Variation In Copy Number Research Articles

Mitochondrial genetics through the lens of single-cell multi-omics.

Mitochondria carry their own genetic information encoding for a subset of protein-coding genes and translational machinery essential for cellular respiration and metabolism. Despite its small size, the mitochondrial genome, its natural genetic variation and molecular phenotypes have been challenging to study using bulk sequencing approaches, due to its variation in cellular copy number, non-Mendelian modes of inheritance and propensity for mutations. Here we highlight emerging strategies designed to capture mitochondrial genetic variation across individual cells for lineage tracing and studying mitochondrial genetics in primary human cells and clinical specimens. We review recent advances surrounding single-cell mitochondrial genome sequencing and its integration with functional genomic readouts, including leveraging somatic mitochondrial DNA mutations as clonal markers that can resolve cellular population dynamics in complex human tissues. Finally, we discuss how single-cell whole mitochondrial genome sequencing approaches can be utilized to investigate mitochondrial genetics and its contribution to cellular heterogeneity and disease.

The Variable Genomic Landscape During Osteosarcoma Progression: Insights From a Longitudinal WGS Analysis.

Osteosarcoma is a primary bone tumor that exhibits a complex genomic landscape characterized by gross chromosomal abnormalities. Osteosarcoma patients often develop metastatic disease, resulting in limited therapeutic options and poor survival rates. To gain knowledge on the mechanisms underlying osteosarcoma heterogeneity and metastatic process, it is important to obtain a detailed profile of the genomic alterations that accompany osteosarcoma progression. We performed WGS on multiple tissue samples from six patients with osteosarcoma, including the treatment naïve biopsy of the primary tumor, resection of the primary tumor after neoadjuvant chemotherapy, local recurrence, and distant metastases. A comprehensive analysis of single-nucleotide variants (SNVs), structural variants, copy number alterations (CNAs), and chromothripsis events revealed the genomic heterogeneity during osteosarcoma progression. SNVs and structural variants were found to accumulate over time, contributing to an increased complexity of the genome of osteosarcoma during disease progression. Phylogenetic trees based on SNVs and structural variants reveal distinct evolutionary patterns between patients, including linear, neutral, and branched patterns. The majority of osteosarcomas showed variable copy number profiles or gained whole-genome doubling in later occurrences. Large proportions of the genome were affected by loss of heterozygosity (LOH), although these regions remain stable during progression. Additionally, chromothripsis is not confined to a single early event, as multiple other chromothripsis events may appear in later occurrences. Together, we provide a detailed analysis of the complex genome of osteosarcomas and show that five of six osteosarcoma genomes are highly dynamic and variable during progression.

Genetics and Pathophysiological Mechanisms of Lipoprotein(a)-Associated Cardiovascular Risk.

Elevated lipoprotein(a) is a genetically transmitted codominant trait that is an independent risk driver for cardiovascular disease. Lipoprotein(a) concentration is heavily influenced by genetic factors, including LPA kringle IV-2 domain size, single-nucleotide polymorphisms, and interleukin-1 genotypes. Apolipoprotein(a) is encoded by the LPA gene and contains 10 subtypes with a variable number of copies of kringle -2, resulting in >40 different apolipoprotein(a) isoform sizes. Genetic loci beyond LPA, such as APOE and APOH, have been shown to impact lipoprotein(a) levels. Lipoprotein(a) concentrations are generally 5% to 10% higher in women than men, and there is up to a 3-fold difference in median lipoprotein(a) concentrations between racial and ethnic populations. Nongenetic factors, including menopause, diet, and renal function, may also impact lipoprotein(a) concentration. Lipoprotein(a) levels are also influenced by inflammation since the LPA promoter contains an interleukin-6 response element; interleukin-6 released during the inflammatory response results in transient increases in plasma lipoprotein(a) levels. Screening can identify elevated lipoprotein(a) levels and facilitate intensive risk factor management. Several investigational, RNA-targeted agents have shown promising lipoprotein(a)-lowering effects in clinical studies, and large-scale lipoprotein(a) testing will be fundamental to identifying eligible patients should these agents become available. Lipoprotein(a) testing requires routine, nonfasting blood draws, making it convenient for patients. Herein, we discuss the genetic determinants of lipoprotein(a) levels, explore the pathophysiological mechanisms underlying the association between lipoprotein(a) and cardiovascular disease, and provide practical guidance for lipoprotein(a) testing.

