Zukunft der Zytogenetik

This issue of the journal “Medizinische Genetik” emphasizes the current development of cytogenetic technologies. Changes in classical banding analysis, which has been a cornerstone of routine human genetics diagnostics for decades, are illustrated by means of quality assurance measures. Several contributions in this issue describe molecular cytogenetic technologies, which are based on fluorescence in situ hybridization (FISH). The introduction of comparative genomic hybridization, especially on various array platforms, revolutionized cytogenetics even further and now allows researchers to address entirely new questions and problems in human genetics. An especial stronghold of cytogenetics that distinguishes it from other molecular technologies is the option to perform analyses on a single-cell level. In this issue, possible future developments in cytogenetics are also discussed.     

Veterinary cytogenetics: past and perspective

Cytogenetics was conceived in the late 1800s and nurtured through the early 1900s by discoveries pointing to the chromosomal basis of inheritance. The relevance of chromosomes to human health and disease was realized more than half a century later when improvements in techniques facilitated unequivocal chromosome delineation. Veterinary cytogenetics has benefited from the information generated in human cytogenetics which, in turn, owes its theoretical and technical advancement to data gathered from plants, insects and laboratory mammals. The scope of this science has moved from the structure and number of chromosomes to molecular cytogenetics for use in research or for diagnostic and prognostic purposes including comparative genomic hybridization arrays, single nucleotide polymorphism array-based karyotyping and automated systems for counting the results of standard FISH preparations. Even though the counterparts to a variety of human diseases and disorders are seen in domestic animals, clinical applications of veterinary cytogenetics will be less well exploited mainly because of the cost-driven nature of demand on diagnosis and treatment which often out-weigh emotional and sentimental attachments. An area where the potential of veterinary cytogenetics will be fully exploited is reproduction since an inherited aberration that impacts on reproductive efficiency can compromise the success achieved over the years in animal breeding. It is gratifying to note that such aberrations can now be tracked and tackled using sophisticated cytogenetic tools already commercially available for RNA expression analysis, chromatin immunoprecipitation, or comparative genomic hybridization using custom-made microarray platforms that allow the construction of microarrays that match veterinary cytogenetic needs, be it for research or for clinical applications. Judging from the technical refinements already accomplished in veterinary cytogenetics since the 1960s, it is clear that the importance of the achievements to date are bound to be matched or out-weighed by what awaits to be accomplished in the not-too-far future.     

Evolutionary Molecular Cytogenetics of Catarrhine Primates: Past, Present and Future. R. Stanyon M. Rocchi(b) F. Bigoni(a) N. Archidiacono(b)

The catarrhine primates were the first group of species studied with comparative molecular cytogenetics. Many of the fundamental techniques and principles of analysis were initially applied to comparisons in these primates, including interspecific chromosome painting, reciprocal chromosome painting and the extensive use of cloned DNA probes for evolutionary analysis. The definition and importance of chromosome syntenies and associations for a correct cladistics analysis of phylogenomic relationships were first applied to catarrhines. These early chromosome painting studies vividly illustrated a striking conservation of the genome between humans and macaques. Contemporarily, it also revealed profound differences between humans and gibbons, a group of species more closely related to humans, making it clear that chromosome evolution did not follow a molecular clock. Chromosome painting has now been applied to more that 60 primate species and the translocation history has been mapped onto the major taxonomic divisions in the tree of primate evolution. In situ hybridization of cloned DNA probes, primarily BAC-FISH, also made it possible to more precisely map breakpoints with spanning and flanking BACs. These studies established marker order and disclosed intrachromosomal rearrangements. When applied comparatively to a range of primate species, they led to the discovery of evolutionary new centromeres as an important new category of chromosome evolution. BAC-FISH studies are intimately connected to genome sequencing, and probes can usually be assigned to a precise location in the genome assembly. This connection ties molecular cytogenetics securely to genome sequencing, assuring that molecular cytogenetics will continue to have a productive future in the multidisciplinary science of phylogenomics.     

Chromosomal phylogeny and evolution of gibbons (Hylobatidae)

