A novel missense variant in HIKESHI: Clinical phenotype, in vitro functional testing, and potential for gene therapy.

A 7-month-old boy presented to our clinic with developmental delay, Magnetic Resonance Imaging (MRI) features of delayed myelination and diffusion restriction, and a homozygous variant of uncertain significance (c.4T>G, p.Phe2Val) in HIKESHI, a gene associated with autosomal-recessive hypomyelinating leukodystrophy 13. We hypothesized that the variant is disease-causing and aimed to rescue the cellular phenotype with vector-mediated gene replacement. HIKESHI mediates heat-induced nuclear accumulation of heat-shock proteins, including HSP70, to protect cells from stress. We generated skin fibroblasts from the proband and proband's mother (heterozygous) to compare protein expression and subcellular localization of HSP70 under heat stress conditions, and the effect of vector-mediated overexpression of HIKESHI in the proband's cells under the same heat stress conditions. Western blot analysis revealed absent HIKESHI protein from proband fibroblasts, contrasted with ample expression in parental cells. Under heat stress conditions, while the mother's cells displayed appropriate nuclear localization of HSP70, the proband's cells displayed impaired nuclear translocalization. When patient fibroblasts were provided exogenous HIKESHI, the transfected proband's cells showed restored heat-induced nuclear translocalization of HSP70 under conditions of heat stress. These functional data establish that the patient's variant is a pathogenic loss-of-function mutation, thus confirming a diagnosis of hypomyelinating leukodystrophy 13 and that vector-mediated gene replacement may be an effective treatment approach for patients with this disorder.

Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance.

Neural progenitor cells within the cerebral cortex undergo a characteristic switch between symmetric self-renewing cell divisions early in development and asymmetric neurogenic divisions later. Yet, the mechanisms controlling this transition remain unclear. Previous work has shown that early but not late neural progenitor cells (NPCs) endogenously express the autism-linked transcription factor Foxp1, and both loss and gain of Foxp1 function can alter NPC activity and fate choices. Here, we show that premature loss of Foxp1 upregulates transcriptional programs regulating angiogenesis, glycolysis, and cellular responses to hypoxia. These changes coincide with a premature destabilization of HIF-1α, an elevation in HIF-1α target genes, including Vegfa in NPCs, and precocious vascular network development. In vitro experiments demonstrate that stabilization of HIF-1α in Foxp1-deficient NPCs rescues the premature differentiation phenotype and restores NPC maintenance. Our data indicate that the endogenous decline in Foxp1 expression activates the HIF-1α transcriptional program leading to changes in the tissue environment adjacent to NPCs, which, in turn, might alter their self-renewal and neurogenic capacities.

Single-cell long-read sequencing-based mapping reveals specialized splicing patterns in developing and adult mouse and human brain.

RNA isoforms influence cell identity and function. However, a comprehensive brain isoform map was lacking. We analyze single-cell RNA isoforms across brain regions, cell subtypes, developmental time points and species. For 72% of genes, full-length isoform expression varies along one or more axes. Splicing, transcription start and polyadenylation sites vary strongly between cell types, influence protein architecture and associate with disease-linked variation. Additionally, neurotransmitter transport and synapse turnover genes harbor cell-type variability across anatomical regions. Regulation of cell-type-specific splicing is pronounced in the postnatal day 21-to-postnatal day 28 adolescent transition. Developmental isoform regulation is stronger than regional regulation for the same cell type. Cell-type-specific isoform regulation in mice is mostly maintained in the human hippocampus, allowing extrapolation to the human brain. Conversely, the human brain harbors additional cell-type specificity, suggesting gain-of-function isoforms. Together, this detailed single-cell atlas of full-length isoform regulation across development, anatomical regions and species reveals an unappreciated degree of isoform variability across multiple axes.

Neural tube defects and epigenetics: role of histone post-translational histone modifications.

Neural tube defects (NTDs) are the most common congenital anomalies of the CNS. It is widely appreciated that both genetic and environmental factors contribute to their etiology. The inability to ascribe clear genetic patterns of inheritance to various NTD phenotypes suggests it is possible that epigenetic mechanisms are involved in the etiology of NTDs. In this context, the contribution of DNA methylation as an underlying contributing factor to the etiology of NTDs has been extensively reviewed. Here, an updated accounting of the evidence linking post-translational histone modifications to these birth defects, relying heavily upon studies in humans, and the possible molecular implications inferred from reports based on cellular and animal models, are presented.

