Genomic Loci Research Articles

Genomic loci associated with grain yield under well-watered and water-stressed conditions in multiple bi-parental maize populations

Smallholder maize farming systems in sub-Saharan Africa (SSA) are vulnerable to drought-induced yield losses, which significantly impact food security and livelihoods within these communities. Mapping and characterizing genomic regions associated with water stress tolerance in tropical maize is essential for future breeding initiatives targeting this region. In this study, three biparental F3 populations composed of 753 families were evaluated in Kenya and Zimbabwe and genotyped with high-density single nucleotide polymorphism (SNP) markers. Quantitative trait loci maping was performed on these genotypes to dissect the genetic architecture for grain yield (GY), plant height (PH), ear height (EH) and anthesis-silking interval (ASI) under well-watered (WW) and water-stressed (WS) conditions. Across the studied maize populations, mean GY exhibited a range of 4.55–8.55 t/ha under WW and 1.29–5.59 t/ha under WS, reflecting a 31–59% reduction range under WS conditions. Genotypic and genotype-by-environment (G × E) variances were significant for all traits except ASI. Overall broad sense heritabilities for GY were low to high (0.25–0.60). For GY, these genetic parameters were decreased under WS conditions. Linkage mapping revealed a significant difference in the number of QTLs detected, with 93 identified under WW conditions and 41 under WS conditions. These QTLs were distributed across all maize chromosomes. For GY, eight and two major effect QTLs (>10% phenotypic variation explained) were detected under WW and WS conditions, respectively. Under WS conditions, Joint Linkage Association Mapping (JLAM) identified several QTLs with minor effects for GY and revealed genomic region overlaps in the studied populations. Across the studied water regimes, five-fold cross-validation showed moderate to high prediction accuracies (−0.15–0.90) for GY and other agronomic traits. Our findings demonstrate the polygenic nature of WS tolerance and highlights the immense potential of using genomic selection in improving genetic gain in maize breeding.

Machine learning unveils an immune-related DNA methylation profile in germline DNA from breast cancer patients

BackgroundThere is an unmet need for precise biomarkers for early non-invasive breast cancer detection. Here, we aimed to identify blood-based DNA methylation biomarkers that are associated with breast cancer.MethodsDNA methylation profiling was performed for 524 Asian Chinese individuals, comprising 256 breast cancer patients and 268 age-matched healthy controls, using the Infinium MethylationEPIC array. Feature selection was applied to 649,688 CpG sites in the training set. Predictive models were built by training three machine learning models, with performance evaluated on an independent test set. Enrichment analysis to identify transcription factors binding to regions associated with the selected CpG sites and pathway analysis for genes located nearby were conducted.ResultsA methylation profile comprising 51 CpGs was identified that effectively distinguishes breast cancer patients from healthy controls achieving an AUC of 0.823 on an independent test set. Notably, it outperformed all four previously reported breast cancer-associated methylation profiles. Enrichment analysis revealed enrichment of genomic loci associated with the binding of immune modulating AP-1 transcription factors, while pathway analysis of nearby genes showed an overrepresentation of immune-related pathways.ConclusionThis study has identified a breast cancer-associated methylation profile that is immune-related to potential for early cancer detection.

Understanding genome structure facilitates the use of wild lentil germplasm for breeding: A case study with shattering loci.

Plant breeders are generally reluctant to cross elite crop cultivars with their wild relatives to introgress novel desirable traits due to associated negative traits such as pod shattering. This results in a genetic bottleneck that could be reduced through better understanding of the genomic locations of the gene(s) controlling this trait. We integrated information on parental genomes, pod shattering data from multiple environments, and high-density genetic linkage maps to identify pod shattering quantitative trait loci (QTLs) in three lentil interspecific recombinant inbred line populations. The broad-sense heritability on a multi-environment basis varied from 0.46 (in LR-70, Lens culinaris × Lens odemensis) to 0.77 (in LR-68, Lens orientalis × L. culinaris). Genetic linkage maps of the interspecific populations revealed reciprocal translocations of chromosomal segments that differed among the populations, and which were associated with reduced recombination. LR-68 had a 2-5 translocation, LR-70 had 1-5, 2-6, and 2-7 translocations, and LR-86 had a 2-7 translocation in one parent relative to the other. Segregation distortion was also observed for clusters of single nucleotide polymorphisms on multiple chromosomes per population, further affecting introgression. Two major QTL, on chromosomes 4 and 7, were repeatedly detected in the three populations and contain several candidate genes. These findings will be of significant value for lentil breeders to strategically access novel superior alleles while minimizing the genetic impact of pod shattering from wild parents.

