Cwc27, associated with retinal degeneration, functions as a splicing factor in vivo.

Previous in vitro studies indicate that CWC27 functions as a splicing factor in the Bact spliceosome complex, interacting with CWC22 to form a landing platform for eIF4A3, a core component of the exon junction complex. However, the function of CWC27 as a splicing factor has not been validated in any in vivo systems. CWC27 variants have been shown to cause autosomal recessive retinal degeneration, in both syndromic and non-syndromic forms. The Cwc27K338fs/K338fs mouse model was shown to have significant retinal dysfunction and degeneration by 6 months of age. In this report, we have taken advantage of the Cwc27K338fs/K338fs mouse model to show that Cwc27 is involved in splicing in vivo in the context of the retina. Bulk RNA and single cell RNA-sequencing of the mouse retina showed that there were gene expression and splicing pattern changes, including alternative splice site usage and intron retention. Positive staining for CHOP suggests that ER stress may be activated in response to the splicing pattern changes and is a likely contributor to the disease mechanism. Our results provide the first evidence that CWC27 functions as a splicing factor in an in vivo context. The splicing defects and gene expression changes observed in the Cwc27K338fs/K338fs mouse retina provide insight to the potential disease mechanisms, paving the way for targeted therapeutic development.

Different treatment options for Takayasu arteritis patients with moderate-to-severe aortic regurgitation: long-term outcomes.

OBJECTIVES: To determine the prognosis of Takayasu arteritis (TA) patients with moderate-to-severe aortic regurgitation treated with surgical vs conservative treatment and to identify independent prognostic factors of long-term outcomes. METHODS: Between January 2002 and January 2017, 101 consecutive TA patients with moderate-to-severe aortic regurgitation treated with either surgical (n = 38) or conservative (n = 63) treatments were investigated in this retrospective observational case-control study. The primary end point was all-cause mortality, and the secondary end point comprised the combined end points of death, non-fatal stroke and cardiac events (non-fatal myocardial infarction and congestive heart failure). Propensity score matching was used to reduce the bias of baseline risk factors. RESULTS: The unadjusted all-cause 10-year mortality in the conservative group was increased compared with the surgical group (28.2% vs 7.4%; log-rank P = 0.036), and the combined end points showed the same trend (52.1% vs 25.3%; log-rank P = 0.005). After an adjustment of baseline risk factors, the conservative treatment was associated with reduced survival rates of both all-cause mortality [hazard ratio (HR): 8.243; 95% CI: 1.069, 63.552; P = 0.007] and combined end points (HR: 6.341; 95% CI: 1.469, 27.375; P = 0.002). Conservative treatment (HR: 3.838, 95% CI: 1.333, 11.053; P = 0.013) and left ventricular end-diastolic diameter (HR: 1.036, 95% CI: 1.001, 1.071; P = 0.042) were risk factors for increased combined end points. CONCLUSION: Surgical treatment improves the outcomes of patients with moderate-to-severe aortic regurgitation due to TA. The dilated left ventricle indicated a worse prognosis.

Noncoding mutation in RPGRIP1 contributes to inherited retinal degenerations.

Purpose: Despite the extensive use of next-generation sequencing (NGS) technology to identify disease-causing genomic variations, a major gap in our understanding of Mendelian diseases is the unidentified molecular lesion in a significant portion of patients. For inherited retinal degenerations (IRDs), although currently close to 300 disease-associated genes have been identified, the mutations in approximately one-third of patients remain unknown. With mounting evidence that noncoding mutations might contribute significantly to disease burden, we aimed to systematically investigate the contributions of noncoding regions in the genome to IRDs. Methods: In this study, we focused on RPGRIP1, which has been linked to various IRD phenotypes, including Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), and macular dystrophy (MD). As several noncoding mutant alleles have been reported in RPGRIP1, and we observed that the mutation carrier frequency of RPGRIP1 is higher in patient cohorts with unsolved IRDs, we hypothesized that mutations in the noncoding regions of RPGRIP1 might be a significant contributor to pathogenicity. To test this hypothesis, we performed whole-genome sequencing (WGS) for 25 patients with unassigned IRD who carry a single mutation in RPGRIP1. Results: Three noncoding variants in RPGRIP1, including a 2,890 bp deletion and two deep-intronic variants (c.2710+233G>A and c.1468-263G>C), were identified as putative second hits of RPGRIP1 in three patients with LCA. The mutant alleles were validated with direct sequencing or in vitro assays. Conclusions: The results highlight the significance of the contribution of noncoding pathogenic variants to unsolved IRD cases.

Inflammation Is Associated With Platelet Coagulation Function Rather Than Enzymatic Coagulation Function in Patients With Takayasu Arteritis.

