iGWAS: Image-based genome-wide association of self-supervised deep phenotyping of retina fundus images.

Existing imaging genetics studies have been mostly limited in scope by using imaging-derived phenotypes defined by human experts. Here, leveraging new breakthroughs in self-supervised deep representation learning, we propose a new approach, image-based genome-wide association study (iGWAS), for identifying genetic factors associated with phenotypes discovered from medical images using contrastive learning. Using retinal fundus photos, our model extracts a 128-dimensional vector representing features of the retina as phenotypes. After training the model on 40,000 images from the EyePACS dataset, we generated phenotypes from 130,329 images of 65,629 British White participants in the UK Biobank. We conducted GWAS on these phenotypes and identified 14 loci with genome-wide significance (p<5×10-8 and intersection of hits from left and right eyes). We also did GWAS on the retina color, the average color of the center region of the retinal fundus photos. The GWAS of retina colors identified 34 loci, 7 are overlapping with GWAS of raw image phenotype. Our results establish the feasibility of this new framework of genomic study based on self-supervised phenotyping of medical images.

SOX7 deficiency causes ventricular septal defects through its effects on endocardial-to-mesenchymal transition and the expression of Wnt4 and Bmp2.

SOX7 is a transcription factor-encoding gene located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with ventricular septal defects (VSDs). We have previously shown that Sox7-/- embryos die of heart failure around E11.5. Here, we demonstrate that these embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. Ablation of Sox7 in the endocardium also resulted in hypocellular endocardial cushions, and we observed VSDs in rare E15.5 Sox7flox/-;Tie2-Cre and Sox7flox/flox;Tie2-Cre embryos that survived to E15.5. In atrioventricular explant studies, we showed that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). RNA-seq studies performed on E9.5 Sox7-/- heart tubes revealed severely reduced Wnt4 transcript levels. Wnt4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of Bmp2 in the myocardium. Both WNT4 and BMP2 have been previously implicated in the development of VSDs in individuals with 46,XX sex reversal with dysgenesis of kidney, adrenals and lungs (SERKAL) syndrome and in individuals with short stature, facial dysmorphism and skeletal anomalies with or without cardiac anomalies 1 (SSFSC1) syndrome, respectively. We now show that Sox7 and Wnt4 interact genetically in the development of VSDs through their additive effects on endocardial cushion development with Sox7+/-;Wnt4+/- double heterozygous embryos having hypocellular endocardial cushions and perimembranous and muscular VSDs not seen in their Sox7+/- and Wnt4+/- littermates. These results provide additional evidence that SOX7, WNT4 and BMP2 function in the same pathway during mammalian septal development and that their deficiency can contribute to the development of VSDs in humans.

Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids.

Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium-retinal organoid-that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.

Single-Cell Transcriptome Analysis Reveals Dynamic Cell Populations and Differential Gene Expression Patterns in Control and Aneurysmal Human Aortic Tissue.

BACKGROUND: Ascending thoracic aortic aneurysm (ATAA) is caused by the progressive weakening and dilatation of the aortic wall and can lead to aortic dissection, rupture, and other life-threatening complications. To improve our understanding of ATAA pathogenesis, we aimed to comprehensively characterize the cellular composition of the ascending aortic wall and to identify molecular alterations in each cell population of human ATAA tissues. METHODS: We performed single-cell RNA sequencing analysis of ascending aortic tissues from 11 study participants, including 8 patients with ATAA (4 women and 4 men) and 3 control subjects (2 women and 1 man). Cells extracted from aortic tissue were analyzed and categorized with single-cell RNA sequencing data to perform cluster identification. ATAA-related changes were then examined by comparing the proportions of each cell type and the gene expression profiles between ATAA and control tissues. We also examined which genes may be critical for ATAA by performing the integrative analysis of our single-cell RNA sequencing data with publicly available data from genome-wide association studies. RESULTS: We identified 11 major cell types in human ascending aortic tissue; the high-resolution reclustering of these cells further divided them into 40 subtypes. Multiple subtypes were observed for smooth muscle cells, macrophages, and T lymphocytes, suggesting that these cells have multiple functional populations in the aortic wall. In general, ATAA tissues had fewer nonimmune cells and more immune cells, especially T lymphocytes, than control tissues did. Differential gene expression data suggested the presence of extensive mitochondrial dysfunction in ATAA tissues. In addition, integrative analysis of our single-cell RNA sequencing data with public genome-wide association study data and promoter capture Hi-C data suggested that the erythroblast transformation-specific related gene(ERG) exerts an important role in maintaining normal aortic wall function. CONCLUSIONS: Our study provides a comprehensive evaluation of the cellular composition of the ascending aortic wall and reveals how the gene expression landscape is altered in human ATAA tissue. The information from this study makes important contributions to our understanding of ATAA formation and progression.