Ribosomal DNA copy number is associated with body mass in humans and other mammals

Body mass results from a complex interplay between genetics and environment. Previous studies of the genetic contribution to body mass have excluded repetitive regions due to the technical limitations of platforms used for population scale studies. Here we apply genome-wide approaches, identifying an association between adult body mass and the copy number (CN) of 47S-ribosomal DNA (rDNA). rDNA codes for the 18 S, 5.8 S and 28 S ribosomal RNA (rRNA) components of the ribosome. In mammals, there are hundreds of copies of these genes. Inter-individual variation in the rDNA CN has not previously been associated with a mammalian phenotype. Here, we show that rDNA CN variation associates with post-pubertal growth rate in rats and body mass index in adult humans. rDNA CN is not associated with rRNA transcription rates in adult tissues, suggesting the mechanistic link occurs earlier in development. This aligns with the observation that the association emerges by early adulthood.

Single-cell RNA-binding protein pattern-mediated molecular subtypes depict the hallmarks of the tumor microenvironment in bladder urothelial carcinoma

Abstract Objectives Bladder carcinoma (BC) is a common malignancy of the urinary tract. As a new hallmark of cancer for drug therapy, RNA-binding proteins (RBPs) are key regulatory factors in alternative splicing events. This work is to uncover the relationship between BC and RBP in order to find drug targets in BC. Methods In this work, data from single-cell RNA-seq GSE1355337, PRJNA662018, and the TCGA-Bladder urothelial carcinoma (BLCA) cohorts are integrated to identify their relationships. A scoring system is constructed according to RBPs gene expression and patients’ survival. A network is constructed to analyze the alternative splicing events and RBP genes. Results A scoring system identified 321 RBPs significantly associated with the prognosis of patients. Subsequent typing of these RBP genes in two single-cell datasets demonstrated that most of the RBP genes had variable copy numbers. Three RBP clusters were identified. Using RBP genes as a signature in BC epithelial cells allows for differentiation between different grades of BC samples. The novel RBP genes-based subtype system reflects BC clinical staging. Notably, CellChat analysis revealed that the RBP genes-associated cell subtypes of T cells had extensive interactions with epithelial cells. Further analysis showed that the ligand-receptor pair MIF-CXCR4 mediated the communication between RBP-associated subtypes of BC epithelial cells and T cells. Conclusions Taken together, RBP genes are associated with BC progress and offer new indicators for precision medicine in BC.

Copy number and sequence variation in rDNA of Daphnia pulex from natural populations: insights from whole-genome sequencing.

Ribosomal DNA (rDNA) has a vital role in ribosome biogenesis as it contains the genes that encode ribosomal RNA (rRNA) separated by intergenic spacers (IGSs). The rRNA genes occur in hundreds to tens of thousands of copies per haploid genome in eukaryotes and are generally highly conserved with low variation within species. Due to the repetitive nature and large size of rDNA arrays, detecting intraindividual variation can be difficult. In this study, we use whole-genome sequences of 169 Daphnia pulex individuals from 10 natural populations to measure the copy number and sequence variation in rDNA. This revealed that variation in rDNA copy number between individuals spans an order of magnitude. We further observed a substantial level of sequence variation within individual genomes. As expected, single-nucleotide polymorphisms occurred in regions of lower functional constraint such as the IGS and expansion segments of the rRNA genes. The presence of strong linkage disequilibrium among variants facilitated identification of haplotypes within each population. Although there was evidence of recombination among haplotypes from different populations, it is insufficient to eliminate linkage disequilibrium within populations. Estimating copy number and haplotype diversity within individuals revealed that the level of intraindividual sequence variation is not strongly correlated with copy number. The observed patterns of variation highlight a complex evolutionary history of rDNA in D. pulex. Future research should explore the functional implications of rDNA copy number and sequence variation on organismal phenotypes.

