Genomic Patterns Research Articles

Genomic Characterization of SARS-CoV-2 Variants from Clinical Isolates during the COVID-19 Epidemic in Mauritania.

The rapid genetic evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the coronavirus disease 2019 (COVID-19) pandemic has greatly challenged public health authorities worldwide, including in Mauritania. Despite the presence of the virus in Mauritania, only one study described its genomic variation during the course of the epidemic. The purpose of the present study was to document the genomic pattern of SARS-CoV-2 variants from clinical isolates during the COVID-19 outbreak in Mauritania, from September to November 2021. The whole genomes from 54 SARS-CoV-2 strains detected in nasopharyngeal swabs with a cycle threshold value ≤ 30 were successfully sequenced using next-generation sequencing (NGS) and the Illumina protocol. The mean genome coverage (±standard deviation) was 96.8% (±3.7). The most commonly identified clade was 21J (57.4%), followed by 21D (16.7%), 20A (11.1%), and 20B (9.2%). At the level of lineages, the majority of the samples were Delta variants with the sub-lineage AY.34 (or B.1.617.2.34). Among the 54 SARS-CoV-2 isolates that were successfully sequenced, 33 (61.1%) came from vaccinated individuals, and 21 (38.9%) were from unvaccinated individuals. Several SARS-CoV-2 variants were present in Mauritania between September and November 2021. As Mauritania, like many West African countries, is resource-limited regarding viral genome sequencing facilities, establishment of mutualized sub-regional sequencing platforms will be necessary to ensure continuous monitoring of mutations in viral genomes and track potential reduction in COVID-19 vaccine efficacy, increased transmissibility, and disease severity.

Evolutionary trajectories of small cell lung cancer under therapy

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1–3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Comparative analysis of the organelle genomes of Aconitum carmichaelii revealed structural and sequence differences and phylogenetic relationships

In this study, we conducted an assembly and analysis of the organelle genomes of Aconitum carmichaelii. Our investigation encompassed the examination of organelle genome structures, gene transfer events, and the environmental selection pressures affecting A. carmichaelii. The results revealed distinct evolutionary patterns in the organelle genomes of A. carmichaelii. Especially, the plastome exhibited a more conserved structure but a higher nucleotide substitution rate (NSR), while the mitogenome displayed a more complex structure with a slower NSR. Through homology analysis, we identified several instances of unidirectional protein-coding genes (PCGs) transferring from the plastome to the mitogenome. However, we did not observe any events which genes moved from the mitogenome to the plastome. Additionally, we observed multiple transposable element (TE) fragments in the organelle genomes, with both organelles showing different preferences for the type of nuclear TE insertion. Divergence time estimation suggested that rapid differentiation occurred in Aconitum species approximately 7.96 million years ago (Mya). This divergence might be associated with the reduction in CO2 levels and the significant uplift of the Qinghai-Tibet Plateau (QTP) during the late Miocene. Selection pressure analysis indicated that the dN/dS values of both organelles were less than 1, suggested that organelle PCGs were subject to purification selection. However, we did not detect any positively selected genes (PSGs) in Subg. Aconitum and Subg. Lycoctonum. This observation further supports the idea that stronger negative selection pressure on organelle genes in Aconitum results in a more conserved amino acid sequence. In conclusion, this study contributes to a deeper understanding of organelle evolution in Aconitum species and provides a foundation for future research on the genetic mechanisms underlying the structure and function of the Aconitum plastome and mitogenome.

A multiplex RPA-CRISPR/Cas12a-based POCT technique and its application in human papillomavirus (HPV) typing assay

