Karyotypic determinants of chromosome instability in aneuploid budding yeast

Recent studies in cancer cells and budding yeast demonstrated that aneuploidy, the state of having abnormal chromosome numbers, correlates with elevated chromosome instability (CIN), i.e. the propensity of gaining and losing chromosomes at a high frequency. Here we have investigated ploidy- and chromosome-specific determinants underlying aneuploidy-induced CIN by observing karyotype dynamics in fully isogenic aneuploid yeast strains with ploidies between 1N and 2N obtained through a random meiotic process. The aneuploid strains exhibited various levels of whole-chromosome instability (i.e. chromosome gains and losses). CIN correlates with cellular ploidy in an unexpected way: cells with a chromosomal content close to the haploid state are significantly more stable than cells displaying an apparent ploidy between 1.5 and 2N. We propose that the capacity for accurate chromosome segregation by the mitotic system does not scale continuously with an increasing number of chromosomes, but may occur via discrete steps each time a full set of chromosomes is added to the genome. On top of such general ploidy-related effect, CIN is also associated with the presence of specific aneuploid chromosomes as well as dosage imbalance between specific chromosome pairs. Our findings potentially help reconcile the divide between gene-centric versus genome-centric theories in cancer evolution.     

Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.     

Aneuploidy,cell delamination and tumorigenesis in Drosophila epithelia

Chromosomal instability (CIN) is a common feature in human cancer, and highly aneuploid tumors are frequently associated with poor prognosis; however, the molecular and cellular mechanisms underlying CIN-induced tumorigenesis are poorly understood. Here we review recent findings about the role of CIN in driving tumor-like growth and host invasiveness in Drosophila epithelia and discuss the commonalities of CIN-induced tumors with other Drosophila-based cancer models. We also discuss possible scenarios that can account for the participation of CIN in tumorigenesis and propose that, alternatively to the classical role of aneuploidy in promoting the accumulation of mutations in cancer cells, aneuploidy can be a source of stress that may contribute to cancer initiation and/or progression.     

Chromosomal composition of aneuploid clones with different DNA contents in head and neck squamous cell carcinomas as determined by combined flow cytometry and fluorescence in situ hybridization.

Studies with DNA flow cytometry (FCM) have shown that DNA contents of aneuploid tumour clones vary in a wide range. The aim of this study was to analyse whether homologous chromosomal changes exist despite the individual differences that may be of general relevance for the development of gross aneuploidy in squamous cell carcinomas of the head and neck. Fluorescence in situ hybridization (FISH) with 13 centromere-specific DNA probes was applied to 3 diploid and 11 aneuploid tumours with DNA indices ranging between 0.8 and 2.2. Disomic and monosomic cell populations were prevalent findings in DNA-diploid tumours. Polysomies were common in aneuploid tumours. Different degrees of aneusomy for identical chromosomes were recurrent features in aneuploid tumours. FISH signal heterogeneity was identified for all chromosomes. The mean number of aneusomic cell populations identified for DNA-aneuploid tumours ranged between 1.6 for chromosome 17 and 3.1 for chromosome 3. Inconsistencies between FISH and FCM data may indicate that centromere-specific DNA probes identify gains and losses of marker DNA due to complex karyotypic rearrangements rather than absolute changes in chromosome numbers. Overall, there was no evidence of the critical involvement of particular chromosomes in the development of different DNA contents.     

Proliferation of aneuploid human cells is limited by a p53-dependent mechanism

Most solid tumors are aneuploid, and it has been proposed that aneuploidy is the consequence of an elevated rate of chromosome missegregation in a process called chromosomal instability (CIN). However, the relationship of aneuploidy and CIN is unclear because the proliferation of cultured diploid cells is compromised by chromosome missegregation. The mechanism for this intolerance of nondiploid genomes is unknown. In this study, we show that in otherwise diploid human cells, chromosome missegregation causes a cell cycle delay with nuclear accumulation of the tumor suppressor p53 and the cyclin kinase inhibitor p21. Deletion of the p53 gene permits the accumulation of nondiploid cells such that CIN generates cells with aneuploid genomes that resemble many human tumors. Thus, the p53 pathway plays an important role in limiting the propagation of aneuploid human cells in culture to preserve the diploid karyotype of the population. These data fit with the concordance of aneuploidy and disruption of the p53 pathway in many tumors, but the presence of aneuploid cells in some normal human and mouse tissues indicates that there are known exceptions to the involvement of p53 in aneuploid cells and that tissue context may be important in how cells respond to aneuploidy.     

