The loci of environmental adaptation in a model eukaryote.

While the underlying genetic changes have been uncovered in some cases of adaptive evolution, the lack of a systematic study prevents a general understanding of the genomic basis of adaptation. For example, it is unclear whether protein-coding or noncoding mutations are more important to adaptive evolution and whether adaptations to different environments are brought by genetic changes distributed in diverse genes and biological processes or concentrated in a core set. We here perform laboratory evolution of 3360 Saccharomyces cerevisiae populations in 252 environments of varying levels of stress. We find the yeast adaptations to be primarily fueled by large-effect coding mutations overrepresented in a relatively small gene set, despite prevalent antagonistic pleiotropy across environments. Populations generally adapt faster in more stressful environments, partly because of greater benefits of the same mutations in more stressful environments. These and other findings from this model eukaryote help unravel the genomic principles of environmental adaptation.

Divergent evolution of male-determining loci on proto-Y chromosomes of the housefly.

Houseflies provide a good experimental model to study the initial evolutionary stages of a primary sex-determining locus because they possess different recently evolved proto-Y chromosomes that contain male-determining loci (M) with the same male-determining gene, Mdmd. We investigate M-loci genomically and cytogenetically revealing distinct molecular architectures among M-loci. M on chromosome V (MV) has two intact Mdmd copies in a palindrome. M on chromosome III (MIII) has tandem duplications containing 88 Mdmd copies (only one intact) and various repeats, including repeats that are XY-prevalent. M on chromosome II (MII) and the Y (MY) share MIII-like architecture, but with fewer repeats. MY additionally shares MV-specific sequence arrangements. Based on these data and karyograms using two probes, one derives from MIII and one Mdmd-specific, we infer evolutionary histories of polymorphic M-loci, which have arisen from unique translocations of Mdmd, embedded in larger DNA fragments, and diverged independently into regions of varying complexity.

[Identification of novel genetic loci associated with major depressive disorder and the hippocampus in a European population using the condFDR method].

OBJECTIVE: To identify additional loci associated with depression and the hippocampus (HIP) through genome-wide association study. METHODS: The depression-related genome-wide association study (GWAS) meta summary data was downloaded from the official website of the Psychiatric Genomics Consortium, which had involved 170 756 cases and 329 443 controls. The left and right hippocampal volume GWAS data sets were downloaded from the UK Biobank, which involved 33 224 participants. The conditional false discovery rate (condFDR) was used to identify novel genetic loci for depression and left and right hippocampal volumes, and a conjunctional false discovery rate (conjFDR) was used to evaluate the enrichment of pleiotropic loci between depression and left and right hippocampal volumes. RESULTS: Respectively, 7, 13, and 12 new loci have been associated with depression, left hippocampal volume and right hippocampal volume, with a significant threshold of condFDR < 0.01. A site of rs1267073 locus was found to be shared by the depression and right hippocampal volume with a threshold of conjFDR < 0.01. CONCLUSION: Above findings have provided more insights into the genetic mechanisms underlying the volume of hippocampus and the risk for depression. The results may also provide evidence for future clinical trials for treating depression.

Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua).

BACKGROUND: The Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod's immune response on a molecular level. RESULTS: A comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5'RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes. CONCLUSIONS: This study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities.

The advent of locus-specific protein recruitment technologies has enabled a new class of studies in chromatin biology. Epigenome editors (EEs) enable biochemical modifications of chromatin at almost any specific endogenous locus. Their locus-specificity unlocks unique information including the functional roles of distinct modifications at specific genomic loci. Given the growing interest in using these tools for biological and translational studies, there are many specific design considerations depending on the scientific question or clinical need. Here, we present and discuss important design considerations and challenges regarding the biochemical and locus specificities of epigenome editors. These include how to: account for the complex biochemical diversity of chromatin; control for potential interdependency of epigenome editors and their resultant modifications; avoid sequestration effects; quantify the locus specificity of epigenome editors; and improve locus-specificity by considering concentration, affinity, avidity, and sequestration effects.

Protocol for DNA Methylation Editing of Imprinted Loci and Assessment of the Effects.

In this chapter, we present an experimental protocol to conduct DNA methylation editing experiments, that is, to induce loss or gain of DNA methylation, targeting Dlk1-Dio3 imprinted domain, a well-studied imprinted locus, in ES cells. In this protocol, plasmid vectors expressing the DNA methylation editing tools, combining the CRISPR/dCas9 system and the SunTag system coupled to a DNA methyltransferase or a TET enzyme, are introduced into cells for transient expression. By employing this strategy, researchers can effectively investigate a distinct DNA methylation signature that has an impact on the imprinting status, including gene expression and histone modifications, across the entire domain. We also describe strategies for allele-specific quantitative analyses of DNA methylation, gene expression, and histone modifications and binding protein levels for assessing the imprinting state of the locus.