Persistent infection with high-risk human papillomavirus (HR-HPV) is the primary and initiating factor for cervical cancer. With over 200 identified HPV types, including 14 high-risk types that integrate into the host cervical epithelial cell DNA, early determination of HPV infection type is crucial for effective risk stratification and management. Presently, on-site immediate testing during the HPV screening stage, known as Point of Care Testing (POCT), remains immature, severely limiting the scope and scenarios of HPV screening. This study, guided by the genomic sequence patterns of HPV, established a multiplex recombinase polymerase amplification (RPA) technology based on the concept of “universal primers.” This approach achieved the multiple amplification of RPA, coupled with the CRISPR/Cas12a system serving as a medium for signal amplification and conversion. The study successfully constructed a POCT combined detection system, denoted as H-MRC12a (HPV—Multiple RPA—CRISPR/Cas12a), and applied it to high-risk HPV typing detection. The system accomplished the typing detection of six high-risk HPV types (16, 18, 31, 33, 35, and 45) can be completed within 40 min, and the entire process, from sample loading to result interpretation, can be accomplished within 45 min, with a detection depth reaching 1 copy/μL for each high-risk type. Validation of the H-MRC12a detection system’s reproducibility and specificity was further conducted through QPCR on 34 clinical samples. Additionally, this study explored and optimized the multiplex RPA amplification system and CRISPR system at the molecular mechanism level. Furthermore, the primer design strategy developed in this study offers the potential to enhance the throughput of H-MRC12a detection while ensuring sensitivity, providing a novel research avenue for high-throughput detection in Point-of-Care molecular pathogen studies.

Multiregion WES of metastatic pancreatic neuroendocrine tumors revealed heterogeneity in genomic alterations, immune microenvironment and evolutionary patterns

Pancreatic neuroendocrine tumors (PanNETs), though uncommon, have a high likelihood of spreading to other body parts. Previously, the genetic diversity and evolutionary patterns in metastatic PanNETs were not well understood. To investigate this, we performed multiregion sampling whole-exome sequencing (MRS-WES) on samples from 10 patients who had not received prior treatment for metastatic PanNETs. This included 29 primary tumor samples, 31 lymph node metastases, and 15 liver metastases. We used the MSK-MET dataset for survival analysis and validation of our findings. Our research indicates that mutations in the MEN1/DAXX genes might trigger the early stages of PanNET development. We categorized the patients based on the presence (MEN1/DAXXmut, n = 7) or absence (MEN1/DAXXwild, n = 3) of these mutations. Notable differences were observed between the two groups in terms of genetic alterations and clinically relevant mutations, confirmed using the MSK-MET dataset. Notably, patients with mutations in MEN1/DAXX/ATRX genes had a significantly longer median overall survival compared to those without these mutations (median not reached vs. 43.63 months, p = 0.047). Multiplex immunohistochemistry (mIHC) analysis showed a more prominent immunosuppressive environment in metastatic tumors, especially in patients with MEN1/DAXX mutations. These findings imply that MEN1/DAXX mutations lead PanNETs through a unique evolutionary path. The disease’s progression pattern indicates that PanNETs can spread early, even before clinical detection, highlighting the importance of identifying biomarkers related to metastasis to guide personalized treatment strategies.

Plastid genome data provide new insights into the dynamic evolution of the tribe Ampelopsideae (Vitaceae)

BackgroundAmpelopsideae J. Wen & Z.L. Nie is a small-sized tribe of Vitaceae Juss., including ca. 47 species from four genera showing a disjunct distribution worldwide across all the continents except Antarctica. There are numerous species from the tribe that are commonly used as medicinal plants with immune-modulating, antimicrobial, and anti-hypertensive properties. The tribe is usually recognized into three clades, i.e., Ampelopsis Michx., Nekemias Raf., and the Southern Hemisphere clade. However, the relationships of the three clades differ greatly between the nuclear and the plastid topologies. There has been limited exploration of the chloroplast phylogenetic relationships within Ampelopsideae, and studies on the chloroplast genome structure of this tribe are only available for a few individuals. In this study, we aimed to investigate the evolutionary characteristics of plastid genomes of the tribe, including their genome structure and evolutionary insights.ResultsWe sequenced, assembled, and annotated plastid genomes of 36 species from the tribe and related taxa in the family. Three main clades were recognized within Ampelopsideae, corresponding to Ampelopsis, Nekemias, and the Southern Hemisphere lineage, respectively, and all with 100% bootstrap supports. The genome sequences and content of the tribe are highly conserved. However, comparative analyses suggested that the plastomes of Nekemias demonstrate a contraction in the large single copy region and an expansion in the inverted repeat region, and possess a high number of forward and palindromic repeat sequences distinct from both Ampelopsis and the Southern Hemisphere taxa.ConclusionsOur results highlighted plastome variations in genome length, expansion or contraction of the inverted repeat region, codon usage bias, and repeat sequences, are corresponding to the three lineages of the tribe, which probably faced with different environmental selection pressures and evolutionary history. This study provides valuable insights into understanding the evolutionary patterns of plastid genomes within the Ampelopsideae of Vitaceae.