Impact of Interactions between Su(Hw)-Dependent Insulators on the Transvection Effect in Drosophila melanogaster.

Transvection is a phenomenon of interallelic communication in which enhancers can activate a specific promoter located on a homologous chromosome. Insulators play a significant role in ensuring functional interactions between enhancers and promoters. In the presented work, we created a model where two or three copies of the insulator are located next to enhancers and promoters localized on homologous chromosomes. Using the Su(Hw) insulator as a model, we showed that the functional interaction between a pair of insulators promotes enhancer-promoter trans-interactions. The interaction between the three insulators, on the contrary, can lead to the formation of chromatin loops that sterically hinder the full enhancer-promoter interaction. The results of the work suggest the participation of insulators in the regulation of homologous chromosome pairing and in communication between distant genomic loci.

Reliable detection of stochastic epigenetic mutations and associations with cardiovascular aging.

Stochastic epigenetic mutations (SEMs) have been proposed as novel aging biomarkers to capture heterogeneity in age-related DNA methylation changes. SEMs are defined as outlier methylation patterns at cytosine-guanine dinucleotide sites, categorized as hypermethylated (hyperSEM) or hypomethylated (hypoSEM) relative to a reference. Because SEMs are defined by their outlier status, it is critical to differentiate extreme values due to technical noise or data artifacts from those due to real biology. Using technical replicate data, we found SEM detection is not reliable: across 3 datasets, 24 to 39% of hypoSEM and 46 to 67% of hyperSEM are not shared between replicates. We identified factors influencing SEM reliability-including blood cell type composition, probe beta-value statistics, genomic location, and presence of SNPs. We used these factors in a training dataset to build a machine learning-based filter that removes unreliable SEMs, and found this filter enhances reliability in two independent validation datasets. We assessed associations between SEM loads and aging phenotypes in the Framingham Heart Study and discovered that associations with aging outcomes were in large part driven by hypoSEMs at baseline methylated probes and hyperSEMs at baseline unmethylated probes, which are the same subsets that demonstrate highest technical reliability. These aging associations were preserved after filtering out unreliable SEMs and were enhanced after adjusting for blood cell composition. Finally, we utilized these insights to formulate best practices for SEM detection and introduce a novel R package, SEMdetectR, which uses parallel programming for efficient SEM detection with comprehensive options for detection, filtering, and analysis.

Genetic mapping across autoimmune diseases reveals shared associations and mechanisms.

Autoimmune and inflammatory diseases are polygenic disorders of the immune system. Many genomic loci harbor risk alleles for several diseases, but the limited resolution of genetic mapping prevents determining whether the same allele is responsible, indicating a shared underlying mechanism. Here, using a collection of 129,058 cases and controls across 6 diseases, we show that ~40% of overlapping associations are due to the same allele. We improve fine-mapping resolution for shared alleles twofold by combining cases and controls across diseases, allowing us to identify more expression quantitative trait loci driven by the shared alleles. The patterns indicate widespread sharing of pathogenic mechanisms but not a single global autoimmune mechanism. Our approach can be applied to any set of traits and is particularly valuable as sample collections become depleted.

Genetic regulation of human brain proteome reveals proteins implicated in psychiatric disorders.

