Effects Of Aneuploidy Research Articles

Inferring replication timing and proliferation dynamics from single-cell DNA sequencing data

Dysregulated DNA replication is a cause and a consequence of aneuploidy in cancer, yet the interplay between copy number alterations (CNAs), replication timing (RT) and cell cycle dynamics remain understudied in aneuploid tumors. We developed a probabilistic method, PERT, for simultaneous inference of cell-specific replication and copy number states from single-cell whole genome sequencing (scWGS) data. We used PERT to investigate clone-specific RT and proliferation dynamics in >50,000 cells obtained from aneuploid and clonally heterogeneous cell lines, xenografts and primary cancers. We observed bidirectional relationships between RT and CNAs, with CNAs affecting X-inactivation producing the largest RT shifts. Additionally, we found that clone-specific S-phase enrichment positively correlated with ground-truth proliferation rates in genomically stable but not unstable cells. Together, these results demonstrate robust computational identification of S-phase cells from scWGS data, and highlight the importance of RT and cell cycle properties in studying the genomic evolution of aneuploid tumors.

Hypermetabolism in mice carrying a near-complete human chromosome 21.

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near-complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are likely due to a combination of increased activity level and sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle and hyperactivity. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.

Abstract P2-11-24: A Novel, Rapid and Economical Prognostic Tool For Adjuvant therapy Decisions in Hormone Positive Breast Cancer

Abstract Several multigene-based, risk scoring methods are available and their use is recommended to support treatment decision making in breast cancer. Although the value of these tests is established, critical challenges remain that limit their use, especially regarding turnaround time for fast decision making and the associated costs. We have developed DigiStain - a novel technology that uses mid-infrared imaging to precisely measure a surrogate of tumour aneuploidy, across unstained biopsy sections. Considering that genomic context shapes the pattern and consequences of aneuploidy during cancer development and progression, aneuploidy may have valuable prognostic or predictive value. Using bespoke software, DigiStain allows a quantitative score ‘DigiStain index’ (DI) to be reproducibly extracted from an objective physical measurement of a cancer i.e., aneuploidy. This information is generated within minutes and is available at the same time as routine H&E staining. We previously showed that DI significantly correlates with tumour grade and survival. To further validate the technology, we have investigated the ability of the DI to predict 5-year survival in a retrospective analysis of patients with oestrogen receptor (ER) positive breast cancer. Methods: Clinical samples were obtained from patients (N=944) treated at the University Hospital of Nottingham UK. Using the DigiStain imager, H&E images were registered before acquiring the DI score for the section. Statistical methodology followed the recommended approach of Royston et al. 2009, The BMJ. A multivariate logistic model modelling the ‘event’ of death by 5 years was generated. The predictive performance of the logistic regression model was assessed using Area Under the receiver operating characteristic Curve (AUC) plots. The predicted vs observed event probability was determined at various cut-offs and the accuracy of the classification was measured by its sensitivity and specificity. Results: Of 944 ER+ patients with follow up information, 77 (8.2%) died in the first 5 years. The age distribution was approximately normal, ranging from 26 to 70 (mean = 54.8 years). Tumour size ranged from 0.2 to 7.5 mm (mean = 1.90 mm and median = 1.70 mm) and had normal distribution when the natural log of size was considered. DI contributed to the prognostic model with (p=0.0018). Odds ratios showed increased odds of death by 5 years with higher DI. The AUC obtained for the model was 0.7722. Due to the number of events the sample was not split into training and validation sets. Instead bootstrapping (n=500) was used to assess performance. When adjusted by bootstrapping the AUC was reduced only slightly to 0.76. Classification tables for sensitivity and specificity across different risk cut-offs and distribution of 5-year predicted probabilities showed the best distinction between groups at low levels of predicted probabilities. As expected, there was a trade-off between sensitivity and specificity when choosing an appropriate cut-off point to define high risk of death by 5 years. Conclusions: The risk score obtained using the DI is a significant prognostic factor for 5-year survival in ER+ breast cancer (p=0.0018) with good diagnostic accuracy. Analysis of 10-year survival data is ongoing and plans to expand the sample collection to include patients from other sites are underway. Citation Format: Hemmel Amrania. A Novel, Rapid and Economical Prognostic Tool For Adjuvant therapy Decisions in Hormone Positive Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-24.