Genome-Wide Identification and Co-Expression Networks of WOX Gene Family in Nelumbo nucifera.

WUSCHEL-related homeobox (WOX) genes are a class of plant-specific transcription factors, regulating the development of multiple tissues. However, the genomic characterizations and expression patterns of WOX genes have not been analyzed in lotus. In this study, 15 NnWOX genes were identified based on the well-annotated reference genome of lotus. According to the phylogenetic analysis, the NnWOX genes were clustered into three clades, i.e., ancient clade, intermediate clade, and WUS clade. Except for the conserved homeobox motif, we further found specific motifs of NnWOX genes in different clades and divergence gene structures, suggesting their distinct functions. In addition, two NnWOX genes in the ancient clade have conserved expression patterns and other NnWOX genes exhibit different expression patterns in lotus tissues, suggesting a low level of functional redundancy in lotus WOX genes. Furthermore, we constructed the gene co-expression networks for each NnWOX gene. Based on weighted gene co-expression network analysis (WGCNA), ten NnWOX genes and their co-expressed genes were assigned to the modules that were significantly related to the cotyledon and seed coat. We further performed RT-qPCR experiments, validating the expression levels of ten NnWOX genes in the co-expression networks. Our study reveals comprehensive genomic features of NnWOX genes in lotus, providing a solid basis for further function studies.

Codon usage bias and phylogenetic analysis of chloroplast genome in 36 gracilariaceae species.

Gracilariaceae is a group of marine large red algae and main source of agar with important economic and ecological value. The codon usage patterns of chloroplast genomes in 36 species from Graciliaceae show that GC range from 0.284 to 0.335, the average GC3 range from 0.135 to 0.243 and the value of ENC range from 35.098 to 42.327, which indicates these genomes are rich in AT and prefer to use codons ending with AT in these species. Nc plot, PR2 plot, neutrality plot analyses and correlation analysis indicate that these biases may be caused by multiple factors, such as natural selection and mutation pressure, but prolonged natural selection is the main driving force influencing codon usage preference. The cluster analysis and phylogenetic analysis show that the differentiation relationship of them is different and indicate that codons with weak or unbiased preferences may also play an irreplaceable role in these species' evolution. In addition, we identified 26 common high-frequency codons and 8-18 optimal codons all ending in A/U in these 36 species. Our results will not only contribute to carrying out transgenic work in Gracilariaceae species to maximize the protein yield in the future, but also lay a theoretical foundation for further exploring systematic classification of them.

A genome-wide study of gastric intramucosal neoplasia based on somatic copy number alterations, gene mutations, and mRNA expression patterns.

We performed comprehensive analyses of somatic copy number alterations (SCNAs) and gene expression profiles of gastric intramucosal neoplasia (IMN) using array-based methods in 97 intestinal-type IMNs, including 39 low-grade dysplasias (LGDs), 37 high-grade dysplasias (HGDs), and 26 intramucosal carcinomas (IMCs) with stromal invasion of the lamina propria to identify the molecular mechanism of IMN. In addition, we examined gene mutations using gene panel analyses. We used cluster analyses for exclusion of arbitrariness to identify SCNA patterns and expression profiles. IMNs were classified into two distinct subgroups (subgroups 1 and 2) based on SCNA patterns. Subgroup 1 showed a genomic stable pattern due to the low frequency of SCNAs, whereas subgroup 2 exhibited a chromosomal instability pattern due to the high frequencies of SCNAs and TP53 mutations. Interestingly, although the frequencies of LGD and HGD were significantly higher in subgroup 1 than in subgroup 2, IMC was commonly found in both types. Although the expression profiles of specific mRNAs could be used to categorise subgroups 1 and 2, no clinicopathological findings correlated with either subgroup. We examined signalling pathways specific to subgroups 1 and 2 to identify the association of each subgroup with signalling pathways based on gene ontology tree visualisation: subgroups 1 and 2 were associated with haem metabolism and chromosomal instability, respectively. These findings reveal a comprehensive genomic landscape that highlights the molecular complexity of IMNs and provide a road map to facilitate our understanding of gastric IMNs.

