Molecular Cytogenetic Methods Research Articles

Clinical and Molecular Cytogenetic Characterisation of Children with Developmental Delay and Dysmorphic Features.

IntroductionDevelopmental delay and dysmorphic features affect 1 – 3 % of paediatric population. In the last few years molecular cytogenetic high resolution techniques (comparative genomic hybridization arrays and single-nucleotide polymorphism arrays) have been proven to be a first-tier choice for clinical diagnostics of developmental delay and dysmorphic features.Methods and resultsIn the present article we describe the clinical advantages of molecular cytogenetic approach (comparative genomic hybridization arrays and single nucleotide polymorphism arrays) in the diagnostic procedure of two children with developmental delay, dysmorphic features and additional morphological phenotypes. Additionally, we demonstrate the necessity of fluorescent in situ hybridization utilisation to identify the localisation and underlying mechanism of detected chromosomal rearrangement.ConclusionsTwo types of chromosomal abnormalities were identified and confirmed using different molecular genetic approaches. Comparative genomic hybridization arrays and single nucleotide polymorphism arrays are hereby presented as important methods to identify chromosomal imbalances in patients with developmental delay and dysmorphic features. We emphasize the importance of molecular genetic testing in patients’ parents for the demonstration of the origin and clinical importance of the aberrations prior determined in the patients. The results obtained using molecular cytogenetic high resolution techniques methods are the cornerstone for proper genetic counselling to the affected families.

Low rate of interchromosomal rearrangements during old radiation of gekkotan lizards (Squamata: Gekkota).

Gekkotan lizards are a highly specious (∼1600 described species) clade of squamate lizards with nearly cosmopolitan distribution in warmer areas. The clade is primarily nocturnal and forms an ecologically dominant part of the world nocturnal herpetofauna. However, molecular cytogenetic methods to study the evolution of karyotypes have not been widely applied in geckos. Our aim here was to uncover the extent of chromosomal rearrangements across the whole group Gekkota and to search for putative synapomorphies supporting the newly proposed phylogenetic relationships within this clade. We applied cross-species chromosome painting with the recently derived whole-chromosomal probes from the gekkonid species Gekko japonicus to members of the major gekkotan lineages. We included members of the families Diplodactylidae, Carphodactylidae, Pygopodidae, Eublepharidae, Phyllodactylidae and Gekkonidae. Our study demonstrates relatively high chromosome conservatism across the ancient group of gekkotan lizards. We documented that many changes in chromosomal shape across geckos can be attributed to intrachromosomal rearrangements. The documented rearrangements are not totally in agreement with the recently newly erected family Phyllodactylidae. The results also pointed to homoplasy, particularly in the reuse of chromosome breakpoints, in the evolution of gecko karyotypes.

Molecular cytogenetics ofAndroctonusscorpions: an oasis of calm in the turbulent karyotype evolution of the diverse family Buthidae

Recent cytogenetic and genomic studies suggest that morphological and molecular evolution is decoupled in the basal arachnid order Scorpiones. Extraordinary karyotype variation has been observed particularly in the family Buthidae, which is unique among scorpions for its holokinetic chromosomes. We analyzed the karyotypes of four geographically distant species of the genus Androctonus Ehrenberg, 1828 (Androctonus australis, Androctonus bourdoni, Androctonus crassicauda, Androctonus maelfaiti) (Scorpiones: Buthidae) using both classic and molecular cytogenetic methods. The mitotic complement of all species consisted of 2n = 24 elements. Fluorescence in situ hybridization with a fragment of the 18S ribosomal RNA gene, a cytogenetic marker well known for its mobility, identified a single interstitial rDNA locus on the largest chromosome pair in all species examined. Our findings thus support the evolutionary stasis of the Androctonus karyotype, which is discussed with respect to current hypotheses on chromosome evolution both within and beyond the family Buthidae. Differences in karyotype dynamics between Androctonus spp. and the other buthids can help us better understand the driving forces behind their chromosome evolution and speciation. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115, 69–76.

Combined use of cytogenetic and molecular methods in prenatal diagnostics of chromosomal abnormalities.