Schizophrenia endothelial cells exhibit higher permeability and altered angiogenesis patterns in patient-derived organoids.

Schizophrenia (SCZ) is a complex neurodevelopmental disorder characterized by the manifestation of psychiatric symptoms in early adulthood. While many research avenues into the origins of SCZ during brain development have been explored, the contribution of endothelial/vascular dysfunction to the disease remains largely elusive. To model the neuropathology of SCZ during early critical periods of brain development, we utilized patient-derived induced pluripotent stem cells (iPSCs) to generate 3D cerebral organoids and define cell-specific signatures of disease. Single-cell RNA sequencing revealed that while SCZ organoids were similar in their macromolecular diversity to organoids generated from healthy controls (CTRL), SCZ organoids exhibited a higher percentage of endothelial cells when normalized to total cell numbers. Additionally, when compared to CTRL, differential gene expression analysis revealed a significant enrichment in genes that function in vessel formation, vascular regulation, and inflammatory response in SCZ endothelial cells. In line with these findings, data from 23 donors demonstrated that PECAM1+ microvascular vessel-like structures were increased in length and number in SCZ organoids in comparison to CTRL organoids. Furthermore, we report that patient-derived endothelial cells displayed higher paracellular permeability, implicating elevated vascular activity. Collectively, our data identified altered gene expression patterns, vessel-like structural changes, and enhanced permeability of endothelial cells in patient-derived models of SCZ. Hence, brain microvascular cells could play a role in the etiology of SCZ by modulating the permeability of the developing blood brain barrier (BBB).

A multi-stem cell basis for craniosynostosis and calvarial mineralization.

Craniosynostosis is a group of disorders of premature calvarial suture fusion. The identity of the calvarial stem cells (CSCs) that produce fusion-driving osteoblasts in craniosynostosis remains poorly understood. Here we show that both physiologic calvarial mineralization and pathologic calvarial fusion in craniosynostosis reflect the interaction of two separate stem cell lineages; a previously identified cathepsin K (CTSK) lineage CSC1 (CTSK+ CSC) and a separate discoidin domain-containing receptor 2 (DDR2) lineage stem cell (DDR2+ CSC) that we identified in this study. Deletion of Twist1, a gene associated with craniosynostosis in humans2,3, solely in CTSK+ CSCs is sufficient to drive craniosynostosis in mice, but the sites that are destined to fuse exhibit an unexpected depletion of CTSK+ CSCs and a corresponding expansion of DDR2+ CSCs, with DDR2+ CSC expansion being a direct maladaptive response to CTSK+ CSC depletion. DDR2+ CSCs display full stemness features, and our results establish the presence of two distinct stem cell lineages in the sutures, with both populations contributing to physiologic calvarial mineralization. DDR2+ CSCs mediate a distinct form of endochondral ossification without the typical haematopoietic marrow formation. Implantation of DDR2+ CSCs into suture sites is sufficient to induce fusion, and this phenotype was prevented by co-transplantation of CTSK+ CSCs. Finally, the human counterparts of DDR2+ CSCs and CTSK+ CSCs display conserved functional properties in xenograft assays. The interaction between these two stem cell populations provides a new biologic interface for the modulation of calvarial mineralization and suture patency.

Single-cell long-read mRNA isoform regulation is pervasive across mammalian brain regions, cell types, and development.

RNA isoforms influence cell identity and function. Until recently, technological limitations prevented a genome-wide appraisal of isoform influence on cell identity in various parts of the brain. Using enhanced long-read single-cell isoform sequencing, we comprehensively analyze RNA isoforms in multiple mouse brain regions, cell subtypes, and developmental timepoints from postnatal day 14 (P14) to adult (P56). For 75% of genes, full-length isoform expression varies along one or more axes of phenotypic origin, underscoring the pervasiveness of isoform regulation across multiple scales. As expected, splicing varies strongly between cell types. However, certain gene classes including neurotransmitter release and reuptake as well as synapse turnover, harbor significant variability in the same cell type across anatomical regions, suggesting differences in network activity may influence cell-type identity. Glial brain-region specificity in isoform expression includes strong poly(A)-site regulation, whereas neurons have stronger TSS regulation. Furthermore, developmental patterns of cell-type specific splicing are especially pronounced in the murine adolescent transition from P21 to P28. The same cell type traced across development shows more isoform variability than across adult anatomical regions, indicating a coordinated modulation of functional programs dictating neural development. As most cell-type specific exons in P56 mouse hippocampus behave similarly in newly generated data from human hippocampi, these principles may be extrapolated to human brain. However, human brains have evolved additional cell-type specificity in splicing, suggesting gain-of-function isoforms. Taken together, we present a detailed single-cell atlas of full-length brain isoform regulation across development and anatomical regions, providing a previously unappreciated degree of isoform variability across multiple scales of the brain.