Compositional features analysis by machine learning in genome represents linear adaptation of monkeypox virus.

Introduction: The global headlines have been dominated by the sudden and widespread outbreak of monkeypox, a rare and endemic zoonotic disease caused by the monkeypox virus (MPXV). Genomic composition based machine learning (ML) methods have recently shown promise in identifying host adaptability and evolutionary patterns of virus. Our study aimed to analyze the genomic characteristics and evolutionary patterns of MPXV using ML methods. Methods: The open reading frame (ORF) regions of full-length MPXV genomes were filtered and 165 ORFs were selected as clusters with the highest homology. Unsupervised machine learning methods of t-distributed stochastic neighbor embedding (t-SNE), Principal Component Analysis (PCA), and hierarchical clustering were performed to observe the DCR characteristics of the selected ORF clusters. Results: The results showed that MPXV sequences post-2022 showed an obvious linear adaptive evolution, indicating that it has become more adapted to the human host after accumulating mutations. For further accurate analysis, the ORF regions with larger variations were filtered out based on the ranking of homology difference to narrow down the key ORF clusters, which drew the same conclusion of linear adaptability. Then key differential protein structures were predicted by AlphaFold 2, which meant that difference in main domains might be one of the internal reasons for linear adaptive evolution. Discussion: Understanding the process of linear adaptation is critical in the constant evolutionary struggle between viruses and their hosts, playing a significant role in crafting effective measures to tackle viral diseases. Therefore, the present study provides valuable insights into the evolutionary patterns of the MPXV in 2022 from the perspective of genomic composition characteristics analysis through ML methods.

Epigenetic Mechanisms in Latent Epstein-Barr Virus Infection and Associated Cancers.

The Epstein-Barr Virus (EBV) is a double-stranded DNA-based human tumor virus that was first isolated in 1964 from lymphoma biopsies. Since its initial discovery, EBV has been identified as a major contributor to numerous cancers and chronic autoimmune disorders. The virus is particularly efficient at infecting B-cells but can also infect epithelial cells, utilizing an array of epigenetic strategies to establish long-term latent infection. The association with histone modifications, alteration of DNA methylation patterns in host and viral genomes, and microRNA targeting of host cell factors are core epigenetic strategies that drive interactions between host and virus, which are necessary for viral persistence and progression of EBV-associated diseases. Therefore, understanding epigenetic regulation and its role in post-entry viral dynamics is an elusive area of EBV research. Here, we present current outlooks of EBV epigenetic regulation as it pertains to viral interactions with its host during latent infection and its propensity to induce tumorigenesis. We review the important epigenetic regulators of EBV latency and explore how the strategies involved during latent infection drive differential epigenetic profiles and host-virus interactions in EBV-associated cancers.

Deep learning infers clinically relevant protein levels and drug response in breast cancer from unannotated pathology images