Extensive protein dosage compensation in aneuploid human cancers.

Aneuploidy is a hallmark of human cancers, but the effects of aneuploidy on protein expression remain poorly understood. To uncover how chromosome copy number changes influence the cancer proteome, we conducted an analysis of hundreds of human cancer cell lines and tumors with matched copy number, RNA expression, and protein expression data. We found that a majority of proteins show dosage compensation and fail to change by the degree expected based on chromosome copy number alone. We uncovered a variety of gene groups that were recurrently buffered upon both chromosome gain and loss, including protein complex subunits and cell cycle genes. Several genetic and biophysical factors were predictive of protein buffering, highlighting complex post-translational regulatory mechanisms that maintain appropriate gene product dosage. Finally, we established that chromosomal aneuploidy has a moderate effect on the expression of oncogenes and tumor suppressors, showing that these key cancer drivers can be subject to dosage compensation as well. In total, our comprehensive analysis of aneuploidy and dosage compensation across cancers will help identify the key driver genes encoded on altered chromosomes and will shed light on the overall consequences of aneuploidy during tumor development.

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

SUMMARYA central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena—from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.

Effects of aneuploidy on cell behaviour and function.

Aneuploidy, a genomic alternation characterized by deviations in the copy number of chromosomes, affects organisms from early development through to aging. Although it is a main cause of human pregnancy loss and a hallmark of cancer, how aneuploidy affects cellular function has been elusive. The last two decades have seen rapid advances in the understanding of the causes and consequences of aneuploidy at the molecular and cellular levels. These studies have uncovered effects of aneuploidy that can be beneficial or detrimental to cells and organisms in an environmental context-dependent and karyotype-dependent manner. Aneuploidy also imposes general stress on cells that stems from an imbalanced genome and, consequently, also an imbalanced proteome. These insights provide the fundamental framework for understanding the impact of aneuploidy in genome evolution, human pathogenesis and drug resistance.

Consequences of aneuploidy in human fibroblasts with trisomy 21

An extra copy of chromosome 21 causes Down syndrome, the most common genetic disease in humans. The mechanisms contributing to aneuploidy-related pathologies in this syndrome, independent of the identity of the triplicated genes, are not well defined. To characterize aneuploidy-driven phenotypes in trisomy 21 cells, we performed global transcriptome, proteome, and phenotypic analyses of primary human fibroblasts from individuals with Patau (trisomy 13), Edwards (trisomy 18), or Down syndromes. On average, mRNA and protein levels were increased by 1.5-fold in all trisomies, with a subset of proteins enriched for subunits of macromolecular complexes showing signs of posttranscriptional regulation. These results support the lack of evidence for widespread dosage compensation or dysregulation of chromosomal domains in human autosomes. Furthermore, we show that several aneuploidy-associated phenotypes are present in trisomy 21 cells, including lower viability and increased dependency on serine-driven lipid synthesis. Our studies establish a critical role of aneuploidy, independent of triplicated gene identity, in driving cellular defects associated with trisomy 21.

SELF-CORRECTION OF MOSAICISM IN HUMAN SELF-ORGANIZING GASTRULOIDS AS POTENTIAL EXPLANATION FOR NORMAL BIRTHS AFTER TRANSFER OF CHROMOSOMAL-ABNORMAL EMBRYOS

To recapitulate in in vitroin self-organizing human “gastruloids”the consequences of aneuploidy in cell fate determination during early human development and investigate a putative aneuploidy-correcting mechanism, suggested by birth of normal offspring following transfer of chromosomal-abnormal embryos.

Mosaic autosomal aneuploidies are detectable from single-cell RNAseq data

BackgroundAneuploidies are copy number variants that affect entire chromosomes. They are seen commonly in cancer, embryonic stem cells, human embryos, and in various trisomic diseases. Aneuploidies frequently affect only a subset of cells in a sample; this is known as “mosaic” aneuploidy. A cell that harbours an aneuploidy exhibits disrupted gene expression patterns which can alter its behaviour. However, detection of aneuploidies using conventional single-cell DNA-sequencing protocols is slow and expensive.MethodsWe have developed a method that uses chromosome-wide expression imbalances to identify aneuploidies from single-cell RNA-seq data. The method provides quantitative aneuploidy calls, and is integrated into an R software package available on GitHub and as an Additional file of this manuscript.ResultsWe validate our approach using data with known copy number, identifying the vast majority of aneuploidies with a low rate of false discovery. We show further support for the method’s efficacy by exploiting allele-specific gene expression levels, and differential expression analyses.ConclusionsThe method is quick and easy to apply, straightforward to interpret, and represents a substantial cost saving compared to single-cell genome sequencing techniques. However, the method is less well suited to data where gene expression is highly variable. The results obtained from the method can be used to investigate the consequences of aneuploidy itself, or to exclude aneuploidy-affected expression values from conventional scRNA-seq data analysis.