The integral changes of coagulation and fibrinolysis, and their relationships with inflammation in patients with Takayasu arteritis (TA) remain undetermined. The purpose of this study was to analyze the changes of coagulation and fibrinolysis process in patients with TA by thrombelastography (TEG).A total of 127 patients with TA and 55 healthy controls were enrolled. Patients with TA were grouped according to disease activity. The routine hematological parameters, traditional coagulation assays, and TEG parameters were summarized retrospectively.A shorter K time, larger alpha angle, and higher levels of MA, MA(A), G, and TPI were found in patients with TA, especially in those at the active stage. The R time, EPL, LY30, and CL30 were similar between patients with TA and healthy controls, as well as TA patients with different disease activity. Spearman's correlation showed that ESR was correlated with PLT (r = 0.206, P = 0.020), K (r = -0.353, P < 0.001), alpha angle (r = 0.328, P < 0.001), MA (r = 0.474, P < 0.001), MA (A) (r = 0.623, P < 0.001), G (r = 0.475, P < 0.001), and TPI (r = 0.458, P < 0.001).In conclusion, inflammation was associated with platelet coagulation function rather than enzymatic coagulation function in patients with TA. Physicians should focus on antiplatelet treatment for improving the prognosis of patients with TA.

To be or not to be - Decoding the Trabecular Meshwork Cell Identity.

The trabecular meshwork within the conventional outflow apparatus is critical in maintaining intraocular pressure homeostasis. In vitro studies employing primary cell cultures of the human trabecular meshwork (hTM) have conventionally served as surrogates for investigating the pathobiology of TM dysfunction. Despite its abundant use, translation of outcomes from in vitro studies to ex vivo and/or in vivo studies remains a challenge. Given the cell heterogeneity, performing single-cell RNA sequencing comparing primary hTM cell cultures to hTM tissue may provide important insights on cellular identity and translatability, as such an approach has not been reported before. In this study, we assembled a total of 14 primary hTM in vitro samples across passages 1-4, including 4 samples from individuals diagnosed with glaucoma. This dataset offers a comprehensive transcriptomic resource of primary hTM in vitro scRNA-seq data to study global changes in gene expression in comparison to cells in tissue in situ. We have performed extensive preprocessing and quality control, allowing the research community to access and utilize this public resource.

Comprehensive single-cell atlas of the mouse retina.

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cellular heterogeneity by characterizing cell types across tissues and species. While several mouse retinal scRNA-seq datasets exist, each dataset is either limited in cell numbers or focused on specific cell classes, thereby hindering comprehensive gene expression analysis across all retina types. To fill the gap, we generated the largest retinal scRNA-seq dataset to date, comprising approximately 190,000 single cells from C57BL/6J mouse retinas, enriched for rare population cells via antibody-based magnetic cell sorting. Integrating this dataset with public datasets, we constructed the Mouse Retina Cell Atlas (MRCA) for wild-type mice, encompassing over 330,000 cells, characterizing 12 major classes and 138 cell types. The MRCA consolidates existing knowledge, identifies new cell types, and is publicly accessible via CELLxGENE, UCSC Cell Browser, and the Broad Single Cell Portal, providing a user-friendly resource for the mouse retina research community.

Comprehensive single-cell atlas of the mouse retina.

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cellular heterogeneity at the single-cell resolution by classifying and characterizing cell types in multiple tissues and species. While several mouse retinal scRNA-seq reference datasets have been published, each dataset either has a relatively small number of cells or is focused on specific cell classes, and thus is suboptimal for assessing gene expression patterns across all retina types at the same time. To establish a unified and comprehensive reference for the mouse retina, we first generated the largest retinal scRNA-seq dataset to date, comprising approximately 190,000 single cells from C57BL/6J mouse whole retinas. This dataset was generated through the targeted enrichment of rare population cells via antibody-based magnetic cell sorting. By integrating this new dataset with public datasets, we conducted an integrated analysis to construct the Mouse Retina Cell Atlas (MRCA) for wild-type mice, which encompasses over 330,000 single cells. The MRCA characterizes 12 major classes and 138 cell types. It captured consensus cell type characterization from public datasets and identified additional new cell types. To facilitate the public use of the MRCA, we have deposited it in CELLxGENE, UCSC Cell Browser, and the Broad Single Cell Portal for visualization and gene expression exploration. The comprehensive MRCA serves as an easy-to-use, one-stop data resource for the mouse retina communities.

Single-cell multiomics of the human retina reveals hierarchical transcription factor collaboration in mediating cell type-specific effects of genetic variants on gene regulation.