The computational pathology has been demonstrated to effectively uncover tumor-related genomic alterations and transcriptomic patterns. Although proteomics has indeed shown great potential in the field of precision medicine, few studies have focused on the computational prediction of protein levels from pathology images. In this paper, we assume that deep learning-based pathological features imply the protein levels of tumor biomarkers that are indicative of prognosis and drug response. For this purpose, we propose wsi2rppa, a weakly supervised contrastive learning framework to infer the protein levels of tumor biomarkers from whole slide images (WSIs) in breast cancer. We first conducted contrastive learning-based pre-training on tessellated tiles to extract pathological features, which are then aggregated by attention pooling and adapted to downstream tasks. We conducted extensive evaluation experiments on the TCGA-BRCA cohort (1978 WSIs of 1093 patients with protein levels of 223 biomarkers) and the CPTAC-BRCA cohort (642 WSIs of 134 patients). The results showed that our method achieved state-of-the-art performance in tumor diagnostic tasks, and also performed well in predicting clinically relevant protein levels and drug response. To show the model interpretability, we spatially visualized the WSIs colored the tiles by their attention scores, and found that the regions with high scores were highly consistent with the tumor and necrotic regions annotated by a 10-year experienced pathologist. Moreover, spatial transcriptomic data further verified that the heatmap generated by attention scores agrees greatly with the spatial expression landscape of two typical tumor biomarker genes. In predicting the response to drug trastuzumab treatment, our method achieved a 0.79 AUC value which is much higher than the previous study reported 0.68. These findings showed the remarkable potential of computational pathology in the prediction of clinically relevant protein levels, drug response, and clinical outcomes.

Genome-wide profiles of DNA damage represent highly accurate predictors of mammalian age.

The identification of novel age-related biomarkers represents an area of intense research interest. Despite multiple studies associating DNA damage with aging, there is a glaring paucity of DNA damage-based biomarkers of age, mainly due to the lack of precise methods for genome-wide surveys of different types of DNA damage. Recently, we developed two techniques for genome-wide mapping of the most prevalent types of DNA damage, single-strand breaks and abasic sites, with nucleotide-level resolution. Herein, we explored the potential of genomic patterns of DNA damage identified by these methods as a source of novel age-related biomarkers using mice as a model system. Strikingly, we found that models based on genomic patterns of either DNA lesion could accurately predict age with higher precision than the commonly used transcriptome analysis. Interestingly, the informative patterns were limited to relatively few genes and the DNA damage levels were positively or negatively correlated with age. These findings show that previously unexplored high-resolution genomic patterns of DNA damage contain useful information that can contribute significantly to both practical applications and basic science.

Whole-genome resequencing provides insights into the diversity and adaptation to desert environment in Xinjiang Mongolian cattle

BackgroundXinjiang Mongolian cattle is an indigenous breed that inhabits the Taklimakan Desert and is characterized by its small body size. However, the genomic diversity, origin, and genetic basis underlying the adaptation to the desert environment have been poorly studied.ResultsWe analyzed patterns of Xinjiang Mongolian cattle genetic variation by sequencing 20 genomes together with seven previously sequenced genomes and comparing them to the 134 genomes of nine representative breeds worldwide. Among the breeds of Bos taurus, we found the highest nucleotide diversity (0.0024) associated with the lower inbreeding coefficient (2.0110-6), the lowest linkage disequilibrium (r2 = 0.3889 at distance of 10 kb), and the highest effective population size (181 at 20 generations ago) in Xinjiang Mongolian cattle. The genomic diversity pattern could be explained by a limited introgression of Bos indicus genes. More importantly, similarly to desert-adapted camel and same-habitat sheep, we also identified signatures of selection including genes, GO terms, and/or KEGG pathways controlling water reabsorption and osmoregulation, metabolic regulation and energy balance, as well as small body size in Xinjiang Mongolian cattle.ConclusionsOur results imply that Xinjiang Mongolian cattle might have acquired distinct genomic diversity by virtue of the introgression of Bos indicus, which helps understand the demographic history. The identification of selection signatures can provide novel insights into the genomic basis underlying the adaptation of Xinjiang Mongolian cattle to the desert environment.

RAS/RAF Comutation and ERBB2 Copy Number Modulates HER2 Heterogeneity and Responsiveness to HER2-directed Therapy in Colorectal Cancer.

