Showing papers on "Karyotype published in 2014"

Single cell sequencing reveals low levels of aneuploidy across mammalian tissues

Abstract: Whole-chromosome copy number alterations, also known as aneuploidy, are associated with adverse consequences in most cells and organisms. However, high frequencies of aneuploidy have been reported to occur naturally in the mammalian liver and brain, fueling speculation that aneuploidy provides a selective advantage in these organs. To explore this paradox, we used single cell sequencing to obtain a genome-wide, high-resolution assessment of chromosome copy number alterations in mouse and human tissues. We find that aneuploidy occurs much less frequently in the liver and brain than previously reported and is no more prevalent in these tissues than in skin. Our results highlight the rarity of chromosome copy number alterations across mammalian tissues and argue against a positive role for aneuploidy in organ function. Cancer is therefore the only known example, in mammals, of altering karyotype for functional adaptation.

Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line

Abstract: Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.

SF3B1 mutations correlated to cytogenetics and mutations in NOTCH1, FBXW7, MYD88, XPO1 and TP53 in 1160 untreated CLL patients

Abstract: We analyzed a large cohort of 1160 untreated CLL patients for novel genetic markers (SF3B1, NOTCH1, FBXW7, MYD88, XPO1) in the context of molecular, immunophenotypic and cytogenetic data. NOTCH1 mutations (mut) (12.3%), SF3B1mut (9.0%) and TP53mut (7.1%) were more frequent than XPO1mut (3.4%), FBXW7mut (2.5%) and MYD88mut (1.5%). SF3B1mut, NOTCH1mut, TP53mut and XPO1mut were highly correlated to unmutated, whereas MYD88mut were associated with mutated IGHV status. Associations of diverse cytogenetic aberrations and mutations emerged: (1) SF3B1mut with del(11q), (2) NOTCH1mut and FBXW7mut with trisomy 12 and nearly exclusiveness of SF3B1mut, (3) MYD88mut with del(13q) sole and low frequencies of SF3B1mut, NOTCH1mut and FBXW7mut. In patients with normal karyotype only SF3B1mut were frequent, whereas NOTCH1mut rarely occurred. An adverse prognostic impact on time to treatment (TTT) and overall survival (OS) was observed for SF3B1mut, NOTCH1mut and TP53 disruption. In multivariate analyses SF3B1mut, IGHV mutational status and del(11q) were the only independent genetic markers for TTT, whereas for OS SF3B1mut, IGHV mutational status and TP53 disruption presented with significant impact. Finally, our data suggest that analysis of gene mutations refines the risk stratification of cytogenetic prognostic subgroups and confirms data of a recently proposed model integrating molecular and cytogenetic data.

The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera

Abstract: Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) ...

Turner syndrome revisited: review of new data supports the hypothesis that all viable 45,X cases are cryptic mosaics with a rescue cell line, implying an origin by mitotic loss

Abstract: We review the data pertinent to the hypothesis we proposed three decades ago, that all embryos that survive gestation as women with Turner syndrome and have an ostensibly non-mosaic 45,X karyotype, actually are cryptic mosaics for a “rescue line” that includes a viable karyotype. Reanalysis of the prevalence and frequency of 45,X in available data on spontaneous abortuses, and livebirths, confirms prior estimates that 1 % to 1.5 % of all recognizable pregnancies start as an apparent non-mosaic 45,X but about 99 % do not survive gestation. From the rates of 45,X in early embryos, which are notably higher than the inferred rate of gametes hypohaploid for a sex chromosome, as well as the negative maternal age association with 45,X of maternal origin we deduce, in agreement with but on independent grounds from Hall et al. (2006), that a very large proportion of 45,X embryos acquired their 45,X line after fertilization. Results of a search for mosaic cell lines in patients with “Turner syndrome” in several reports indicate that not only does the detection rate of a mosaic line depend upon the number and sensitivity of the markers used, and the number of different tissues examined, but also upon the severity of the phenotype of those cases studied, and the number of cells karyotyped initially. Such factors may explain variation in the extent of detected “cryptic” mosaicism in 45,X individuals (currently at least 50 %). We note a report by Urbach and Benvenitsy (2009) of a gene necessary for placental function, PSF2RA, which lies in the pseudoautosomal-one region of the X and Y chromosomes. Deletion of such a gene could account for the high embryonic lethality in 45,X conceptions, and a rescue line in the placenta could account for embryonic and fetal survival of those cases in which a cryptic mosaic line cannot be found in the usual studies of blood and other tissues from affected individuals. Our primary conclusions are 1) all 45,X individuals with Turner syndrome are cryptic mosaics, 2) absence of the X chromosome in 45,X embryos is caused primarily by mitotic factors, and 3) the placenta is a strong candidate for the location of the rescue line in apparently non-mosaic 45,X individuals.