Aneuploidy Detection and mtDNA Quantification in Bovine Embryos with Different Cleavage Onset Using a Next-Generation Sequencing-Based Protocol

Bovine embryos are now routinely used in agricultural systems as a means of disseminating superior genetics worldwide, ultimately with the aim of feeding an ever-growing population. Further investigations, common for human IVF embryos, thus have priority to improve cattle IVF, as has screening for aneuploidy (abnormal chromosome number). Although the incidence and consequences of aneuploidy are well documented in human preimplantation embryos, they are less well known for the embryos of other animals. To address this, we assessed aneuploidy levels in thirty-one 2-cell bovine embryos derived from early- and late-cleaving zygotes. Contemporary approaches ( Whole Genome Amplification and next-generation sequencing) allowed aneuploidy assessment for all chromosomes in oocytes from donors aged 4-7 years. We also quantified mitochondrial DNA (mtDNA) levels in all blastomeres assessed, thereby testing the hypothesis that they are related to levels of aneuploidy. The overall incidence of aneuploidy in this cohort of bovine embryos was 41.1% and correlated significantly with the timing of cleavage (77.8% in late-cleaving vs. 31.8% in early-cleaving embryos). Moreover, based on mtDNA sequence read counts, we observed that the median mtDNA quantity is significantly lower in late-cleaving embryos. These findings further reinforce the use of the bovine system as a model for human IVF studies.

Mechanisms and consequences of aneuploidy and chromosome instability in the aging brain

Aneuploidy and polyploidy are a form of Genomic Instability (GIN) known as Chromosomal Instability (CIN) characterized by sporadic abnormalities in chromosome copy numbers. Aneuploidy is commonly linked to pathological states. It is a hallmark of spontaneous abortions and birth defects and it is observed virtually in every human tumor, therefore being generally regarded as detrimental for the development or the maturation of tissues under physiological conditions. Polyploidy however, occurs as part of normal physiological processes during maturation and differentiation of some mammalian cell types. Surprisingly, high levels of aneuploidy are present in the brain, and their frequency increases with age suggesting that the brain is able to maintain its functionality in the presence of high levels of mosaic aneuploidy. Because somatic aneuploidy with age can reach exceptionally high levels, it is likely to have long-term adverse effects in this organ. We describe the mechanisms accountable for an abnormal DNA content with a particular emphasis on the CNS where cell division is limited. Next, we briefly summarize the types of GIN known to date and discuss how they interconnect with CIN. Lastly we highlight how several forms of CIN may contribute to genetic variation, tissue degeneration and disease in the CNS.

Bub3 promotes Cdc20-dependent activation of the APC/C in S. cerevisiae.

The spindle checkpoint ensures accurate chromosome segregation by sending a signal from an unattached kinetochore to inhibit anaphase onset. Numerous studies have described the role of Bub3 in checkpoint activation, but less is known about its functions apart from the spindle checkpoint. In this paper, we demonstrate that Bub3 has an unexpected role promoting metaphase progression in budding yeast. Loss of Bub3 resulted in a metaphase delay that was not a consequence of aneuploidy or the activation of a checkpoint. Instead, bub3Δ cells had impaired binding of the anaphase-promoting complex/cyclosome (APC/C) with its activator Cdc20, and the delay could be rescued by Cdc20 overexpression. Kinetochore localization of Bub3 was required for normal mitotic progression, and Bub3 and Cdc20 colocalized at the kinetochore. Although Bub1 binds Bub3 at the kinetochore, bub1Δ cells did not have compromised APC/C and Cdc20 binding. The results demonstrate that Bub3 has a previously unknown function at the kinetochore in activating APC/C-Cdc20 for normal mitotic progression.

Unanticipated success stories: an interview with Angelika Amon.