Aim:The aim of prenatal diagnostics is to provide information of the genetic abnormalities of the fetus early enough for the termination of pregnancy to be possible. Chromosomal abnormalities can be detected in an unborn child through the use of cytogenetic, molecular- cytogenetic and molecular methods. In between them, central spot is still occupied by cytogenetic methods. In cases where use of such methods is not informative enough, one or more molecular cytogenetic methods can be used for further clarification. Combined use of the mentioned methods improves the quality of the final findings in the diagnostics of chromosomal abnormalities, with classical cytogenetic methods still occupying the central spot.Material and methods:Conducted research represent retrospective-prospective study of a four year period, from 2008 through 2011. In the period stated, 1319 karyotyping from amniotic fluid were conducted, along with 146 FISH analysis.Results:Karyotyping had detected 20 numerical and 18 structural aberrations in that period. Most common observed numerical aberration were Down syndrome (75%), Klinefelter syndrome (10%), Edwards syndrome, double Y syndrome and triploidy (5% each). Within observed structural aberrations more common were balanced chromosomal aberrations then non balanced ones. Most common balanced structural aberrations were as follows: reciprocal translocations (60%), Robertson translocations (13.3%), chromosomal inversions, duplications and balanced de novo chromosomal rearrangements (6.6% each).Conclusion:With non- balanced aberrations observed in the samples of amniotic fluid, non- balanced translocations, deletions and derived chromosomes were equally represented. Number of detected aneuploidies with FISH, prior to obtaining results with karyotyping, were 6.

The ASXL1 Gene Alterations in Patients with Myeloid Diseases and Chromosomal 20q Deletion

Deletion of the long arm of chromosome 20 - del(20q) - is a recurrent abnormality observed in various myeloid disorders, including myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), or acute myeloid leukemia (AML). It is a primary cytogenetic aberration, occurring often as a sole abnormality or the first abnormality in complex karyotypes, therefore it is assumed to play a key role in pathogenesis of myeloid malignancies. The proximal breakpoints of the deletion are consistently located in the 20q11.21 band, and the distal breakpoints span from 20q13.13 to band 20q13.33. In our previous study we showed a fusion of the ASXL1 and TSHZ2 genes resulting from an isochromosome of a deleted 20q in a patient with MDS (Brezinova et al Br J Haematol 2014). The ASXL1 gene is one of the most frequently mutated genes in myeloid disorders, mutations are generally associated with more aggressive course of the disease and poor clinical outcome. The aim of this study was to determine the frequency of ASXL1 breakpoints and/or ASXL1/TSHZ2 fusion in del(20q) cases.Fluorescence in situ hybridizations (FISH) with locus specific probes for 20q11 and 20q12 regions (Abbott, Des Plaines, IL; Kreatech Diagnostics, Amsterdam, The Netherlands) confirmed the cytogenetically observed deletions of 20q in a cohort of 20 patients with myeloid malignancies (MDS 13x, MPN 3x, AML 2x, myelofibrosis 1x, thrombocytopenia 1x). In 15 patients deletion of 20q was a sole aberration, in 2 patients a variant of del(20q), an isochromosome of deleted 20q, was detected by FISH with a subtelomeric probe, Vysis ToTel 20p/20q (Abbott). Metaphase FISH mapping with a set of 7 bacterial artificial chromosome (BAC) probes (BlueGnome, Cambridge, UK) distributed in 20q11.21 and 20q13.2, with a chromosome-20-specific centromeric probe (Kreatech Diagnostics) as a control was used for determination of the breakpoints. Array comparative genomic hybridization (CytoChip Cancer 180K, BlueGnome) was performed on DNA samples of bone marrow cells of 11 patients with suspected ASXL1 gene deletion to find out the gene copy number variation.A weak signal of RP11-358N2 BAC probe (30.93-31.12 Mb from telomere on the short arm, 20q11.21) was observed in 6 patients(30%), suggesting the proximal breakpoint of the deletion in the ASXL1 gene (30.95-31.03 Mb). However, the distal breakpoint in the TSHZ2 gene (51.80-52.11 Mb; 20q13.2) was found in one patient only, as previously reported. In 6 patients (30%) the signal of RP11-358N2 BAC probe was not present on the derivative chromosome confirming the ASXL1 gene deletion. In the remaining 8 patients the proximal breakpoint of the deletion was determined distally to the ASXL1 gene, in 3 patients into 9.3 Mb region between the MAPRE and the PTPRT genes. None but 1 patient had the distal breakpoint localized in the TSHZ2 gene. Three patients died in a group of 12 patients with ASXL1 gene alteration (deletion or partial deletion).Using combination of molecular cytogenetic methods we proved that the extent of 20q deletions varied among the patients with a frequent proximal breakpoint in the ASXL1 gene. In our cohort, the ASXL1 gene was altered (deleted or partially deleted) in totally 60% of patients. The determination of the ASXL1 gene alteration in del(20q) cases may have a clinical and prognostic impact, but the relations with clinical data should be studied in a larger cohort of patients.Supported by MHCR project for conceptual development of research organization 00023736, RVO-VFN64165/2012, and GACR P302/12/G157/1. DisclosuresNo relevant conflicts of interest to declare.

