Neuroblastomas (NB) are highly heterogeneous pediatric extracranial solid tumors in children with variable epigenetic, biological, and clinical characteristics. However, predictive models that can accurately classify patient risks and predict prognoses are currently limited. We analyzed a metabolism-related genes network perturbation using machine learning algorithms to construct a model to assess the risk and prognosis of patients with NB. Metabolism-related gene expression data for patients with NB were obtained from the Gene Expression Omnibus (GEO) (GSE49710, N = 498), ArrayExpress (E-MTAB-8248, N = 228), and TARGET (TARGET-NBL, N = 150) databases. An individual-specific gene interaction perturbation network was constructed using the Reactome Pathway Database. Unsupervised clusters and principal components analysis were analyzed using the R package “Consensus Cluster Plus”. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the R package “Cluster Profiler”. Gene set variation analysis (GSVA) and single-sample gene set enrichment analysis (ssGSEA) were performed to evaluate immune cell infiltration in patients with NB using the HALLMARK database. Ten machine learning methods (Lasso, Enet, plsRcox, CoxBoost, StepCox, gradient boosting machine [GBM], Ridge, random survival forests [RSF], survival-support vector machine [SVM], and super principal component [PC]) were used with 110 machine learning algorithms to screen for a metabolism-related signature to predict NB prognosis in GSE49710 and other cohorts (E-MTAB-8248 and GSE76427). Finally, Immunohistochemical staining (IHC) was performed to validate the UCK2 expression levels in human tissues. Gene network perturbation analysis of 948 metabolism-related genes revealed distinct discriminability, and patients with NB were classified into three differentiated subtypes. Kaplan-Meier survival analysis revealed that the prognoses were best for patients with subtype C3, followed by those with subtypes C1 and C2. Correlation analysis of clinical information indicated that subtypes C2 and C3 were associated with higher and lower percentages of high-degree malignancies, respectively, whereas C3 subtype showed a lower percentage of high-degree malignancies. The KRAS and myogenesis pathways were upregulated, and the levels of MYC targets were downregulated in patients with subtype C3; those with C2 subtype exhibited opposite trends. Patients with C3- and C2-subtypes exhibited immune-activated and immune-suppressed phenotypes, respectively. The combination of the StepCox [forward] and RSF algorithms provided the most accurate prognostic predictions for patients with NB. The importance score was highest for UCK2 among all subtypes. IHC staining further confirmed that UCK2 expression was substantially higher in the tissues of patients with NB than in those of controls. The machine learning-based prognostic prediction model that analyzes metabolism-related gene interaction perturbation networks supports the development of personalized management strategies for patients with NB.

The trabecular meshwork (TM) and ciliary body (CB) regulate aqueous humor dynamics and intraocular pressure (IOP), and TM/Schlemm's canal (SC) dysfunction underlies glaucoma. Here, we present a spatially resolved multi-omics atlas of human TM and CB, integrating snRNA-seq, scRNA-seq, and snATAC-seq from over one million cells and nuclei across 112 donors with Xenium spatial transcriptomics. We identified 9 major cell classes and 21 cell types, revealing heterogeneity, including undercharacterized fibroblast and epithelial subpopulations. Spatial mapping supported TM fibroblast zonation and CB epithelial organization. Regulatory analyses identified cell type-specific programs, including OTX/PAX networks in CB epithelium and SMAD3/TGF-β signaling in fibroblasts. Integration with glaucoma loci showed enrichment of non-coding variants in regulatory elements associated with POAG and PACG. Age- and ancestry-associated remodeling revealed divergent fibroblast aging with increased PIEZO1, suggesting impaired outflow and elevated IOP. Together, this high-resolution atlas links cellular, regulatory, and genetic variation to anterior segment function and glaucoma susceptibility.

Foveal hypoplasia causes visual impairment across congenital eye disorders, yet the genetic programmes governing foveal development remain poorly characterised and no tractable model exists for foveal disease. In the first genome-wide association study of foveal hypoplasia, we identified 42 sentinel variants mapping to 54 effector genes supported by ≥ 2 criteria from a variant-to-gene framework incorporating developmental multi-omics. Disruption of six effector genes using mutant lines and CRISPR knockouts in the zebrafish high acuity zone recapitulates structural, functional, and ultrastructural hallmarks of foveal hypoplasia, establishing the first vertebrate disease model. Integration with human foetal single-cell and spatial transcriptomics reveals two temporal waves of effector gene expression and identifies Müller glia as critical mediators of foveal patterning. Phenome-wide analyses reveal foveal variants are pleiotropic with refractive, lenticular, and metabolic traits, connecting foveal development to anterior segment and systemic disease biology. These findings should inform mechanistic studies of macular disease.