Co-expression in tissue-specific gene networks links genes in cancer-susceptibility loci to known somatic driver genes.

BACKGROUND: The genetic background of cancer remains complex and challenging to integrate. Many somatic mutations within genes are known to cause and drive cancer, while genome-wide association studies (GWAS) of cancer have revealed many germline risk factors associated with cancer. However, the overlap between known somatic driver genes and positional candidate genes from GWAS loci is surprisingly small. We hypothesised that genes from multiple independent cancer GWAS loci should show tissue-specific co-regulation patterns that converge on cancer-specific driver genes. RESULTS: We studied recent well-powered GWAS of breast, prostate, colorectal and skin cancer by estimating co-expression between genes and subsequently prioritising genes that show significant co-expression with genes mapping within susceptibility loci from cancer GWAS. We observed that the prioritised genes were strongly enriched for cancer drivers defined by COSMIC, IntOGen and Dietlein et al. The enrichment of known cancer driver genes was most significant when using co-expression networks derived from non-cancer samples of the relevant tissue of origin. CONCLUSION: We show how genes within risk loci identified by cancer GWAS can be linked to known cancer driver genes through tissue-specific co-expression networks. This provides an important explanation for why seemingly unrelated sets of genes that harbour either germline risk factors or somatic mutations can eventually cause the same type of disease.

Analysis of Fluorescent Proteins for Observing Single Gene Locus in a Live and Fixed Escherichia coli Cell.

Fluorescent proteins (FPs) are essential tools for advanced microscopy techniques such as super-resolution imaging, single-particle tracking, and quantitative single-molecule counting. Various FPs fused to DNA-binding proteins have been used to observe the subcellular location and movement of specific gene loci in living and fixed bacterial cells. However, quantitative assessments of the properties of FPs for gene locus measurements are still lacking. Here, we assessed various FPs to observe specific gene loci in live and fixed Escherichia coli cells using a fluorescent repressor-operator binding system (FROS), tet operator-Tet repressor proteins (TetR). Tsr-fused FPs were used to assess the intensity and photostability of various FPs (five red FPs: mCherry2, FusionRed, mRFP, mCrimson3, and dKatushka; and seven yellow FPs: SYFP2, Venus, mCitrine, YPet, mClover3, mTopaz, and EYFP) at the single-molecule level in living cells. These FPs were then used for gene locus measurements using FROS. Our results indicate that TetR-mCrimson3 (red) and TetR-EYFP (yellow) had better properties for visualizing gene loci than the other TetR-FPs. Furthermore, fixation procedures affected the clustering of diffusing TetR-FPs and altered the locations of the TetR-FP foci. Fixation with formaldehyde consistently disrupted proper DNA locus observations using TetR-FPs. Notably, the foci measured using TetR-mCrimson3 remained close to their original positions in live cells after glyoxal fixation. This in vivo study provides a cell-imaging guide for the use of FPs for gene-locus observation in E. coli and a scheme for evaluating the use of FPs for other cell-imaging purposes.

Genome-wide association study identifies novel susceptible loci and evaluation of polygenic risk score for chronic obstructive pulmonary disease in a Taiwanese population.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) describes a group of progressive lung diseases causing breathing difficulties. While COPD development typically involves a complex interplay between genetic and environmental factors, genetics play a role in disease susceptibility. This study used genome-wide association studies (GWAS) and polygenic risk score (PRS) to elucidate the genetic basis for COPD in Taiwanese patients. RESULTS: GWAS was performed on a Taiwanese COPD case-control cohort with a sample size of 5,442 cases and 17,681 controls. Additionally, the PRS was calculated and assessed in our target groups. GWAS results indicate that although there were no single nucleotide polymorphisms (SNPs) of genome-wide significance, prominent COPD susceptibility loci on or nearby genes such as WWTR1, EXT1, INTU, MAP3K7CL, MAMDC2, BZW1/CLK1, LINC01197, LINC01894, and CFAP95 (C9orf135) were identified, which had not been reported in previous studies. Thirteen susceptibility loci, such as CHRNA4, AFAP1, and DTWD1, previously reported in other populations were replicated and confirmed to be associated with COPD in Taiwanese populations. The PRS was determined in the target groups using the summary statistics from our base group, yielding an effective association with COPD (odds ratio [OR] 1.09, 95% confidence interval [CI] 1.02-1.17, p = 0.011). Furthermore, replication a previous lung function trait PRS model in our target group, showed a significant association of COPD susceptibility with PRS of Forced Expiratory Volume in one second (FEV1)/Forced Vital Capacity (FCV) (OR 0.89, 95% CI 0.83-0.95, p = 0.001). CONCLUSIONS: Novel COPD-related genes were identified in the studied Taiwanese population. The PRS model, based on COPD or lung function traits, enables disease risk estimation and enhances prediction before suffering. These results offer new perspectives on the genetics of COPD and serve as a basis for future research.