FEMALE PATIENT WITH A VARIANT OF TURNER SYNDROME

AbstractThe X chromosome contains determinants necessary to assure normal structural growth. Deletions of proportions of the X chromosome have been reported in a huge number of patients. We report on a mosaic short arm deletion of X chromosome (Xp-) in 14-years-old female patient with short stature, low set ears and no secondary sexual characteristics, a rare Turner syndrome variant. Chromosomal analysis using GTG-banding showed in 85% of cells and 46, X, del(Xp) in 15% of cells. The Fluorescence in situ hybridization technique of the cultured cells helped to perceive low grade cell line. Hence, molecular cytogenetics methods are essential to detect low level mosaicism which is important in better counseling.

Epigenetic Regulation of “Aged” Heterochromatin by Peptide Bioregulator Cortagen

Peptide bioregulator Cortagen (Ala-Glu-Asp-Pro) in humans demonstrated a pronounced therapeutic effect up on the structural and functional recovery of the damaged peripheral nerve tissue. The synthetic peptide Cortagen was obtained by directed synthesis based on amino acid analysis of natural brain cortex peptide preparation Cortexin. The effect of synthetic peptide bioregulator Cortagen on total, constitutive [pericentromeric, nucleolus organizer regions (NOR)] heterochromatin and facultative (transcriptionally inactive euchromatin) heterochromatin has been studied. We used a molecular-cytogenetic methods: differential scanning calorimetry; activity of ribosomal genes of acrocentric chromosome satellite stalks—NORs; polymorphism of structural pericentromeric C-heterochromatin; variability of the facultative heterochromatin (FH) in cultivated lymphocytes from individuals at the age of 80 and older. We show that peptide bioregulator Cortagen induces unrolling deheterochromatinization (decondensation) of total heterochromatin; activates synthetic processes of ribosomal genes as a result of deheterochromatinization of satellite stalks of acrocentric chromosomes; does not induce deheterochromatinization of pericentromeric structural heterochromatin; and releases repressed genes because of the condensation of euchromatic regions forming FH. Our data are important because it provides new information on the remodeling effects of FH induced by peptide bioregulator Cortagen in aging and aging pathologies, and may lead to the development of a therapeutic treatment.

Improvement of the agronomic traits of a wheat-barley centric fusion by introgressing the 3HS.3BL translocation into a modern wheat cultivar.

The 3HS.3BL spontaneous Robertsonian translocation obtained from the progenies of wheat-barley (Chinese Spring × Betzes) hybrids backcrossed with wheat line Mv9kr1 was transferred into the modern Martonvásár wheat cultivar Mv Bodri. The translocation was identified with molecular cytogenetic methods. The inheritance of the translocation was traced using genomic in situ hybridization. Fluorescence in situ hybridization using barley subtelomeric (HvT01) and centromere-specific [(AGGGAG)4] repetitive DNA probes confirmed that the complete barley chromosome arm was involved in the Robertsonian translocation. The wheat-specific repetitive DNA probes identified the presence of the whole wheat genome, except the short arm of the 3B chromosome. Genotypes homozygous for the centric fusion were selected, after which morphological analysis was performed on the plants and the yield components were measured in the field during two consecutive vegetative seasons. The introgression of the 3HS.3BL translocation into the modern wheat cultivar Mv Bodri significantly reduced the plant height due to the incorporation of the dwarfing allele RhtD1b. The presence of the 3HS.3BL translocation in the Mv9kr1 and Mv Bodri wheat background improved tillering and seeds per plant productivity in field experiments carried out in Martonvásár and Keszthely, Hungary.