THE Genetics Society of America Medal is awarded to an individual for outstanding contributions to the field of genetics in the past 15 years. Recipients of the GSA Medal are recognized for elegant and highly meaningful contributions to modern genetics. The 2014 recipient, Angelika B. Amon, has uncovered key principles governing the cell cycle and was the first to demonstrate a connection between the physical completion of anaphase and the initiation of mitotic exit. More recently, her research has focused on the consequences of aneuploidy. GENETICS spoke with Dr. Amon about her approach to science and what is next on the horizon.

Chromosome instability induced by Mps1 and p53 mutation generates aggressive lymphomas exhibiting aneuploidy-induced stress.

Aneuploidy is a hallmark of human solid cancers that arises from errors in mitosis and results in gain and loss of oncogenes and tumor suppressors. Aneuploidy poses a growth disadvantage for cells grown in vitro, suggesting that cancer cells adapt to this burden. To understand better the consequences of aneuploidy in a rapidly proliferating adult tissue, we engineered a mouse in which chromosome instability was selectively induced in T cells. A flanked by Lox mutation was introduced into the monopolar spindle 1 (Mps1) spindle-assembly checkpoint gene so that Cre-mediated recombination would create a truncated protein (Mps1(DK)) that retained the kinase domain but lacked the kinetochore-binding domain and thereby weakened the checkpoint. In a sensitized p53(+/-) background we observed that Mps1(DK/DK) mice suffered from rapid-onset acute lymphoblastic lymphoma. The tumors were highly aneuploid and exhibited a metabolic burden similar to that previously characterized in aneuploid yeast and cultured cells. The tumors nonetheless grew rapidly and were lethal within 3-4 mo after birth.

Aneuploidy in pluripotent stem cells and implications for cancerous transformation.

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.

Cell Cycle Regulation of Microtubule Interactomes: Multi-layered Regulation Is Critical for the Interphase/Mitosis Transition

Microtubules dramatically change their dynamics and organization at the entry into mitosis. Although this change is mediated by microtubule-associated proteins (MAPs), how MAPs themselves are regulated is not well understood. Here we used an integrated multi-level approach to establish the framework and biological significance of MAP regulation critical for the interphase/mitosis transition. Firstly, we applied quantitative proteomics to determine global cell cycle changes in the profiles of MAPs in human and Drosophila cells. This uncovered a wide range of cell cycle regulations of MAPs previously unidentified. Secondly, systematic studies of human kinesins highlighted an overlooked aspect of kinesins: most mitotic kinesins suppress their affinity to microtubules or reduce their protein levels in interphase in combination with nuclear localization. Thirdly, in-depth analysis of a novel Drosophila MAP (Mink) revealed that the suppression of the microtubule affinity of this mitotic MAP in combination with nuclear localization is essential for microtubule organization in interphase, and phosphorylation of Mink is needed for kinetochore-microtubule attachment in mitosis. Thus, this first comprehensive analysis of MAP regulation for the interphase/mitosis transition advances our understanding of kinesin biology and reveals the prevalence and importance of multi-layered MAP regulation.

Causes and consequences of aneuploidy in cancer

Genetic instability, which includes both numerical and structural chromosomal abnormalities, is a hallmark of cancer. Whereas the structural chromosome rearrangements have received substantial attention, the role of whole-chromosome aneuploidy in cancer is much less well-understood. Here we review recent progress in understanding the roles of whole-chromosome aneuploidy in cancer, including the mechanistic causes of aneuploidy, the cellular responses to chromosome gains or losses and how cells might adapt to tolerate these usually detrimental alterations. We also explore the role of aneuploidy in cellular transformation and discuss the possibility of developing aneuploidy-specific therapies.

Loss of A-type lamin expression compromises nuclear envelope integrity in breast cancer