Deletion of the Murine Ortholog of the Human 9p21.3 Locus Leads to Insulin Resistance and Obesity in Hypercholesterolemic Mice.

The 9p21.3 genomic locus is a hot spot for disease-associated single-nucleotide polymorphisms (SNPs), and its strongest associations are with coronary artery disease (CAD). The disease-associated SNPs are located within the sequence of a long noncoding RNA ANRIL, which potentially contributes to atherogenesis by regulating vascular cell stress and proliferation, but also affects pancreatic ß-cell proliferation. Altered expression of a neighboring gene, CDKN2B, has been also recognized to correlate with obesity and hepatic steatosis in people carrying the risk SNPs. In the present study, we investigated the impact of 9p21.3 on obesity accompanied by hyperlipidemia in mice carrying a deletion of the murine ortholog for the 9p21.3 (Chr4Δ70/Δ70) risk locus in hyperlipidemic Ldlr-/-ApoB100/100 background. The Chr4Δ70/Δ70 mice showed decreased mRNA expression of insulin receptors in white adipose tissue already at a young age, which developed into insulin resistance and obesity by aging. In addition, the Sirt1-Ppargc1a-Ucp2 pathway was downregulated together with the expression of Cdkn2b, specifically in the white adipose tissue in Chr4Δ70/Δ70 mice. These results suggest that the 9p21.3 locus, ANRIL lncRNA, and their murine orthologues may regulate the key energy metabolism pathways in a white adipose tissue-specific manner in the presence of hypercholesterolemia, thus contributing to the pathogenesis of metabolic syndrome.

Accelerated evolution in the human lineage led to gain and loss of transcriptional enhancers in the RBFOX1 locus.

A long-standing goal of evolutionary biology is to decode how changes in gene regulatory networks contribute to human-specific traits. Human accelerated regions (HARs) are prime candidates for driving gene regulatory modifications in human development. The RBFOX1 locus is densely populated with HARs, providing a set of potential regulatory elements that could have changed its expression in the human lineage. Here, we examined the role of RBFOX1-HARs using transgenic zebrafish reporter assays and identified 15 transcriptional enhancers that are active in the developing nervous system, 9 of which displayed differential activity between the human and chimpanzee sequences. The engineered loss of two selected RBFOX1-HARs in knockout mouse models modified Rbfox1 expression at specific developmental stages and tissues in the brain, influencing the expression and splicing of a high number of Rbfox1 target genes. Our results provided insight into the spatial and temporal changes in gene expression driven by RBFOX1-HARs.

Genome-Wide Identification of Specific Genetic Loci Common to Sheep and Goat.

Sheep and goat may become carriers of some zoonotic diseases. They are important livestock and experimental model animals for human beings. The fast and accurate identification of genetic materials originating from sheep and goat can prevent and inhibit the spread of some zoonotic diseases, monitor market product quality, and maintain the stability of animal husbandry and food industries. This study proposed a methodology for identifying sheep and goat common specific sites from a genome-wide perspective. A total of 150 specific sites were selected from three data sources, including the coding sequences of single copy genes from nine species (sheep, goat, cow, pig, dog, horse, human, mouse, and chicken), the dbSNPs for these species, and human 100-way alignment data. These 150 sites exhibited low intraspecific heterogeneity in the resequencing data of 1450 samples from five species (sheep, goat, cow, pig, and chicken) and high interspecific divergence in the human 100-way alignment data after quality control. The results were proven to be reliable at the data level. Using the process proposed in this study, specific sites of other species can be screened, and genome-level species identification can be performed using the screened sites.

Preliminary study of identified novel susceptibility loci for HAPE risk in a Chinese male Han population.