Sex chromosome composition revealed in Characidium fishes (Characiformes: Crenuchidae) by molecular cytogenetic methods

The W chromosome of the fishes Characidium cf. fasciatum, Characidium sp. and Characidium cf. gomesi is heterochromatic, as is usually seen in most Characidium species. Samples of W-chromatin were collected by mechanical microdissection and amplified by DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction), to be used as painting probes (DCg and CgW) and for sequence analysis. FISH (fluorescence in situ hybridization) with DCg probe painted the whole W chromosome, the pericentromeric region of Z chromosomes and the terminal region of B chromosomes. DOP-PCR-generated fragments were cloned, sequenced and tested by in situ hybridization, but only CgW4 produced positive hybridization signals. Clone sequence analysis recovered seven distinct sequences, of which six did not reveal any similarity to other known sequences in the GenBank or GIRI databases. Only CgW9 clone sequence was recognized as probably derived from a Helitron-transposon similar to that found in the genome of the zebrafish Danio rerio .O ur results show that the composition of Characidium's W chromosome does seem rich in repetitive sequences as well as other W chromosomes found in several species with a ZW sex-determining mechanism.

Analysis of receptor tyrosine kinase gene amplification on the example of FGFR1.

FISH (fluorescent in situ hybridization) is a molecular cytogenetic method to detect large-scale genetic alterations in tissue and/or cells. Numerical aberrations (deletions and amplifications) and structural aberrations (translocations and fusions) are detectable. Probes bind complementary to the DNA strand of the region of interest. Subsequently, the probes are detected via fluorochromes and appear as colored dots that can be assessed under the fluorescence microscope.In situ hybridization is divided into three steps: pretreatment, hybridization, and posthybridization. Pretreatment opens up the cell membranes for hybridization, so that the probe can bind to the complementary DNA target. Posthybridization includes washing steps to remove excessive probes and detection of probes via secondary marked fluorochromes. DAPI stains nuclei and serves as mounting media.

GENETICS FOR DIAGNOSTICS IN PREVENTIVE MEDICINE

The sequencing of first human genome followed by rapid development of technologies, that led to significant lowering of costs for genetic analyze and its fast performing, made possible a broad invention of genetic diagnostics methods into clinical practice. Contemporary methods of molecular genetics make possible to research on inherited factors on chromosome level with molecular cytogenetics methods, and on the level of local mutations with the use or polymeraze chain reaction, microchips and sequencing. Temps of the next generation sequencing methods provide the opportunity to predict soon inclusion in practice of the personalized medical analysis of large genetic data massive, that can be used for the disease outcome prediction, estimation of its course, and for the prescription and correction of pharmacotherapy. In this review, different (including novel) approaches to genetic diagnostics are explored for the rare as common diseases, their benefits and restrictions.

Plant biosystematics with the help of cytology and cytogenetics

The importance of cytological and cytogenetic information in the investigation of evolutionary history of species, i.e. biosystematics (cytotaxonomy) and cytogeography, is surveyed. The main issues considered are the numbers and morphology of the chromosomes, the distribution and amount of the different types of chromatin in chromosomes, nuclei at interphase stained with classical and molecular cytogenetic methods, e.g. fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and bacterial artificial chromosome (BAC), painting chromosomes, the isolation of large DNA segments using e.g. pulse-field gel electrophoresis, and chromosome disposition in the cell nucleus. A wide ranging biosystematic interest puts emphasis with the help of cytological and cytogenetic methods on getting to know chromosome variation within the family, genus and species connected with the phenomena of hybridization and polyploidization. Chromosome variation among taxa includes differences in the morphology and numbers of chromosomes, the amount of DNA, the sizes of chromosomes, inner chromosome structure, and DNA sequence, often shown in chromosome bands or various proportions of AT and GC pairs. On this basis we can track the course of evolution among taxa and determine reciprocal relationships. One of the main aims of our work, apart from getting to know evolutionary processes, is to create a taxonomic system of organisms.