Psychiatric disorders are highly heritable yet polygenic, potentially involving hundreds of risk genes. Genome-wide association studies have identified hundreds of genomic susceptibility loci with susceptibility to psychiatric disorders; however, the contribution of these loci to the underlying psychopathology and etiology remains elusive. Here we generated deep human brain proteomics data by quantifying 11,608 proteins across 268 subjects using 11-plex tandem mass tag coupled with two-dimensional liquid chromatography-tandem mass spectrometry. Our analysis revealed 788 cis-acting protein quantitative trait loci associated with the expression of 883 proteins at a genome-wide false discovery rate <5%. In contrast to expression at the transcript level and complex diseases that are found to be mainly influenced by noncoding variants, we found protein expression level tends to be regulated by non-synonymous variants. We also provided evidence of 76 shared regulatory signals between gene expression and protein abundance. Mediation analysis revealed that for most (88%) of the colocalized genes, the expression levels of their corresponding proteins are regulated by cis-pQTLs via gene transcription. Using summary data-based Mendelian randomization analysis, we identified 4 proteins and 19 genes that are causally associated with schizophrenia. We further integrated multiple omics data with network analysis to prioritize candidate genes for schizophrenia risk loci. Collectively, our findings underscore the potential of proteome-wide linkage analysis in gaining mechanistic insights into the pathogenesis of psychiatric disorders.

Retrotransposons in embryogenesis and neurodevelopment.

Retrotransposable elements (RTEs) are genetic elements that can replicate and insert new copies into different genomic locations. RTEs have long been identified as 'parasitic genes', as their mobilization can cause mutations, DNA damage, and inflammation. Interestingly, high levels of retrotransposon activation are observed in early embryogenesis and neurodevelopment, suggesting that RTEs may possess functional roles during these stages of development. Recent studies demonstrate that RTEs can function as transcriptional regulatory elements through mechanisms such as chromatin organization and noncoding RNAs. It is clear, however, that RTE expression and activity must be restrained at some level during development, since overactivation of RTEs during neurodevelopment is associated with several developmental disorders. Further investigation is needed to understand the importance of RTE expression and activity during neurodevelopment and the balance between RTE-regulated development and RTE-mediated pathogenesis.

Risk Factors for Intracerebral Hemorrhage: Genome-Wide Association Study and Mendelian Randomization Analyses.

The genetic and nongenetic causes of intracerebral hemorrhage (ICH) remain obscure. The present study aimed to uncover the genetic and modifiable risk factors for ICH. We meta-analyzed genome-wide association study data from 3 European biobanks, involving 7605 ICH cases and 711 818 noncases, to identify the genomic loci linked to ICH. To uncover the potential causal associations of cardiometabolic and lifestyle factors with ICH, we performed Mendelian randomization analyses using genetic instruments identified in previous genome-wide association studies of the exposures and ICH data from the present genome-wide association study meta-analysis. We performed multivariable Mendelian randomization analyses to examine the independent associations of the identified risk factors with ICH and evaluate potential mediating pathways. We identified 1 ICH risk locus, located at the APOE genomic region. The lead variant in this locus was rs429358 (chr19:45411941), which was associated with an odds ratio of ICH of 1.17 (95% CI, 1.11-1.20; P=6.01×10-11) per C allele. Genetically predicted higher levels of body mass index, visceral adiposity, diastolic blood pressure, systolic blood pressure, and lifetime smoking index, as well as genetic liability to type 2 diabetes, were associated with higher odds of ICH after multiple testing corrections. Additionally, a genetic increase in waist-to-hip ratio and liability to smoking initiation were consistently associated with ICH, albeit at the nominal significance level (P<0.05). Multivariable Mendelian randomization analysis showed that the association between body mass index and ICH was attenuated on adjustment for type 2 diabetes and further that type 2 diabetes may be a mediator of the body mass index-ICH relationship. Our findings indicate that the APOE locus contributes to ICH genetic susceptibility in European populations. Excess adiposity, elevated blood pressure, type 2 diabetes, and smoking were identified as the chief modifiable cardiometabolic and lifestyle factors for ICH.