Chromosome Preparation From Cultured Cells

Abstract: Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births(1,2), 60-80% of all miscarriages(3,4), 10% of stillbirths(2,5), 13% of individuals with congenital heart disease(6), 3-6% of infertility cases(2), and in many patients with developmental delay and birth defects(7). Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance(8,9). Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents(10-13). Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy's fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)(14,15).

Anolis sex chromosomes are derived from a single ancestral pair.

Abstract: To explain the frequency and distribution of heteromorphic sex chromosomes in the lizard genus Anolis, we compared the relative roles of sex chromosome conservation versus turnover of sex-determining mechanisms. We used model-based comparative methods to reconstruct karyotype evolution and the presence of heteromorphic sex chromosomes onto a newly generated Anolis phylogeny. We found that heteromorphic sex chromosomes evolved multiple times in the genus. Fluorescent in situ hybridization (FISH) of repetitive DNA showed variable rates of Y chromosome degeneration among Anolis species and identified previously undetected, homomorphic sex chromosomes in two species. We confirmed homology of sex chromosomes in the genus by performing FISH of an X-linked bacterial artificial chromosome (BAC) and quantitative PCR of X-linked genes in multiple Anolis species sampled across the phylogeny. Taken together, these results are consistent with long-term conservation of sex chromosomes in the group. Our results pave the way to address additional questions related to Anolis sex chromosome evolution and describe a conceptual framework that can be used to evaluate the origins and evolution of heteromorphic sex chromosomes in other clades.

Chromosomal translocations and karyotype complexity in chronic lymphocytic leukemia: a systematic reappraisal of classic cytogenetic data.

Abstract: The significance of chromosomal translocations (CTRAs) and karyotype complexity (KC) in chronic lymphocytic leukemia (CLL) remains uncertain. To gain insight into these issues, we evaluated a series of 1001 CLL cases with reliable classic cytogenetic data obtained within 6 months from diagnosis before any treatment. Overall, 320 cases were found to carry 1 CTRAs. The most frequent chromosome breakpoints were 13q, followed by 14q, 18q, 17q, and 17p; notably, CTRAs involving chromosome 13q showed a wide spectrum of translocation partners. KC (3 aberrations) was detected in 157 cases and significantly (P<0.005) associated with unmutated IGHV genes and aberrations of chromosome 17p. Furthermore, it was identified as an independent prognostic factor for shorter time-to-first-treatment. CTRAs were assigned to two categories (i) CTRAs present in the context of KC, often with involvement of chromosome 17p aberrations, occurring mostly in CLL with unmutated IGHV genes; in such cases, we found that KC rather than the presence of CTRAs per se negatively impacts on survival; (ii) CTRAs in cases without KC, having limited if any impact on survival. On this evidence, we propose that all CTRAs in CLL are not equivalent but rather develop by different processes and are associated with distinct clonal behavior. Am. J. Hematol. 89:249-255, 2014. (c) 2013 Wiley Periodicals, Inc.

Unique features of the transcriptional response to model aneuploidy in human cells

Abstract: Aneuploidy, a karyotype deviating from multiples of a haploid chromosome set, affects the physiology of eukaryotes. In humans, aneuploidy is linked to pathological defects such as developmental abnormalities, mental retardation or cancer, but the underlying mechanisms remain elusive. There are many different types and origins of aneuploidy, but whether there is a uniform cellular response to aneuploidy in human cells has not been addressed so far. Here we evaluate the transcription profiles of eleven trisomic and tetrasomic cell lines and two cell lines with complex aneuploid karyotypes. We identify a characteristic aneuploidy response pattern defined by upregulation of genes linked to endoplasmic reticulum, Golgi apparatus and lysosomes, and downregulation of DNA replication, transcription as well as ribosomes. Strikingly, complex aneuploidy elicits the same transcriptional changes as trisomy. To uncover the triggers of the response, we compared the profiles with transcription changes in human cells subjected to stress conditions. Interestingly, we found an overlap only with the response to treatment with the autophagy inhibitor bafilomycin A1. Finally, we identified 23 genes whose expression is significantly altered in all aneuploids and which may thus serve as aneuploidy markers. Our analysis shows that despite the variability in chromosome content, aneuploidy triggers uniform transcriptional response in human cells. A common response independent of the type of aneuploidy might be exploited as a novel target for cancer therapy. Moreover, the potential aneuploidy markers identified in our analysis might represent novel biomarkers to assess the malignant potential of a tumor.

