Search

PIK3CA-related overgrowth spectrum (PROS) disorders are caused by somatic mosaic variants that result in constitutive activation of the phosphatidylinositol-3-kinase/AKT/mTOR pathway. Promising responses to molecularly targeted therapy have been reported, although identification of an appropriate agent can be hampered by the mosaic nature and corresponding low variant allele frequency of the causal variant. Moreover, our understanding of the molecular consequences of these variants-for example how they affect gene expression profiles-remains limited. Here we describe in vitro expansion of a human capillary malformation followed by molecular characterization using exome sequencing, single cell gene expression, and targeted long-read single cell RNA-sequencing in a patient with clinical features consistent with Megalencephaly-Capillary Malformation Syndrome (MCAP, a PROS condition). These approaches identified a targetable PIK3CA variant with expression restricted to PAX3+ fibroblast and undifferentiated keratinocyte populations. This study highlights the innovative combination of next-generation single cell sequencing methods to better understand unique transcriptomic profiles and cell types associated with MCAP, revealing molecular intricacies of this genetic syndrome., (© 2024. The Author(s).) more...

Somatic mosaicism is a known cause of neurological disorders, including developmental brain malformations and epilepsy. Brain mosaicism is traditionally attributed to post-zygotic genetic alterations arising in fetal development. Here we describe post-zygotic rescue of meiotic errors as an alternate origin of brain mosaicism in patients with focal epilepsy who have mosaic chromosome 1q copy number gains. Genomic analysis showed evidence of an extra parentally derived chromosome 1q allele in the resected brain tissue from five of six patients. This copy number gain is observed only in patient brain tissue, but not in blood or buccal cells, and is strongly enriched in astrocytes. Astrocytes carrying chromosome 1q gains exhibit distinct gene expression signatures and hyaline inclusions, supporting a novel genetic association for astrocytic inclusions in epilepsy. Further, these data demonstrate an alternate mechanism of brain chromosomal mosaicism, with parentally derived copy number gain isolated to brain, reflecting rescue in other tissues during development., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.) more...

Oncolytic viruses, modified for tumor-restricted infection, are a promising cancer immunotherapeutic, yet much remains to be understood about factors driving their activity and outcome in the tumor microenvironment. Here, we report that oncolytic herpes simplex virus C134, previously found to exert T cell-dependent efficacy in mouse models of glioblastoma, exerts T cell-independent efficacy in mouse models of medulloblastoma, indicating this oncolytic virus uses different mechanisms in different tumors. We investigated C134's behavior in mouse medulloblastomas, using single cell RNA sequencing to map C134-induced gene expression changes across cell types, timepoints, and medulloblastoma subgroup models at whole-transcriptome resolution. Our work details substantial oncolytic virus-induced transcriptional remodeling of medulloblastoma-infiltrating immune cells, 10 subpopulations of monocytes and macrophages collectively demonstrating M1-like responses to C134, and suggests C134 be investigated as a potential new therapy for medulloblastoma., Competing Interests: The authors in full transparency disclose the following commercial interactions. E.R.M. reports PACT Pharma LLC, scientific advisory board membership, honorarium, and stock options; Scorpion Therapeutics, LLC, scientific advisory board membership, honorarium, and stock options; Qiagen N.V., supervisory board membership, stock; Singular Genomics, Inc., board of directors membership and stock. K.A.C. reports stock in Mustang Bio licensure., (© 2023 The Author(s).) more...

Somatic variants are a major cause of human disease, including neurological disorders like focal epilepsies, but can be challenging to study due to their mosaicism in bulk tissue biopsies. Coupling single-cell genotype and transcriptomic data has potential to provide insight into the role somatic variants play in disease etiology, such as by determining what cell types are affected or how the mutations affect gene expression. Here, we asked whether commonly used single-nucleus 3'- or 5'-RNA-sequencing assays can be used to derive single-nucleus genotype data for a priori known variants that are located near to either end of a transcript. To that end, we compared performance of commercially available single-nuclei 3'- and 5'- gene expression kits using resected brain samples from three pediatric patients with focal epilepsy. We quantified the ability to detect genetic variants in single-nucleus datasets depending on distance from the transcript end. Finally, we demonstrated the ability to identify affected cell types in a patient with a RHEB somatic variant causing an epilepsy-associated cortical malformation. Our results demonstrate that single-nuclei 3' or 5'-RNA-sequencing data can be used to identify known somatic variants in single-nuclei when they are expressed within proximity to a transcript end., (© 2023. The Author(s).) more...

Rasmussen encephalitis (RE) is a rare childhood neurological disease characterized by progressive unilateral loss of function, hemispheric atrophy and drug-resistant epilepsy. Affected brain tissue shows signs of infiltrating cytotoxic T-cells, microglial activation, and neuronal death, implicating an inflammatory disease process. Recent studies have identified molecular correlates of inflammation in RE, but cell-type-specific mechanisms remain unclear. We used single-nucleus RNA-sequencing (snRNA-seq) to assess gene expression across multiple cell types in brain tissue resected from two children with RE. We found transcriptionally distinct microglial populations enriched in RE compared to two age-matched individuals with unaffected brain tissue and two individuals with Type I focal cortical dysplasia (FCD). Specifically, microglia in RE tissues demonstrated increased expression of genes associated with cytokine signaling, interferon-mediated pathways, and T-cell activation. We extended these findings using spatial proteomic analysis of tissue from four surgical resections to examine expression profiles of microglia within their pathological context. Microglia that were spatially aggregated into nodules had increased expression of dynamic immune regulatory markers (PD-L1, CD14, CD11c), T-cell activation markers (CD40, CD80) and were physically located near distinct CD4+ and CD8+ lymphocyte populations. These findings help elucidate the complex immune microenvironment of RE., (© 2022. The Author(s).) more...