The Prrx1 homeodomain transcription factor plays a central role in pancreatic regeneration and carcinogenesis.

Pancreatic exocrine cell plasticity can be observed during development, pancreatitis with subsequent regeneration, and also transformation. For example, acinar-ductal metaplasia (ADM) occurs during acute pancreatitis and might be viewed as a prelude to pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC) development. To elucidate regulatory processes that overlap ductal development, ADM, and the progression of normal cells to PanIN lesions, we undertook a systematic approach to identify the Prrx1 paired homeodomain Prrx1 transcriptional factor as a highly regulated gene in these processes. Prrx1 annotates a subset of pancreatic ductal epithelial cells in Prrx1creER(T2)-IRES-GFP mice. Furthermore, sorted Prrx1(+) cells have the capacity to self-renew and expand during chronic pancreatitis. The two isoforms, Prrx1a and Prrx1b, regulate migration and invasion, respectively, in pancreatic cancer cells. In addition, Prrx1b is enriched in circulating pancreatic cells (Pdx1cre;LSL-Kras(G12D/+);p53(fl/+);R26YFP). Intriguingly, the Prrx1b isoform, which is also induced in ADM, binds the Sox9 promoter and positively regulates Sox9 expression. This suggests a new hierarchical scheme whereby a Prrx1-Sox9 axis may influence the emergence of acinar-ductal metaplasia and regeneration. Furthermore, our data provide a possible explanation of why pancreatic cancer is skewed toward a ductal fate.

The apical complex protein Pals1 is required to maintain cerebellar progenitor cells in a proliferative state.

Through their biased localization and function within the cell, polarity complex proteins are necessary to establish the cellular asymmetry required for tissue organization. Well-characterized germinal zones, mitogenic signals and cell types make the cerebellum an excellent model for addressing the crucial function of polarity complex proteins in the generation and organization of neural tissues. Deletion of the apical polarity complex protein Pals1 in the developing cerebellum results in a remarkably undersized cerebellum with disrupted layers in poorly formed folia and strikingly reduced granule cell production. We demonstrate that Pals1 is not only essential for cerebellum organogenesis, but also for preventing premature differentiation and thus maintaining progenitor pools in cerebellar germinal zones, including cerebellar granule neuron precursors in the external granule layer. In the Pals1 mouse mutants, the expression of genes that regulate the cell cycle was diminished, correlating with the loss of the proliferating cell population of germinal zones. Furthermore, enhanced Shh signaling through activated Smo cannot overcome impaired cerebellar cell generation, arguing for an epistatic role of Pals1 in proliferation capacity. Our study identifies Pals1 as a novel intrinsic factor that regulates the generation of cerebellar cells and Pals1 deficiency as a potential inhibitor of overactive mitogenic signaling.

Inhibition of pancreatic cancer Panc1 cell migration by omeprazole is dependent on aryl hydrocarbon receptor activation of JNK.