High altitude pulmonary edema (HAPE) is a life-threatening form of non-cardiogenic pulmonary edema. In recent years, association studies have become the main method for identifying HAPE genetic loci. A genome-wide association study (GWAS) of HAPE risk-associated loci was performed in Chinese male Han individuals (164 HAPE cases and 189 healthy controls) by the Precision Medicine Diversity Array Chip with 2,771,835 loci (Applied Biosystems Axiom™). Eight overlapping candidate loci in CCNG2, RP11-445O3.2, NUPL1 and WWOX were finally selected. In silico functional analyses displayed the PPI network, functional enrichment and signal pathways related to CCNG2, NUPL1, WWOX and NRXN1. This study provides data supplements for HAPE susceptibility gene loci and new insights into HAPE susceptibility.

A set of circulating microRNAs belonging to the 14q32 chromosome locus identifies two subgroups of individuals with recent-onset type 1 diabetes.

Circulating microRNAs (miRNAs) are linked to the onset and progression of type 1 diabetes mellitus (T1DM), thus representing potential disease biomarkers. In this study, we employed a multiplatform sequencing approach to analyze circulating miRNAs in an extended cohort of prospectively evaluated recent-onset T1DM individuals from the INNODIA consortium. Our findings reveal that a set of miRNAs located within T1DM susceptibility chromosomal locus 14q32 distinguishes two subgroups of individuals. To validate our results, we conducted additional analyses on a second cohort of T1DM individuals, confirming the identification of these subgroups, which we have named cluster A and cluster B. Remarkably, cluster B T1DM individuals, who exhibit increased expression of a set of 14q32 miRNAs, show better glycemic control and display a different blood immunomics profile. Our findings suggest that this set of circulating miRNAs can identify two different T1DM subgroups with distinct blood immunomics at baseline and clinical outcomes during follow-up.

Genome-wide association study of the common retinal disorder epiretinal membrane: Significant risk loci in each of three American populations.

Epiretinal membrane (ERM) is a common retinal condition characterized by the presence of fibrocellular tissue on the retinal surface, often with visual distortion and loss of visual acuity. We studied European American (EUR), African American (AFR), and Latino (admixed American, AMR) ERM participants in the Million Veteran Program (MVP) for genome-wide association analysis-a total of 38,232 case individuals and 557,988 control individuals. We completed a genome-wide association study (GWAS) in each population separately, and then results were meta-analyzed. Genome-wide significant (GWS) associations were observed in all three populations studied: 31 risk loci in EUR subjects, 3 in AFR, and 2 in AMR, with 48 in trans-ancestry meta-analysis. Many results replicated in the FinnGen sample. Several GWS variants associate to alterations in gene expression in the macula. ERM showed significant genetic correlation to multiple traits. Pathway enrichment analyses implicated collagen and collagen-adjacent mechanisms, among others. This well-powered ERM GWAS identified novel genetic associations that point to biological mechanisms for ERM.

Sequential breakdown of the Cf-9 leaf mould resistance locus in tomato by Fulvia fulva.

Leaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which encodes five paralogous receptor-like proteins. Two of these proteins confer resistance: Cf-9C recognises the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that can overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B. Comparative genomics, transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a world-wide strain collection. A strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. This research showcases how F. fulva has evolved to sequentially break down the Cf-9 locus and stresses the urgent need for commercial tomato cultivars that carry novel, stacked resistance genes active against this pathogen.

Genome-wide analyses reveal a potential role for the MAPT, MOBP, and APOE loci in sporadic frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most common cause of early-onset dementia after Alzheimer disease (AD). Efforts in the field mainly focus on familial forms of disease (fFTDs), while studies of the genetic etiology of sporadic FTD (sFTD) have been less common. In the current work, we analyzed 4,685 sFTD cases and 15,308 controls looking for common genetic determinants for sFTD. We found a cluster of variants at the MAPT (rs199443; p = 2.5 × 10-12, OR = 1.27) and APOE (rs6857; p = 1.31 × 10-12, OR = 1.27) loci and a candidate locus on chromosome 3 (rs1009966; p = 2.41 × 10-8, OR = 1.16) in the intergenic region between RPSA and MOBP, contributing to increased risk for sFTD through effects on expression and/or splicing in brain cortex of functionally relevant in-cis genes at the MAPT and RPSA-MOBP loci. The association with the MAPT (H1c clade) and RPSA-MOBP loci may suggest common genetic pleiotropy across FTD and progressive supranuclear palsy (PSP) (MAPT and RPSA-MOBP loci) and across FTD, AD, Parkinson disease (PD), and cortico-basal degeneration (CBD) (MAPT locus). Our data also suggest population specificity of the risk signals, with MAPT and APOE loci associations mainly driven by Central/Nordic and Mediterranean Europeans, respectively. This study lays the foundations for future work aimed at further characterizing population-specific features of potential FTD-discriminant APOE haplotype(s) and the functional involvement and contribution of the MAPT H1c haplotype and RPSA-MOBP loci to pathogenesis of sporadic forms of FTD in brain cortex.