Single-cell sequencing has revolutionized the scale and resolution of molecular profiling of tissues and organs. Here we present an integrated dual-modal reference atlas of the most accessible portion of the mammalian central nervous system, the retina. We compiled around 3.9 million cells from 125 donors of diverse ancestral backgrounds, including 8 published studies and 2.7 million unpublished data points, to create a comprehensive human retina cell atlas (HRCA) with more than 130 cell types identified. We annotated each cluster, identified marker genes and characterized cis-regulatory elements and gene regulatory networks. Our analysis uncovered differences in transcriptome, chromatin and gene regulatory networks across cell types. We modeled changes in gene expression and chromatin accessibility across age, ancestry and tissue region. This integrated atlas enhanced the fine-mapping of genome-wide association study and expression quantitative trait loci variants. Accessible through interactive browsers, this multimodal multidonor and multilab HRCA can facilitate a better understanding of retinal function and pathology. Single-cell RNA sequencing and assay for transposase-accessible chromatin using sequencing profiling of human retinal samples from diverse ancestries create an epitranscriptomic atlas characterizing over 130 cell types. Integration with genome-wide association study and expression quantitative trait loci data provides further insights into gene regulation and disease etiology.

BackgroundRhesus macaques are increasingly used to model inherited sensory disorders, yet the phenotypic impact of naturally occurring variants in primate colonies often remains undefined.MethodsWe identified five rhesus macaques homozygous for a missense variant in WHRN (p.Val495Met in exon 7), which encodes whirlin and is implicated in human Usher syndrome type 2D, and compared them with five age and sex matched wild-type controls (WHRN homozygotes: mean age 9.4 ± 1.8 years; controls: mean age 8.6 ± 1.1 years) using standardized ocular and auditory phenotyping, including comprehensive ophthalmic examination, A-scan ocular biometry, intraocular pressure measurement, cycloplegic refraction, macular optical coherence tomography with retinal layer thickness quantification, full-field electroretinography, and brainstem auditory evoked response testing.ResultsWHRN homozygotes showed a consistent shift in ocular component dimensions, with significantly reduced lens thickness, while refractive error remained centered near emmetropia. By contrast, fundus examination and macular optical coherence tomography showed preserved retinal morphology, electroretinography waveforms were comparable between groups, and brainstem auditory evoked responses did not show evidence of overt hearing impairment under the recording conditions used.ConclusionThese findings define a subtle ocular biometry phenotype associated with WHRN p. Val495Met homozygosity in rhesus macaques, while retinal structure and function were preserved at the time of testing; more comprehensive auditory phenotyping across frequencies and thresholds will be needed to assess whether subtle, frequency specific hearing deficits consistent with an atypical Usher presentation are present.

The retinal pigment epithelium and choroid are critical for supporting the function and maintaining the homeostasis of the outer retina, and their dysfunction underlies a range of inherited and complex ocular diseases. To comprehensively characterize the cellular, transcriptomic, and epigenomic heterogeneity and dynamics within these tissues, we assembled an integrated multi-omics reference atlas comprising 719,813 single-cell/single-nucleus transcriptomes and 234,007 snATAC-seq profiles from 102 ancestrally diverse donors spanning 0 to 99 years of age, including cells from both the macula and periphery. This atlas resolves 48 distinct cell types or states and catalogs 448,567 open chromatin regions. Specifically, we resolved five distinct RPE subpopulations organized along a central-to-peripheral spatial axis, alongside two distinct stress/senescence states. We reconstructed the transcriptomic and epigenetic zonation of endothelial cells and expanded choroidal stromal heterogeneity by characterizing 11 fibroblast and two pericyte types. Age-associated compositional analysis revealed a significant fractional depletion of melanocytes, PI16+ fibroblasts, and venule endothelial cells with age, alongside a modest relative loss of central RPE and a corresponding increase in far-peripheral RPE. Cell-type-specific aging transcriptomics uncovered shared pathways related to inflammatory responses alongside distinct cell-type-specific signatures. Notably, significant age-associated epigenetic changes concentrated in the macula during the transition from early-to-middle adulthood and remained stable into old age, with transcription factors from the AP-1/bZIP family emerging as the dominant enriched motifs. Finally, integrating this atlas with AMD GWAS data provides novel variant-to-gene evidence implicating LIPG and COL4A3 in AMD pathogenesis. Together, this multi-omics atlas serves as both an invaluable community reference and a powerful discovery engine that translates genetic risk signals into localized target cells and candidate mechanisms, laying a foundation for understanding RPE/choroid biology in health and disease.

