Single-cell transcriptome of the mouse retinal pigment epithelium in response to a low-dose of doxorubicin.

Cellular senescence of the retinal pigment epithelium (RPE) is thought to play an important role in vision-threatening retinal degenerative diseases, such as age-related macular degeneration (AMD). However, the single-cell RNA profiles of control RPE tissue and RPE tissue exhibiting cellular senescence are not well known. We have analyzed the single-cell transcriptomes of control mice and mice with low-dose doxorubicin (Dox)-induced RPE senescence (Dox-RPE). Our results have identified 4 main subpopulations in the control RPE that exhibit heterogeneous biological activities and play roles in ATP synthesis, cell mobility/differentiation, mRNA processing, and catalytic activity. In Dox-RPE mice, cellular senescence mainly occurs in the specific cluster, which has been characterized by catalytic activity in the control RPE. Furthermore, in the Dox-RPE mice, 6 genes that have not previously been associated with senescence also show altered expression in 4 clusters. Our results might serve as a useful reference for the study of control and senescent RPE.

Geographical contrasts of Y-chromosomal haplogroups from wild and domestic goats reveal ancient migrations and recent introgressions.

By their paternal transmission, Y-chromosomal haplotypes are sensitive markers of population history and male-mediated introgression. Previous studies identified biallelic single-nucleotide variants in the SRY, ZFY and DDX3Y genes, which in domestic goats identified four major Y-chromosomal haplotypes, Y1A, Y1B, Y2A and Y2B, with a marked geographical partitioning. Here, we extracted goat Y-chromosomal variants from whole-genome sequences of 386 domestic goats (75 breeds) and seven wild goat species, which were generated by the VarGoats goat genome project. Phylogenetic analyses indicated domestic haplogroups corresponding to Y1B, Y2A and Y2B, respectively, whereas Y1A is split into Y1AA and Y1AB. All five haplogroups were detected in 26 ancient DNA samples from southeast Europe or Asia. Haplotypes from present-day bezoars are not shared with domestic goats and are attached to deep nodes of the trees and networks. Haplogroup distributions for 186 domestic breeds indicate ancient paternal population bottlenecks and expansions during migrations into northern Europe, eastern and southern Asia, and Africa south of the Sahara. In addition, sharing of haplogroups indicates male-mediated introgressions, most notably an early gene flow from Asian goats into Madagascar and the crossbreeding that in the 19th century resulted in the popular Boer and Anglo-Nubian breeds. More recent introgressions are those from European goats into the native Korean goat population and from Boer goat into Uganda, Kenya, Tanzania, Malawi and Zimbabwe. This study illustrates the power of the Y-chromosomal variants for reconstructing the history of domestic species with a wide geographical range.

Comparative genomics and selection analysis of Yeonsan Ogye black chicken with whole-genome sequencing.

Yeonsan Ogye (OGYE; Gallus gallus domesticus) is a rare indigenous chicken breed that inhabits the Korean Peninsula. This breed has completely black coloring, including plumage, skin, eyes, beak, and internal organs. Despite these unique morphological characteristics, the population of OGYE has declined without in-depth research into their genome research. Therefore, this study aimed to compare the whole genome of OGYE to 12 other chicken populations, including ancestral breed, commercial breeds, Chinese indigenous breeds, and Korean native chickens. We focused on revealing the selection signature of OGYE, which has occurred through environmental pressures in the Korean Peninsula. Genome-wide selection analysis has identified local adaptation traits, such as egg development, that contribute to fetal viability and innate immune response to prevent viral and microbes infection in OGYE. In particular, SPP1 (Secreted Phosphoprotein 1), HSP90AA1 (Heat Shock Protein 90 Alpha Family Class A Member 1), and P2RX4 (Purinergic Receptor P2X 4) could have considerable involvement in egg development and RNASEL (Ribonuclease L), BRIP1 (BRCA1 Interacting Protein C-terminal Helicase 1), and TLR4 (Toll-Like Receptor 4) are crucial for the determination of the innate immune response. This study revealed the unique genetic diversity of OGYE at the genome-wide level. Furthermore, we emphasized the sustainable management of genetic resources and formulated breeding strategies for livestock on the Korean Peninsula.