Five autism-associated transcriptional regulators target shared loci proximal to brain-expressed genes.

Many autism spectrum disorder (ASD)-associated genes act as transcriptional regulators (TRs). Chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify the regulatory targets of ARID1B, BCL11A, FOXP1, TBR1, and TCF7L2, ASD-associated TRs in the developing human and mouse cortex. These TRs shared substantial overlap in the binding sites, especially within open chromatin. The overlap within a promoter region, 1-2,000 bp upstream of the transcription start site, was highly predictive of brain-expressed genes. This signature was observed in 96 out of 102 ASD-associated genes. In vitro CRISPRi against ARID1B and TBR1 delineated downstream convergent biology in mouse cortical cultures. After 8 days, NeuN+ and CALB+ cells were decreased, GFAP+ cells were increased, and transcriptomic signatures correlated with the postmortem brain samples from individuals with ASD. We suggest that functional convergence across five ASD-associated TRs leads to shared neurodevelopmental outcomes of haploinsufficient disruption.

A common flanking variant is associated with enhanced stability of the FGF14-SCA27B repeat locus.

The factors driving or preventing pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14 (GAA)·(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a common 5'-flanking variant in 70.34% of alleles analyzed (3,463/4,923) that represents the phylogenetically ancestral allele and is present on all major haplotypes. This common sequence variation is present nearly exclusively on nonpathogenic alleles with fewer than 30 GAA-pure triplets and is associated with enhanced stability of the repeat locus upon intergenerational transmission and increased Fiber-seq chromatin accessibility.

Identification of novel genetic susceptibility loci for calcium-containing kidney stone disease by genome-wide association study and polygenic risk score in a Taiwanese population.

Approximately 80% of kidney stone diseases contain calcium. Inherited genetic factors are among the variables that influence the development of calcium-containing kidney stone diseases (CKSD). Previous genome-wide association studies (GWAS) on stone diseases have been reported worldwide; however, these are not focused on calcium-containing stones. We conducted a GWAS to identify germline genetic polymorphisms associated with CKSD in a Medical Center in Taiwan; hence, this study was based primarily on a hospital-based database. CKSD was diagnosed using the chart records. Patients infected with urea-splitting-microorganisms and those with at least two urinary pH value below 5.5 were excluded. None of the patients had cystic stones based on stone analysis. Those over 40 years of age with no history of CKSD and no microscopic hematuria on urinalysis were considered as controls. The DNA isolated from the blood of 14,934 patients (63.7% male and 36.3% female) with CKSD and 29,868 controls (10,830 men and 19,038 women) at a medical center was genotyped for approximately 714,457 single nucleotide polymorphisms (SNPs) with minor allele frequency of ≥ 0.05. We used PLINK 1.9 to calculate the polygenic risk score (PRS) to investigate the association between CKSD and controls. The accuracy of the PRS was verified by dividing it into the training and testing groups. The statistical analyses were calculated with the area under the curve (AUC) using IBM SPSS version 22. We identified 432 susceptibility loci that reached a genome-wide threshold of P < 1.0 × 10- 5. A total of 132 SNPs reached a threshold of P < 5 × 10- 8 using a stricter definition of significance on chromosomes 4, 13, 16, 17, and 18. At the top locus of our study, SNPs in DGKH, PDILT, BCAS3, and ABCG2 have been previously reported. RN7SKP27, HDAC4, PCDH15, AP003068.2, and NFATC1 were novel findings in this study. PRS was adjusted for sex and age, resulting in an AUC of 0.65. The number of patients in the top quartile of PRS was 1.39 folds in the risk of CKSD than patients in the bottom quartile. Our data identified the significance of GWAS for patients with CKSD in a hospital-based study. The PRS also had a high AUC for discriminating patients with CKSD from controls. A total of 132 SNP loci of SNPs significantly associated with the development of CKSD. This first survey, which focused on patients with CKSD, will provide novel insights specific to CKSD and its potential clinical biomarkers.