Autosomal dominant optic atrophy (ADOA) is an inherited optic neuropathy primarily caused by mutations in OPA1 . We identified and defined a spontaneous nonhuman primate (NHP) model of ADOA using rhesus macaques heterozygous for a missense mutation ( OPA1 A8S). With ocular examinations, ophthalmic imaging, electroretinography, histopathology, immunohistochemistry, and transmission electron microscopy (TEM), we documented retinal nerve fiber layer (RNFL) thinning, retinal ganglion cell (RGC) loss and dysfunction, OPA1 mislocalization, and reduced axonal mitochondrial density in affected macaques. Our investigation revealed substantial phenotypic variability among affected macaques, shedding light on the pathogenesis of ADOA. The retinas were evaluated using techniques such as spectral-domain optical coherence tomography and fundus photography facilitating observation of structural changes in the retina and optic nerve. Thinning of the RNFL and optic nerve head degeneration, hallmark features of ADOA, were observed in affected macaques. Decreased RGC function in the OPA1 heterozygotes was demonstrated with pattern electroretinography. Histopathological analysis and immunohistochemical staining of postmortem retinal tissue suggested RGC loss in the papillomacular bundle, with reduced OPA1 and mitochondria in the RGC axons, indicating dysfunctional mitochondrial dynamics and reduced function consistent with ADOA. Ultrastructural changes were evident with TEM including dysmorphic mitochondria, axonal loss, myelin disruption, and hypertrophic astrocytic processes. The observed similar pattern of RGC loss and dysfunction coupled with phenotypic heterogeneity in our NHP model reflects the clinical variability observed in human ADOA patients indicating that therapeutic interventions in this foveate model will likely translate to the human condition.

BACKGROUND:As the most common malignant renal tumor in children, the progression of Wilms' tumor is frequently driven by abnormal alternative splicing (AS), cell death imbalance, and an immunosuppressive microenvironment. However, the precise regulatory chain connecting these three critical elements remains largely unexplored. This study aimed to systematically construct and characterize an "AS-cell death-immunity" regulatory network in Wilms' tumor. METHODS:We performed a comprehensive in silico analysis using matched paired Wilms' tumor and adjacent normal RNA-seq data from the GSE138869 cohort. The SUVA algorithm was employed to identify cell death-related regulated alternative splicing events (RASEs). A tripartite regulatory network was constructed via correlation analysis to link these RASEs with upstream differentially expressed splicing factors (DESFs). Immune cell infiltration was quantified using CIBERSORT. Finally, the HNRNPD knockout and FLASH-seq multi-omics dataset (GSE212767) was utilized to computationally validate the predicted regulatory axis. RESULTS:Our analysis identified 118 cell death-related host genes undergoing significant alternative splicing in Wilms' tumor. Network integration highlighted a critical regulatory axis where the overexpressed splicing factor HNRNPD is strongly correlated with an aberrant AS event (clualt5p51764) in the apoptosis-related kinase MAP4K4. Further immune deconvolution demonstrated that both HNRNPD upregulation and the MAP4K4 splicing shift were significantly correlated with increased monocyte infiltration in the tumor microenvironment. Moreover, cross-validation utilizing the GSE212767 dataset confirmed that HNRNPD perturbation directly alters MAP4K4 splicing. CONCLUSIONS:Our computational framework proposes that the HNRNPD-MAP4K4 splicing axis links apoptotic dysregulation to immune microenvironment remodeling in Wilms' tumor. These correlative in silico findings provide a robust, hypothesis-generating basis for discovering novel prognostic biomarkers and developing targeted therapeutic strategies directed at the splicing machinery.

Osteoarthritis is a leading cause of chronic pain and disability, which lacks disease-modifying treatment. Given the complex multi-tissue and multifactorial drivers behind disease progression, effective treatments will require simultaneously targeting several mechanisms underlying joint degeneration and pain. Here, we developed and evaluated a combinatorial gene therapy, consisting of a high-capacity adenoviral vector carrying two therapeutic genes to target distinct pathological mechanisms: inflammation (IL-1Ra) and chondrocyte health (PRG4). Intra-articular delivery of this treatment improved functional, structural, and pain outcomes in murine and equine osteoarthritis models. In addition, treatment normalized inflammatory environments in joint tissues, as well as in the dorsal root ganglia (DRG) known to harbor joint-innervating sensory neurons. Moreover, gene therapy reversed OA-induced molecular signatures of neural hyperexcitability, suggesting amelioration of peripheral sensitization. Collectively, these findings support combinatorial gene therapy as a promising treatment for osteoarthritis, while identifying neuroinflammatory signatures for correction of disease progression and pain. One Sentence Summary:A single intra-articular injection of a combinatorial gene therapy slows OA progression and reduces pain in small and large animal models.

