundance in the tish brain. Developmental electrographic characterization of the tish rat demonstrated spontaneous spike-wave discharge (SWD) bursts beginning as early as postnatal day (P) 17. A dihybrid cross demonstrated that the mutantEml1 allele segregates with the observed dysplastic cortex and SWD bursts in monogenic autosomal recessive frequencies. Our data link the development of the bilateral, heterotopic dysplastic cortex of the tish rat to a mutation in Eml1 and provide a novel rat model of MCD.
Project description:Cell type-specificity of mosaic chromosome 1q gain resolved by single-nucleus RNA-seq in a case of pharmacoresistant epilepsy associated with hyaline protoplasmic astrocytopathy
Project description: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.
Project description:Genome wide DNA methylation profiling of human normal and epileptic brain tissue. The Illumina Infinium 850K Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 850.000 CpGs in formalin-fixed paraffin-embedded surgical brain samples. Samples included 316 cases diagnosed with malformations of cortical development (MCD), non-MCD epilepsy or no-epilepsy autopsy controls.
Project description:Protoplasmic astrocytes in layers II to VI of the mammalian neocortex have historically been thought to comprise a homogeneous population. Given that layer-specific neuronal subtypes play essential roles in cortical circuitry, astrocytes might also be expected to support and modify this circuitry in a layer-specific manner. In order to investigate whether protoplasmic astrocytes exhibit layer-specific heterogeneity, we compared the gene expression profiles of astrocytes between upper layers (layers II to IV) and deep layers (layers V and VI). Although most genes known to be preferentially expressed in astrocytes (astrocyte-enriched genes) were equally expressed between upper-layer astrocytes and deep-layer astrocytes, some such genes (astrocyte-enriched genes or genes with known function in astrocytes) were significantly enriched in upper-layer astrocytes or deep-layer astrocytes.
Project description:Astrocytes are broadly categorized as protoplasmic or fibrous, with the former localized to synaptically dense gray matter regions and the latter associated with axons in the white matter. Much of what we know about astrocyte form and function is derived from the study of protoplasmic astrocytes, whereas fibrous astrocytes remain relatively unexplored. We investigated the transcriptome of uninjured fibrous astrocytes in the mouse from three regions: unmyelinated optic nerve head (ONH), myelinated optic nerve proper (ONP), and corpus callosum (CC).
Project description:Analysis of biopsy hippocampal tissue of patients with pharmacoresistant temporal lobe epilepsy (TLE) undergoing neurosurgical removal of the epileptogenic focus for seizure control. Chronic TLE goes along with focal hyperexcitability. Results provide insight into molecular mechanisms that may play a role in seizure propensity
Project description:Focal cortical dysplasia (FCD) is a heterogeneous group of cortical developmental malformations that constitute a common cause of medically intractable epilepsy. Multiomic integration was conducted via single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assays for transposase-accessible chromatin sequencing (snATAC-seq) to analyse cell type-specific alterations in chromatin accessibility and correlate them with gene expression changes in the epileptogenic cortex of FCD type IIIa (FCD IIIa).
Project description:Focal cortical dysplasia (FCD) is a heterogeneous group of cortical developmental malformations that constitute a common cause of medically intractable epilepsy. Multiomic integration was conducted via single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assays for transposase-accessible chromatin sequencing (snATAC-seq) to analyse cell type-specific alterations in chromatin accessibility and correlate them with gene expression changes in the epileptogenic cortex of FCD type IIIa (FCD IIIa).