ERBB2-amplified colorectal cancer is a distinct molecular subtype with expanding treatments. Implications of concurrent oncogenic RAS/RAF alterations are not known. Dana-Farber and Foundation Medicine Inc. Colorectal cancer cohorts with genomic profiling were used to identify ERBB2-amplified cases [Dana-Farber, n = 47/2,729 (1.7%); FMI, n = 1857/49,839 (3.7%)]. Outcomes of patients receiving HER2-directed therapies are reported (Dana-Farber, n = 9; Flatiron Health-Foundation Medicine clinicogenomic database, FH-FMI CGDB, n = 38). Multisite HER2 IHC and genomic profiling were performed to understand HER2 intratumoral and interlesional heterogeneity. The impact of concurrent RAS comutations on the effectiveness of HER2-directed therapies were studied in isogenic colorectal cancer cell lines and xenografts. ERBB2 amplifications are enriched in left-sided colorectal cancer. Twenty percent of ERBB2-amplified colorectal cancers have co-occurring oncogenic RAS/RAF alterations. While RAS/RAF WT colorectal cancers typically have clonal ERBB2 amplification, colorectal cancers with co-occurring RAS/RAF alterations have lower level ERRB2 amplification, higher intratumoral heterogeneity, and interlesional ERBB2 discordance. These distinct genomic patterns lead to differential responsiveness and patterns of resistance to HER2-directed therapy. ERBB2-amplified colorectal cancer with RAS/RAF alterations are resistant to trastuzumab-based combinations, such as trastuzumab/tucatinib, but retain sensitivity to trastuzumab deruxtecan in in vitro and murine models. Trastuzumab deruxtecan shows clinical efficacy in cases with high-level ERBB2-amplified RAS/RAF coaltered colorectal cancer. Co-occurring RAS/RAF alterations define a unique subtype of ERBB2-amplified colorectal cancer that has increased intratumoral heterogeneity, interlesional discordance, and resistance to trastuzumab-based combinations. Further examination of trastuzumab deruxtecan in this previously understudied cohort of ERBB2-amplified colorectal cancer is warranted.

Real-world genomic testing patterns in patients with prostate cancer.

97 Background: The armamentarium of therapies for metastatic prostate cancer (PCa) has now expanded to include genomically targeted agents and CPIs. Men with both somatic and germline alterations in the DNA damage repair (DDR) pathway, most commonly BRCA2, are candidates for PARP inhibitors. Recent data from the PROPEL and MAGNITUDE trials led to approval of PARP inhibitors in combination with novel hormonal therapies earlier in the PCa continuum, in the first-line metastatic castrate-resistant space. Some PCas harbor genomic alterations, such as TMB>10 m/Mb and MSI high, which predispose to increased responses to CPIs, leading to tumor agnostic approval of pembrolizumab in these patient populations. However, it is imperative to perform timely genomic testing to assess for presence of these alterations. Methods: We conducted a 15-question survey using the Qualtrics XM software to assess genomic testing patterns of various providers in academic and community settings managing PCa patients. The survey link was distributed electronically to providers predominantly in the Western United States. Results: A total of 47 survey responses were received, with 59.6% (28/47) self-identifying as medical oncologists (Onc), 21.3% (10/47) as urologists (Uro), 4.3% (2/47) as radiation oncologists, 8.5% (4/47) as advanced practitioners, and 6.4% (3/47) as other. Academic Onc comprised 23.4 % (11/47), community Onc comprised 36.2% (17/47), academic Uro comprised 17.0% (8/47), and community Uro comprised 4.6 % (2/47) of providers. Timing of ordering of germline and somatic genomic testing by selected provider type is outlined in the Table. Conclusions: Given availability of targeted therapies in PCa management, ordering germline and somatic germline genomic testing is imperative. Despite the small sample size, several trends emerge from this dataset. Oncs at academic centers tend to order genomic testing earlier than Oncs at community practices, while Uros were less likely to order genomic testing when compared to Oncs in general. [Table: see text]

Comparative analysis of codon usage patterns in the chloroplast genomes of nine forage legumes