Purpose:To define the genetic architecture of foveal morphology and explore its relevance to foveal hypoplasia (FH), a hallmark of developmental macular disorders. Methods:We applied deep-learning algorithms to quantify foveal pit depth from central optical coherence tomography (OCT) B-scans in 61,269 UK Biobank participants. A genome-wide association study (GWAS) was conducted using REGENIE, adjusting for age, sex, height, and ancestry. Rare coding variants (frequency <1%) were analyzed in an exome-wide rare-variant association study (RVAS). Candidate genes were prioritized using integrative mapping; pathway, cross-ancestry, and genetic-correlation analyses were exploratory. Results:GWAS identified 126 sentinel variants, including 47 novel associations. Integrative mapping prioritized 129 putative causal genes, with 64 not previously implicated in foveal biology. Enriched pathways included retinoic acid metabolism (e.g., CYP26A1), photoreceptor differentiation (e.g., VSX2), extracellular matrix organization, and pigmentation. RVAS identified missense variants in ACTN3 and ESYT3 (P < 5 × 10-⁹) associated with FH features. Polygenic scores were predictive across African and South Asian ancestries. Overlap was observed with monogenic FH genes (TYR, OCA2, PAX6, AHR) and with genes underlying systemic diseases (COL11A1, KIF11, TUBB4B, PHYH). Re-examination of OCTs in affected individuals confirmed FH in select cases, including those with recurrent TUBB4B p.(Arg390Trp) variants. Conclusions:This is the first GWAS of human foveal morphology. Our findings redefine the genetic and biological framework underlying normal foveal development and foveal hypoplasia (FH). By linking common variation to rare monogenic disease, we establish a continuum model of FH with implications for future mechanistic and clinical investigation.

The vast majority of protein-coding genes in the human genome produce multiple mRNA isoforms through alternative splicing, significantly enhancing the complexity of the transcriptome and proteome. To establish an efficient method for characterizing transcript isoforms within tissue samples, we conducted a systematic comparison between single-cell long-read and conventional short-read RNA sequencing techniques. The transcriptome of approximately 30,000 mouse retina cells was profiled using 1.54 billion Illumina short reads and 1.40 billion Oxford Nanopore Technologies long reads. Consequently, we identify 44,325 transcript isoforms, with a notable 38% previously uncharacterized and 17% expressed exclusively in distinct cellular subclasses. We observe that long-read sequencing not only matches the gene expression and cell-type annotation performance of short-read sequencing but also excel in the precise identification of transcript isoforms. While transcript isoforms are often shared across various cell types, their relative abundance shows considerable cell type-specific variation. The data generated from our study significantly enhance the existing repertoire of transcript isoforms, thereby establishing a resource for future research into the mechanisms and implications of alternative splicing within retinal biology and its links to related diseases.

11-cis-Retinal is essential for light perception in mammalian photoreceptors (PRs), and aberrations in retinoid transformations cause severe retinal diseases. Understanding these processes is crucial for combating blinding diseases. The visual cycle, operating within PRs and the retinal pigment epithelium (RPE), regenerates 11-cis-retinal to sustain light sensitivity. Retinoids are also present in Müller glia (MG), hypothesized to supply 11-cis-retinol to cone PRs and retinal ganglion cells (RGCs). To trace retinoid movement through retinal cell types, we used cell-specific knockin of lecithin:retinol acyltransferase (LRAT), which converts retinols into stable retinyl esters (REs). Ectopic LRAT expression in murine PRs, MG, and RGCs resulted in RE synthesis, with REs differing in abundance and isomeric composition across cell types under genetic and light-based perturbations. PR inner segments showed high 11-cis-RE content, suggesting a constant 11-cis-retinoid supply for pigment regeneration. In MG expressing LRAT, all-trans-REs were detected, contrasting with 11-cis-REs in PRs. The MG-specific LRAT phenotype mirrored the RE-rich human neural retina, suggesting human MG may utilize LRAT to maintain retinoid reservoirs. Our findings reveal tightly controlled retinoid flux throughout the mammalian retina that supports sustained vision, expanding understanding of the visual cycle to combat retinal diseases.