Although human and gibbons are classified in the same primate superfamily (Hominoidae), their karyotypes differ by extensive chromosome reshuffling. To date, there is still limited understanding of the events that shaped extant gibbon karyotypes. Further, the phylogeny and evolution of the twelve or more extant gibbon species (lesser apes, Hylobatidae) is poorly understood, and conflicting phylogenies have been published. We present a comprehensive analysis of gibbon chromosome rearrangements and a phylogenetic reconstruction of the four recognized subgenera based on molecular cytogenetics data. We have used two different approaches to interpret our data: (1) a cladistic reconstruction based on the identification of ancestral versus derived chromosome forms observed in extant gibbon species; (2) an approach in which adjacent homologous segments that have been changed by translocations and intra-chromosomal rearrangements are treated as discrete characters in a parsimony analysis (PAUP). The orangutan serves as an "outgroup", since it has a karyotype that is supposed to be most similar to the ancestral form of all humans and apes. Both approaches place the subgenus Bunopithecus as the most basal group of the Hylobatidae, followed by Hylobates, with Symphalangus and Nomascus as the last to diverge. Since most chromosome rearrangements observed in gibbons are either ancestral to all four subgenera or specific for individual species and only a few common derived rearrangements at subsequent branching points have been recorded, all extant gibbons may have diverged within relatively short evolutionary time. In general, chromosomal rearrangements produce changes that should be considered as unique landmarks at the divergence nodes. Thus, molecular cytogenetics could be an important tool to elucidate phylogenies in other species in which speciation may have occurred over very short evolutionary time with not enough genetic (DNA sequence) and other biological divergence to be picked up.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

荧光原位杂交技术及其在植物研究中的应用

荧光原位杂交(FISH)是20世纪生物学领域的一项新技术。FISH应用细胞遗传学和分子生物学的基本原理,作为架设细胞遗传学与分子生物学之间的桥梁,现已被广泛应用于植物学各方面的研究。本文就FISH的基本原理、技术发展及其在植物遗传育种、起源进化、染色体物理图谱构建方面的应用及发展趋势进行了综述。     

染色体微分离和微克隆技术

染色体微分离和微克隆技术是将细胞遗传学和分子遗传学二紧密结合的一项技术,目前已广泛应用于遗传学、医学等研究领域,具有广阔的应用前景。本综述了该技术发展过程中所应用的不同方法,详细介绍了各种方法的步骤及其优缺点,最后探讨了该技术的应用及展望。     

Whose Turn? Chromosome Research and the Study of the Human Genome

A common account sees the human genome sequencing project of the 1990s as a “natural outgrowth” of the deciphering of the double helical structure of DNA in the 1950s. The essay aims to complicate this neat narrative by putting the spotlight on the field of human chromosome research that flourished at the same time as molecular biology. It suggests that we need to consider both endeavors – the human cytogeneticists who collected samples and looked down the microscope and the molecular biologists who probed the molecular mechanisms of gene function – to understand the rise of the human genome sequencing project and the current genomic practices. In particular, it proposes that what has often been described as the “molecularization” of cytogenetics could equally well be viewed as the turn of molecular biologists to human and medical genetics – a field long occupied by cytogeneticists. These considerations also have implications for the archives that are constructed for future historians and policy makers.     

Primary pleuropulmonary synovial sarcoma diagnosed by fine needle aspiration with cytogenetic confirmation: a case report

BACKGROUND: Pleuropulmonary synovial sarcomas (PPSSs) are rare neoplasins that have been well described in recent years, although there are only very infrequent reports within the cytology literature. Such lesions present a diagnostic challenge on fine needle aspiration (ENA) due to several factors, particularly when the aspirate material displays monophasic, small cell or poorly differentiated morphology. Immunoperoxidase studies on cell block material and confirmation with molecular cytogenetics are important tools to establish the diagnosis and determine appropriate therapy. We report a case of PPSS in a 27-year-old man diagnosed by computed tomography (CT)-guided FNA with confirmation by conventional and molecular cytogenetics. CASE: A 27-year-old man presented with several rapidly enlarging, pleura-based masses following a several-month history of recurrent hemopneumothorax. Previous surgical pathology on decorticated pleura was interpreted as a reactive mesothelial proliferation at another institution. Upon referral, CT-guided transthoracic FNA was performed. Smears revealed a highly cellular, dispersed "small round blue cell" neoplasm in a hemorrhagic background. The cytomorphology, in conjunction with a select immunoperoxidase panel, was diagnostic of PPSS. Conventional and molecular cytogenetics subsequently provided confirmation of the diagnosis. CONCLUSION: PPSSs are uncommon neoplasms seldom diagnosed by FNA, with only very rare reports in the cytology literature. Although their cytomorphology has been well described, monophasic tumors and other morphologic variants present a diagnostic challenge and may be difficult to discern from a variety of neoplastic and reactive/reparative processes. Emphasis should be placed upon securing material at the time of aspiration for immunoperoxidase studies (cell block or core biopsy). In equivocal cases, conventional and/or molecular cytogenetic studies may be needed.     