A RAD-tag Genetic Map for the Platyfish (Xiphophorus maculatus) Reveals Mechanisms of Karyotype Evolution Among Teleost Fish

Abstract: Mammalian genomes can vary substantially in haploid chromosome number even within a small taxon (e.g., 3–40 among deer alone); in contrast, teleost fish genomes are stable (24–25 in 58% of teleosts), but we do not yet understand the mechanisms that account for differences in karyotype stability. Among perciform teleosts, platyfish (Xiphophorus maculatus) and medaka (Oryzias latipes) both have 24 chromosome pairs, but threespine stickleback (Gasterosteus aculeatus) and green pufferfish (Tetraodon nigroviridis) have just 21 pairs. To understand the evolution of teleost genomes, we made a platyfish meiotic map containing 16,114 mapped markers scored on 267 backcross fish. We tiled genomic contigs along the map to create chromosome-length genome assemblies. Genome-wide comparisons of conserved synteny showed that platyfish and medaka karyotypes remained remarkably similar with few interchromosomal translocations but with numerous intrachromosomal rearrangements (transpositions and inversions) since their lineages diverged ∼120 million years ago. Comparative genomics with platyfish shows how reduced chromosome numbers in stickleback and green pufferfish arose by fusion of pairs of ancestral chromosomes after their lineages diverged from platyfish ∼195 million years ago. Zebrafish and human genomes provide outgroups to root observed changes. These studies identify likely genome assembly errors, characterize chromosome fusion events, distinguish lineage-independent chromosome fusions, show that the teleost genome duplication does not appear to have accelerated the rate of translocations, and reveal the stability of syntenies and gene orders in teleost chromosomes over hundreds of millions of years.

Aneuploidy across individual chromosomes at the embryonic level in trophectoderm biopsies: changes with patient age and chromosome structure

Abstract: Purpose To characterize each chromosome’s risk for being involved in embryonic aneuploidy.

Acute lymphoblastic leukemia with low hypodiploid/near triploid karyotype is a specific clinical entity and exhibits a very high TP53 mutation frequency of 93%

Abstract: B lymphoblastic leukemia/lymphoma (ALL) are subdivided by the WHO classification into five subgroups defined by specific translocations and two further subgroups defined by the number of chromosomes. The hypodiploid subgroup is heterogeneous and comprises ALL with a chromosome number of <46. To characterize a specific subset with low hypodiploid karyotype, we performed chromosome banding analysis, FISH, array comparative genomic hybridization, and mutational analyses of FBXW7, NOTCH1, KRAS, NRAS, TP53, and IKZF1 in 29 cases. We observed a nonrandom pattern of chromosome losses, including chromosomes 3, 7, 13, 15, 16, and 17. A deletion encompassing the CDKN2A/B locus was the only recurrent structural abnormality. A duplication of the low hypodiploid karyotype occurred frequently, resulting in a near triploid karyotype based on the definition by merely counting chromosomes but in fact was a very low tetraploid chromosome set. Mutational analyses revealed no mutations in IKZF1, FBXW7, NOTCH1, and KRAS and only one mutation in NRAS. However, we discovered a high frequency of TP53 mutations in 93% (27/29) of cases. In 26/27 cases with TP53 mutation, the second TP53 allele was lost due to monosomy 17. Median overall survival was short (18.5 months), which might be related to the high frequency of TP53 alterations. Therefore, ALL with low hypodiploidy is characterized by a typical pattern of chromosome losses and a remarkably high TP53 mutation frequency. Our data suggest the introduction of a novel WHO entity within the B lymphoblastic leukemia/lymphoma group showing low hypodiploid/very low tetraploid karyotype and concomitant TP53 mutation. © 2014 Wiley Periodicals, Inc.