CIN and Aneuploidy: Different Concepts,Different Consequences

Chromosomal instability (CIN) and aneuploidy are similar concepts but not synonymous. CIN is the process that leads to chromosome copy number alterations, and aneuploidy is the result. While CIN and resulting aneuploidy often cause growth defects, they are also selected for in cancer cells. Although such contradicting fates may seem paradoxical at first, they can be better understood when CIN and aneuploidy are assessed separately, taking into account the in vitro or in vivo context, the rate of CIN, and severity of the aneuploid karyotype. As CIN can only be measured in living cells, which proves to be technically challenging in vivo, aneuploidy is more frequently quantified. However, CIN rates might be more predictive for tumor outcome than assessing aneuploidy rates alone. In reviewing the literature, we therefore conclude that there is an urgent need for new models in which we can monitor chromosome mis‐segregation and its consequences in vivo. Also see the video abstract here: https://youtu.be/fL3LxZduchg     

DIAPH1 regulates chromosomal instability of cancer cells by controlling microtubule dynamics

Chromosomal instability (CIN) is a hallmark of cancer, resulting from misalignment and missegregation of chromosomes during meta- and anaphase, due to non-precise regulation of spindle-MT dynamics. Diaphanous Related Formin 1 (DIAPH1) is an actin nucleator and also binds microtubule (MT) with high affinity. In this study, we analyzed the role of DIAPH1 in regulation of spindle MT-dynamics and CIN in HT29 and HCT-116 colorectal cancer (CRC) cells. Our data show that down-regulation of DIAPH1 in these cell lines decreased spindle-MT speed by 50 % and the fraction of cells with misaligned and missegregated chromosomes was significantly increased. Furthermore, in HCT-116 DIAPH1 depleted cells deviation of chromosome number was elevated and the number of cells with micronuclei and cytosolic DNA was increased in both DIAPH1-knock down cell lines. In line with these results, database analysis revealed a significant correlation with low DIAPH1 mRNA expression and aneuploidy. Thus, DIAPH1 is substantially involved in the control of CIN in CRC cells. Since in vitro, DIAPH1 directly increased MT-polymerization, we assume that DIAPH1 controls CIN by regulating spindle-MT dynamics.     

CINister thoughts

Chromosome instability (CIN) is the process that leads to aneuploidy, a known hallmark of human tumours for over a century. Nowadays, it is believed that CIN promotes tumorigenesis by shuffling the genome into a malignant order through translocations, amplifications, deletions (structural CIN), and gains and losses of whole chromosomes (numerical CIN or nCIN). The present review focuses on the causes and consequences of nCIN. Several roads can lead to nCIN, including a compromised spindle assembly checkpoint, cohesion defects, p53 deficiency and flawed microtubule-kinetochore attachments. Whereas the link between nCIN and tumorigenesis is becoming more evident, indications have emerged recently that nCIN can suppress tumour formation as well. To understand these paradoxical findings, novel reagents and more sophisticated mouse models are needed. This will provide us with a better understanding of nCIN and eventually with therapies that exploit this characteristic of human tumours.     

Dynamic karyotype,dynamic proteome: buffering the effects of aneuploidy

Despite its ubiquity in cancer, link with other pathologies, and role in promoting adaptive evolution, the effects of aneuploidy or imbalanced chromosomal content on cellular physiology have remained incompletely characterized. Significantly, it appears that the detrimental as well as beneficial effects of aneuploidy are due to the altered gene expression elicited by the aneuploid state. In this review we examine the correlation between chromosome copy number changes and gene expression in aneuploid cells. We discuss the primary effects of aneuploidy on gene expression and describe the cellular response to altered mRNA and protein levels. Moreover, we consider compensatory mechanisms that may ameliorate imbalanced gene expression and restore protein homeostasis in aneuploid cells. Finally, we propose a novel hypothesis to explain the hitherto enigmatic abundance compensation of proteins encoded on supernumerary chromosomes.     