Genetic Differences between Physical Injury Patients With and Without Post-traumatic Syndrome: Focus on Secondary Findings and Potential Variants Revealed by Whole Exome Sequencing.

Objective: Sudden traumatic physical injuries often cause psychological distress, which may be associated with chronic disability. Although considerable effort has been expended to identify genetic predictors of post-traumatic stress disorder (PTSD) after traumatic events, genetic predictors of psychological distress in response to severe physical injuries have been yet to be elucidated using whole exome sequencing (WES). Here, the genetic architecture of post-traumatic syndrome (PTS), which encompasses a broad range of psychiatric disorders after traumatic events including depression, anxiety disorder, acute stress disorder, and PTSD, was explored using WES in severely physically injured patients, focusing on secondary findings and potential PTS-related variants. Methods: In total, 141 severely physically injured patients were consecutively recruited, and PTS was evaluated within 1 month of the injury. Secondary findings were analyzed according to PTS status. To identify PTS-related variants, genome-wide association analyses and the optimal sequencing kernel association test were performed. Results: Of the 141 patients, 88 (62%) experienced PTS. There were 108 disease-causing variants in severely physically injured patients. As secondary findings, the stress- and inflammation-related signaling pathways were enriched in the PTS patients, while the glucose metabolism pathway was enriched in those without PTS. However, no significant PTS-related variants were identified. Conclusion: Our findings suggest that genetic alterations in stress and inflammatory pathways might increase the likelihood of PTS immediately after severe physical injury. Future studies with larger samples and longitudinal designs are needed.

Targeting senescent retinal pigment epithelial cells facilitates retinal regeneration in mouse models of age-related macular degeneration.

Although age-related macular degeneration (AMD) is a multifactorial disorder with angiogenic, immune, and inflammatory components, the most common clinical treatment strategies are antiangiogenic therapies. However, these strategies are only applicable to neovascular AMD, which accounts for less than 20% of all AMD cases, and there are no FDA-approved drugs for the treatment of dry AMD, which accounts for ~ 80% of AMD cases. Here, we report that the elimination of senescent cells is a potential novel therapeutic approach for the treatment of all types of AMD. We identified senescent retinal pigment epithelium (RPE) cells in animal models of AMD and determined their contributions to retinal degeneration. We further confirmed that the clearance of senescent RPE cells with the MDM2-p53 inhibitor Nutlin-3a ameliorated retinal degeneration. These findings provide new insights into the use of senescent cells as a therapeutic target for the treatment of AMD.

Whole Exome Sequencing Identifies Novel De Novo Variants Interacting with Six Gene Networks in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a highly heritable condition caused by a combination of environmental and genetic factors such as de novo and inherited variants, as well as rare or common variants among hundreds of related genes. Previous genome-wide association studies have identified susceptibility genes; however, most ASD-associated genes remain undiscovered. This study aimed to examine rare de novo variants to identify genetic risk factors of ASD using whole exome sequencing (WES), functional characterization, and genetic network analyses of identified variants using Korean familial dataset. We recruited children with ASD and their biological parents. The clinical best estimate diagnosis of ASD was made according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5TM), using comprehensive diagnostic instruments. The final analyses included a total of 151 individuals from 51 families. Variants were identified and filtered using the GATK Best Practices for bioinformatics analysis, followed by genome alignments and annotation to the reference genome assembly GRCh37 (liftover to GRCh38), and further annotated using dbSNP 154 build databases. To evaluate allele frequencies of de novo variants, we used the dbSNP, gnomAD exome v2.1.1, and genome v3.0. We used Ingenuity Pathway Analysis (IPA, Qiagen) software to construct networks using all identified de novo variants with known autism-related genes to find probable relationships. We identified 36 de novo variants with potential relations to ASD; 27 missense, two silent, one nonsense, one splice region, one splice site, one 5' UTR, and one intronic SNV and two frameshift deletions. We identified six networks with functional relationships. Among the interactions between de novo variants, the IPA assay found that the NF-κB signaling pathway and its interacting genes were commonly observed at two networks. The relatively small cohort size may affect the results of novel ASD genes with de novo variants described in our findings. We did not conduct functional experiments in this study. Because of the diversity and heterogeneity of ASD, the primary purpose of this study was to investigate probable causative relationships between novel de novo variants and known autism genes. Additionally, we based functional relationships with known genes on network analysis rather than on statistical analysis. We identified new variants that may underlie genetic factors contributing to ASD in Korean families using WES and genetic network analyses. We observed novel de novo variants that might be functionally linked to ASD, of which the variants interact with six genetic networks.