Frequency of prenatal determination of 13, 18 and 21 trisomies and link with risk factors

The study was conducted in the Laboratory of the Hospital of Lithuanian University of Health Sciences Kaunas Clinic, in the Laboratory of Clinical Chemistry and Genetics and in the Department of Biology of Vytautas Magnus University. Abnormalities of number of chromosomes are diagnosed from amniotic fluid, which is taken by using an invasive prenatal method – amniocentesis, and modern diagnostic methods such as molecular cytogenetic analysis (FISH) and cytogenetic method, diagnosis of karyotype, by using G-staining method. In order to analyse the main risk factors that determine the frequency of Patau, Edwards and Down syndromes in the analysed group and to evaluate the variety of changeovers of chromosomes structure, the retrospective analysis of patient’s case records data was performed. The aforementioned patients were examined in the Hospital of Lithuanian University of Health Sciences Kaunas Clinic in 2007–2011. The received data is processed by using Statistical Package for Social Sciences (T-Test). The aim of the study is to evaluate, by using cytogenetic and molecular cytogenetic research methods, the frequency of prenatal trisomies determination of the thirteenth (13), the eighteenth (18), and the twenty first (21) chromosomes for the patients, who during 2007–2011 had genetic consultations in the Hospital of Lithuanian University of Health Sciences Kaunas Clinics and to determine the connection between these trisomies and risk factors. During 2007–2011 1 156 patients were examined and 49 (4.2%) alterations of karyotype were determined, out of which 30 (61%) are related to Down syndrome, 14 (29%) to Edwards syndrome and 5 (10%) to Patau syndrome. After studying the data of questionnaires and case records, on the basis of the statistical reliability of data analysis, the influence of the main reproductive factors (abortions, contraceptives) and other risk or environmental factors (smoking, genetic diseases and use of medicines) to fetal anomalies was determined.

Molecular cytogenetic analysis of chromosomal aberrations in cells of low grade gliomas and its contribution for tumour classification

Low-grade gliomas represent a heterogeneous group of primary brain malignancies. The current diagnostics of these tumors rely strongly on histological classification. With the development of molecular cytogenetic methods several genetic markers were described, contributing to a better distinction of glial subtypes. The aim of this study was to assess the frequency of acquired chromosomal aberrations in lowgrade gliomas and to search for new genomic changes associated with higher risk of tumor progression. We analysed biopsy specimens from 41 patients with histological dia-gnosis of low-grade glioma using interphase fluorescence in situ hybridization (I FISH) and single nucleotide polymorphism (SNP) array techniques (19 females and 22 males, medium age 42 years). Besides notorious and most frequent finding of combined deletion of 1p/ 19q (81.25% patients) several other recurrent aberrations were described in patients with oligodendrogliomas: deletions of p and q arms of chromosome 4 (25% patients), deletions of the short arms of chromosome 9 (18.75% patients), deletions of the long arms of chromosome 13 and monosomy of chromosome 18 (18.75% patients). In bio-psy specimens from patients with astrocytomas, we often observed deletion of 1p (24% patients), amplification of the long arms of chromosome 7 (16% patients), deletion of the long arm of chromosome 13 (20% patients), segmental uniparental disomy (UPD) of the short arms of chromosome 17 (60% patients) and deletion of the long arms of chromosome 19 (28% patients). In one patient we detected a shuttered chromosome 10 resulting from chromothripsis. Using a combination of I FISH and SNP array, we detected not only known chromosomal changes but also new or less frequent recur-rent aberrations. Their role in cancer cell progression and their impact on low grade gliomas classification remains to be elucidated in a larger cohort of patients.

ABL1 gene involvement within a complex three-way translocation (2;9;4) in perineurioma characterized by molecular cytogenetic methods

Perineuriomas are rare peripheral nerve sheath tumors with one or few chromosomal rearrangements or numerical changes. Two main types and three subtypes have been defined but with few specific genetic associations. Chromosome 10 aberrations have been found in three cases of the sclerosing perineurioma subtype. Chromosome 22 abnormalities have been described in different types of perineurioma. None of these aberrations has been described at the molecular level. We report on a complex rearrangement characterized by fluorescence in situ hybridization and array-comparative genome hybridization, which revealed submicroscopic deletions at 2p23 and 9q34 that involved the ABL1 gene in a soft tissue perineurioma case.

Genetic alterations of chromosomes, p53 and p16 genes in low- and high-grade bladder cancer.