Etude de l'aneuploïdie dans différents naissains d'Ostreidae (Bivalvia)

Study of aneuploidy in spats of Ostreidae (Bivalvia). Chromosomes of cells from gill tissue of juveniles (5–10 mm) from four different populations of Ostrea edulis and five different populations of Crassostrea gigas were examined in order to study aneuploidy and its significance. Mitotic chromosome counts were made for a sample of spats in each population. Cells with the normal diploid complement (2n=20) and with aneuploid complements (2n different from 20) were scored. The total percentage of aneuploid cells in the studied populations of the two species varies from 9% to 34%. Individual patterns of aneuploidy were examined in three populations of Crassostrea gigas. Some animals showed only normal diploid cells, others having both normal and aneuploid cells in variable proportions. The relationship between aneuploidy and growth rate was studied in experimental spats. Mitotic chromosome counts were made on individuals from two groups of animals: the first group was collected at a precise date at a control size of 8 mm, the second group reaching the same size of 8 mm only one month later. The percentage of animals showing aneuploid cells is much greater in the second group. There is a relationship between occurrence of aneuploid cells and growth rate. The karyotypes of 18 aneuploid sets of Ostrea edulis from different spats were analyzed. Chromosome loss was observed only in the submetacentric chromosome pairs. This loss of chromosomes could have an effect on the percentage of homozygotes observed in electrophoregrams. Thus, the percentage of homozygotes would be higher in juveniles showing the greatest number of aneuploid cells. An excess of homozygosity (=heterozygote deficiency) has been reported at a number of enzyme loci in over two dozen bivalve species (including Ostrea and Crassostrea). We suggest the hypothesis that this excess of homozygosity could be related to the occurrence of aneuploid cells. The relationship between aneuploidy and growth rate may prove to be a genetic factor of importance for oyster culturing.     

Elevated Tolerance to Aneuploidy in Cancer Cells: Estimating the Fitness Effects of Chromosome Number Alterations by In Silico Modelling of Somatic Genome Evolution

An unbalanced chromosome number (aneuploidy) is present in most malignant tumours and has been attributed to mitotic mis-segregation of chromosomes. However, recent studies have shown a relatively high rate of chromosomal mis-segregation also in non-neoplastic human cells, while the frequency of aneuploid cells remains low throughout life in most normal tissues. This implies that newly formed aneuploid cells are subject to negative selection in healthy tissues and that attenuation of this selection could contribute to aneuploidy in cancer. To test this, we modelled cellular growth as discrete time branching processes, during which chromosome gains and losses were generated and their host cells subjected to selection pressures of various magnitudes. We then assessed experimentally the frequency of chromosomal mis-segregation as well as the prevalence of aneuploid cells in human non-neoplastic cells and in cancer cells. Integrating these data into our models allowed estimation of the fitness reduction resulting from a single chromosome copy number change to an average of ≈30% in normal cells. In comparison, cancer cells showed an average fitness reduction of only 6% (p = 0.0008), indicative of aneuploidy tolerance. Simulations based on the combined presence of chromosomal mis-segregation and aneuploidy tolerance reproduced distributions of chromosome aberrations in >400 cancer cases with higher fidelity than models based on chromosomal mis-segregation alone. Reverse engineering of aneuploid cancer cell development in silico predicted that aneuploidy intolerance is a stronger limiting factor for clonal expansion of aneuploid cells than chromosomal mis-segregation rate. In conclusion, our findings indicate that not only an elevated chromosomal mis-segregation rate, but also a generalised tolerance to novel chromosomal imbalances contribute to the genomic landscape of human tumours.     