The genetics and clinical outcomes in 151 cases of fetal growth restriction: A Chinese single-center study

ObjectiveTo determine the detection rate of chromosomal abnormalities and pregnancy outcomes in fetuses with intrauterine growth restriction.Study designA total of 151 fetal samples with intrauterine growth restriction were divided into the isolated fetal growth restriction (FGR) group, FGR group with structural malformation, and FGR group with non-structural malformation, according to ultrasound abnormalities. The enrolled patients were divided into an early onset FGR group (<32 weeks) and a late-onset FGR group (≥32 weeks). Chromosomal karyotype and microarray analyses were performed and pregnancy outcomes were monitored.ResultsThe karyotypes of 122 patients were analyzed. Four patients exhibited abnormal chromosome numbers or structures. Variations in copy number were detected in 151 cases; 19 cases were found to have chromosomal abnormalities, with a positivity rate of 12.6 %. There was one trisomy in 18 cases, one trisomy in 21 cases, eight pathogenic copy number variations (CNVs), and nine CNVs of unknown clinical significance. The detection rate of FGR combined with structural malformation was significantly higher than that of isolated FGR group. The detection rate of FGR with structural malformations was significantly higher than that with non-structural malformations. The positive detection rate in the FGR group was similar to that in the FGR group with non-structural malformations, with no statistical significance. Chromosomal abnormalities were detected in 17 patients with early onset FGR, with a positivity rate of 13.8 %. Two cases of chromosomal abnormalities were detected in the late-onset FGR group, with a positive rate of 7.1 %, with no statistical significance. A total of 151 fetuses with FGR were followed up for pregnancy outcomes, resulting in 36 cases of pregnancy termination and 13 cases of loss to follow-up. Among the 102 delivered fetuses, six exhibited delayed growth and development, one presented with hypospadias, and another failed the hearing screening. The remaining 94 fetuses demonstrated normal growth and development.ConclusionsThis study confirms the value of CNV detection in fetuses and dynamic ultrasound monitoring for fetuses with intrauterine growth restriction.

Characterization of zinc finger protein 536, a neuroendocrine regulator, using pan-cancer analysis

BackgroundPrevious studies suggested that zinc finger protein 536 (ZNF536) was abundant in the central brain and regulated neuronal differentiation. However, the role of ZNF536 in cancer has remained unclear.MethodsZNF536 mutation, copy number alteration, DNA methylation, and RNA expression were explored using public portals. Data from The Cancer Genome Atlas (TCGA) were utilized to analyze pathways and tumor microenvironment (TME), with a focus on prognosis in both TCGA and immunotherapy pan-cancer cohorts. Methylated ZNF536 from small cell lung cancer (SCLC) cell lines were utilized to train with probes for conducting enrichment analysis. Single-cell RNA profile demonstrated the sublocalization and co-expression of ZNF536, and validated its targets by qPCR.ResultsGenetic alterations in ZNF536 were found to be high-frequency and a single sample could harbor different variations. ZNF536 at chromosome 19q12 exerted a bypass effect on CCNE1, supported by CRISPR data. For lung cancer, ZNF536 mutation was associated with longer survival in primary lung adenocarcinoma (LUAD), but its prognosis was poor in metastatic LUAD and SCLC. Importantly, ZNF536 mutation and amplification had opposite prognoses in Stand Up To Cancer-Mark Foundation (SU2C-MARK) LUAD cohort. ZNF536 mutation altered the patterns of genomic alterations in tumors, and had distinct impacts on the signaling pathways and TME compared to ZNF536 amplification. Additionally, ZNF536 expression was predominantly in endocrine tumors and brain tissues. High-dimensional analysis supported this finding and further revealed regulators of ZNF536. Considering that the methylation of ZNF536 was involved in the synaptic pathway associated with neuroendocrine neoplasms, demonstrating both diagnostic and prognostic value. Moreover, we experimentally verified ZNF536 upregulated neuroendocrine markers.ConclusionsOur results showed that ZNF536 alterations in cancer, including variations in copy number, mutation, and methylation. We proved the involvement of ZNF536 in neuroendocrine regulation, and identified highly altered ZNF536 as a potential biomarker for immunotherapy.