A majority of patients with bladder cancer present with superficial disease and subsequently, some patients show progression to muscle invasive or metastatic disease. Bladder cancer has a complex genetic process and identification of the genetic alterations which occur during progression may lead to the understanding of the nature of the disease and provide the possibility of early treatment. The aim of the present study was to compare the structural and numerical chromosomal differences and changes in the p16 and p53 genes between low-grade (LG) and high-grade (HG) bladder cancer (BC) using cytogenetic and molecular cytogenetic methods. Between March 2009 and March 2010, cytogenetic analyses were carried out on tumor and blood samples in 34 patients with transitional cell type BC, and on blood samples of 34 healthy patients as a control group. Fluorescence in situ hybridization probes for the p16 and p53 genes were also used to screen the alterations in these genes in 32 patients with BC. The patients were divided into two groups (LG and HG) and the findings were compared. A total of 11 (32.3%) patients exhibited LGBC, 22 (64.7%) exhibited HGBC and one (3%) patient exhibited carcinoma in situ. There were no differences between the LGBC and HGBC groups according to the number of chromosomal aberrations (P=0.714); however, differences between alterations of the p16 and p53 genes were significant (P=0.002 and P=0.039). Almost all structural abnormalities were found to be located to the 1q21, 1q32, 3p21 and 5q31 regions in patients with HG tumors. In conclusion, the p16 and p53 genes were altered more prominently in patients with HG tumors compared with LG tumors. The structural abnormalities in the 1q21, 1q32, 3p21 and 5q31 regions were observed more frequently in patients with HG tumors. These regions may play significant roles in the progression of BC, but further studies are required to find candidate genes for a panel of BC.

Ritka genomikai betegségek azonosítása array komparatív genomhibridizációs módszerrel – elsőként Magyarországon

In the past decade the study of genomic disorders has received more interest. Array comparative genome hybridization is a widely spread diagnostic method in the research of genomic disorders. This method was implemented in the laboratory of the authors in 2012. This molecular cytogenetic method was first used to examine patients with complex developmental disorders in whom no genetic background was identified by traditional methods. The authors complemented traditional diagnostic methods with array comparative genome hybridization, which has not been used in routine diagnostics in Hungary so far. Using this novel method the authors were able to identify genomic alterations in 7 out of 18 patients with complex developmental disorders. They found de novo alterations in 6 out of 7 patients, which were most likely causative in the development of the phenotype, while in one case they detected a familial genomic alteration. This method helped the authors to determine the breakpoint of genomic variation in their patients and delineate the affected genes contributing to the phenotype. These results call attention to the usefulness of next generation diagnostic methods available in the laboratory of the authors.

Chromosomal evolution: Molecular cytogenetic evolution of mammals

This volume reviews and synthesizes the cytogenetic results obtained in the numerous studies of mammalian chromosome evolution including the recent advances in molecular cytogenetics. The study of animal karyotypes showed that animals have a great diversity in number and morphology of chromosomes despite that their genomes are remarkably conserved. How can this conservation be resolved? This volume try to answer to this question. The first contribution of the volume, written by the Editors with a bringing of MA Ferguson-Smith, is an overview and a history of the cytogenetic techniques used in the analysis of mammalian karyotypes including the results obtained, with an emphasis on chromosome painting in mammals. Chromosome banding (Q-, G-, R-, and C-banding) allowed the workers looking for between-species homologies. However, the homologies established between distantly related species remained speculative until the advent of molecular cytogenetics and especially chromosome painting in the late 1980s and early 1990s. Then it became possible to search for ancestral karyotypes, and to offer original perspectives on genome evolution in mammals and in other vertebrates, as well as on syntenic and associational evolution. The second contribution is devoted to genome size as the evolution of the main genome signature is its size. However, the genome size of several taxa vary over a broad range and do not correlate with the complexity of the organisms (the C-value paradox). The biology of transposable elements may explain some of the molecular mechanisms that are involved in genome size variation and novelties. The other 13 contributions of the volume, written by leading experts in the field, details the cytogenetic findings in Marsupialia and Monotreme, Xenarthra, Afrotheria, Eulipotyphla, Chiroptera, Carnivora and Pholidota, Cetartiodactyla, Perissodactyla, Sciuromorpha and Lagomorpha, Non-Sciuromorpha Rodentia, Strepsirrhine Primates, Dermoptera and Scandentia, new-world monkeys (Platyrrhini), and Catarrhine Primates (old-world monkeys, apes and humans). In this volume, a consistent view of chromosome evolution in mammals is proposed. However, more work is needed as there is an insufficiency of taxa sampling. Even in the more studied mammalian orders, primates and carnivores, only a few species have been studied. The study of more species for enormous orders such as rodents and bats should be done as well for other species all over the mammalian tree, including smaller clades such as Afrotheria and Xenarthra. Painting comparisons of humans and other primates displayed that there is no molecular clock for chromosome evolution. Different rates of chromosome evolution were reported, for example, the high chromosome conservation in felids and whales, and the rapid chromosome evolution in canids, skunks, pigs, and equids. The molecular cytogenetic method commonly used is chromosome painting. Few species, mostly primates, have been studied with higher resolution methods such as BAC-FISH (fluorescent in situ hybridization using BAC), which are able to define intrachromosomal rearrangements and inversions. There is much work to be done at this level. However, chromosome painting has allowed a large sampling of taxa, whereas sequencing studies have been performed mainly in species of economic interest or in species that are of peculiar significance for understanding human evolution such as the higher primates. Nonetheless, with the next-generation sequencing, the number of vertebrate species sequenced will highly increase. It is probable that the integration of cytogenetic and sequencing information will be extremely important for resolving issues in cytogenetics such as the reconstruction of ancestral karyotypes, the elimination of homoplasy that can plague cytogenetic reconstructions, and the distinction of convergent associations and homologous associations. Those searching for an answer to the question what makes us human, will not find it in this volume. However, one can imagine that understanding the forces responsible for different rates of chromosome evolution and the peculiar characteristics of the mammalian genome will be possible at the sequencing level in the near future. Will this mean that cytogenetics is no more relevant in evolutionary studies? The Editors of this volume do not think so, they think that a closer integration between cytogenetics and sequencing efforts will be necessary if cytogeneticists want to maintain their relevance in evolutionary studies. This book, available in hardback and in electronic format, is recommended to those interested in the evolution of the vertebrate genome and for researchers involved in mammalian evolution, comparative cytogenetics, molecular cytogenetics, comparative genomics, primatology, and zoology▪

