Chronic ultraviolet irradiation induces memory deficits via dysregulation of the dopamine pathway.

The effects of ultraviolet (UV) radiation on brain function have previously been investigated; however, the specific neurotransmitter-mediated mechanisms responsible for UV radiation-induced neurobehavioral changes remain elusive. In this study, we aimed to explore the mechanisms underlying UV radiation-induced neurobehavioral changes. In a mouse model, we observed that UV irradiation of the skin induces deficits in hippocampal memory, synaptic plasticity, and adult neurogenesis, as well as increased dopamine levels in the skin, adrenal glands, and brain. Chronic UV exposure altered the expression of genes involved in dopaminergic neuron differentiation. Furthermore, chronic peripheral dopamine treatments resulted in memory deficits. Systemic administration of a dopamine D1/D5 receptor antagonist reversed changes in memory, synaptic plasticity, adult neurogenesis, and gene expression in UV-irradiated mice. Our findings provide converging evidence that chronic UV exposure alters dopamine levels in the central nervous system and peripheral organs, including the skin, which may underlie the observed neurobehavioral shifts, such as hippocampal memory deficits and impaired neurogenesis. This study underscores the importance of protection from UV exposure and introduces the potential of pharmacological approaches targeting dopamine receptors to counteract the adverse neurological impacts of UV exposure.

CWAS-Plus: Estimating category-wide association of rare noncoding variation from whole-genome sequencing data with cell-type-specific functional data.

Variants in cis-regulatory elements link the noncoding genome to human brain pathology; however, detailed analytic tools for understanding the association between cell-level brain pathology and noncoding variants are lacking. CWAS-Plus, adapted from a Python package for category-wide association testing (CWAS) employs both whole-genome sequencing and user-provided functional data to enhance noncoding variant analysis, with a faster and more efficient execution of the CWAS workflow. Here, we used single-nuclei assay for transposase-accessible chromatin with sequencing to facilitate CWAS-guided noncoding variant analysis at cell-type specific enhancers and promoters. Examining autism spectrum disorder whole-genome sequencing data (n = 7,280), CWAS-Plus identified noncoding de novo variant associations in transcription factor binding sites within conserved loci. Independently, in Alzheimer's disease whole-genome sequencing data (n = 1,087), CWAS-Plus detected rare noncoding variant associations in microglia-specific regulatory elements. These findings highlight CWAS-Plus's utility in genomic disorders and scalability for processing large-scale whole-genome sequencing data and in multiple-testing corrections. CWAS-Plus and its user manual are available at https://github.com/joonan-lab/cwas/ and https://cwas-plus.readthedocs.io/en/latest/, respectively.

Short tandem repeat expansions in cortical layer-specific genes implicate in phenotypic severity and adaptability of autism spectrum disorder.

AIM: Short tandem repeats (STRs) are repetitive DNA sequences and highly mutable in various human disorders. While the involvement of STRs in various genetic disorders has been extensively studied, their role in autism spectrum disorder (ASD) remains largely unexplored. In this study, we aimed to investigate genetic association of STR expansions with ASD using whole genome sequencing (WGS) and identify risk loci associated with ASD phenotypes. METHODS: We analyzed WGS data of 634 ASD families and performed genome-wide evaluation for 12,929 STR loci. We found rare STR expansions that exceeded normal repeat lengths in autism cases compared to unaffected controls. By integrating single cell RNA and ATAC sequencing datasets of human postmortem brains, we prioritized STR loci in genes specifically expressed in cortical development stages. A deep learning method was used to predict functionality of ASD-associated STR loci. RESULTS: In ASD cases, rare STR expansions predominantly occurred in early cortical layer-specific genes involved in neurodevelopment, highlighting the cellular specificity of STR-associated genes in ASD risk. Leveraging deep learning prediction models, we demonstrated that these STR expansions disrupted the regulatory activity of enhancers and promoters, suggesting a potential mechanism through which they contribute to ASD pathogenesis. We found that individuals with ASD-associated STR expansions exhibited more severe ASD phenotypes and diminished adaptability compared to non-carriers. CONCLUSION: Short tandem repeat expansions in cortical layer-specific genes are associated with ASD and could potentially be a risk genetic factor for ASD. Our study is the first to show evidence of STR expansion associated with ASD in an under-investigated population.