Regulatory landscape enrichment analysis (RLEA): a computational toolkit for non-coding variant enrichment and cell type prioritization

BackgroundAs genomic studies continue to implicate non-coding sequences in disease, testing the roles of these variants requires insights into the cell type(s) in which they are likely to be mediating their effects. Prior methods for associating non-coding variants with cell types have involved approaches using linkage disequilibrium or ontological associations, incurring significant processing requirements. GaiaAssociation is a freely available, open-source software that enables thousands of genomic loci implicated in a phenotype to be tested for enrichment at regulatory loci of multiple cell types in minutes, permitting insights into the cell type(s) mediating the studied phenotype.ResultsIn this work, we present Regulatory Landscape Enrichment Analysis (RLEA) by GaiaAssociation and demonstrate its capability to test the enrichment of 12,133 variants across the cis-regulatory regions of 44 cell types. This analysis was completed in 134.0 ± 2.3 s, highlighting the efficient processing provided by GaiaAssociation. The intuitive interface requires only four inputs, offers a collection of customizable functions, and visualizes variant enrichment in cell-type regulatory regions through a heatmap matrix. GaiaAssociation is available on PyPi for download as a command line tool or Python package and the source code can also be installed from GitHub at https://github.com/GreallyLab/gaiaAssociation.ConclusionsGaiaAssociation is a novel package that provides an intuitive and efficient resource to understand the enrichment of non-coding variants across the cis-regulatory regions of different cells, empowering studies seeking to identify disease-mediating cell types.

Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-PtprcaPepcb/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5′-tail to allow for simultaneous identification of the Ptprca and Ptprcb alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.

ESKEMAP: exact sketch-based read mapping

BackgroundGiven a sequencing read, the broad goal of read mapping is to find the location(s) in the reference genome that have a “similar sequence”. Traditionally, “similar sequence” was defined as having a high alignment score and read mappers were viewed as heuristic solutions to this well-defined problem. For sketch-based mappers, however, there has not been a problem formulation to capture what problem an exact sketch-based mapping algorithm should solve. Moreover, there is no sketch-based method that can find all possible mapping positions for a read above a certain score threshold.ResultsIn this paper, we formulate the problem of read mapping at the level of sequence sketches. We give an exact dynamic programming algorithm that finds all hits above a given similarity threshold. It runs in O(|t|+|p|+ℓ2)\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mathcal {O} (|t| + |p| + \\ell ^2)$$\\end{document} time and O(ℓlogℓ)\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mathcal {O} (\\ell \\log \\ell )$$\\end{document} space, where |t| is the number of k\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$k$$\\end{document}-mers inside the sketch of the reference, |p| is the number of k\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$k$$\\end{document}-mers inside the read’s sketch and ℓ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\ell$$\\end{document} is the number of times that k\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$k$$\\end{document}-mers from the pattern sketch occur in the sketch of the text. We evaluate our algorithm’s performance in mapping long reads to the T2T assembly of human chromosome Y, where ampliconic regions make it desirable to find all good mapping positions. For an equivalent level of precision as minimap2, the recall of our algorithm is 0.88, compared to only 0.76 of minimap2.

Quercetin Exhibits Preferential Binding Interaction by Selectively Targeting HRAS1 I-Motif DNA-Forming Promoter Sequences.

I-Motif (iM) DNA structures represent among the most significant noncanonical nucleic acid configurations. iM-forming DNA sequences are found in an array of vital genomic locations and are particularly frequent in the promoter islands of various oncogenes. Thus, iM DNA is a crucial candidate for anticancer medicines; therefore, binding interactions between iM DNA and small molecular ligands, such as flavonoids, are critically important. Extensive sets of spectroscopic strategies and thermodynamic analysis were utilized in the present investigation to find out the favorable interaction of quercetin (Que), a dietary flavonoid that has various health-promoting characteristics, including anticancer properties, with noncanonical iM DNA structure. Spectroscopic studies and thermal analysis revealed that Que interacts preferentially with HRAS1 iM DNA compared with VEGF, BCL2 iM, and duplex DNA. Que, therefore, emerged as a suitable natural-product-oriented antagonist for targeting HRAS1 iM DNA. The innovative spectroscopic as well as mechanical features of Que and its specific affinity for HRAS1 iM may be useful for therapeutic applications and provide crucial insights for the design of compounds with remarkable medicinal properties.