Several aryl hydrocarbon receptor (AhR)-active pharmaceuticals were screened as inhibitors of pancreatic cancer cell invasion and identified two compounds, omeprazole, that inhibited invasion. Inhibition of highly invasive Panc1 cell invasion by omeprazole involves an AhR-dependent non-genomic pathway, and omeprazole-mediated inhibition of Panc1 cell invasion was dependent on Jun-N-terminal kinase (JNK) and mitogen-activated kinase kinase 7 (MKK7). The failure of omeprazole to induce nuclear translocation of the AhR was not due to overexpression of cytosolic AhR partner proteins Hsp90 or XAP2, and results of DNA sequencing show that the AhR expressed in Panc1 cells was not mutated. Results of RNAseq studies indicate that omeprazole induced an AhR-dependent downregulation of several pro-invasion factors including activated leukocyte cell adhesion molecule (ALCAM), long chain fatty acid CoA-synthase (CSL4), stathmin 3 (STMN3) and neuropillin 2 (NRP2), and the specific functions of these genes are currently being investigated.

Omeprazole Inhibits Pancreatic Cancer Cell Invasion through a Nongenomic Aryl Hydrocarbon Receptor Pathway.

Omeprazole and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are aryl hydrocarbon receptor (AhR) agonists that inhibit the invasion of breast cancer cells through inhibition of CXCR4 transcription. Treatment of highly invasive Panc1 pancreatic cancer cells with TCDD, omeprazole, and seven other AhR-active pharmaceuticals showed that only omeprazole and tranilast, but not TCDD, inhibited invasion in a Boyden chamber assay. Similar results were observed in MiaPaCa2 cells, another quasimensenchymal pancreatic ductal adenocarcinoma (QM-PDA) pancreatic cancer cell line, whereas invasion was not observed with BxPC3 or L3.6pL cells, which are classified as classical (less invasive) pancreatic cancer cells. It was also observed in QM-PDA cells that TCDD, omeprazole, and tranilast did not induce CYP1A1 or CXCR4 and that treatment with these compounds did not result in nuclear uptake of AhR. In contrast, treatment of BxPC3 and L3.6pL cells with these AhR ligands resulted in induction of CYP1A1 (by TCDD) and nuclear uptake of AhR, which was similar to that observed for Ah-responsive MDA-MB-468 breast and HepG2 liver cancer cell lines. Results of AhR and AhR nuclear translocator (Arnt) knockdown experiments in Panc1 and MiaPaCa2 cells demonstrated that omeprazole- and tranilast-mediated inhibition of invasion was AhR-dependent but Arnt-independent. These results demonstrate that in the most highly invasive subtype of pancreatic cancer cells (QM-PDA) the selective AhR modulators omeprazole and tranilast inhibit invasion through a nongenomic AhR pathway.

Genomic predictors for recurrence patterns of hepatocellular carcinoma: model derivation and validation.

BACKGROUND: Typically observed at 2 y after surgical resection, late recurrence is a major challenge in the management of hepatocellular carcinoma (HCC). We aimed to develop a genomic predictor that can identify patients at high risk for late recurrence and assess its clinical implications. METHODS AND FINDINGS: Systematic analysis of gene expression data from human liver undergoing hepatic injury and regeneration revealed a 233-gene signature that was significantly associated with late recurrence of HCC. Using this signature, we developed a prognostic predictor that can identify patients at high risk of late recurrence, and tested and validated the robustness of the predictor in patients (n = 396) who underwent surgery between 1990 and 2011 at four centers (210 recurrences during a median of 3.7 y of follow-up). In multivariate analysis, this signature was the strongest risk factor for late recurrence (hazard ratio, 2.2; 95% confidence interval, 1.3-3.7; p = 0.002). In contrast, our previously developed tumor-derived 65-gene risk score was significantly associated with early recurrence (p = 0.005) but not with late recurrence (p = 0.7). In multivariate analysis, the 65-gene risk score was the strongest risk factor for very early recurrence (<1 y after surgical resection) (hazard ratio, 1.7; 95% confidence interval, 1.1-2.6; p = 0.01). The potential significance of STAT3 activation in late recurrence was predicted by gene network analysis and validated later. We also developed and validated 4- and 20-gene predictors from the full 233-gene predictor. The main limitation of the study is that most of the patients in our study were hepatitis B virus-positive. Further investigations are needed to test our prediction models in patients with different etiologies of HCC, such as hepatitis C virus. CONCLUSIONS: Two independently developed predictors reflected well the differences between early and late recurrence of HCC at the molecular level and provided new biomarkers for risk stratification. Please see later in the article for the Editors' Summary.