Genetic variation in aneuploidy prevalence and tolerance across Saccharomyces cerevisiae lineages

Individuals carrying an aberrant number of chromosomes can vary widely in their expression of aneuploidy phenotypes. A major unanswered question is the degree to which an individual’s genetic makeup influences its tolerance of karyotypic imbalance. Here we investigated within-species variation in aneuploidy prevalence and tolerance, using Saccharomyces cerevisiae as a model for eukaryotic biology. We analyzed genotypic and phenotypic variation recently published for over 1,000 S. cerevisiae strains spanning dozens of genetically defined clades and ecological associations. Our results show that the prevalence of chromosome gain and loss varies by clade and can be better explained by differences in genetic background than ecology. The relationships between lineages with high aneuploidy frequencies suggest that increased aneuploidy prevalence emerged multiple times in S. cerevisiae evolution. Separate from aneuploidy prevalence, analyzing growth phenotypes revealed that some genetic backgrounds—such as the European Wine lineage—show fitness costs in aneuploids compared to euploids, whereas other clades with high aneuploidy frequencies show little evidence of major deleterious effects. Our analysis confirms that chromosome gain can produce phenotypic benefits, which could influence evolutionary trajectories. These results have important implications for understanding genetic variation in aneuploidy prevalence in health, disease, and evolution.     

Aurea Mediocritas: The Importance of a Balanced Genome

Aneuploidy, defined as an abnormal number of chromosomes, is a hallmark of cancer. Paradoxically, aneuploidy generally has a negative impact on cell growth and fitness in nontransformed cells. In this work, we review recent progress in identifying how aneuploidy leads to genomic and chromosomal instability, how cells can adapt to the deleterious effects of aneuploidy, and how aneuploidy contributes to tumorigenesis in different genetic contexts. Finally, we also discuss how aneuploidy might be a target for anticancer therapies.As Horace famously wrote in his Odes, the “golden mean” is the secret to a happy, balanced life. Recent work, reviewed here, emphasizes the importance of this kind of balance for the genetics of human cells.Maintaining a stable genome is critical for the preservation of genetic information during the life span of an organism. Despite mechanisms designed to ensure a diploid karyotype, errors can and do occur during chromosome segregation that result in the gain and loss of whole chromosomes. In vitro estimates suggest that normal, diploid cells missegregate a chromosome once every 100 cell divisions (Thompson and Compton 2008). The in vivo rate of chromosome missegregation is unknown, but could vary between different cell types. Even if the rate is low, an abnormal number of chromosomes, or aneuploidy, could have a significant impact on normal cell physiology, as well as tumorigenesis.Aneuploidy, at the level of the organism, is detrimental and generally incompatible with life. In humans, only three aneuploidies—trisomy 13, 18, and 21—are viable, and only trisomy 21 is compatible with a life span beyond infancy (Hassold et al. 2007). Despite the deleterious consequences of aneuploidy in normal physiological contexts, an abnormal number of chromosomes is one of the hallmarks of cancer cells. Aneuploidy is found in ∼90% of solid tumors and >50% of blood cancers (Beroukhim et al. 2010; Mitelman et al. 2013). Whether aneuploidy is a cause or consequence of cell transformation is a frequent topic of debate. The challenge for establishing a causal relationship stems from the complexity of cancer cells, in which numerical chromosome abnormalities are rarely found in isolation but are usually accompanied by other genomic alterations, such as point mutations, translocations, and microsatellite instability. This complexity makes it difficult to define the initiating event(s) in tumorigenesis.This review focuses on whole-chromosome aneuploidy, although it has been shown that the gain and loss of chromosome arms is also a common occurrence in cancer cells (Beroukhim et al. 2010; Mitelman et al. 2013). We review the molecular pathways leading to aneuploidy, the effects of aneuploidy on cellular physiology, and the links between aneuploidy and tumorigenesis. Finally, we also explore the exciting concept of targeting aneuploidy as a novel therapeutic approach in treating cancer.     

Oncogenic Adenomatous Polyposis Coli Mutants Impair the Mitotic Checkpoint through Direct Interaction with Mad2

The majority of colorectal tumors are aneuploid because of the underlying chromosome instability (CIN) phenotype, in which a defective mitotic checkpoint is implicated. Adenomatous polyposis coli (APC), a tumor suppressor gene that is commonly mutated in colon cancers, has been suggested in causing CIN; however, the molecular mechanism remains unresolved. In this study, we report an interaction of tumor-associated N-terminal APC fragments (N-APC) with Mad2, an essential mitotic checkpoint protein, providing a direct molecular support for linking APC mutations to the generation of CIN. N-APC interacts with Mad2 in Xenopus egg extracts, colon cancer cells, and in vitro with purified components. The interaction between N-APC and Mad2 decreases the soluble pool of Mad2, which is essential for Mad2 cycling and releasing from unattached kinetochores to produce a diffusible |P`wait anaphase|P' signal. Addition of such an N-APC mutant of egg extracts inactivates the mitotic checkpoint. Expressing a tumor-associated N-APC mutant in mammalian cells with an intact mitotic checkpoint produces premature anaphase onset with missegregated chromosomes.     