Activation of RhoC by regulatory ubiquitination is mediated by LNX1 and suppressed by LIS1.

Regulation of Rho GTPases remains a topic of active investigation as they are essential participants in cell biology and the pathophysiology of many human diseases. Non-degrading ubiquitination (NDU) is a critical regulator of the Ras superfamily, but its relevance to Rho proteins remains unknown. We show that RhoC, but not RhoA, is a target of NDU by E3 ubiquitin ligase, LNX1. Furthermore, LNX1 ubiquitination of RhoC is negatively regulated by LIS1 (aka, PAFAH1B1). Despite multiple reports of functional interaction between LIS1 and activity of Rho proteins, a robust mechanism linking the two has been lacking. Here, LIS1 inhibition of LNX1 effects on RhoGDI-RhoC interaction provides a molecular mechanism underpinning the enhanced activity of Rho proteins observed upon reduction in LIS1 protein levels. Since LNX1 and RhoC are only found in vertebrates, the LIS1-LNX1-RhoC module represents an evolutionarily acquired function of the highly conserved LIS1. While these nearly identical proteins have several distinct RhoA and RhoC downstream effectors, our data provide a rare example of Rho-isoform specific, upstream regulation that opens new therapeutic opportunities.

CIC missense variants contribute to susceptibility for spina bifida.

Neural tube defects (NTDs) are congenital malformations resulting from abnormal embryonic development of the brain, spine, or spinal column. The genetic etiology of human NTDs remains poorly understood despite intensive investigation. CIC, homolog of the Capicua transcription repressor, has been reported to interact with ataxin-1 (ATXN1) and participate in the pathogenesis of spinocerebellar ataxia type 1. Our previous study demonstrated that CIC loss of function (LoF) variants contributed to the cerebral folate deficiency syndrome by downregulating folate receptor 1 (FOLR1) expression. Given the importance of folate transport in neural tube formation, we hypothesized that CIC variants could contribute to increased risk for NTDs by depressing embryonic folate concentrations. In this study, we examined CIC variants from whole-genome sequencing (WGS) data of 140 isolated spina bifida cases and identified eight missense variants of CIC gene. We tested the pathogenicity of the observed variants through multiple in vitro experiments. We determined that CIC variants decreased the FOLR1 protein level and planar cell polarity (PCP) pathway signaling in a human cell line (HeLa). In a murine cell line (NIH3T3), CIC loss of function variants downregulated PCP signaling. Taken together, this study provides evidence supporting CIC as a risk gene for human NTD.

Astrocytes derived from ASD individuals alter behavior and destabilize neuronal activity through aberrant Ca2+ signaling.

The cellular mechanisms of autism spectrum disorder (ASD) are poorly understood. Cumulative evidence suggests that abnormal synapse function underlies many features of this disease. Astrocytes regulate several key neuronal processes, including the formation of synapses and the modulation of synaptic plasticity. Astrocyte abnormalities have also been identified in the postmortem brain tissue of ASD individuals. However, it remains unclear whether astrocyte pathology plays a mechanistic role in ASD, as opposed to a compensatory response. To address this, we combined stem cell culturing with transplantation techniques to determine disease-specific properties inherent to ASD astrocytes. We demonstrate that ASD astrocytes induce repetitive behavior as well as impair memory and long-term potentiation when transplanted into the healthy mouse brain. These in vivo phenotypes were accompanied by reduced neuronal network activity and spine density caused by ASD astrocytes in hippocampal neurons in vitro. Transplanted ASD astrocytes also exhibit exaggerated Ca2+ fluctuations in chimeric brains. Genetic modulation of evoked Ca2+ responses in ASD astrocytes modulates behavior and neuronal activity deficits. Thus, this study determines that astrocytes derived from ASD iPSCs are sufficient to induce repetitive behavior as well as cognitive deficit, suggesting a previously unrecognized primary role for astrocytes in ASD.

Familial colloid cysts: not a chance occurrence.