Tat-activating regulatory DNA-binding protein regulates glycolysis in hepatocellular carcinoma by regulating the platelet isoform of phosphofructokinase through microRNA 520.

UNLABELLED: Metabolic changes are common features of many cancer cells and are frequently associated with the clinical outcome of patients with various cancers, including hepatocellular carcinoma (HCC). Thus, aberrant metabolic pathways in cancer cells are attractive targets for cancer therapy. However, our understanding of cancer-specific regulatory mechanisms of cell metabolism is still very limited. We found that Tat-activating regulatory DNA-binding protein (TARDBP) is a novel regulator of glycolysis in HCC cells. TARDBP regulates expression of the platelet isoform of phosphofructokinase (PFKP), the rate-limiting enzyme of glycolysis that catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate. Silencing of TARDBP expression in multiple HCC cell lines leads to impaired glucose metabolism and inhibition of in vitro and in vivo growth of HCC cells. Notably, the microRNA 520 (miR-520) family is an intermediate regulator of TARDBP-mediated regulation of glycolysis. Mechanistically, TARDBP suppressed expression of the miR-520 family, which, in turn, inhibited expression of PFKP. We further showed that expression of TARDBP is significantly associated with the overall survival of patients with HCC. CONCLUSION: Our study provides new mechanistic insights into the regulation of glycolysis in HCC cells and reveals TARDBP as a potential therapeutic target for HCC.

Cross-species hybridization of microarrays for studying tumor transcriptome of brain metastasis.

Although the importance of the cellular microenvironment (soil) during invasion and metastasis of cancer cells (seed) has been well-recognized, technical challenges have limited the ability to assess the influence of the microenvironment on cancer cells at the molecular level. Here, we show that an experimental strategy, competitive cross-species hybridization of microarray experiments, can characterize the influence of different microenvironments on cancer cells by independently extracting gene expression data of cancer and host cells when human cancer cells were xenografted into different organ sites of immunocompromised mice. Surprisingly, the analysis of gene expression data showed that the brain microenvironment induces complete reprogramming of metastasized cancer cells, resulting in a gain of neuronal cell characteristics and mimicking neurogenesis during development. We also show that epigenetic changes coincide with transcriptional reprogramming in cancer cells. These observations provide proof of principle for competitive cross-species hybridization of microarray experiments to characterize the effect of the microenvironment on tumor cell behavior.

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.

Sixty-five gene-based risk score classifier predicts overall survival in hepatocellular carcinoma.

UNLABELLED: Clinical application of the prognostic gene expression signature has been delayed due to the large number of genes and complexity of prediction algorithms. In the current study we aimed to develop an easy-to-use risk score with a limited number of genes that can robustly predict prognosis of patients with hepatocellular carcinoma (HCC). The risk score was developed using Cox coefficient values of 65 genes in the training set (n = 139) and its robustness was validated in test sets (n = 292). The risk score was a highly significant predictor of overall survival (OS) in the first test cohort (P = 5.6 × 10(-5), n = 100) and the second test cohort (P = 5.0 × 10(-5) , n = 192). In multivariate analysis, the risk score was a significant risk factor among clinical variables examined together (hazard ratio [HR], 1.36; 95% confidence interval [CI], 1.13-1.64; P = 0.001 for OS). CONCLUSION: The risk score classifier we have developed can identify two clinically distinct HCC subtypes at early and late stages of the disease in a simple and highly reproducible manner across multiple datasets.

Prognostic gene expression signature associated with two molecularly distinct subtypes of colorectal cancer.