Analysis of clinical Candida parapsilosis isolates reveals copy number variation in key fluconazole resistance genes.

We used whole-genome sequencing to analyze a collection of 35 fluconazole-resistant and 7 susceptible Candida parapsilosis isolates together with coverage analysis and GWAS techniques to identify new mechanisms of fluconazole resistance. Phylogenetic analysis shows that although the collection is diverse, two persistent clinical lineages were identified. We identified copy number variation (CNV) of two genes, ERG11 and CDR1B, in resistant isolates. Two strains have a CNV at the ERG11 locus; the entire ORF is amplified in one, and only the promoter region is amplified in the other. We show that the annotated telomeric gene CDR1B is actually an artifactual in silico fusion of two highly similar neighboring CDR genes due to an assembly error in the C. parapsilosis CDC317 reference genome. We report highly variable copy numbers of the CDR1B region across the collection. Several strains have increased the expansion of the two genes into a tandem array of new chimeric genes. Other strains have experienced a deletion between the two genes creating a single gene with a reciprocal chimerism. We find translocations, duplications, and gene conversion across the CDR gene family in the C. parapsilosis species complex, showing that it is a highly dynamic family.

Single-cell mtDNA dynamics in tumors is driven by coregulation of nuclear and mitochondrial genomes.

The extent of cell-to-cell variation in tumor mitochondrial DNA (mtDNA) copy number and genotype, and the phenotypic and evolutionary consequences of such variation, are poorly characterized. Here we use amplification-free single-cell whole-genome sequencing (Direct Library Prep (DLP+)) to simultaneously assay mtDNA copy number and nuclear DNA (nuDNA) in 72,275 single cells derived from immortalized cell lines, patient-derived xenografts and primary human tumors. Cells typically contained thousands of mtDNA copies, but variation in mtDNA copy number was extensive and strongly associated with cell size. Pervasive whole-genome doubling events in nuDNA associated with stoichiometrically balanced adaptations in mtDNA copy number, implying that mtDNA-to-nuDNA ratio, rather than mtDNA copy number itself, mediated downstream phenotypes. Finally, multimodal analysis of DLP+ and single-cell RNA sequencing identified both somatic loss-of-function and germline noncoding variants in mtDNA linked to heteroplasmy-dependent changes in mtDNA copy number and mitochondrial transcription, revealing phenotypic adaptations to disrupted nuclear/mitochondrial balance.

Diagnostic yield of the chromosomal microarray analysis in turkish patients with unexplained development delay/ıntellectual disability(ID), autism spectrum disorders and/or multiple congenital anomalies and new clinical findings.

Chromosomal microarray analysis is an essential tool for copy number variants detection in patients with unexplained developmental delay/intellectual disability, autism spectrum disorders, and multiple congenital anomalies. The study aims to determine the clinical significance of chromosomal microarray analysis in this patient group. Another crucial aspect is the evaluation of copy number variants detected in terms of the diagnosis of patients. A Chromosomal microarray analysis was was conducted on a total of 1227 patients and phenotype-associated etiological diagnosis was established in 135 patients. Phenotype-associated copy number variants were detected in 11% of patients. Among these, 77 patients 77 (57%, 77/135) were diagnosed with well-recognized genetic syndromes and phenotype-associated copy number variants were found in 58 patients (42.9%, 58/135). The study was designed to collect data of patients in Kocaeli Derince Training and Research Hospital retrospectively. In our study, we examined 135 cases with clinically significant copy number variability among all patients. In this study, chromosomal microarray analysis revealed pathogenic de novo copy number variants with new clinical features. Chromosomal microarray analysis in the Turkish population has been reported in the largest patient cohort to date.