Gene Expression Profile in Similar Tissues Using Transcriptome Sequencing Data of Whole-Body Horse Skeletal Muscle.

Horses have been studied for exercise function rather than food production, unlike most livestock. Therefore, the role and characteristics of tissue landscapes are critically understudied, except for certain muscles used in exercise-related studies. In the present study, we compared RNA-Seq data from 18 Jeju horse skeletal muscles to identify differentially expressed genes (DEGs) between tissues that have similar functions and to characterize these differences. We identified DEGs between different muscles using pairwise differential expression (DE) analyses of tissue transcriptome expression data and classified the samples using the expression values of those genes. Each tissue was largely classified into two groups and their subgroups by k-means clustering, and the DEGs identified in comparison between each group were analyzed by functional/pathway level using gene set enrichment analysis and gene level, confirming the expression of significant genes. As a result of the analysis, the differences in metabolic properties like glycolysis, oxidative phosphorylation, and exercise adaptation of the groups were detected. The results demonstrated that the biochemical and anatomical features of a wide range of muscle tissues in horses could be determined through transcriptome expression analysis, and provided proof-of-concept data demonstrating that RNA-Seq analysis can be used to classify and study in-depth differences between tissues with similar properties.

GMStool: GWAS-based marker selection tool for genomic prediction from genomic data.

The increased accessibility to genomic data in recent years has laid the foundation for studies to predict various phenotypes of organisms based on the genome. Genomic prediction collectively refers to these studies, and it estimates an individual's phenotypes mainly using single nucleotide polymorphism markers. Typically, the accuracy of these genomic prediction studies is highly dependent on the markers used; however, in practice, choosing optimal markers with high accuracy for the phenotype to be used is a challenging task. Therefore, we present a new tool called GMStool for selecting optimal marker sets and predicting quantitative phenotypes. The GMStool is based on a genome-wide association study (GWAS) and heuristically searches for optimal markers using statistical and machine-learning methods. The GMStool performs the genomic prediction using statistical and machine/deep-learning models and presents the best prediction model with the optimal marker-set. For the evaluation, the GMStool was tested on real datasets with four phenotypes. The prediction results showed higher performance than using the entire markers or the GWAS-top markers, which have been used frequently in prediction studies. Although the GMStool has several limitations, it is expected to contribute to various studies for predicting quantitative phenotypes. The GMStool written in R is available at www.github.com/JaeYoonKim72/GMStool .

The clonal repopulation of HSPC gene modified with anti-HIV-1 RNAi is not affected by preexisting HIV-1 infection.