Whole-genome doubling is a double-edged sword: the heterogeneous role of whole-genome doubling in various cancer types.

Whole-genome doubling (WGD), characterized by the duplication of an entire set of chromosomes, is commonly observed in various tumors, occurring in approximately 30-40% of patients with different cancer types. The effect of WGD on tumorigenesis varies depending on the context, either promoting or suppressing tumor progression. Recent advances in genomic technologies and large-scale clinical investigations have led to the identification of the complex patterns of genomic alterations underlying WGD and their functional consequences on tumorigenesis progression and prognosis. Our comprehensive review aims to summarize the causes and effects of WGD on tumorigenesis, highlighting its dualistic influence on cancer cells. We then introduce recent findings on WGD-associated molecular signatures and genetic aberrations and a novel subtype related to WGD. Finally, we discuss the clinical implications of WGD in cancer subtype classification and future therapeutic interventions. Overall, a comprehensive understanding of WGD in cancer biology is crucial to unraveling its complex role in tumorigenesis and identifying novel therapeutic strategies. [BMB Reports 2024; 57(3): 125-134].

A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of non-coding elements in vivo.

The function of regulatory elements is highly dependent on the cellular context, and thus for understanding the function of elements associated with psychiatric diseases these would ideally be studied in neurons in a living brain. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3'UTR MPRA library with genomic elements containing variants from autism patients, we developed a method to achieve reproducible measurements of element effects in vivo in a cell type-specific manner, using excitatory cortical neurons and striatal medium spiny neurons as test cases. This targeted technique should enable robust, functional annotation of genetic elements in the cellular contexts most relevant to psychiatric disease.

Systematic evaluation of genome sequencing for the diagnostic assessment of autism spectrum disorder and fetal structural anomalies.

Short-read genome sequencing (GS) holds the promise of becoming the primary diagnostic approach for the assessment of autism spectrum disorder (ASD) and fetal structural anomalies (FSAs). However, few studies have comprehensively evaluated its performance against current standard-of-care diagnostic tests: karyotype, chromosomal microarray (CMA), and exome sequencing (ES). To assess the clinical utility of GS, we compared its diagnostic yield against these three tests in 1,612 quartet families including an individual with ASD and in 295 prenatal families. Our GS analytic framework identified a diagnostic variant in 7.8% of ASD probands, almost 2-fold more than CMA (4.3%) and 3-fold more than ES (2.7%). However, when we systematically captured copy-number variants (CNVs) from the exome data, the diagnostic yield of ES (7.4%) was brought much closer to, but did not surpass, GS. Similarly, we estimated that GS could achieve an overall diagnostic yield of 46.1% in unselected FSAs, representing a 17.2% increased yield over karyotype, 14.1% over CMA, and 4.1% over ES with CNV calling or 36.1% increase without CNV discovery. Overall, GS provided an added diagnostic yield of 0.4% and 0.8% beyond the combination of all three standard-of-care tests in ASD and FSAs, respectively. This corresponded to nine GS unique diagnostic variants, including sequence variants in exons not captured by ES, structural variants (SVs) inaccessible to existing standard-of-care tests, and SVs where the resolution of GS changed variant classification. Overall, this large-scale evaluation demonstrated that GS significantly outperforms each individual standard-of-care test while also outperforming the combination of all three tests, thus warranting consideration as the first-tier diagnostic approach for the assessment of ASD and FSAs.