Fifty years of cytogenetics: a parallel view of the evolution of cytogenetics and genotoxicology

A parallelism exists between human cytogenetics and cytogenetic toxicology. The breakthroughs, mostly coming from and used in clinical genetics, are widely used in genetic toxicology. The birth of human cytogenetics occurred in 1956 when it was published that the diploid number of chromosomes in humans is 46. The first stage in chromosome-induced mutagenesis began in 1938 when Sax published the effects of X-rays on the chromosomes of Drosophila. In 1959, the cytogenetic anomalies for Down, Klinefelter, and Turner syndromes were described, and parallelly in 1960, the first publication on chromosomal aberrations in man caused by ionizing radiation appeared. The cytogenetic analysis of chromosomal aberrations in cell cultures is considered one of the primary methods to evaluate induced mutagenesis. At the end of the 1960s, banding techniques allowed chromosomes to be individually identified, in parallel, the sister chromatid exchange analysis technology was described. Another milestone in the history of induced mutagenesis was the discovery that mutagenic agents were able to alter chromosomal division and segregation in gonads inducing meiotic nondisjunction. Here we review new approaches and applications such as biological dosimetry, translocation scoring using FISH, and micronucleus test. Chromosomal aberrations and micronucleus test are now effective cytogenetic biomarkers of early effect used as cancer predictors. Human cytogenetics has proven to be effective over its 50-year lifespan and, although each new technique that has appeared seemed to announce its end, the fact is that the current state of cytogenetics is in reality a collection of techniques that, while common, are cheap, fast, and wide-ranging. Therefore, in genotoxicology, they continue to be useful to identify mutagenic agents as well as to evaluate and analyze exposed populations.     

Multiplex-FISH (M-FISH): technique, developments and applications

Multiplex FISH (M-FISH) represents one of the most significant developments in molecular cytogenetics of the past decade. Originally designed to generate 24 colour karyotyping, the technique has spawned many variations and an equally diverse range of applications. In tumour and leukaemia cytogenetics, the two groups that have been targeted represent both ends of the cytogenetic spectrum: those with an apparently normal karyotype (suspected of harbouring small rearrangements not detectable by conventional cytogenetics) and those with a complex aberrant karyotype (which are difficult to karyotype accurately due to the sheer number of aberrations). In research, mouse M-FISH provides a powerful tool to characterize mouse models of a disease. In addition, the ability to accurately karyotype single metaphases without selection makes M-FISH the perfect tool in chromosome breakage studies and for characterizing clonal evolution of tumours. Finally, M-FISH has emerged as the perfect partner for the developing genomic microarray (array CGH) technologies, providing a powerful approach to gene discovery.     

适应辐射类群穇属的系统学研究进展

综合花序拓扑学、比较形态学、分子系统发育、细胞遗传学等资料,对适应辐射类群穆属(Eleusine Gaertn.)的系统学研究进展进行了述评.穆属系统位置--Eleusiinae亚族成员得到分子系统发育证据的支持.该属具有3种花序类型、7个基因组类型、多倍体均由二倍体杂交起源、C4植物高度适应半湿润-半干旱镶嵌气候等特征.据可靠化石记载和现代地理分布推断,穆属很可能起源于东非,时间是晚中新世,而适应辐射则发生在上新世-中新世间隔.总的来说,分子系统发育、细胞遗传学、古地质、古气候数据的整合研究能够为穆属多倍体起源和谱系多样化历史提供令人信服的证据.     

Perspectives on the formation of radiation-induced exchange aberrations

Controversy surrounding the proposed mechanism of radiation-induced translocation has existed virtually since the inception of radiation genetics/cytogenetics, some 75 years ago. Chief among these controversies is how close chromosomes have to be to one another at the time of exposure for an exchange to occur. An historically related issue, and one that continues to generate lively debate, is whether both chromosomes participating in an exchange must sustain radiation damage, or whether instead a single damaged site on one chromosome is sufficient. The intent of this paper is to present one person's perspective as we revisit these two long-standing issues, armed with more recent knowledge in three key areas. These include a new-found appreciation for the complexity of chromosome rearrangements; molecular processes of recombination that are likely to be involved; and the architecture of the nucleus regarding the relationship among chromosomes during interphase.     

Molecular cytogenetics and taxonomy of insects,with particular reference to the coleoptera

We assess and review the impact of the new cytogenetic techniques in insects for their roles in taxonomy and for a better knowledge of their chromosomal structure. Particular emphasis is given to molecular cytogenetics by fluorescent in situ hybridization, localization of AT- or GC-rich regions with fluorochromes, and restriction endonuclease banding in beetle chromosomes. The main features of the cytogenetics of Coleoptera are treated in detail, taking into account the range of variation in chromosome number and genome size, and our in-depth findings on the constitutive heterochromatin and satellite DNAs of tenebrionid beetles. Some other topics of interest for insect cytogenetics, such as the meiotic association of sex chromosomes, the nucleolar organizing regions (NORs), and the molecular constitution of telomeres, are also discussed from the taxonomic and structural viewpoints.     