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection

BackgroundMost recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors.ResultsIn micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct.ConclusionsFor the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

Haplotype-resolved genomes of octoploid species in Phyllanthaceae family reveal a critical role for polyploidization and hybridization in speciation.

The Phyllanthaceae family comprises a diverse range of plants with medicinal, edible, and ornamental value, extensively cultivated worldwide. Polyploid species commonly occur in Phyllanthaceae. Due to the rather complex genomes and evolutionary histories, their speciation process has been still lacking in research. In this study, we generated chromosome-scale haplotype-resolved genomes of two octoploid species (Phyllanthus emblica and Sauropus spatulifolius) in Phyllanthaceae family. Combined with our previously reported one tetraploid (Sauropus androgynus) and one diploid species (Phyllanthus cochinchinensis) from the same family, we explored their speciation history. The three polyploid species were all identified as allopolyploids with subgenome A/B. Each of their two distinct subgenome groups from various species was uncovered to independently share a common diploid ancestor (Ancestor-AA and Ancestor-BB). Via different evolutionary routes, comprising various scenarios of bifurcating divergence, allopolyploidization (hybrid polyploidization), and autopolyploidization, they finally evolved to the current tetraploid S. androgynus, and octoploid S. spatulifolius and P. emblica, respectively. We further discuss the variations in copy number of alleles and the potential impacts within the two octoploids. In addition, we also investigated the fluctuation of metabolites with medical values and identified the key factor in its biosynthesis process in octoploids species. Our study reconstructed the evolutionary history of these Phyllanthaceae species, highlighting the critical roles of polyploidization and hybridization in their speciation processes. The high-quality genomes of the two octoploid species provide valuable genomic resources for further research of evolution and functional genomics.

Revisiting soil fungal biomarkers and conversion factors: Interspecific variability in phospholipid fatty acids, ergosterol and rDNA copy numbers

Refined conversion factors for soil fungal biomarkers are proposed.High interspecific variability is present in all fungal biomarkers.A modeling approach supports the validity of biomarker estimates in diverse soils.ITS1 copies vary strongly, but are fungal-specific with least phylogenetic bias.A combination of fungal biomarkers will reveal soil fungal physiology and activity.The abundances of fungi and bacteria in soil are used as simple predictors for carbon dynamics, and represent widely available microbial traits. Soil biomarkers serve as quantitative estimates of these microbial groups, though not quantifying microbial biomass per se. The accurate conversion to microbial carbon pools, and an understanding of its comparability among soils is therefore needed. We refined conversion factors for classical fungal biomarkers, and evaluated the application of quantitative PCR (qPCR, rDNA copies) as a biomarker for soil fungi. Based on biomarker contents in pure fungal cultures of 30 isolates tested here, combined with comparable published datasets, we propose average conversion factors of 95.3 g fungal C g−1 ergosterol, 32.0 mg fungal C µmol−1 PLFA 18:2ω6,9 and 0.264 pg fungal C ITS1 DNA copy−1. As expected, interspecific variability was most pronounced in rDNA copies, though qPCR results showed the least phylogenetic bias. A modeling approach based on exemplary agricultural soils further supported the hypothesis that high diversity in soil buffers against biomarker variability, whereas also phylogenetic biases impact the accuracy of comparisons in biomarker estimates. Our analyses suggest that qPCR results cover the fungal community in soil best, though with a variability only partly offset in highly diverse soils. PLFA 18:2ω6,9 and ergosterol represent accurate biomarkers to quantify Ascomycota and Basidiomycota. To conclude, the ecological interpretation and coverage of biomarker data prior to their application in global models is important, where the combination of different biomarkers may be most insightful.