Dysregulation of the Wnt/ß-catenin signaling pathway via Rnf146 upregulation in a VPA-induced mouse model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder associated with impaired social behavior and communication, repetitive behaviors, and restricted interests. In addition to genetic factors, environmental factors such as prenatal drug exposure contribute to the development of ASD. However, how those prenatal factors induce behavioral deficits in the adult stage is not clear. To elucidate ASD pathogenesis at the molecular level, we performed a high-resolution mass spectrometry-based quantitative proteomic analysis on the prefrontal cortex (PFC) of mice exposed to valproic acid (VPA) in utero, a widely used animal model of ASD. Differentially expressed proteins (DEPs) in VPA-exposed mice showed significant overlap with ASD risk genes, including differentially expressed genes from the postmortem cortex of ASD patients. Functional annotations of the DEPs revealed significant enrichment in the Wnt/ß-catenin signaling pathway, which is dysregulated by the upregulation of Rnf146 in VPA-exposed mice. Consistently, overexpressing Rnf146 in the PFC impaired social behaviors and altered the Wnt signaling pathway in adult mice. Furthermore, Rnf146-overexpressing PFC neurons showed increased excitatory synaptic transmission, which may underlie impaired social behavior. These results demonstrate that Rnf146 is critical for social behavior and that dysregulation of Rnf146 underlies social deficits in VPA-exposed mice.

Angiopoietin-2-Dependent Spatial Vascular Destabilization Promotes T-cell Exclusion and Limits Immunotherapy in Melanoma.

T-cell position in the tumor microenvironment determines the probability of target encounter and tumor killing. CD8+ T-cell exclusion from the tumor parenchyma is associated with poor response to immunotherapy, and yet the biology that underpins this distinct pattern remains unclear. Here we show that the vascular destabilizing factor angiopoietin-2 (ANGPT2) causes compromised vascular integrity in the tumor periphery, leading to impaired T-cell infiltration to the tumor core. The spatial regulation of ANGPT2 in whole tumor cross-sections was analyzed in conjunction with T-cell distribution, vascular integrity, and response to immunotherapy in syngeneic murine melanoma models. T-cell exclusion was associated with ANGPT2 upregulation and elevated vascular leakage at the periphery of human and murine melanomas. Both pharmacologic and genetic blockade of ANGPT2 promoted CD8+ T-cell infiltration into the tumor core, exerting antitumor effects. Importantly, the reversal of T-cell exclusion following ANGPT2 blockade not only enhanced response to anti-PD-1 immune checkpoint blockade therapy in immunogenic, therapy-responsive mouse melanomas, but it also rendered nonresponsive tumors susceptible to immunotherapy. Therapeutic response after ANGPT2 blockade, driven by improved CD8+ T-cell infiltration to the tumor core, coincided with spatial TIE2 signaling activation and increased vascular integrity at the tumor periphery where endothelial expression of adhesion molecules was reduced. These data highlight ANGPT2/TIE2 signaling as a key mediator of T-cell exclusion and a promising target to potentiate immune checkpoint blockade efficacy in melanoma. SIGNIFICANCE: ANGPT2 limits the efficacy of immunotherapy by inducing vascular destabilization at the tumor periphery to promote T-cell exclusion.

Characterization of De Novo Promoter Variants in Autism Spectrum Disorder with Massively Parallel Reporter Assays.

Autism spectrum disorder (ASD) is a common, complex, and highly heritable condition with contributions from both common and rare genetic variations. While disruptive, rare variants in protein-coding regions clearly contribute to symptoms, the role of rare non-coding remains unclear. Variants in these regions, including promoters, can alter downstream RNA and protein quantity; however, the functional impacts of specific variants observed in ASD cohorts remain largely uncharacterized. Here, we analyzed 3600 de novo mutations in promoter regions previously identified by whole-genome sequencing of autistic probands and neurotypical siblings to test the hypothesis that mutations in cases have a greater functional impact than those in controls. We leveraged massively parallel reporter assays (MPRAs) to detect transcriptional consequences of these variants in neural progenitor cells and identified 165 functionally high confidence de novo variants (HcDNVs). While these HcDNVs are enriched for markers of active transcription, disruption to transcription factor binding sites, and open chromatin, we did not identify differences in functional impact based on ASD diagnostic status.