Leguminosae is one of the three largest families of angiosperms after Compositae and Orchidaceae. It is widely distributed and grows in a variety of environments, including plains, mountains, deserts, forests, grasslands, and even waters where almost all legumes can be found. It is one of the most important sources of starch, protein and oil in the food of mankind and also an important source of high-quality forage material for animals, which has important economic significance. In our study, the codon usage patterns and variation sources of the chloroplast genome of nine important forage legumes were systematically analyzed. Meanwhile, we also constructed a phylogenetic tree based on the whole chloroplast genomes and protein coding sequences of these nine forage legumes. Our results showed that the chloroplast genomes of nine forage legumes end with A/T bases, and seven identical high-frequency (HF) codons were detected among the nine forage legumes. ENC-GC3s mapping, PR2 analysis, and neutral analysis showed that the codon bias of nine forage legumes was influenced by many factors, among which natural selection was the main influencing factor. The codon usage frequency showed that the Nicotiana tabacum and Saccharomyces cerevisiae can be considered as receptors for the exogenous expression of chloroplast genes of these nine forage legumes. The phylogenetic relationships of the chloroplast genomes and protein coding genes were highly similar, and the nine forage legumes were divided into three major clades. Among the clades Melilotus officinalis was more closely related to Medicago sativa, and Galega officinalis was more closely related to Galega orientalis. This study provides a scientific basis for the molecular markers research, species identification and phylogenetic studies of forage legumes.

DNA methylation impacts soybean early development by modulating hormones and metabolic pathways.

Genomic DNA methylation patterns play a crucial role in the developmental processes of plants and mammals. In this study, we aimed to investigate the significant effects of epigenetic mechanisms on the development of soybean seedlings and metabolic pathways. Our analyses show that 5-azaC-treatment affects radicle development from two Days After Imbibition (DAI), as well as both shoot and root development. We examined the expression levels of key genes related to DNA methylation and demethylation pathways, such as DRM2, which encodes RNA-directed DNA Methylation (RdDM) pathway, SAM synthase, responsible for methyl group donation, and ROS1, a DNA demethylase. In treated seedling roots, we observed an increase in DRM2 expression and a decrease in ROS1 expression. Additionally, 5-azaC treatment altered protein accumulation, indicating epigenetic control over stress response while inhibiting nitrogen assimilation, urea cycle, and glycolysis-related proteins. Furthermore, it influenced the levels of various phytohormones and metabolites crucial for seedling growth, such as ABA, IAA, ethylene, polyamines (PUT and Cad), and free amino acids, suggesting that epigenetic changes may shape soybean responses to pathogens, abiotic stress, and nutrient absorption. Our results assist in understanding how hypomethylation shapes soybean responses to pathogens, abiotic stress, and nutrient absorption crucial for seedling growth, suggesting that the plant's assimilation of carbon and nitrogen, along with hormone pathways, may be influenced by epigenetic changes.

Correlation of urinary comprehensive genomic profile with risk of recurrence of BCG-unresponsive non-muscle invasive bladder cancer treated with atezolizumab in SWOG S1605.

529 Background: Radical cystectomy is recommended for patients with BCG-unresponsive (BU) non-muscle invasive bladder cancer (NMIBC) due to high risk of progression. Improved methods for assessing these risks would greatly facilitate the potential for bladder preservation. Here, we tested the capacity of the urinary comprehensive genomic profile (uCGP) to predict event-free survival (EFS) in patients with BU-NMIBC treated with atezolizumab in the single arm phase 2 trial SWOG S1605. Methods: Urine was collected from patients with BU NMIBC (CIS, Ta, T1) treated with at least one dose of intravenous atezolizumab at baseline and before the 5th cycle of therapy (3 months). The uCGP was assessed using UroAmp (Convergent Genomics). Risk scores for recurrence at baseline and at 3 months were calculated by a prespecified machine learning algorithm incorporating alterations in 60 genes and low pass whole genome sequencing, and categorized as high versus low. Molecular response was classified based on change in uCGP between the two time points. Risk scores and molecular response were submitted to SWOG for clinical correlation with EFS using a Cox model, adjusting for CIS status. Time to event was calculated from date of study entry (baseline risk) or from the 2nd collection time (3-month risk) to first high grade (HG) recurrence or persistent CIS at 3 months. Death unrelated to bladder cancer and patients last known to be alive without HG recurrence were censored at date of last visit. Results: Samples were provided at baseline in 89 patients and before the 5th cycle in 77; 68 had both samples available for paired analysis. The risk score at baseline was classified as high in 69% of samples (73% for CIS ±Ta/T1 and 62% for Ta/T1). At 12 and 18 months the EFS probabilities were 26% and 23% for high-risk and 67% and 51% for low-risk patients, respectively, with a HR of 2.82 (95% CI: 1.58, 5.03; p<0.001). The risk score at the 3-month timepoint was classified as high in 81% of samples, and the EFS probabilities at 12 and 18 months after urine collection were 26% and 22% for high-risk and 80% and 72% for low-risk patients, respectively, with a HR of 3.39 (95% CI: 1.41, 8.13; p<0.006). Molecular response to treatment was classified as complete (CR) in 8%, partial (PR) in 14%, stable (SD) in 25% and progression (PD) in 46%, with 7% having no detectable genomic abnormalities at both time points. Clinical recurrence was observed by 18 months in 0/6 patients with CR, 7/9 (78%) with PR, 11/14 (79%) with SD and 25/33 (76%) with PD by genomic profile. Conclusions: This study suggests that uCGP at baseline and after 4 cycles of treatment can identify genomic patterns associated with an increased risk of HG persistence, recurrence or progression in BU NMIBC treated with immune checkpoint inhibition. Future studies will determine if this can be used to guide early treatment intensification.