PURPOSE: Colloid cysts are rare, benign brain tumors of the third ventricle with an estimated population prevalence of 1 in 5800. Sudden deterioration and death secondary to obstructive hydrocephalus are well-described presentations in patients with a colloid cyst. Although historically conceptualized as driven by sporadic genetic events, a growing body of literature supports the possibility of an inherited predisposition. METHODS: A prospective registry of patients with colloid cysts was maintained between 1996 and 2021. Data pertaining to a family history of colloid cyst was collected retrospectively; self-reporting was validated in each case by medical record or imaging review. Frequency of patients with a documented first-degree family member with a colloid cyst based on self-reporting was calculated. The rate of familial co-occurrence within our series was then compared to a systematic literature review and aggregation of familial case studies, as well as population-based prevalence rates of sporadic colloid cysts. RESULTS: Thirteen cases with affected first-degree relatives were identified in our series. Of the entire cohort, 19/26 were symptomatic from the lesion (73%), 12/26 (46.2%) underwent resection, and 2/26 (7.7%) had sudden death from presumed obstructive hydrocephalus. The majority of transmission patterns were between mother and child (9/13). Compared with the estimated prevalence of colloid cysts, our FCC rate of 13 cases in 383 (3.4%) estimates a greater-than-chance rate of co-occurrence. CONCLUSION: Systematic screening for FCCs may facilitate early recognition and treatment of indolent cysts, thereby preventing the rapid deterioration that can occur with an unrecognized third ventricular tumor. Furthermore, identifying a transmission pattern may yield more insight into the molecular and genetic underpinnings of colloid cysts.

Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue.

Single-nuclei RNA sequencing characterizes cell types at the gene level. However, compared to single-cell approaches, many single-nuclei cDNAs are purely intronic, lack barcodes and hinder the study of isoforms. Here we present single-nuclei isoform RNA sequencing (SnISOr-Seq). Using microfluidics, PCR-based artifact removal, target enrichment and long-read sequencing, SnISOr-Seq increased barcoded, exon-spanning long reads 7.5-fold compared to naive long-read single-nuclei sequencing. We applied SnISOr-Seq to adult human frontal cortex and found that exons associated with autism exhibit coordinated and highly cell-type-specific inclusion. We found two distinct combination patterns: those distinguishing neural cell types, enriched in TSS-exon, exon-polyadenylation-site and non-adjacent exon pairs, and those with multiple configurations within one cell type, enriched in adjacent exon pairs. Finally, we observed that human-specific exons are almost as tightly coordinated as conserved exons, implying that coordination can be rapidly established during evolution. SnISOr-Seq enables cell-type-specific long-read isoform analysis in human brain and in any frozen or hard-to-dissociate sample.

Adult-onset Niemann-Pick disease type C masquerading as spinocerebellar ataxia.

BACKGROUND: Adult-onset Nieman-Pick disease type C (NPC) is a rare progressive ataxia caused by lysosomal accumulation of unesterified cholesterol resulting in severe disability and death. The diagnosis of NPC can be challenging as clinical features overlap with other more common hereditary ataxias. This study pursued the molecular genetic basis of adult-onset cerebellar ataxia manifesting in two siblings. A prior diagnosis of spinocerebellar ataxia type 2 (SCA2) based on an ataxia gene panel was questioned when the younger sibling developed similar symptoms but had discordant genetic results. METHODS: Neurologic examination, whole exome sequence (WES), targeted sequence to establish genome phasing, and cytochemical and biochemical studies of fibroblast cultures were employed. RESULTS: The pedigree and neurological examinations suggested a recessive or possibly dominant cerebellar ataxia. WES showed the siblings were both compound heterozygous for two rare variants in the NPC1 gene-one pathogenic, stop gain at p.Arg934Ter (NM_000271.4), and a missense change, p.Pro471Leu (NM_000271.4), of uncertain significance. Filipin staining of fibroblast cultures showed lysosomal cholesterol accumulation and biochemical assay demonstrated impaired cholesterol esterification. CONCLUSIONS: The study established the correct molecular diagnosis of biallelic, adult-onset NPC in a patient initially diagnosed with SCA. Additionally, the p.Pro471Leu variant was identified as likely pathogenic. Inaccurate molecular diagnosis will deprive NPC patients of treatment options. Investigation using WES is justified when a detected expansion size is in the borderline range for pathogenicity.

Systems biology analysis of human genomes points to key pathways conferring spina bifida risk.