Cell-autonomous correction of ring chromosomes in human induced pluripotent stem cells

Abstract: Ring chromosomes are structural aberrations commonly associated with birth defects, mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome, and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes, no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division, ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations, enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of 'chromosome therapy' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition, our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control, which is of critical relevance to human development and disease.

Chromosome damage studies in the onion plant Allium cepa L.

Abstract: The onion plant (Allium cepa L.) is a suitable indicator plant for the determination of potential genotoxic agents in the samples taken from the environment. The genotoxic level of the agent under study is reflected by structural changes of the chromosomes and their changed numbers. The chromosomes under study are taken from the meristem cells of the young growing onion roots. A healthy normal onion cell has 16 (2n = 16) chromosomes. They are relatively large and so very appropriate for the detection of morphological changes. Prior to the chromosome study the root tip cells were immersed in a 0.1% aquatic solution of colchicine which stopped the mitotic cycle continuing beyond metaphase. The changes in morphology varied from a single distortion of a single chromosome up to several morphological changes observed on many chromosomes. We identified 15 categories of morphological aberrations which are classified into three groups: chromatid damage (CtD), centromere damage (CmD) and chromosome damage (CsD). Ct...

Ploidy changes and genome stability in yeast

Abstract: Eukaryotic organisms maintain karyotypes with constant chromosome number, but polyploid cells that contain more than two sets of chromosomes can frequently be found. On the one hand, polyploidization is likely to provide some beneficial effects, as naturally occurring polyploid cells can be readily found. On the other hand, polyploidization profoundly affects cell physiology, which may be detrimental to cells. Additionally, polyploidy leads often to aneuploidy and diversification of genetic information; therefore, it has always been considered a prominent driving force in evolution. Recently tetraploid-derived aneuploidy was suggested as a possible mechanism for resistance to fungicides. Another prominent example of the effects of tetraploid-derived aneuploidy is cancer, in which up to one-third of tumours likely originate through tetraploid intermediates. Studying the cellular consequences of polyploidization in human cells is challenging. In contrast, polyploid and aneuploid cells can be easily generated and analysed in the budding yeast Saccharomyces cerevisiae as well as in other yeast species. This, together with the naturally occurring yeast polyploids and aneuploids, provides a valuable model to study the effects of abnormal chromosome numbers on cellular physiology. Thus, the yeast model may provide novel insights into the general mechanisms of genomic instability in eukaryotes and improve our understanding of the consequences of ploidy changes and their relevance for disease.

Highly differentiated ZW sex microchromosomes in the Australian Varanus species evolved through rapid amplification of repetitive sequences.

Abstract: Transitions between sex determination systems have occurred in many lineages of squamates and it follows that novel sex chromosomes will also have arisen multiple times. The formation of sex chromosomes may be reinforced by inhibition of recombination and the accumulation of repetitive DNA sequences. The karyotypes of monitor lizards are known to be highly conserved yet the sex chromosomes in this family have not been fully investigated. Here, we compare male and female karyotypes of three Australian monitor lizards, Varanus acanthurus, V. gouldii and V. rosenbergi, from two different clades. V. acanthurus belongs to the acanthurus clade and the other two belong to the gouldii clade. We applied C-banding and comparative genomic hybridization to reveal that these species have ZZ/ZW sex micro-chromosomes in which the W chromosome is highly differentiated from the Z chromosome. In combination with previous reports, all six Varanus species in which sex chromosomes have been identified have ZZ/ZW sex chromosomes, spanning several clades on the varanid phylogeny, making it likely that the ZZ/ZW sex chromosome is ancestral for this family. However, repetitive sequences of these ZW chromosome pairs differed among species. In particular, an (AAT)n microsatellite repeat motif mapped by fluorescence in situ hybridization on part of W chromosome in V. acanthurus only, whereas a (CGG)n motif mapped onto the W chromosomes of V. gouldii and V. rosenbergi. Furthermore, the W chromosome probe for V. acanthurus produced hybridization signals only on the centromeric regions of W chromosomes of the other two species. These results suggest that the W chromosome sequences were not conserved between gouldii and acanthurus clades and that these repetitive sequences have been amplified rapidly and independently on the W chromosome of the two clades after their divergence.