Despite advances in hematopoietic stem/progenitor cell (HSPC) transplant for HIV-1-infected patients, the impact of a preexisting HIV-1 infection on the engraftment and clonal repopulation of HSPCs remains poorly understood. We have developed a long terminal repeat indexing-mediated integration site sequencing (LTRi-Seq) method that provides a multiplexed clonal quantitation of both anti-HIV-1 RNAi (RNA interference) gene-modified and control vector-modified cell populations, together with HIV-1-infected cells-all within the same animal. In our HIV-1-preinfected humanized mice, both therapeutic and control HSPCs repopulated efficiently without abnormalities. Although the HIV-1-mediated selection of anti-HIV-1 RNAi-modified clones was evident in HIV-1-infected mice, the organ-to-organ and intra-organ clonal distributions in infected mice were indistinguishable from those in uninfected mice. HIV-1-infected cells showed clonal patterns distinct from those of HSPCs. Our data demonstrate that, despite the substantial impact of HIV-1 infection on CD4+ T cells, HSPC repopulation remains polyclonal, thus supporting the use of HSPC transplant for anti-HIV treatment.

Genetically, Dietary Sodium Intake Is Causally Associated with Salt-Sensitive Hypertension Risk in a Community-Based Cohort Study: a Mendelian Randomization Approach.

PURPOSE OF REVIEW: Excessive dietary salt intake is associated with an increased risk of hypertension. Salt sensitivity, i.e., an elevation in blood pressure in response to high dietary salt intake, has been associated with a high risk of cardiovascular disease and mortality. We investigated whether a causal association exists between dietary sodium intake and hypertension risk using Mendelian randomization (MR). RECENT FINDINGS: We performed an MR study using data from a large genome-wide association study comprising 15,034 Korean adults in a community-based cohort study. A total of 1282 candidate single nucleotide polymorphisms associated with dietary sodium intake, such as rs2960306, rs4343, and rs1937671, were selected as instrumental variables. The inverse variance weighted method was used to assess the evidence for causality. Higher dietary sodium intake was associated with salt-sensitive hypertension risk. The variants of SLC8E1 rs2241543 and ADD1 rs16843589 were strongly associated with increased blood pressure. In the logistic regression model, after adjusting for age, gender, smoking, drinking, exercise, and body mass index, the GRK4 rs2960306TT genotype was inversely associated with hypertension risk (OR, 0.356; 95% CI, 0.236-0.476). However, the 2350GG genotype (ACE rs4343) exhibited a 2.11-fold increased hypertension risk (OR, 2.114; 95% CI, 2.004-2.224) relative to carriers of the 2350AA genotype, after adjusting for confounders. MR analysis revealed that the odds ratio for hypertension per 1 mg/day increment of dietary sodium intake was 2.24 in participants with the PRKG1 rs12414562 AA genotype. Our findings suggest that dietary sodium intake may be causally associated with hypertension risk.

Bioinformatics services for analyzing massive genomic datasets.

The explosive growth of next-generation sequencing data has resulted in ultra-large-scale datasets and ensuing computational problems. In Korea, the amount of genomic data has been increasing rapidly in the recent years. Leveraging these big data requires researchers to use large-scale computational resources and analysis pipelines. A promising solution for addressing this computational challenge is cloud computing, where CPUs, memory, storage, and programs are accessible in the form of virtual machines. Here, we present a cloud computing-based system, Bio-Express, that provides user-friendly, cost-effective analysis of massive genomic datasets. Bio-Express is loaded with predefined multi-omics data analysis pipelines, which are divided into genome, transcriptome, epigenome, and metagenome pipelines. Users can employ predefined pipelines or create a new pipeline for analyzing their own omics data. We also developed several web-based services for facilitating downstream analysis of genome data. Bio-Express web service is freely available at https://www.bioexpress.re.kr/.

Genome-wide association and epistatic interactions of flowering time in soybean cultivar.