Prognosis and immunological characteristics of HDAC family in pan-cancer through integrative multi-omic analysis

BackgroundThe histone deacetylase (HDAC) family plays a significant role in a variety of biological processes related to cancer, such as metabolism and cancer progression, and recognized as a target of anti-cancer drugs. Nevertheless, there has been limited systematic research available regarding HDAC family in human cancers using multi-omics approaches.ObjectiveTo evaluate the prognostic implications and immunological features of the HDAC family across multiple cancer types through comprehensive multi-omics analysis.MethodsWe studied the associations of HDAC activity and a variety of factors, encompassing immune checkpoint genes, the tumor microenvironment (TME), instability of microsatellites (MSI), inherited mutations, variation in copy number, tumor mutation burden (TMB), and sensitivity to drugs in a variety of cancer types. Moreover, we analyzed the link between the degree of HDAC activity and the effectiveness of immunotherapy in several cohorts, providing significant details about the possible impact of HDACs on immunotherapeutic responses. Furthermore, we explored potential signaling pathways of HDACs in bladder cancer (BC) using gene set enrichment analysis (GSEA). Immune infiltration analysis in bladder cancer was performed based on HDACs expression, copy number or somatic mutations. Furthermore, the Human Protein Atlas (HPA) dataset has been used to validate the expression of the HDACs protein in BC.ResultsThe HDACs expression exhibits significant heterogeneity across different cancer types and is significantly correlated with patient prognosis, clinical traits, mutations, TME, TMB, MSI, immune checkpoint genes, and objective responses in immunotherapy. The drug sensitivity of cancers was found to be correlated with the gene expression of specific HDAC familial members. In BC, GSEA demonstrates enrichment of multiple immune-related functions and pathways; moreover, there are significant associations between genomic variations in HDACs and the degree of typical immunological cell recruitment. Furthermore, findings from differential expression analysis in BC are validated by protein expression analysis obtained from the HPA database.ConclusionsThese findings reveal the significance of HDAC family in survival prognosis and treatment guidance of human cancers, and some of them have the potential to be used as immunotherapy biomarkers.

A rapid PCR-free next-generation sequencing method for comprehensive diagnosis of chromosome disease syndromes in prenatal samples.

The aim of this study is to investigate the application performance of rapid copy number variation sequencing (rCNV-seq) technology for the detection of chromosomal abnormalities during prenatal diagnosis. Samples were collected from 424 pregnant women who were at high-risk for noninvasive prenatal screening in Kunming Maternal and Child Care Hospital from January 2018 to May 2022. rCNV-seq technique was used to detect fetal chromosome abnormalities and compare the results with that of chromosomal karyotype analysis. The Result showed that 330 (77.83%, 330/424) cases indicated chromosomal abnormalities among 424 high-risk pregnant women who underwent rCNV-seq. Moreover, 94 (22.17%, 94/424) cases were discovered to have copy number variations. Among the 330 fetuses with chromosomal abnormalities, common autosomal aneuploidy was observed in 203 cases (47.87%, 203/424) and sex chromosome aneuploidy was observed in 91 cases (21.46%, 91/424). Moreover, the abnormalities in multiple chromosomes were discovered in 33 cases (7.78%, 33/424), and the rare autosomal aneuploidy was observed in 3 cases (0.71%, 3/424). There were 63 fetuses (14.86%, 63/424) with pathogenic CNVs among the 94 fetuses with variable copy numbers. Of the 245 pregnant women who voluntarily selected G-band karyotyping, 1 fetus with copy number variation had normal karyotype results, and the remaining women were consistent with rCNV-seq. Our study revealed that rCNV-seq has higher accuracy in detecting common trisomy and can also detect chromosomal microdeletions or microduplications that cannot be detected by G-banding karyotype analysis. There is no effective treatment for chromosomal diseases, so it is particularly important to prevent chromosomal diseases through genetic counseling and prenatal diagnosis of chromosomal diseases.