Identification of the linkage group of the Z sex chromosomes of the sand lizard (Lacerta agilis, Lacertidae) and elucidation of karyotype evolution in lacertid lizards

Abstract: The sand lizard (Lacerta agilis, Lacertidae) has a chromosome number of 2n = 38, with 17 pairs of acrocentric chromosomes, one pair of microchromosomes, a large acrocentric Z chromosome, and a micro-W chromosome. To investigate the process of karyotype evolution in L. agilis, we performed chromosome banding and fluorescent in situ hybridization for gene mapping and constructed a cytogenetic map with 86 functional genes. Chromosome banding revealed that the Z chromosome is the fifth largest chromosome. The cytogenetic map revealed homology of the L. agilis Z chromosome with chicken chromosomes 6 and 9. Comparison of the L. agilis cytogenetic map with those of four Toxicofera species with many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) showed highly conserved linkage homology of L. agilis chromosomes (LAG) 1, 2, 3, 4, 5(Z), 7, 8, 9, and 10 with macrochromosomes and/or macrochromosome segments of the four Toxicofera species. Most of the genes located on the microchromosomes of Toxicofera were localized to LAG6, small acrocentric chromosomes (LAG11–18), and a microchromosome (LAG19) in L. agilis. These results suggest that the L. agilis karyotype resulted from frequent fusions of microchromosomes, which occurred in the ancestral karyotype of Toxicofera and led to the disappearance of microchromosomes and the appearance of many small macrochromosomes.

Single origin of sex chromosomes and multiple origins of B chromosomes in fish genus Characidium

Abstract: Chromosome painting with DNA probes obtained from supernumerary (B) and sex chromosomes in three species of fish genus Characidium (C. gomesi, C. pterostictum and C. oiticicai) showed a close resemblance in repetitive DNA content between B and sex chromosomes in C. gomesi and C. pterostictum. This suggests an intraspecific origin for B chromosomes in these two species, probably deriving from sex chromosomes. In C. oiticicai, however, a DNA probe obtained from its B chromosome hybridized with the B but not with the A chromosomes, suggesting that the B chromosome in this species could have arisen interspecifically, although this hypothesis needs further investigation. A molecular phylogenetic analysis performed on nine Characidium species, with two mtDNA genes, showed that the presence of heteromorphic sex chromosomes in these species is a derived condition, and that their origin could have been unique, a conclusion also supported by interspecific chromosome painting with a CgW probe derived from the W chromosome in C. gomesi. Summing up, our results indicate that whereas heteromorphic sex chromosomes in the genus Characidium appear to have had a common and unique origin, B chromosomes may have had independent origins in different species. Our results also show that molecular phylogenetic analysis is an excellent complement for cytogenetic studies by unveiling the direction of evolutionary chromosome changes.

Comparative Chromosomal Mapping of Microsatellites in Leporinus Species (Characiformes, Anostomidae): Unequal Accumulation on the W Chromosomes

Abstract: Approximately 90 species in the genus Leporinus (Characiformes, Anostomidae) are known, and most of them do not present differentiated sex chromosomes. However, there is a group of 7 species that share a heteromorphic ZW sex system. In all of these species, the W chromosome is the largest one in the karyotype and is mostly heterochromatic. We investigated the distribution of several microsatellites in the genome of 4 Leporinus species that possess ZW chromosomes. Our results showed a very large accumulation of mostly microsatellites on the W chromosomes. This finding supports the presence of an interconnection between heterochromatinization and the accumulation of repetitive sequences, which has been proposed for sex chromosome evolution, and suggests that heterochromatinization is the earlier of the 2 processes. In spite of the common origin that has been proposed for W chromosomes in all of the studied species, the microsatellites followed different evolutionary trajectories in each species, which indicates a high plasticity for sex chromosome differentiation.