Through advances in technology, the genetic basis of cancer has been investigated at the genomic level, and many fundamental questions have begun to be addressed. Among several key unresolved questions in cancer biology, the molecular basis for the link between nuclear deformation and malignancy has not been determined. Another hallmark of human cancer is aneuploidy; however, the causes and consequences of aneuploidy are unanswered and are hotly contested topics. We found that nuclear lamina proteins lamin A/C are absent in a significant fraction (38%) of human breast cancer tissues. Even in lamin A/C–positive breast cancer, lamin A/C expression is heterogeneous or aberrant (such as non-nuclear distribution) in the population of tumor cells, as determined by immunohistology and immunofluorescence microscopy. In most breast cancer cell lines, a significant fraction of the lamin A/C– negative population was observed. To determine the consequences of the loss of lamin A/C, we suppressed their expression by shRNA in non-cancerous primary breast epithelial cells. Down-regulation of lamin A/C in breast epithelial cells led to morphological deformation, resembling that of cancer cells, as observed by immunofluorescence microscopy. The lamin A/C–suppressed breast epithelial cells developed aneuploidy as determined by both flow Cytometry and fluorescence in situ hybridization. We conclude that the loss of nuclear envelope structural proteins lamin A/C in breast cancer underlies the two hallmarks of cancer aberrations in nuclear morphology and aneuploidy.

Abstract A79: Genome-wide RNAi screen for tetraploid-specific lethality in cancer cells

Abstract Cancer cells are often genetically unstable, resulting in aneuploid genomes. The consequences of aneuploidy are poorly understood, but have been suggested to play roles in tumor aggressiveness, chemotherapy resistance, and metastasis. One important route to an aneuploid genome may be through doubling of the genome, leading to unstable tetraploid cells. Many cell division errors are ultimately manifest as a failure of cytokinesis. Especially in cells defective in p53 function, tetraploid cells proliferate, but generate both high rates of whole chromosome aneuploidy and defects in the maintenance of chromosome structural integrity. Our laboratory has demonstrated that, in p53-primary cells, tetraploidy promotes tumor development and the resulting tumors display markedly abnormal genomes (Fujiwara T. et al. Nature 2005, 437: 1043). It is now known that several human cancer-mutations — e.g. Rb loss or loss of the adenomatous polyposis coli tumor suppressor — also predispose cytokinesis failure and tetraploidy. These findings highlight a need to understand in detail the physiological changes that accompany tetraploidy. To gain a comprehensive understanding of physiological alterations associated with tetraploidy in cancer cells, we have established tetraploid cell lines derived from a near diploid human colorectal cancer cell line HCT116. Using these paired tetraploid and diploid cell lines, we have performed a genome-wide RNAi screen to identify genes whose knock down selectively affect the viability of either diploids or tetraploid cells. We are currently validating the result of our ploidy-specific lethality screen and we would like to update our progress at this conference. Citation Information: Cancer Res 2009;69(23 Suppl):A79.

Association between sperm DNA fragmentation and aneuploidy rate in assisted reproduction patients

OBJECTIVE: Damage to sperm chromatin, such as DNA fragmentation that has been associated with male infertility, unsuccessful conception and poor sustained pregnancy. However few studies have shown that fragmented DNA can be associated with chromosomal aberrations in sperm. Therefore the aim of this study was to analyze the frequency of sex-chromosomal aneuploidy in spermatozoa of non-fragmented (NF) and fragmented (F) assisted reproduction treatment (ART) patients, and to correlate aneuploidy rate between each group. DESIGN: Prospective laboratory study. MATERIALS AND METHODS: Sperm DNA fragmentation rate (SDFR) was assessed by sperm chromatin dispersion (SCD) test and patients were divided in NF (SDFR ≤16%) and F (SDFR>16%). Aneuploidy rate evaluated by Fluorescence in situ hybridization (FISH) with direct label probes specific for chromosome 18, X and Y (Kreatech Diagnosis, Netherlands). A total of 500 spermatozoa were analyzed per sample. Statistical analysis was performed with Mann-Whitney test (median; range), Spearman's correlation test and binary logistic regression. Every calculation was done by Graph Pad Prism 4 program. RESULTS: Twenty samples from ART patients were assigned to F (n=10) and NF (n=10). The rate of gonosomal aneuploidy was lower in NF (11.5; 5-43) than in F (28; 14-56; P<0.05). Likelihood for aneuploidy in spermatozoa DNA is 2-fold increased in men with fragmented sperm DNA (odds ratio: 2.01; 95% confidence interval=1.6-2.4; p<0.001). Aneuploidy rate and fragmentation of sperm DNA were significantly correlated (r=0.56; P<0.01). CONCLUSIONS: Sperm DNA fragmentation is strongly correlated to aneuploidy rate and may be a consequence of aneuploidy during sperm maturation. Additionally our findings suggest that males with fragmented sperm DNA have risk of fathering aneuploid offspring.