Fusobacterium nucleatum subsp. polymorphum recovered from malignant and potentially malignant oral disease exhibit heterogeneity in adhesion phenotypes and adhesin gene copy number, shaped by inter-subspecies horizontal gene transfer and recombination-derived mosaicism.

Fusobacterium nucleatum is an anaerobic commensal of the oral cavity associated with periodontitis and extra-oral diseases, including colorectal cancer. Previous studies have shown an increased relative abundance of this bacterium associated with oral dysplasia or within oral tumours. Using direct culture, we found that 75 % of Fusobacterium species isolated from malignant or potentially malignant oral mucosa were F. nucleatum subsp. polymorphum. Whole genome sequencing and pangenome analysis with Panaroo was carried out on 76 F. nucleatum subsp. polymorphum genomes. F. nucleatum subsp. polymorphum was shown to possesses a relatively small core genome of 1604 genes in a pangenome of 7363 genes. Phylogenetic analysis based on the core genome shows the isolates can be separated into three main clades with no obvious genotypic associations with disease. Isolates recovered from healthy and diseased sites in the same patient are generally highly related. A large repertoire of adhesins belonging to the type V secretion system (TVSS) could be identified with major variation in repertoire and copy number between strains. Analysis of intergenic recombination using fastGEAR showed that adhesin complement is shaped by horizontal gene transfer and recombination. Recombination events at TVSS adhesin genes were not only common between lineages of subspecies polymorphum, but also between different subspecies of F. nucleatum. Strains of subspecies polymorphum with low copy numbers of TVSS adhesin encoding genes tended to have the weakest adhesion to oral keratinocytes. This study highlights the genetic heterogeneity of F. nucleatum subsp. polymorphum and provides a new framework for defining virulence in this organism.

Plasmid-mediated phenotypic noise leads to transient antibiotic resistance in bacteria

The rise of antibiotic resistance is a critical public health concern, requiring an understanding of mechanisms that enable bacteria to tolerate antimicrobial agents. Bacteria use diverse strategies, including the amplification of drug-resistance genes. In this paper, we showed that multicopy plasmids, often carrying antibiotic resistance genes in clinical bacteria, can rapidly amplify genes, leading to plasmid-mediated phenotypic noise and transient antibiotic resistance. By combining stochastic simulations of a computational model with high-throughput single-cell measurements of blaTEM-1 expression in Escherichia coli MG1655, we showed that plasmid copy number variability stably maintains populations composed of cells with both low and high plasmid copy numbers. This diversity in plasmid copy number enhances the probability of bacterial survival in the presence of antibiotics, while also rapidly reducing the burden of carrying multiple plasmids in drug-free environments. Our results further support the tenet that multicopy plasmids not only act as vehicles for the horizontal transfer of genetic information between cells but also as drivers of bacterial adaptation, enabling rapid modulation of gene copy numbers. Understanding the role of multicopy plasmids in antibiotic resistance is critical, and our study provides insights into how bacteria can transiently survive lethal concentrations of antibiotics.

Abstract 2268: CREDAC: Copy number-based reference-free expression deconvolution analysis of cancers