Estimating Tempo and Mode of Y Chromosome Turnover: Explaining Y Chromosome Loss With the Fragile Y Hypothesis

Abstract: Chromosomal sex determination is phylogenetically widespread, having arisen independently in many lineages. Decades of theoretical work provide predictions about sex chromosome differentiation that are well supported by observations in both XY and ZW systems. However, the phylogenetic scope of previous work gives us a limited understanding of the pace of sex chromosome gain and loss and why Y or W chromosomes are more often lost in some lineages than others, creating XO or ZO systems. To gain phylogenetic breadth we therefore assembled a database of 4724 beetle species’ karyotypes and found substantial variation in sex chromosome systems. We used the data to estimate rates of Y chromosome gain and loss across a phylogeny of 1126 taxa estimated from seven genes. Contrary to our initial expectations, we find that highly degenerated Y chromosomes of many members of the suborder Polyphaga are rarely lost, and that cases of Y chromosome loss are strongly associated with chiasmatic segregation during male meiosis. We propose the “fragile Y” hypothesis, that recurrent selection to reduce recombination between the X and Y chromosome leads to the evolution of a small pseudoautosomal region (PAR), which, in taxa that require XY chiasmata for proper segregation during meiosis, increases the probability of aneuploid gamete production, with Y chromosome loss. This hypothesis predicts that taxa that evolve achiasmatic segregation during male meiosis will rarely lose the Y chromosome. We discuss data from mammals, which are consistent with our prediction.

Genes on B chromosomes of vertebrates

Abstract: There is a growing body of evidence that B chromosomes, once regarded as totally heterochromatic and genetically inert, harbor multiple segmental duplications containing clusters of ribosomal RNA genes, processed pseudogenes and protein-coding genes. Application of novel molecular approaches further supports complex composition and possible phenotypic effects of B chromosomes. Here we review recent findings of gene-carrying genomic segments on B chromosomes from different vertebrate groups. We demonstrate that the genetic content of B chromosomes is highly heterogeneous and some B chromosomes contain multiple large duplications derived from various chromosomes of the standard karyotype. Although B chromosomes seem to be mostly homologous to each other within a species, their genetic content differs between species. There are indications that some genomic regions are more likely to be located on B chromosomes. The discovery of multiple autosomal genes on B chromosomes opens a new discussion about their possible effects ranging from sex determination to fitness and adaptation, their complex interactions with host genome and role in evolution.

A geometric approach to fully automatic chromosome segmentation

Abstract: A fundamental task in human chromosome analysis is chromosome segmentation. Segmentation plays an important role in chromosome karyotyping. The first step in segmentation is to remove intrusive objects such as stain debris and other noises. The next step is detection of touching and overlapping chromosomes, and the final step is separation of such chromosomes. Common methods for separation between touching chromosomes are interactive and require human intervention for correct separation between touching and overlapping chromosomes. In this paper, a geometric-based method is used for automatic detection of touching and overlapping chromosomes and separating them. The proposed scheme performs segmentation in two phases. In the first phase, chromosome clusters are detected using three geometric criteria, and in the second phase, chromosome clusters are separated using a cut-line. Most of earlier methods did not work properly in case of chromosome clusters that contained more than two chromosomes. Our method, on the other hand, is quite efficient in separation of such chromosome clusters. At each step, one separation will be performed and this algorithm is repeated until all individual chromosomes are separated. Another important point about the proposed method is that it uses the geometric features of chromosomes which are independent of the type of images and it can easily be applied to any type of images such as binary images and does not require multispectral images as well. We have applied our method to a database containing 62 touching and partially overlapping chromosomes and a success rate of 91.9% is achieved.

The Staurotypus turtles and aves share the same origin of sex chromosomes but evolved different types of heterogametic sex determination.

Abstract: Reptiles have a wide diversity of sex-determining mechanisms and types of sex chromosomes. Turtles exhibit temperature-dependent sex determination and genotypic sex determination, with male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) sex chromosomes. Identification of sex chromosomes in many turtle species and their comparative genomic analysis are of great significance to understand the evolutionary processes of sex determination and sex chromosome differentiation in Testudines. The Mexican giant musk turtle (Staurotypus triporcatus, Kinosternidae, Testudines) and the giant musk turtle (Staurotypus salvinii) have heteromorphic XY sex chromosomes with a low degree of morphological differentiation; however, their origin and linkage group are still unknown. Cross-species chromosome painting with chromosome-specific DNA from Chinese soft-shelled turtle (Pelodiscus sinensis) revealed that the X and Y chromosomes of S. triporcatus have homology with P. sinensis chromosome 6, which corresponds to the chicken Z chromosome. We cloned cDNA fragments of S. triporcatus homologs of 16 chicken Z-linked genes and mapped them to S. triporcatus and S. salvinii chromosomes using fluorescence in situ hybridization. Sixteen genes were localized to the X and Y long arms in the same order in both species. The orders were also almost the same as those of the ostrich (Struthio camelus) Z chromosome, which retains the primitive state of the avian ancestral Z chromosome. These results strongly suggest that the X and Y chromosomes of Staurotypus turtles are at a very early stage of sex chromosome differentiation, and that these chromosomes and the avian ZW chromosomes share the same origin. Nonetheless, the turtles and birds acquired different systems of heterogametic sex determination during their evolution.