Abstract PO3-24-06: Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers

Abstract Background Pathogenic BRCA1 and BRCA2 (BRCA1/2) germline mutations contribute to hereditary breast cancer and are linked with increased risk of other cancers. Paradoxically, bi-allelic inactivation of BRCA1/2 (bBRCA1/2) is embryonically lethal. Although TP53 loss can at least in part mitigated the deleterious impact of Brca1 deficiency in mice, this is insufficient in human cells to compensate for decreased proliferative capacity. What other compensatory mechanisms facilitate oncogenesis remains unclear. Methods We performed an integrative analysis of cancer genomes from bBRCA1/2 tumors and CRISPR/Cas9 screens from cellular models to identify novel gene candidates that facilitate synthetic viability. First, we analyzed cancer genomes from ovarian and breast cancer derived from TCGA and identified genomic loci, which were enriched for recurrent copy number alterations (CNAs) in bBRCA1/2 tumors. We subsequently identified genes from these CNA loci that had corresponding changes in gene expression. Then, we used high-throughput genome-wide CRISPR/Cas9 knockout screens in BRCA1/2−/− cell lines (BRCA1−/- in RPE1 cells, BRCA2−/− in DLD1 cells) to identify which of these genes increased cell proliferation in vitro. Lastly, we experimentally validated these synthetic viable interactions using an orthogonal MCF12a system to evaluate changes in colony formation with a clonogenic cell survival assay. Results In human cancer genomes, we identified that bBRCA1/2 tumors harbored recurrent large-scale genetic deletions significantly more frequent than histologically matched control tumors. We identified 148 cytobands (23 distinct genomic loci) frequently deleted in bBRCA1/2 ovarian cancers (N=92), and 90 cytobands (15 distinct genomic loci) in bBRCA1/2 ER+ breast cancers (N=30). Subsequent computational analysis of gene expression data identified transcriptionally decreased genes in the frequently deleted loci. Cross-referencing with CRISPR/Cas9 screens in BRCA1/2−/− cells, we ultimately identified 277 and 218 genes that were altered in human tumors and putatively enhanced viability in BRCA1/2-null models but not in isogenic wild-type cell lines in BRCA1 and BRCA2 models, respectively. We subsequently selected 17 genes that either increased viability in BRCA1, BRCA2, or both contexts for experimental validation. Of these, about 40% (7 of 17) were experimentally validated (RIC8A, GNA12, ATMIN, IPO7, ATXN2, KDM1A, and NUP98) and had evidence of synthetic viability in this cellular context. Interestingly, RIC8A and GNA12 interact with each other, are both involved in G protein-coupled signaling pathway, and are synthetically viable with both BRCA1−/− and BRCA2−/− cells. In a re-analysis of a recently published genomic dataset on metastatic breast cancer, we found that homologous recombination deficient tumors developed additional loss of function mutations in RIC8A in the metastatic setting. Conclusions This study provides insights into the oncogenesis of BRCA1/2 malignancies and describes a high-throughput framework to identify synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers. Citation Format: Yingjie Zhu, Xin Pei, Ardijana Novaj, Jeremy Setton, Fatemeh Derakhshan, Pier Selenica, Niamh McDermott, Sonali Sinha, Atif Khan, Simon Powell, Jorge Reis-Filho, Nadeem Riaz. Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-06.

Abstract PO1-14-01: Clonal Architecture of circulating tumor DNA predicts Early Progression on Tamoxifen With or Without Palbociclib in Hormone Receptor-Positive Advanced Breast Cancer: NCCH1607/PATHWAY trial