Cntnap2-dependent molecular networks in autism spectrum disorder revealed through an integrative multi-omics analysis.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder in which patients present with core symptoms of social communication impairment, restricted interest, and repetitive behaviors. Although various studies have been performed to identify ASD-related mechanisms, ASD pathology is still poorly understood. CNTNAP2 genetic variants have been found that represent ASD genetic risk factors, and disruption of Cntnap2 expression has been associated with ASD phenotypes in mice. In this study, we performed an integrative multi-omics analysis by combining quantitative proteometabolomic data obtained with Cntnap2 knockout (KO) mice with multi-omics data obtained from ASD patients and forebrain organoids to elucidate Cntnap2-dependent molecular networks in ASD. To this end, a mass spectrometry-based proteometabolomic analysis of the medial prefrontal cortex in Cntnap2 KO mice led to the identification of Cntnap2-associated molecular features, and these features were assessed in combination with multi-omics data obtained on the prefrontal cortex in ASD patients to identify bona fide ASD cellular processes. Furthermore, a reanalysis of single-cell RNA sequencing data obtained from forebrain organoids derived from patients with CNTNAP2-associated ASD revealed that the aforementioned identified ASD processes were mainly linked to excitatory neurons. On the basis of these data, we constructed Cntnap2-associated ASD network models showing mitochondrial dysfunction, axonal impairment, and synaptic activity. Our results may shed light on the Cntnap2-dependent molecular networks in ASD.

Chronic skin ultraviolet irradiation induces transcriptomic changes associated with microglial dysfunction in the hippocampus.

Recent evidence indicates that ultraviolet (UV) exposure of the skin can affect brain functions such as learning and memory, addictive behavior, and hippocampal neurogenesis. These changes are closely associated with hippocampal function, which plays a pivotal role in learning and memory formation. However, the molecular mechanisms underlying these UV-induced skin-brain interactions remain unclear. To elucidate the molecular signature associated with UV-induced neurobehavioral changes, we analyzed the hippocampal transcriptome in a well-established mouse skin aging model, which showed thickened skin and impaired hippocampal memory. Transcriptome analysis revealed that significantly downregulated genes in UV-irradiated mice are enriched in neuroimmune-related signaling pathways. Furthermore, cell-type analysis showed that DEGs are also enriched in microglia. Consistently, immunofluorescence imaging showed an increased number of Iba1-positive microglia in the hippocampi of UV-irradiated mice. Collectively, our findings highlight that chronic UV irradiation of the skin causes significant changes in the neuroimmune system in the hippocampus, accompanied by microglial dysfunction and cognitive impairment.

Brief Report: Risk Variants Could Inform Early Neurodevelopmental Outcome in Children with Developmental Disabilities.

The aim of this study was to examine genetic variations underlying the early neurodevelopmental outcome of developmental disabilities (DDs). The study cohort consisted of 191 children with DDs. Diagnosis was assessed at baseline and at the index age (72-84 months). Exome sequencing was conducted and putative risk variants were identified. According to the diagnostic improvement, children were categorized into the improvement group (n = 19) and the non-improvement group (n = 172). Compared to the non-improvement group, the improvement group had lower number of risk variants in known DD genes and haploinsufficient genes, and lower number of overall putative risk variants. Our results may serve as a preliminary basis for developing a model that informs clinical outcome by sequencing analysis.

Augmentation of the RNA m6A reader signature is associated with poor survival by enhancing cell proliferation and EMT across cancer types.

N6-Methyladenosine (m6A) RNA modification plays a critical role in the posttranscriptional regulation of gene expression. Alterations in cellular m6A levels and m6A-related genes have been reported in many cancers, but whether they play oncogenic or tumor-suppressive roles is inconsistent across cancer types. We investigated common features of alterations in m6A modification and m6A-related genes during carcinogenesis by analyzing transcriptome data of 11 solid tumors from The Cancer Genome Atlas database and our in-house gastric cancer cohort. We calculated m6A writer (W), eraser (E), and reader (R) signatures based on corresponding gene expression. Alterations in the W and E signatures varied according to the cancer type, with a strong positive correlation between the W and E signatures in all types. When the patients were divided according to m6A levels estimated by the ratio of the W and E signatures, the prognostic effect of m6A was inconsistent according to the cancer type. The R and especially the R2 signatures (based on the expression of IGF2BPs) were upregulated in all cancers. Patients with a high R2 signature exhibited poor prognosis across types, which was attributed to enrichment of cell cycle- and epithelial-mesenchymal transition-related pathways. Our study demonstrates common features of m6A alterations across cancer types and suggests that targeting m6A R proteins is a promising strategy for cancer treatment.