Spina bifida (SB) is a debilitating birth defect caused by multiple gene and environment interactions. Though SB shows non-Mendelian inheritance, genetic factors contribute to an estimated 70% of cases. Nevertheless, identifying human mutations conferring SB risk is challenging due to its relative rarity, genetic heterogeneity, incomplete penetrance, and environmental influences that hamper genome-wide association studies approaches to untargeted discovery. Thus, SB genetic studies may suffer from population substructure and/or selection bias introduced by typical candidate gene searches. We report a population based, ancestry-matched whole-genome sequence analysis of SB genetic predisposition using a systems biology strategy to interrogate 298 case-control subject genomes (149 pairs). Genes that were enriched in likely gene disrupting (LGD), rare protein-coding variants were subjected to machine learning analysis to identify genes in which LGD variants occur with a different frequency in cases versus controls and so discriminate between these groups. Those genes with high discriminatory potential for SB significantly enriched pathways pertaining to carbon metabolism, inflammation, innate immunity, cytoskeletal regulation, and essential transcriptional regulation consistent with their having impact on the pathogenesis of human SB. Additionally, an interrogation of conserved noncoding sequences identified robust variant enrichment in regulatory regions of several transcription factors critical to embryonic development. This genome-wide perspective offers an effective approach to the interrogation of coding and noncoding sequence variant contributions to rare complex genetic disorders.

Genome-wide investigation identifies a rare copy-number variant burden associated with human spina bifida.

PURPOSE: Next-generation sequencing has implicated some risk variants for human spina bifida (SB), but the genome-wide contribution of structural variation to this complex genetic disorder remains largely unknown. We examined copy-number variant (CNV) participation in the genetic architecture underlying SB risk. METHODS: A high-confidence ensemble approach to genome sequences (GS) was benchmarked and employed for systematic detection of common and rare CNVs in two separate ancestry-matched SB case-control cohorts. RESULTS: SB cases were enriched with exon disruptive rare CNVs, 44% of which were under 10 kb, in both ancestral populations (P = 6.75 × 10-7; P = 7.59 × 10-4). Genes containing these disruptive CNVs fall into molecular pathways, supporting a role for these genes in SB. Our results expand the catalog of variants and genes with potential contribution to genetic and gene-environment interactions that interfere with neurulation, useful for further functional characterization. CONCLUSION: This study underscores the need for genome-wide investigation and extends our previous threshold model of exonic, single-nucleotide variation toward human SB risk to include structural variation. Since GS data afford detection of CNVs with greater resolution than microarray methods, our results have important implications toward a more comprehensive understanding of the genetic risk and mechanisms underlying neural tube defect pathogenesis.

A spatially resolved brain region- and cell type-specific isoform atlas of the postnatal mouse brain.

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 ( www.isoformAtlas.com ). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity.

Clinical and genomic characteristics of LAMA2 related congenital muscular dystrophy in a patients' cohort from Qatar. A population specific founder variant.

Congenital LAMA2 related muscular dystrophy (LAMA2-RD), the most commonly recognized type of congenital muscular dystrophies, has been described in patients' cohorts from Europe and the UK but not from Middle-Eastern. This study aimed to reveal the prevalence, clinical and genomic characteristics of congenital LAMA2-RD in a patient's cohort of 17 families (21 patients) from the Gulf and Middle East. Affected subjects exhibited the classic phenotype of generalized hypotonia, developmental delay, and progressive muscular weakness. Despite the homogeneous background of most of our patients, clinical variability was evident; however, none of our patients was able to achieve independent ambulation. The associated features of nephrocalcinosis, infantile-onset osteopenia, and cardiac arrest were first described in this study. LAMA2 mutations constituted 48% of the genetic causes underlying congenital muscular dystrophies (CMDs) in our patients. We estimated a point prevalence of 0.8 in 100.000 for LAMA2-RD in Qatar, relatively higher compared to that described in Europe's studies. The founder mutation and high rate of consanguinity are potential contributors. This study identified five LAMA2 truncating variants, two novel and three recurrent, of which the c.6488delA-frameshift that was found in 12 unrelated Qatari families, highlighting a founder mutation in Qatari patients. The two novel variants involved an acceptor splice site and N-terminus deletion that removes the LAMA2 promoter, exon1, and part of intron1. The "residual" expression of LAMA2 transcript and protein associated with this large N-terminus deletion suggested an alternative promoter that, while seems to be activated, acts less efficiently.