Abstract Objective: Some patients with poor prognosis hormone receptor-positive, human epidermal growth factor receptor 2-negative (HR+/HER2-) advanced breast cancer (ABC) experience early disease progression on the combination of CDK4/6 inhibitors and endocrine therapy. We aimed to determine whether clonal architecture assessed by circulating tumor DNA (ctDNA) could identify patients at higher risk of early progression on tamoxifen therapy with or without palbociclib. Methods: PATHWAY trial is a randomized, placebo-controlled, Asian international, double-blind, phase 3 study evaluating palbociclib plus tamoxifen (PAL+TAM) versus placebo plus tamoxifen (PLB+TAM) in pre/perimenopausal and postmenopausal women with HR+/HER2- ABC (NCT03423199). The study was conducted as a Clinical Research Collaboration with the National Cancer Center Hospital as the regulatory study sponsor and Pfizer providing drug and financial support. The primary endpoint was investigator assessed progression-free survival (PFS). Genomic profiling by next-generation sequencing was an exploratory endpoint to characterize molecular alterations in ctDNA and correlate these with clinical outcomes. Plasma samples were collected at cycle 1-day 1 (pre-treatment) and cycle 2-day 15 and sequenced with the Guardant360 assay (Guardant Health). Patients were included if they had a minimum of 14 days of treatment in the first cycle. The variant allele frequency (VAF) was defined as number of mutant molecules at a specific nucleotide location over the total number of molecules present in the background at a specific given genomic location. We assessed the gene with highest VAF as the dominant clone allele frequency (DCAF). The association of changes in ctDNA alterations with PFS was evaluated to identify biomarkers of early progression. Results: Pre-treatment plasma was successfully sequenced in 177 of the 184 patients; 88 patients treated with PAL+TAM, and 89 with PLB+TAM. ctDNA was detected in 144 (81.4%) patients; the median number of genetic abnormalities was 2 (range, 0-19). PIK3CA, TP53, ERBB2, ESR1, AKT1, EGFR and PTEN were the genes with highest VAF as the dominant clone (24.3%, 11.9%, 4.5%, 4.5%, 3.4%, 3.4%, 3.4%, respectively). The median DCAF was 1.79 (range, 0.1-81.23); PAL+TAM, 2.53 (range, 0.1-63.5) and PLB+TAM, 1.65 (range, 0.08-81.23). Patients with ≥ 2 detectable genomic alterations and DCAF ≥ 1.8% had worse PFS (hazard ratio (HR) 1.93 [95% confidence interval (CI): 1.375 to 2.721], p = 0.0003 and HR 1.95 [95% CI: 1.345 to 2.834], p &amp;lt; 0.0001, respectively) compared to all other patients. In addition, we evaluated whether changes of clonal architecture in ctDNA at early treatment time point (ie. Cycle 2 Day 15) are associated with PFS. Patients with increased DCAF had worse PFS on both PAL+TAM (HR 2.80 [95% CI: 0.552 to 14.15], p = 0.04) and PLB+TAM (HR 2.02 [95% CI: 1.022 to 3.990], p = 0.01). All patients with high pretreatment DCAF (≥ 1.8%) and increase in DCAF progressed within 1 year (n=5, 2 patients treated with PAL+TAM, and 3 with PLB+TAM). Conclusion: Evaluation of clonal architecture in ctDNA could support identification of HR+/HER2- ABC patients with poorer prognosis, who may be at higher risk of early progression, regardless of CDK4/6 inhibitor use. Citation Format: Yoon-Sim Yap, Yuki Kojima, Akinobu Hamada, Hirofumi Mukai, Yen-Shen Lu, Joo Hyuk Sohn, Yoshiko Umeyama, Emi Noguchi, Kazuki Sudo, Tomomi Hata, Aya Kuchiba, Taro Shibata, Shigehiro Yagishita, Masayuki Yoshida, Shinji Kohsaka, Kouya Shiraishi, Kenichi Nakamura, Kenji Tamura, Kan Yonemori. Clonal Architecture of circulating tumor DNA predicts Early Progression on Tamoxifen With or Without Palbociclib in Hormone Receptor-Positive Advanced Breast Cancer: NCCH1607/PATHWAY trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-01.

Investigating the nature of prokaryotic genomic island locations within a genome.