Non-coding de novo mutations in chromatin interactions are implicated in autism spectrum disorder.

Three-dimensional chromatin interactions regulate gene expressions. The significance of de novo mutations (DNMs) in chromatin interactions remains poorly understood for autism spectrum disorder (ASD). We generated 813 whole-genome sequences from 242 Korean simplex families to detect DNMs, and identified target genes which were putatively affected by non-coding DNMs in chromatin interactions. Non-coding DNMs in chromatin interactions were significantly involved in transcriptional dysregulations related to ASD risk. Correspondingly, target genes showed spatiotemporal expressions relevant to ASD in developing brains and enrichment in biological pathways implicated in ASD, such as histone modification. Regarding clinical features of ASD, non-coding DNMs in chromatin interactions particularly contributed to low intelligence quotient levels in ASD probands. We further validated our findings using two replication cohorts, Simons Simplex Collection (SSC) and MSSNG, and showed the consistent enrichment of non-coding DNM-disrupted chromatin interactions in ASD probands. Generating human induced pluripotent stem cells in two ASD families, we were able to demonstrate that non-coding DNMs in chromatin interactions alter the expression of target genes at the stage of early neural development. Taken together, our findings indicate that non-coding DNMs in ASD probands lead to early neurodevelopmental disruption implicated in ASD risk via chromatin interactions.

Infantile esotropia in a family with TUBB3 mutation associated congenital fibrosis of extraocular muscles.

BACKGROUND: The TUBB3 gene has been reported to be associated with type 3 congenital fibrosis of the extraocular muscles (CFEOM). The clinical features of CFEOM3 that are linked to TUBB3 mutations are diverse, ranging from mild ptosis and limitation of extraocular movement to severe ocular motility problems and central nervous system abnormalities. MATERIALS AND METHODS: This was a single retrospective case report. RESULT: This case report describes a patient with infantile esotropia, who had a heterozygous variant in TUBB3 c.904 G > A (p.A302T) known to cause CFEOM3 and her family members, who presented with manifestations associated with CFEOM3. CONCLUSION: Given the diverse clinical features of CFEOM3, the possibility of the occurrence of CFEOM3 should be considered when there is a congenital abnormality of extraocular muscle movement and a positive family history.

High-throughput characterization of the role of non-B DNA motifs on promoter function.

lternative DNA conformations, termed non-B DNA structures, can affect transcription, but the underlying mechanisms and their functional impact have not been systematically characterized. Here, we used computational genomic analyses coupled with massively parallel reporter assays (MPRAs) to show that certain non-B DNA structures have a substantial effect on gene expression. Genomic analyses found that non-B DNA structures at promoters harbor an excess of germline variants. Analysis of multiple MPRAs, including a promoter library specifically designed to perturb non-B DNA structures, functionally validated that Z-DNA can significantly affect promoter activity. We also observed that biophysical properties of non-B DNA motifs, such as the length of Z-DNA motifs and the orientation of G-quadruplex structures relative to transcriptional direction, have a significant effect on promoter activity. Combined, their higher mutation rate and functional effect on transcription implicate a subset of non-B DNA motifs as major drivers of human gene-expression-associated phenotypes.

Developmental dynamics of voltage-gated sodium channel isoform expression in the human and mouse brain.