Here we report the first genome-wide association study of foveal pit depth. In a cohort of 61,269 individuals, we identified 123 genome-wide significant loci associated with pit depth, including 47 novel associations not previously linked to macular traits. Using 12 complementary variant-to-gene mapping strategies, we prioritised 128 putative causal genes, 64 of which have not previously been implicated in foveal development. Our findings reveal previously unrecognised biological influences on foveal morphogenesis, including retinoic acid metabolism (implicating CYP26A1 for the first time in human foveal development), extracellular matrix and cytoskeletal dynamics, and retinal cell fate determination. In addition, rare-variant analysis uncovered two further gene associations, including ESYT3, a gene not previously linked to foveal structure. Together, these results provide new insights into the genetic architecture and molecular pathways underlying human foveal development, and offer a foundation for future functional studies aimed at characterising foveal development and disease.

Non-apoptotic regulated cell death (RCD) of tumor cells profoundly affects tumor progression and plays critical roles in determining response to immune checkpoint inhibitors (ICIs). Prognosis-distinctive HCC subtypes were identified by consensus cluster analysis based on the expressions of 507 non-apoptotic RCD genes obtained from databases and literature. Meanwhile, a set of bioinformatic tools was integrated to analyze the differences of the tumor immune microenvironment infiltration, genetic mutation, copy number variation, and epigenetics alternations within two subtypes. Finally, a non-apoptotic RCDRS signature was constructed and its reliability was evaluated in HCC patients' tissues. The high-RCDRS HCC subgroup showed a significantly lower overall survival and less sensitivity to ICIs compared to low-RCDRS subgroup, but higher sensitivity to cisplatin, paclitaxel, and sorafenib. Overall, we established an RCDRS panel consisting of four non-apoptotic RCD genes, which might be a promising predictor for evaluating HCC prognosis, guiding therapeutic decision-making, and ultimately improving patient outcomes.

Understanding and treating human diseases require valid animal models. Leveraging the genetic diversity in rhesus macaque populations across eight primate centers in the United States, we conduct targeted-sequencing on 1845 individuals for 374 genes linked to inherited human retinal and neurodevelopmental diseases. We identify over 47,000 single nucleotide variants, a substantial proportion of which are shared with human populations. By combining rhesus and human allele frequencies with established variant prediction methods, we develop a machine learning-based score that outperforms established methods in predicting missense variant pathogenicity. Remarkably, we find a marked number of loss-of-function variants and putative deleterious variants, which may lead to the development of rhesus disease models. Through phenotyping of macaques carrying a pathogenic OPA1:p.A8S variant, we identify a genetic model of autosomal dominant optic atrophy. Finally, we present a public website housing variant and genotype data from over two thousand rhesus macaques.

Background: Cancer-associated fibroblasts (CAFs) are the key components of the immune barrier in liver cancer. Therefore, gaining a deeper understanding of the heterogeneity and intercellular communication of CAFs holds utmost importance in boosting immunotherapy effectiveness and improving clinical outcomes. Methods: A comprehensive analysis by combing single-cell, bulk, and spatial transcriptome profiling with multiplexed immunofluorescence was conducted to unravel the complexities of CAFs in liver cancer. Results: Through an integrated approach involving 235 liver cancer scRNA-seq samples encompassing over 1.2 million cells, we found that CAFs were particularly increased in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). FAP(+) fibroblasts were identified as the dominant subtype of CAFs, and which were mainly involved in extracellular matrix organization and angiogenesis. These CAFs were enriched in the tumor boundary of HCC, but diffusely scattered within ICC. The DAB2(+) and SPP1(+) tumor-associated macrophages (TAMs) reinforce the function of FAP(+) CAFs through signals such as TGF-beta, PDGF, and ADM. Notably, the interaction between DAB2(+) TAMs and FAP(+) CAFs promoted the formation of immune barrier and correlated with poorer patient survival, non-response to immunotherapy in HCC. High FAP and DAB2 immunohistochemical scores predicted shorter survival and higher serum AFP concentration in a local clinical cohort of 90 HCC patients. Furthermore, this communication pattern might be applicable to other solid malignancies as well. Conclusions: The interaction between DAB2(+) TAMs and FAP(+) CAFs appears crucial in shaping the immune barrier. Strategies aimed at disrupting this communication or inhibiting the functions of FAP(+) CAFs could potentially enhance immunotherapy effectiveness and improve clinical outcomes.