Aneuploidy, the primary cause of the multilateral genomic instability of neoplastic and preneoplastic cells

Cancers have a clonal origin, yet their chromosomes and genes are non-clonal or heterogeneous due to an inherent genomic instability. However, the cause of this genomic instability is still debated. One theory postulates that mutations in genes that are involved in DNA repair and in chromosome segregation are the primary causes of this instability. But there are neither consistent correlations nor is there functional proof for the mutation theory. Here we propose aneuploidy, an abnormal number of chromosomes, as the primary cause of the genomic instability of neoplastic and preneoplastic cells. Aneuploidy destabilizes the karyotype and thus the species, independent of mutation, because it corrupts highly conserved teams of proteins that segregate, synthesize and repair chromosomes. Likewise it destabilizes genes. The theory explains 12 of 12 specific features of genomic instability: (1) Mutagenic and non-mutagenic carcinogens induce genomic instability via aneuploidy. (2) Aneuploidy coincides and segregates with preneoplastic and neoplastic genomic instability. (3) Phenotypes of genomically unstable cells change and even revert at high rates, compared to those of diploid cells, via aneuploidy-catalyzed chromosome rearrangements. (4) Idiosyncratic features of cancers, like immortality and drug-resistance, derive from subspecies within the 'polyphyletic' diversity of individual cancers. (5) Instability is proportional to the degree of aneuploidy. (6) Multilateral chromosomal and genetic instabilities typically coincide, because aneuploidy corrupts multiple targets simultaneously. (7) Gene mutation is common, but neither consistent nor clonal in cancer cells as predicted by the aneuploidy theory. (8) Cancers fall into a near-diploid (2 N) class of low instability, a near 1.5 N class of high instability, or a near 3 N class of very high instability, because aneuploid fitness is maximized either by minimally unstable karyotypes or by maximally unstable, but adaptable karyotypes. (9) Dominant phenotypes, because of aneuploid genotypes. (10) Uncertain developmental phenotypes of Down and other aneuploidy syndromes, because supply-sensitive, diploid programs are destabilized by products from aneuploid genes supplied at abnormal concentrations; the maternal age-bias for Down's would reflect age-dependent defects of the spindle apparatus of oocytes. (11) Non-selective phenotypes, e.g., metastasis, because of linkage with selective phenotypes on the same chromosomes. (12) The target, induction of genomic instability, is several 1000-fold bigger than gene mutation, because it is entire chromosomes. The mutation theory explains only a few of these features. We conclude that the transition of stable diploid to unstable aneuploid cell species is the primary cause of preneoplastic and neoplastic genomic instability and of cancer, and that mutations are secondary.     

Aneuploidy and inbreeding depression in random mating and self-fertilizing autotetraploid populations

Summary Almost all autotetraploids produce aneuploid progeny because of irregularities at meiosis. Aneuploid plants produce high frequencies of aneuploids. If it were not for selection against aneuploid gametes and sporophytes the amount of aneuploidy would increase every generation. Most experimental and theoretical studies on population genetics and heterosis in autotetraploids have neglected aneuploidy as a factor. To take aneuploidy into account experimentally requires the cytological identification of all chromosomes and to consider it theoretically requires a huge amount of computations. Consequently, microcomputer programs have been devised to show the effects of random mating and self-fertilization in autotetraploid populations. According to the model aneuploidy rapidly increases in randomly mated and self-fertilized autotetraploid populations until they achieve an equilibrium where the amount of aneuploidy introduced into the population is balanced by the amount of aneuploidy removed from the population by selection. The model suggests that self-fertilized populations have greater frequencies of aneuploid gametes and zygotes than do randomly mated populations and therefore aneuploidy may be a significant cause of the great inbreeding depressions found in autotetraploids.Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 9998     