BACKGROUND: Genetic variants in the voltage-gated sodium channels SCN1A, SCN2A, SCN3A, and SCN8A are leading causes of epilepsy, developmental delay, and autism spectrum disorder. The mRNA splicing patterns of all four genes vary across development in the rodent brain, including mutually exclusive copies of the fifth protein-coding exon detected in the neonate (5N) and adult (5A). A second pair of mutually exclusive exons is reported in SCN8A only (18N and 18A). We aimed to quantify the expression of individual exons in the developing human brain. METHODS: RNA-seq data from 783 human brain samples across development were analyzed to estimate exon-level expression. Developmental changes in exon utilization were validated by assessing intron splicing. Exon expression was also estimated in RNA-seq data from 58 developing mouse neocortical samples. RESULTS: In the mature human neocortex, exon 5A is consistently expressed at least 4-fold higher than exon 5N in all four genes. For SCN2A, SCN3A, and SCN8A, a brain-wide synchronized 5N to 5A transition occurs between 24 post-conceptual weeks (2nd trimester) and 6 years of age. In mice, the equivalent 5N to 5A transition begins at or before embryonic day 15.5. In SCN8A, over 90% of transcripts in the mature human cortex include exon 18A. Early in fetal development, most transcripts include 18N or skip both 18N and 18A, with a transition to 18A inclusion occurring from 13 post-conceptual weeks to 6 months of age. No other protein-coding exons showed comparably dynamic developmental trajectories. CONCLUSIONS: Exon usage in SCN1A, SCN2A, SCN3A, and SCN8A changes dramatically during human brain development. These splice isoforms, which alter the biophysical properties of the encoded channels, may account for some of the observed phenotypic differences across development and between specific variants. Manipulation of the proportion of splicing isoforms at appropriate stages of development may act as a therapeutic strategy for specific mutations or even epilepsy in general.

Integrative Multi-Omics Approaches in Cancer Research: From Biological Networks to Clinical Subtypes.

Multi-omics approaches are novel frameworks that integrate multiple omics datasets generated from the same patients to better understand the molecular and clinical features of cancers. A wide range of emerging omics and multi-view clustering algorithms now provide unprecedented opportunities to further classify cancers into subtypes, improve the survival prediction and therapeutic outcome of these subtypes, and understand key pathophysiological processes through different molecular layers. In this review, we overview the concept and rationale of multi-omics approaches in cancer research. We also introduce recent advances in the development of multi-omics algorithms and integration methods for multiple-layered datasets from cancer patients. Finally, we summarize the latest findings from large-scale multi-omics studies of various cancers and their implications for patient subtyping and drug development.

Kdm3b haploinsufficiency impairs the consolidation of cerebellum-dependent motor memory in mice.

Histone modifications are a key mechanism underlying the epigenetic regulation of gene expression, which is critically involved in the consolidation of multiple forms of memory. However, the roles of histone modifications in cerebellum-dependent motor learning and memory are not well understood. To test whether changes in histone methylation are involved in cerebellar learning, we used heterozygous Kdm3b knockout (Kdm3b+/-) mice, which show reduced lysine 9 on histone 3 (H3K9) demethylase activity. H3K9 di-methylation is significantly increased selectively in the granule cell layer of the cerebellum of Kdm3b+/- mice. In the cerebellum-dependent optokinetic response (OKR) learning, Kdm3b+/- mice show deficits in memory consolidation, whereas they are normal in basal oculomotor performance and OKR acquisition. In addition, RNA-seq analyses revealed that the expression levels of several plasticity-related genes were altered in the mutant cerebellum. Our study suggests that active regulation of histone methylation is critical for the consolidation of cerebellar motor memory.

Genetic architecture of autism spectrum disorder: Lessons from large-scale genomic studies.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a strong genetic component. Recently developed genomic technologies, including microarray and next-generation sequencing (NGS), have enabled researchers to genetic analyses aimed at identifying genetic variations associated with ASD and to elucidate the genetic architecture of the disorder. Large-scale microarray, exome sequencing analyses, and robust statistical methods have resulted in successful gene discovery and identification of high-confidence ASD genes from among de novo and inherited variants. Efforts have been made to understand the genetic architecture of ASD using whole-genome sequencing and genome-wide association studies aimed at identifying noncoding mutations and common variants associated with ASD. In addition, the development of systems biology approaches has resulted in the integration of genetic findings with functional genomic datasets, thereby providing a unique insight into the functional convergence of ASD risk genes and their neurobiology. In this review, we summarize the latest findings of ASD genetic studies involving large cohorts and discuss their implications in ASD neurobiology and in clinical practice.