Differences and homologies of chromosomal alterations within and between breast cancer cell lines: a clustering analysis.

Abstract: The MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism. A range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines. Our study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines. The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level.

Genetic evaluation of mesenchymal stem cells by G-banded karyotyping in a Cell Technology Center.

Abstract: OBJECTIVE: To present the initial results of first three years of implementation of a genetic evaluation test for bone marrow-derived mesenchymal stem cells in a Cell Technology Center. METHODS: A retrospective study was carried out of 21 candidates for cell therapy. After the isolation of bone marrow mononuclear cells by density gradient, mesenchymal stem cells were cultivated and expanded at least until the second passage. Cytogenetic analyses were performed before and after cell expansion (62 samples) using G-banded karyotyping. RESULTS: All the samples analyzed, before and after cell expansion, had normal karyotypes, showing no clonal chromosomal changes. Signs of chromosomal instability were observed in 11 out of 21 patients (52%). From a total of 910 analyzed metaphases, five chromatid gaps, six chromatid breaks and 14 tetraploid cells were detected giving as total of 25 metaphases with chromosome damage (2.75%). CONCLUSION: The absence of clonal chromosomal aberrations in our results for G-banded karyotyping shows the maintenance of chromosomal stability of bone marrow-derived mesenchymal stem cells until the second passage; however, signs of chromosomal instability such as chromatid gaps, chromosome breaks and tetraploidy indicate that the long-term cultivation of these cells can provide an intermediate step for tumorigenesis.

Comparative chromosome mapping of U2 snRNA and 5S rRNA genes in Gymnotus species (Gymnotiformes, Gymnotidae): evolutionary dynamics and sex chromosome linkage in G . pantanal.

Abstract: A comparative mapping of U2 small nuclear RNA (snRNA) and 5S ribosomal RNA (rRNA) genes was performed in 6 Gymnotus species. All species analyzed presented the U2 snDNA organized in conspicuous blocks and not co-located with rRNA genes. In addition, 5 species showed the U2 snDNA located in a single pair of chromosomes, which seems to be a conserved trait in this genus. Conversely, G. pantanal was the only species displaying several terminal signals in different chromosome pairs, including the X1 sex chromosome but not the Y chromosome. This is the first report of U2 snRNA genes in sex chromosomes of fishes. The absence of sites in the Y chromosome of G. pantanal indicates a possible loss of terminal segments of the chromosomes involved in the Y formation.

Molecular cytogenetic characterization of multiple intrachromosomal rearrangements in two representatives of the genus Turdus (Turdidae, Passeriformes).

Abstract: Turdus rufiventris and Turdus albicollis, two songbirds belonging to the family Turdidae (Aves, Passeriformes) were studied by C-banding, 18S rDNA, as well as the use of whole chromosome probes derived from Gallus gallus (GGA) and Leucopternis albicollis (LAL). They showed very similar karyotypes, with 2n = 78 and the same pattern of distribution of heterochromatic blocks and hybridization patterns. However, the analysis of 18/28S rDNA has shown differences in the number of NOR-bearing chromosomes and ribosomal clusters. The hybridization pattern of GGA macrochromosomes was similar to the one found in songbirds studied by Fluorescent in situ hybridization, with fission of GGA 1 and GGA 4 chromosomes. In contrast, LAL chromosome paintings revealed a complex pattern of intrachromosomal rearrangements (paracentric and pericentric inversions) on chromosome 2, which corresponds to GGA1q. The first inversion changed the chromosomal morphology and the second and third inversions changed the order of chromosome segments. Karyotype analysis in Turdus revealed that this genus has derived characteristics in relation to the putative avian ancestral karyotype, highlighting the importance of using new tools for analysis of chromosomal evolution in birds, such as the probes derived from L. albicollis, which make it possible to identify intrachromosomal rearrangements not visible with the use of GGA chromosome painting solely.