The Polymorphism of Cytogenetic Markers in the Farm and Wild-Living Raccoon Dog (Nyctereutes Procyonoides) / Polimorfizm Markerów Cytogenetycznych U Jenota (Nyctereutes Procyonoides) W Populacjach Hodowlanych I Dziko Żyjących

Abstract The raccoon dog (Nyctereutes procyonoides) is a mammalian species that belongs to Canidae family, order Carnivora. This species represents both animals living in the wild and farm animals used in the fur industry. Raccoon dogs have the most ‘primitive’ karyotype among Canidae family. The Chinese raccoon dog (Nyctereutes procyonoides procyonoides) is characterised by a variable number of chromosomes (2n = 54 + 0-4 B). B chromosomes are supernumerary chromosomes occurring in addition to the basic set of A chromosomes in the cells of many organisms. The function and origin of these additional chromosomes is not clear. The aim of this work was to determine possible karyotypic differences between wild-living and farm populations, using methods of classical and molecular cytogenetics. The most useful cytogenetic markers to analyse karyotype polymorphism of canine are the number of B chromosomes and nucleolar organizer regions. A variation was identified in the number of B chromosomes and nucleolar organizer regions (NORs) in cells between wild-living and breeding populations.

Oncofuse: a computational framework for the prediction of the oncogenic potential of gene fusions

Gene fusions resulting from chromosomal aberrations are an important cause of cancer. The complexity of genomic changes in certain cancer types has hampered the identification of gene fusions by molecular cytogenetic methods, especially in carcinomas. This is changing with the advent of next-generation sequencing, which is detecting a substantial number of new fusion transcripts in individual cancer genomes. However, this poses the challenge of identifying those fusions with greater oncogenic potential amid a background of 'passenger' fusion sequences. In the present work, we have used some recently identified genomic hallmarks of oncogenic fusion genes to develop a pipeline for the classification of fusion sequences, namely, Oncofuse. The pipeline predicts the oncogenic potential of novel fusion genes, calculating the probability that a fusion sequence behaves as 'driver' of the oncogenic process based on features present in known oncogenic fusions. Cross-validation and extensive validation tests on independent datasets suggest a robust behavior with good precision and recall rates. We believe that Oncofuse could become a useful tool to guide experimental validation studies of novel fusion sequences found during next-generation sequencing analysis of cancer transcriptomes. Oncofuse is a naive Bayes Network Classifier trained and tested using Weka machine learning package. The pipeline is executed by running a Java/Groovy script, available for download at www.unav.es/genetica/oncofuse.html.