AIMS: Despite continual efforts to develop prognostic and predictive models of colorectal cancer by using clinicopathological and genetic parameters, a clinical test that can discriminate between patients with good or poor outcome after treatment has not been established. Thus, the authors aim to uncover subtypes of colorectal cancer that have distinct biological characteristics associated with prognosis and identify potential biomarkers that best reflect the biological and clinical characteristics of subtypes. METHODS: Unsupervised hierarchical clustering analysis was applied to gene expression data from 177 patients with colorectal cancer to determine a prognostic gene expression signature. Validation of the signature was sought in two independent patient groups. The association between the signature and prognosis of patients was assessed by Kaplan-Meier plots, log-rank tests and the Cox model. RESULTS: The authors identified a gene signature that was associated with overall survival and disease-free survival in 177 patients and validated in two independent cohorts of 213 patients. In multivariate analysis, the signature was an independent risk factor (HR 3.08; 95% CI 1.33 to 7.14; p=0.008 for overall survival). Subset analysis of patients with AJCC (American Joint Committee on Cancer) stage III cancer revealed that the signature can also identify the patients who have better outcome with adjuvant chemotherapy (CTX). Adjuvant chemotherapy significantly affected disease-free survival in patients in subtype B (3-year rate, 71.2% (CTX) vs 41.9% (no CTX); p=0.004). However, such benefit of adjuvant chemotherapy was not significant for patients in subtype A. CONCLUSION: The gene signature is an independent predictor of response to chemotherapy and clinical outcome in patients with colorectal cancer.

Observations of the delayed-choice quantum eraser using coherent photons.

Quantum superposition is the cornerstone of quantum mechanics, where interference fringes originate in the self-interference of a single photon via indistinguishable photon characteristics. Wheeler's delayed-choice experiments have been extensively studied for the wave-particle duality over the last several decades to understand the complementarity theory of quantum mechanics. The heart of the delayed-choice quantum eraser is in the mutually exclusive quantum feature violating the cause-effect relation. Here, we experimentally demonstrate the quantum eraser using coherent photon pairs by the delayed choice of a polarizer placed out of the interferometer. Coherence solutions of the observed quantum eraser are derived from a typical Mach-Zehnder interferometer, where the violation of the cause-effect relation is due to selective measurements of basis choice.

Revisiting self-interference in Young's double-slit experiments.

Quantum superposition is the heart of quantum mechanics as mentioned by Dirac and Feynman. In an interferometric system, single photon self-interference has been intensively studied over the last several decades in both quantum and classical regimes. In Born rule tests, the Sorkin parameter indicates the maximum number of possible quantum superposition allowed to the input photons entering an interferometer, where multi-photon interference fringe is equivalent to that of a classical version by a laser. Here, an attenuated laser light in a quantum regime is investigated for self-interference in a Mach-Zehnder interferometer, and the results are compared with its classical version. The equivalent result supports the Born rule tests, where the classical interference originates in the superposition of individual single-photon self-interferences. This understanding sheds light on the fundamental physics of quantum features between bipartite systems.

A Spontaneous Nonhuman Primate Model of Myopic Foveoschisis.

Purpose: Foveoschisis involves the pathologic splitting of retinal layers at the fovea, which may occur congenitally in X-linked retinoschisis (XLRS) or as an acquired complication of myopia. XLRS is attributed to functional loss of the retinal adhesion protein retinoschisin 1 (RS1), but the pathophysiology of myopic foveoschisis is unclear due to the lack of animal models. Here, we characterized a novel nonhuman primate model of myopic foveoschisis through clinical examination and multimodal imaging followed by morphologic, cellular, and transcriptional profiling of retinal tissues and genetic analysis. Methods: We identified a rhesus macaque with behavioral and anatomic features of myopic foveoschisis, and monitored disease progression over 14 months by fundus photography, fluorescein angiography, and optical coherence tomography (OCT). After necropsy, we evaluated anatomic and cellular changes by immunohistochemistry and transcriptomic changes using single-nuclei RNA-sequencing (snRNA-seq). Finally, we performed Sanger and whole exome sequencing with focus on the RS1 gene. Results: Affected eyes demonstrated posterior hyaloid traction and progressive splitting of the outer plexiform layer on OCT. Immunohistochemistry showed increased GFAP expression in Müller glia and loss of ramified Iba-1+ microglia, suggesting macro- and microglial activation with minimal photoreceptor alterations. SnRNA-seq revealed gene expression changes predominantly in cones and retinal ganglion cells involving chromatin modification, suggestive of cellular stress at the fovea. No defects in the RS1 gene or its expression were detected. Conclusions: This nonhuman primate model of foveoschisis reveals insights into how acquired myopic traction leads to phenotypically similar morphologic and cellular changes as congenital XLRS without alterations in RS1.