BACKGROUND: Systematic characterization of how  genetic variation modulates gene regulation in a cell type-specific context is essential for understanding complex traits. To address this question, we profile gene expression and chromatin accessibility in cells from healthy retinae of 20 human donors through single-cell multiomics and genomic sequencing. RESULTS: We map eQTL, caQTL, allelic-specific expression, and allelic-specific chromatin accessibility in major retinal cell types. By integrating these results, we identify and characterize regulatory elements and genetic variants effective on gene regulation in individual cell types. The majority of identified sc-eQTLs and sc-caQTLs display cell type-specific effects, while the cis-elements containing genetic variants with cell type-specific effects are often accessible in multiple cell types. Furthermore, the transcription factors whose binding sites are perturbed by genetic variants tend to have higher expression levels in the cell types where the variants exert their effects, compared to the cell types where the variants have no impact. We further validate our findings with high-throughput reporter assays. Lastly, we identify the enriched cell types, candidate causal variants and genes, and cell type-specific regulatory mechanism underlying GWAS loci. CONCLUSIONS: Overall, genetic effects on gene regulation are highly context dependent. Our results suggest that cell type-dependent genetic effect is driven by precise modulation of both trans-factor expression and chromatin accessibility of cis-elements. Our findings indicate hierarchical collaboration among transcription factors plays a crucial role in mediating cell type-specific effects of genetic variants on gene regulation.

A multi-omics atlas of the human retina at single-cell resolution.

Cell classes in the human retina are highly heterogeneous with their abundance varying by several orders of magnitude. Here, we generated and integrated a multi-omics single-cell atlas of the adult human retina, including more than 250,000 nuclei for single-nuclei RNA-seq and 137,000 nuclei for single-nuclei ATAC-seq. Cross-species comparison of the retina atlas among human, monkey, mice, and chicken revealed relatively conserved and non-conserved types. Interestingly, the overall cell heterogeneity in primate retina decreases compared with that of rodent and chicken retina. Through integrative analysis, we identified 35,000 distal cis-element-gene pairs, constructed transcription factor (TF)-target regulons for more than 200 TFs, and partitioned the TFs into distinct co-active modules. We also revealed the heterogeneity of the cis-element-gene relationships in different cell types, even from the same class. Taken together, we present a comprehensive single-cell multi-omics atlas of the human retina as a resource that enables systematic molecular characterization at individual cell-type resolution.

Single-nucleotide variant calling in single-cell sequencing data with Monopogen.

Single-cell omics technologies enable molecular characterization of diverse cell types and states, but how the resulting transcriptional and epigenetic profiles depend on the cell's genetic background remains understudied. We describe Monopogen, a computational tool to detect single-nucleotide variants (SNVs) from single-cell sequencing data. Monopogen leverages linkage disequilibrium from external reference panels to identify germline SNVs and detects putative somatic SNVs using allele cosegregating patterns at the cell population level. It can identify 100 K to 3 M germline SNVs achieving a genotyping accuracy of 95%, together with hundreds of putative somatic SNVs. Monopogen-derived genotypes enable global and local ancestry inference and identification of admixed samples. It identifies variants associated with cardiomyocyte metabolic levels and epigenomic programs. It also improves putative somatic SNV detection that enables clonal lineage tracing in primary human clonal hematopoiesis. Monopogen brings together population genetics, cell lineage tracing and single-cell omics to uncover genetic determinants of cellular processes.

DeMixSC: a deconvolution framework that uses single-cell sequencing plus a small benchmark dataset for improved analysis of cell-type ratios in complex tissue samples.

Bulk deconvolution with single-cell/nucleus RNA-seq data is critical for understanding heterogeneity in complex biological samples, yet the technological discrepancy across sequencing platforms limits deconvolution accuracy. To address this, we introduce an experimental design to match inter-platform biological signals, hence revealing the technological discrepancy, and then develop a deconvolution framework called DeMixSC using the better-matched, i.e., benchmark, data. Built upon a novel weighted nonnegative least-squares framework, DeMixSC identifies and adjusts genes with high technological discrepancy and aligns the benchmark data with large patient cohorts of matched-tissue-type for large-scale deconvolution. Our results using a benchmark dataset of healthy retinas suggest much-improved deconvolution accuracy. Further analysis of a cohort of 453 patients with age-related macular degeneration supports the broad applicability of DeMixSC. Our findings reveal the impact of technological discrepancy on deconvolution performance and underscore the importance of a well-matched dataset to resolve this challenge. The developed DeMixSC framework is generally applicable for deconvolving large cohorts of disease tissues, and potentially cancer.

Integrated multi-omics single cell atlas of the human retina.