Horizontal gene transfer (HGT) is a powerful evolutionary force that considerably shapes the structure of prokaryotic genomes and is associated with genomic islands (GIs). A GI is a DNA segment composed of transferred genes that can be found within a prokaryotic genome, obtained through HGT. Much research has focused on detecting GIs in genomes, but here we pursue a new course, which is identifying possible preferred locations of GIs in the prokaryotic genome. Here, we identify the locations of the GIs within prokaryotic genomes to examine patterns in those locations. Prokaryotic GIs were analyzed according to the genome structure that they are located in, whether it be a circular or a linear genome. The analytical investigations employed are: (1) studying the GI locations in relation to the origin of replication (oriC); (2) exploring the distances between GIs; and (3) determining the distribution of GIs across the genomes. For each of the investigations, the analysis was performed on all of the GIs in the data set. Moreover, to void bias caused by the distribution of the genomes represented, the GIs in one genome from each species and the GIs of the most frequent species are also analyzed. Overall, the results showed that there are preferred sites for the GIs in the genome. In the linear genomes, these sites are usually located in the oriC region and terminus region, while in the circular genomes, they are located solely in the terminus region. These results also showed that the distance distribution between the GIs is almost exponential, which proves that GIs have preferred sites within genomes. The oriC and termniuns are preferred sites for the GIs and a possible natural explanation for this could be connected to the content of the oriC region. Moreover, the content of the GIs in terms of its protein families was studied and the results demonstrated that the majority of frequent protein families are close to identical in each section.

Abstract PO2-08-03: Developing a polygenic risk score for precision prevention of estrogen receptor-positive (ER+)/HER2- breast cancer in Brazilian women

Abstract Polygenic risk scores (PRS) quantify the genetic risk of diseases based on risk alleles in genomic loci identified in genome-wide association studies (GWAS). As individuals of European ancestry are overrepresented in GWAS, PRS performance may vary in individuals of different ancestries. Brazil is the largest and most populated country in Latin America, with around 43% of admixed individuals, and data on breast cancer PRS performance for this population is lacking. We aimed to evaluate the performance of a breast cancer PRS with 313 SNPs generated with Europeans in Brazilian population. In this case-control study, we expect to include 1000 women age &amp;gt;= 18 years old with current or previous diagnosis of ER+/HER2- breast cancer. Germline DNA samples are genotyped with Illumina Infinium Global Screening Array-24 v3.0 chip. Controls are healthy woman previously genotyped with Global Screening Array-24 v1.0 chip. We selected the 313 SNPs PRS for ER+/HER2- breast cancer from PGS Catalog for evaluation. Quality control, imputation, PRS calculation and statistical analysis were performed in PLINK v2.0, Michigan server, PGS Catalog and R, respectively. We present the preliminary results for 1347 women (504 cases and 843 controls) included until March of 2023. After quality control and imputation, we calculated PRS for 1212 subjects (475 cases and 737 controls). Out of 313 SNPs, 211 were validated in our samples. For ER+/HER2- breast cancer, the odds ratio per SD of the 211-variant PRS (PRS211) was 1.363 (95% confidence interval [CI]: 1.211 to 1.537). Compared with women with average risk (40th-60th PRS percentile), women in the top 10% of PRS211 had a 1.623-fold increased risk (95% CI: 1.047 to 2.522). The area under the receiver operating characteristic curve of 0.581. Our preliminary results indicate that a PRS derived from European-ancestry population correlates with ER+/HER2- breast cancer in admixed Brazilian population, although with attenuated performance. Further work is needed to better characterize PRS risk stratification in admixed populations. Citation Format: Marina Candido Visontai Cormedi, Álison Luís Eburnêo Pereira, Fernanda Borchers Coeli-Lacchini, Sabrina Baroni, Lívia Mara Torres Bueno, Evelyn Ayumi Onga, Victor Girelli Piloto De Oliveira, Fabiana De Oliveira Buono, Aline Fares, Tatiana Strava Correa, Iasmin Alves Chirichela, Fernanda Peria, Romulado Barroso-Sousa, Rodrigo Santa Cruz Guindalini, Taisa Manuela Bonfim Machado Lopes, Rosimeire Aparecida Roela, Maria Aparecida Azevedo Koike Folgueira, Daniel Guimarães Tiezzi, Leandro Machado Colli, Liane Rapatoni. Developing a polygenic risk score for precision prevention of estrogen receptor-positive (ER+)/HER2- breast cancer in Brazilian women [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-08-03.