Genome-wide association studies (GWAS) have enabled the discovery of candidate markers that play significant roles in various complex traits in plants. Recently, with increased interest in the search for candidate markers, studies on epistatic interactions between single nucleotide polymorphism (SNP) markers have also increased, thus enabling the identification of more candidate markers along with GWAS on single-variant-additive-effect. Here, we focused on the identification of candidate markers associated with flowering time in soybean (Glycine max). A large population of 2,662 cultivated soybean accessions was genotyped using the 180k Axiom® SoyaSNP array, and the genomic architecture of these accessions was investigated to confirm the population structure. Then, GWAS was conducted to evaluate the association between SNP markers and flowering time. A total of 93 significant SNP markers were detected within 59 significant genes, including E1 and E3, which are the main determinants of flowering time. Based on the GWAS results, multilocus epistatic interactions were examined between the significant and non-significant SNP markers. Two significant and 16 non-significant SNP markers were discovered as candidate markers affecting flowering time via interactions with each other. These 18 candidate SNP markers mapped to 18 candidate genes including E1 and E3, and the 18 candidate genes were involved in six major flowering pathways. Although further biological validation is needed, our results provide additional information on the existing flowering time markers and present another option to marker-assisted breeding programs for regulating flowering time of soybean.

Dissection of soybean populations according to selection signatures based on whole-genome sequences.

BACKGROUND: Domestication and improvement processes, accompanied by selections and adaptations, have generated genome-wide divergence and stratification in soybean populations. Simultaneously, soybean populations, which comprise diverse subpopulations, have developed their own adaptive characteristics enhancing fitness, resistance, agronomic traits, and morphological features. The genetic traits underlying these characteristics play a fundamental role in improving other soybean populations. RESULTS: This study focused on identifying the selection signatures and adaptive characteristics in soybean populations. A core set of 245 accessions (112 wild-type, 79 landrace, and 54 improvement soybeans) selected from 4,234 soybean accessions was re-sequenced. Their genomic architectures were examined according to the domestication and improvement, and accessions were then classified into 3 wild-type, 2 landrace, and 2 improvement subgroups based on various population analyses. Selection and gene set enrichment analyses revealed that the landrace subgroups have selection signals for soybean-cyst nematode HG type 0 and seed development with germination, and that the improvement subgroups have selection signals for plant development with viability and seed development with embryo development, respectively. The adaptive characteristic for soybean-cyst nematode was partially underpinned by multiple resistance accessions, and the characteristics related to seed development were supported by our phenotypic findings for seed weights. Furthermore, their adaptive characteristics were also confirmed as genome-based evidence, and unique genomic regions that exhibit distinct selection and selective sweep patterns were revealed for 13 candidate genes. CONCLUSIONS: Although our findings require further biological validation, they provide valuable information about soybean breeding strategies and present new options for breeders seeking donor lines to improve soybean populations.

Korean soybean core collection: Genotypic and phenotypic diversity population structure and genome-wide association study.

A core collection is a subset that represents genetic diversity of the total collection. Soybean (Glycine max (L.) Merr.) is one of major food and feed crops. It is the world's most cultivated annual herbaceous legume. Constructing a core collection for soybean could play a pivotal role in conserving and utilizing its genetic variability for research and breeding programs. To construct and evaluate a Korean soybean core collection, genotypic and phenotypic data as well as population structure, were analyzed. The Korean soybean core collection consisted of 430 accessions selected from 2,872 collections based on Affymetrix Axiom® 180k SoyaSNP array data. The core collection represented 99% of genotypic diversity of the total collection. Analysis of population structure clustered the core collection into five subpopulations. Accessions from South Korea and North Korea were distributed across five subpopulations. Analysis of molecular variance indicated that only 2.01% of genetic variation could be explained by geographic origins while 16.18% of genetic variation was accounted for by subpopulations. Genome-wide association study (GWAS) for days to flowering, flower color, pubescent color, and growth habit confirmed that the core collection had the same genetic diversity for tested traits as the total collection. The Korean soybean core collection was constructed based on genotypic information of the 180k SNP data. Size and phenotypic diversity of the core collection accounted for approximately 14.9% and 18.1% of the total collection, respectively. GWAS of core and total collections successfully confirmed loci associated with tested traits. Consequently, the present study showed that the Korean soybean core collection could provide fundamental and practical material and information for both soybean genetic research and breeding programs.