Abstract Introduction: Analyses of bulk RNA sequencing data are central to most large-scale tumor sequencing studies. Bulk expression data represent population averages and its interpretation is confounded by both normal cell contamination and somatic copy number alterations. Several computational methods to deconvolve have been developed recently. However, these methods often rely on a set of cell-type-specific reference signatures and ignore the effect of copy number changes. Methods: To address these issues, we have developed a method that formalizes the relationship between allele-specific copy number, expression and sample purity to deconvolve the expression profiles of tumor and normal cells from bulk RNA-seq in an unbiased manner. Our method was applied to sequencing data produced by the TRACERx consortium, a longitudinal study with multi-region whole-exome and RNA-seq of non-small-cell lung cancers. A total of 414 primary tumor regions and 140 adjacent normal tissue samples from 140 patients with matched DNA and RNA-seq data were processed. Results: We were able to directly deconvolve a median of ~2,000 genes per sample and indirectly infer tumor and normal expression profiles of ~10,000 genes using an scaled and weighted mean approach. The accuracy of the deconvolution was validated using (1) in-silico mixtures of patient-derived tumor and normal cells, (2) pseudo-bulk scRNAseq and (3) in regions with loss of heterogeneity (LOH) directly on the bulk sequencing data, where the total fraction of expression attributed to tumor cells can be computed directly using somatic mutations. CREDAC allows for single sample bulk-level tumor-normal differential expression analysis and revealed a strong and constitutive genome-wide overexpression in cancer compared to admixed normal cells, with a more pronounced overexpression in lung squamous cell carcinoma than lung adenocarcinoma (p&amp;lt;0.001). CREDAC adjusts for variations in copy number, thus facilitating the investigation of cancer-specific dosage compensatory responses triggered by aneuploidy. We revealed that a majority of genes (~65%) exhibit proportionality to copy number alterations, while a substantial proportion (~35%) shows anti-scaling or dosage compensation strategies. We show an enrichment of genes involved in cell cycle and expression processes within the dosage compensated group and revealed methylation as a key mechanism driving this compensatory process. Conclusion: Overall, our results suggest that CREDAC is able to accurately disentangle the expression of tumor and normal cells from bulk RNA-seq without any previous knowledge. It has potential applications in many studies that include matched RNA-seq and copy number data and can provide new insights functional characterization, the taxonomy of cancer, and tumor evolution. Citation Format: Carla Castignani, Jonas Demeulemeester, Oriol Pich, Tom Lesluyes, Robert E. Hynds, David R. Pearce, Elizabeth Larose Cadieux, Stefan C. Dentro, TRACERx Consortium, Nnenna Kanu, Charles Swanton, Peter Van Loo. CREDAC: Copy number-based reference-free expression deconvolution analysis of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2268.

Abstract 4338: A pan-cancer single-cell analysis of intratumoral copy number diversity and evolution

Abstract Aneuploidy is a hallmark of human cancers and studies have shown that cancer patients harbor many differences in their copy number aberrations (CNAs) reflecting interpatient heterogeneity (IPH). While IPH has been studied extensively, the intratumoral heterogeneity (ITH) of CNAs within a tumor remains understudied. Here, we conducted a pan-cancer analysis of 94 human tumor samples at single cell genomic resolution, representing seven major cancer types: bladder, breast, colon, glioblastoma, kidney, lung, and ovary. In total, 62,646 aneuploid cells were analyzed by single cell copy number profiling and bulk exome sequencing was performed. In most cancer types, increased subclonal diversity was associated with higher CNA burden, whole genome doubling (WGD) events, and TP53 mutations. In all tumors, our data show that cancer cells share a clonal set of truncal CNAs or mutations, suggesting these tumors evolved from a single ancestral cell. In 18/94 cases we identified subclonal WGD events, which resulted in a higher number of copy number losses after tetraploidization. Macro-spatial sampling showed that increased clonal diversity also correlated with increased spatial geographical diversity. We found that many cancers evolved CNAs in short punctuated bursts of evolution, and that this was not restricted to a specific cancer type. Collectively, these data show that copy number ITH is universally associated with molecular features across these human cancer types. Citation Format: Hanghui Ye, Thomas McDonald, Emi Sei, Darlan Conterno Minussi, Min Hu, Chenling Tang, Junke Wang, Kaile Wang, Anna Casasent, Hui Chen, Franziska Michor, Nicholas Navin. A pan-cancer single-cell analysis of intratumoral copy number diversity and evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4338.