Single-cell sequencing has revolutionized the scale and resolution of molecular profiling of tissues and organs. Here, we present an integrated multimodal reference atlas of the most accessible portion of the mammalian central nervous system, the retina. We compiled around 2.4 million cells from 55 donors, including 1.4 million unpublished data points, to create a comprehensive human retina cell atlas (HRCA) of transcriptome and chromatin accessibility, unveiling over 110 types. Engaging the retina community, we annotated each cluster, refined the Cell Ontology for the retina, identified distinct marker genes, and characterized cis-regulatory elements and gene regulatory networks (GRNs) for these cell types. Our analysis uncovered intriguing differences in transcriptome, chromatin, and GRNs across cell types. In addition, we modeled changes in gene expression and chromatin openness across gender and age. This integrated atlas also enabled the fine-mapping of GWAS and eQTL variants. Accessible through interactive browsers, this multimodal cross-donor and cross-lab HRCA, can facilitate a better understanding of retinal function and pathology.

Bi-order multimodal integration of single-cell data.

Integration of single-cell multiomics profiles generated by different single-cell technologies from the same biological sample is still challenging. Previous approaches based on shared features have only provided approximate solutions. Here, we present a novel mathematical solution named bi-order canonical correlation analysis (bi-CCA), which extends the widely used CCA approach to iteratively align the rows and the columns between data matrices. Bi-CCA is generally applicable to combinations of any two single-cell modalities. Validations using co-assayed ground truth data and application to a CAR-NK study and a fetal muscle atlas demonstrate its capability in generating accurate multimodal co-embeddings and discovering cellular identity.

Microparticles from Endothelial Cells and Immune Cells in Patients with Takayasu Arteritis.

AIM: This study was designed to analyze microparticles (MPs) from endothelial cells (EMPs) and immune cells from healthy individuals and paitents with Takayasu arteritis (TA), and any possible relationships between MPs and TA acitivity. METHODS: MPs derived from the plasma of 51 subjects were analyzed, including 32 patients with TA and 19 healthy individuals. Flow cytometry was performed with Annexin (Anx)-V and antibodies against surface markers of endothelial cells (CD144), T cells (CD3), B cells (CD19), and monocytes (CD14). RESULTS: The concentrations of total EMPs, AnxV＋ EMPs and AnxV- EMPs were significantly increased when comparing patients with TA and healthy controls (54×103 vs. 32×103 MPs /ml, P=0.0004; 22×103 vs. 12×103 MPs /ml, P=0.0006; and 31×103 vs. 19×103 MPs /ml, P=0.0005), and comparing active TA patients with remission ones (85×103 vs. 45×103 MPs /ml, P=0.016; 39×103 vs. 14×103 MPs /ml, P=0.0092; and 47×103 vs.29×103 MPs /ml, P=0.0371). In addition, the concentrations of total EMPs (odds ratio [OR]=1.024, 95% confidence interval [CI]: 1.001 to 1.048, P=0.037), AnxV＋(OR=1.089, 95%CI: 1.011 to 1.172, P=0.024), and AnxV- EMPs (OR=1.029, 95% CI: 1.002 to 1.056, P=0.034) were positively related to TA activity. With multiple linear regression analysis, platelet was associated with both total and AnxV- EMP concentrations independently, while erythrocyte sedimentation rate was independently correlated with AnxV＋EMPs. CONCLUSION: Concentrations of endothelial microparticles are correlated with inflammation in Takayasu arteritis and may be useful markers to assess disease activity.

Long-term outcomes of coronary artery bypass grafting versus percutaneous coronary intervention for Takayasu arteritis patients with coronary artery involvement.

OBJECTIVE: Coronary artery involvement significantly increases mortality of patients with Takayasu arteritis (TA); however, the optimal revascularization strategy for this condition has not been established. We aimed to compare the long-term outcomes of TA patients with coronary artery involvement treated with coronary artery bypass grafting (CABG) and percutaneous coronary intervention with stenting (PCI). METHODS: Data from 46 TA patients with coronary artery involvement were analyzed according to their revascularization strategies. The resulting events included myocardial infarction, repeated revascularization, cardiac death, and the major adverse cardiac events (MACE), which is a combination of the former events. RESULTS: The risk of MACE was significantly higher in the PCI group than in the CABG group during a median of 41.0 months follow-up (P < 0.001), especially in those who underwent revascularization at the active stage of TA (P = 0.001), whereas no difference was found between PCI and CABG groups in patients who underwent revascularization at the stable stage of TA (P = 0.138). The incidence of MACE was higher in TA patients at the active stage than those at the stable stage in all patients (P < 0.001). For patients at the active stage, the risk of MACE was significantly lower in patients with than those without usage of prednisone (P = 0.028); while no difference was found between patients who were stable not requiring prednisone and patients who were stable on prednisone (P = 0.525). CONCLUSION: With regard to MACE, CABG is superior to PCI despite medical therapy in TA patients with coronary artery involvement. In TA patients at the stable stage, PCI is similar with CABG in prognosis. For patients at the active stage, if emergency revascularization is necessary, CABG is ideal; if not, receiving medical therapy until disease remission and then undergoing PCI may be an alternative choice of CABG.