Discovery of Genomic Characteristics and Selection Signatures in Korean Indigenous Goats Through Comparison of 10 Goat Breeds.

Indigenous breeds develop their own genomic characteristics by adapting to local environments or cultures over long periods of time. Most of them are not particularly productive in commercial terms, but they have abilities to survive in harsh environments or tolerate to specific diseases. Their adaptive characteristics play an important role as genetic materials for improving commercial breeds. As a step toward this goal, we analyzed the genome of Korean indigenous goats within 10 goat breeds. We collected 136 goat individuals by sequencing 46 new goats and employing 90 publicly available goats. Our whole-genome data was comprised of three indigenous breeds (Korean indigenous goat, Iranian indigenous goat, and Moroccan indigenous goat; n = 29, 18, 20), six commercial breeds (Saanen, Boer, Anglo-Nubian, British Alpine, Alpine, and Korean crossbred; n = 16, 11, 5, 5, 2, 13), and their ancestral species (Capra aegagrus; n = 17). We identified that the Iranian indigenous goat and the Moroccan indigenous goat have relatively similar genomic characteristics within a large category of genomic diversity but found that the Korean indigenous goat has unique genomic characteristics distinguished from the other nine breeds. Through population analysis, we confirmed that these characteristics have resulted from a near-isolated environment with strong genetic drift. The Korean indigenous goat experienced a severe genetic bottleneck upon entering the Korean Peninsula about 2,000 years ago, and has subsequently rarely experienced genetic interactions with other goat breeds. From selection analysis and gene-set enrichment analysis, we revealed selection signals for Salmonella infection and cardiomyopathy in the genome of the Korean indigenous goat. These adaptive characteristics were further identified with genomic-based evidence. We uncovered genomic regions of selective sweeps in the LBP and BPI genes (Salmonella infection) and the TTN and ITGB6 genes (cardiomyopathy), among several candidate genes. Our research presents unique genomic characteristics and distinctive selection signals of the Korean indigenous goat based on the extensive comparison. Although the adaptive traits require further validation through biological experiments, our findings are expected to provide a direction for future biodiversity conservation strategies and to contribute another option to genomic-based breeding programmes for improving the viability of Capra hircus.

Investigation of Gene Expression and DNA Methylation From Seven Different Brain Regions of a Crab-Eating Monkey as Determined by RNA-Seq and Whole-Genome Bisulfite Sequencing.

The crab-eating monkey is widely used in biomedical research for pharmacological experiments. Epigenetic regulation in the brain regions of primates involves complex patterns of DNA methylation. Previous studies of methylated CpG-binding domains using microarray technology or peak identification of sequence reads mostly focused on developmental stages or disease, rather than normal brains. To identify correlations between gene expression and DNA methylation levels that may be related to transcriptional regulation, we generated RNA-seq and whole-genome bisulfite sequencing data from seven different brain regions from a single crab-eating monkey. We identified 92 genes whose expression levels were significantly correlated, positively or negatively, with DNA methylation levels. Among them, 11 genes exhibited brain region-specific characteristics, and their expression patterns were strongly correlated with DNA methylation level. Nine genes (SLC2A5, MCM5, DRAM1, TTC12, DHX40, COR01A, LRAT, FLVCR2, and PTER) had effects on brain and eye function and development, and two (LHX6 and MEST) were previously identified as genes in which DNA methylation levels change significantly in the promoter region and are therefore considered brain epigenetic markers. Furthermore, we characterized DNA methylation of repetitive elements at the whole genome through repeat annotation at single-base resolution. Our results reveal the diverse roles of DNA methylation at single-base resolution throughout the genome and reflect the epigenetic variations in adult brain tissues.