Genomic profiling of bladder cancer in waterpipe and cigarette smokers: Implications for carcinogenicity and genetic landscape.

657 Background: While cigarette smoking is a well-established risk factor for bladder cancer (BC), limited research has explored the contribution of waterpipe smoking (WPS) to BC. WPS is gaining popularity among Middle Eastern youth, often perceived as a safer alternative to cigarettes. Alarmingly, Lebanese males had an age-standardized incidence rate of 54.0 per 100,000 in 2019, the highest in the Arab World and five times the global average, prompting a critical investigation into potential risk factors. Mounting evidence suggests that WPS exposes users to toxicants and carcinogens that can induce chromosomal damage contributing to cancer. This study aims to assess the genomic and mutational profiles of BC in waterpipe smokers compared to cigarette smokers. Methods: This cross-sectional study enrolled adults with urothelial carcinoma (UC) who exclusively smoked waterpipe at the American University of Beirut Medical Center (AUBMC) between April 2017 and September 2019. Next-generation sequencing by MSK-IMPACT characterized mutational profiles, tumor mutation burden, and microsatellite instability (MSI) status in tumor-normal pairs. For comparison, a cohort from Memorial Sloan Kettering Cancer Center (MSKCC), comprising previously profiled BC tumors from cigarette smokers, was used. Results: A total of 14 exclusively WPS-BC patients were enrolled, with 12 males (85.7%) and a median age at diagnosis of 62.9 years (range: 30.7 to 78.9). Among these patients, 9 (64.3%) had non-muscle invasive BC, with 8 (57.2%) classified as high-grade UC. MSK-IMPACT sequencing successfully analyzed 13 WPS samples, revealing genomic patterns similar to the cigarette smoking cohort. Common genetic alterations in the WPS cohort included mutations in TERT promoter (62%), TP53 and FGFR3 (46% each), and PIK3CA (38%). Mutations in chromatin modifiers were less prevalent, with the highest frequencies observed in KDM6A (31%) and KMT2C (15%). The tumor mutation burden was higher in the WPS group (22 per MB) compared to the cigarette smoking cohort (9 per MB), with no definite evidence of MSI. PD-L1 expression was generally low, with only 2 samples exhibiting combined positivity scores (CPS) of 15 and 10, primarily associated with immune cells PD-L1 expression. Only one tumor demonstrated 3% PD-L1 expression on tumor cells, while 7 tumors displayed no expression on either tumor or immune cells (CPS of 0). Conclusions: This study underscores the genomic similarities between BC in waterpipe and cigarette smokers. The preliminary findings, limited by sample size, highlight the need for further investigations to elucidate the potential role of WPS in BC development. Future efforts will focus on cohort expansion to validate initial observations, raising awareness of health risks associated with WPS, and emphasizing the importance of continued research to inform preventive strategies.