2n or not 2n: Aneuploidy,polyploidy and chromosomal instability in primary and tumor cells

Mitotic defects leading to aneuploidy have been recognized as a hallmark of tumor cells for over 100 years. Current data indicate that ∼85% of human cancers have missegregated chromosomes to become aneuploid. Some maintain a stable aneuploid karyotype, while others consistently missegregate chromosomes over multiple divisions due to chromosomal instability (CIN). Both aneuploidy and CIN serve as markers of poor prognosis in diverse human cancers. Despite this, aneuploidy is generally incompatible with viability during development, and some aneuploid karyotypes cause a proliferative disadvantage in somatic cells. In vivo, the intentional introduction of aneuploidy can promote tumors, suppress them, or do neither. Here, we summarize current knowledge of the effects of aneuploidy and CIN on proliferation and cell death in nontransformed cells, as well as on tumor promotion, suppression, and prognosis.     

Culture expansion induces non-tumorigenic aneuploidy in adipose tissue-derived mesenchymal stromal cells

Background aimsAdipose tissue-derived mesenchymal stromal cells (ASCs) are of interest as a cell therapeutic agent for immunologic and degenerative diseases. During in vitro expansion, ASCs may be at risk for genetic alterations, and genetic screening is a prerequisite. We examined the presence of aneuploidy in ASCs and its origin and development during culture and evaluated the implications of aneuploidy for therapeutic use of ASCs.MethodsAdipose tissue of healthy individuals was used for isolation and expansion of ASCs. Chromosome copy numbers were studied using fluorescence in situ hybridization analysis. Aneuploidy was studied in freshly isolated ASCs, in ASCs cultured for 0–16 passages and in senescent cultures. To evaluate the plasticity of ploidy, ASCs were cloned, and the variation of ploidy in the clones was examined. Tumorigenicity was studied by subcutaneous injection of aneuploid ASCs in immunodeficient NOD/SCID mice.ResultsNo aneuploidy was detected in freshly isolated ASCs. In low passages (passages 0–4), aneuploidy was detected in 3.4% of ASCs. Prolonged culture expansion of ASCs (passages 5–16) resulted in a significant increase of aneuploidy to 7.1%. With senescence, aneuploidy increased further to 19.8%. Aneuploidy was observed in clones of diploid ASCs, demonstrating the de novo development of aneuploidy. No transformation of ASCs was observed, and in contrast to cancer cell lines, aneuploid ASCs were incapable of tumor formation in immunodeficient mice.ConclusionsASC cultures contain a stable percentage of aneuploid cells. Aneuploidy was not a predecessor of transformation or tumor formation. This finding indicates that aneuploidy is culture-induced but unlikely to compromise clinical application of ASCs.     

An analysis of frequencies of chromosome configurations in wheat and wheat hybrids

Presynaptic association of homologous chromosomes is a prerequisite to the sequence of events that lead to chiasma formation. This association of homologous chromosomes, as entire units, occurs with probability a, and chiasma formation occurs independently in opposite chromosome arms with probability c. a and c have been estimated from frequencies of different chromosome configurations at metaphase I of euhexaploid wheat and several derived lines. In the euploid, a is essentially unity and c is of the order of 95%. All changes in the aneuploidy studied involved c rather than a, whereas the change induced by colchicine application primarily involved a.—Observed and expected frequencies of configurations were compared in wheat hybrids in which only homoeologues were present. The expected frequencies of configurations were estimated from the data, based on a being unity for entire groups of homoeologues and c being the probability of chiasma formation between random homoeologous arms. Observed and expected frequences of configurations were in general agreement; however, an excess of observed closed bivalents at the expense of multivalents is interpreted to mean that not all homoeologues are effectively associated in all cells.—In euhexaploid wheat, we suggest that homologues associate with almost certainty, whereas homoeologous pairs of chromosomes associate less efficiently. The aneuploidy examined in this study does not appear to affect the association of chromosomes, but rather the number of chiasmata that eventuate and, in the case of deficiency of chromosome 5B, the distribution of chiasmata within homoelogues, perhaps by way of rendering sites for chiasma formation of homoelogues more similar.