Retinal organoids derived from rhesus macaque iPSCs undergo accelerated differentiation compared to human stem cells.

PURPOSE: To compare the timing and efficiency of the development of Macaca mulatta, a nonhuman primate (NHP), induced pluripotent stem cell (rhiPSC) derived retinal organoids to those derived from human embryonic stem cells (hESCs). RESULTS: Generation of retinal organoids was achieved from both human and several NHP pluripotent stem cell lines. All rhiPSC lines resulted in retinal differentiation with the formation of optic vesicle-like structures similar to what has been observed in hESC retinal organoids. NHP retinal organoids had laminated structure and were composed of mature retinal cell types including cone and rod photoreceptors. Single-cell RNA sequencing was conducted at two time points; this allowed identification of cell types and developmental trajectory characterization of the developing organoids. Important differences between rhesus and human cells were measured regarding the timing and efficiency of retinal organoid differentiation. While the culture of NHP-derived iPSCs is relatively difficult compared to that of human stem cells, the generation of retinal organoids from NHP iPSCs is feasible and may be less time-consuming due to an intrinsically faster timing of retinal differentiation. CONCLUSIONS: Retinal organoids produced from rhesus monkey iPSCs using established protocols differentiate through the stages of organoid development faster than those derived from human stem cells. The production of NHP retinal organoids may be advantageous to reduce experimental time for basic biology studies in retinogenesis as well as for preclinical trials in NHPs studying retinal allograft transplantation.

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.

Assessing Variant Causality and Severity Using Retinal Pigment Epithelial Cells Derived from Stargardt Disease Patients.

Purpose: Modern molecular genetics has revolutionized gene discovery, genetic diagnoses, and precision medicine yet many patients remain unable to benefit from these advances as disease-causing variants remain elusive for up to half of Mendelian genetic disorders. Patient-derived induced pluripotent stem (iPS) cells and transcriptomics were used to identify the fate of unsolved ABCA4 alleles in patients with Stargardt disease. Methods: Multiple independent iPS lines were generated from skin biopsies of three patients with Stargardt disease harboring a single identified pathogenic ABCA4 variant. Derived retinal pigment epithelial cells (dRPE) from a normal control and patient cells were subjected to RNA-Seq on the Novaseq6000 platform, analyzed using DESeq2 with calculation of allele specific imbalance from the pathogenic or a known linked variant. Protein analysis was performed using the automated Simple Western system. Results: Nine dRPE samples were generated, with transcriptome analysis on eight. Allele-specific expression indicated normal transcripts expressed from splice variants albeit at low levels, and missense transcripts expressed at near-normal levels. Corresponding protein was not easily detected. Patient phenotype correlation indicated missense variants expressed at high levels have more deleterious outcomes. Transcriptome analysis suggests mitochondrial membrane biodynamics and the unfolded protein response pathway may be relevant in Stargardt disease. Conclusions: Patient-specific iPS-derived RPE cells set the stage to assess non-expressing variants in difficult-to-detect genomic regions using easily biopsied tissue. Translational Relevance: This "Disease in a Dish" approach is likely to enhance the ability of patients to participate in and benefit from clinical trials while providing insights into perturbations in RPE biology.