Cellular landscape of adrenocortical carcinoma at single-nuclei resolution

Adrenocortical carcinoma (ACC) is a rare yet devastating tumour of the adrenal gland with a molecular pathology that remains incompletely understood. To gain novel insights into the cellular landscape of ACC, we generated single-nuclei RNA sequencing (snRNA-seq) data sets from twelve ACC tumour samples and analysed these alongside snRNA-seq data sets from normal adrenal glands (NAGs). We find the ACC tumour microenvironment to be relatively devoid of immune cells compared to NAG tissues, consistent with known high tumour purity values for ACC as an immunologically “cold” tumour. Our analysis identifies three separate groups of ACC samples that are characterised by different relative compositions of adrenocortical cell types. These include cell populations that are specifically enriched in the most clinically aggressive and hormonally active tumours, displaying hallmarks of reorganised cell mechanobiology and dysregulated steroidogenesis, respectively. We also identified and validated a population of mitotically active adrenocortical cells that strongly overexpress genes POLQ, DIAPH3 and EZH2 to support tumour expansion alongside an LGR4+ progenitor-like or cell-of-origin candidate for adrenocortical carcinogenesis. Trajectory inference suggests the fate adopted by malignant adrenocortical cells upon differentiation is associated with the copy number or allelic balance state of the imprinted DLK1/MEG3 genomic locus, which we verified by assessing bulk tumour DNA methylation status. In conclusion, our results therefore provide new insights into the clinical and cellular heterogeneity of ACC, revealing how genetic perturbations to healthy adrenocortical renewal and zonation provide a molecular basis for disease pathogenesis.

Diverse nucleotide substitutions in rice base editing mediated by novel TadA variants

CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution in rapid genetic improvement of crops. The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes. Compared to the well-developed adenine base editors (ABEs), cytosine base editors (CBEs) and dual base editors suffer from unstable editing efficiency and pattern at different genomic loci in rice, significantly limiting their application. Here, the activities of multiple evolved TadA8e variants in base editing have been comprehensively examined in rice. We found that both TadA-CDd and TadA-E27R/N46L achieve more robust C-to-T editing than previously reported hyperactive hAID*Δ, whereas TadA-CDd outperformed TadA-E27R/N46L. Also, the C-to-G base editor (CGBE) engineered with TadA-CDd and OsUNG performs highly efficient C-to-G editing in rice compared to TadA-N46P. In addition, the dual base editor, which was constructed with a single protein TadDE, enables simultaneously edit C-to-T and A-to-G at high efficiency in rice. Collectively, our study demonstrates that TadA8e derivatives improve both CBE and dual base editor in rice, provide a powerful way in inducing diverse nucleotide substitutions for plant genome editing.

A new G3BP1-GFP reporter system for assessing skin toxicity by real-time monitoring of stress granules in vitro

The skin, the organ with the largest surface area in the body, is the most susceptible to chemical exposure from the external environment. In this study, we aimed to establish an in vitro skin toxicity monitoring system that utilizes the mechanism of stress granule (SG) formation induced by various cellular stresses. In HaCaT cells, a keratinocyte cell line that comprises the human skin, a green fluorescent protein (GFP) was knocked in at the C-terminal genomic locus of Ras GTPase-activating protein-binding protein 1 (G3BP1), a representative component of SGs. The G3BP1-GFP knock-in HaCaT cells and wild-type (WT) HaCaT cells formed SGs containing G3BP1-GFP upon exposure to arsenite and household chemicals, such as bisphenol A (BPA) and benzalkonium chloride (BAC), in real-time. In addition, the exposure of G3BP1-GFP knock-in HaCaT cells to BPA and BAC promoted the phosphorylation of eukaryotic initiation factor 2 alpha and protein kinase R-like endoplasmic reticulum kinase, which are cell signaling factors involved in SG formation, similar to WT HaCaT cells. In conclusion, this novel G3BP1-GFP knock-in human skin cell system can monitor SG formation in real-time and be utilized to assess skin toxicity to various substances.