Centromere repositioning in mammals

The evolutionary history of chromosomes can be tracked by the comparative hybridization of large panels of bacterial artificial chromosome clones. This approach has disclosed an unprecedented phenomenon: 'centromere repositioning', that is, the movement of the centromere along the chromosome without marker order variation. The occurrence of evolutionary new centromeres (ENCs) is relatively frequent. In macaque, for instance, 9 out of 20 autosomal centromeres are evolutionarily new; in donkey at least 5 such neocentromeres originated after divergence from the zebra, in less than 1 million years. Recently, orangutan chromosome 9, considered to be heterozygous for a complex rearrangement, was discovered to be an ENC. In humans, in addition to neocentromeres that arise in acentric fragments and result in clinical phenotypes, 8 centromere-repositioning events have been reported. These 'real-time' repositioned centromere-seeding events provide clues to ENC birth and progression. In the present paper, we provide a review of the centromere repositioning. We add new data on the population genetics of the ENC of the orangutan, and describe for the first time an ENC on the X chromosome of squirrel monkeys. Next-generation sequencing technologies have started an unprecedented, flourishing period of rapid whole-genome sequencing. In this context, it is worth noting that these technologies, uncoupled from cytogenetics, would miss all the biological data on evolutionary centromere repositioning. Therefore, we can anticipate that classical and molecular cytogenetics will continue to have a crucial role in the identification of centromere movements. Indeed, all ENCs and human neocentromeres were found following classical and molecular cytogenetic investigations.     

Molecular Cytogenetics and Cytogenomics of Brain Diseases

Molecular cytogenetics is a promising field of biomedical research that has recently revolutionized our thinking on genome structure and behavior. This is in part due to discoveries of human genomic variations and their contribution to biodiversity and disease. Since these studies were primarily targeted at variation of the genome structure, it appears apposite to cover them by molecular cytogenomics. Human brain diseases, which encompass pathogenic conditions from severe neurodegenerative diseases and major psychiatric disorders to brain tumors, are a heavy burden for the patients and their relatives. It has been suggested that most of them, if not all, are of genetic nature and several recent studies have supported the hypothesis assuming them to be associated with genomic instabilities (i.e. single-gene mutations, gross and subtle chromosome imbalances, aneuploidy). The present review is focused on the intriguing relationship between genomic instability and human brain diseases. Looking through the data, we were able to conclude that both interindividual and intercellular genomic variations could be pathogenic representing, therefore, a possible mechanism for human brain malfunctioning. Nevertheless, there are still numerous gaps in our knowledge concerning the link between genomic variations and brain diseases, which, hopefully, will be filled by forthcoming studies. In this light, the present review considers perspectives of this dynamically developing field of neurogenetics and genomics.     

Comparative painting of mammalian chromosomes

Comparative chromosome painting has shown that synteny has been conserved for large segments of the genome in various placental mammals. Advances such as spectral karyotyping and multicolour ‘bar coding’ lend speed and precision to comparative molecular cytogenetics. Reciprocal chromosome painting and hybridisations with probes such as yeast artificial chromosomes, cosmids, and fibre fluorescence in situ hybridisation allow subchromosomal assignments of chromosome regions and can identify breakpoints of rearranged chromosomes. Advances in molecular cytogenetics can now be used to test the hypothesis that chromosome rearrangement breakpoints in human pathology and in evolution are correlated.     

Image analysis as a simple and useful tool for evaluating the constitutive heterochromatin (C-banding) in human chromosomes

Image analysis can contribute to those fields of cytogenetics that are influenced most by subjectivity, especially evaluation of chromosome regions that are histochemically polymorphic. C-banding of human or primate chromosomes may be used as a typical example of this concept. In addition, using it quantitatively it is useful in studies of population cytogenetics of man and Primates. Therefore, the aim of this study was to determine the best conditions for measurement by image analysis of C-positive regions in a sample of 29 normal subjects. We looked for relationships of chromosomal C-positive areas with the dimensions of the corresponding metaphases. Finally, we suggest some criteria for potential use of C-banding in population and comparative studies.     

Chromosome painting in mammals as an approach to comparative genomics

Chromosome painting has become a routine tool in comparative cytogenetics. The utility of interspecies chromosome painting has been demonstrated in taxa characterized by highly rearranged karyotypes such as in rodents and lesser apes. Chromosome painting also provides a new level of precision in comparative genome analysis for eliminating errors of confounding convergence with homology. Recent results hold promise that molecular cytogenetics will make a significant contribution to the understanding of the major features of genome evolution.     

Canine cytogenetics--from band to basepair

Humans and dogs have coexisted for thousands of years, during which time we have developed a unique bond, centered on companionship. Along the way, we have developed purebred dog breeds in a manner that has resulted unfortunately in many of them being affected by serious genetic disorders, including cancers. With serendipity and irony the unique genetic architecture of the 21st century genome of Man's best friend may ultimately provide many of the keys to unlock some of nature's most intriguing biological puzzles. Canine cytogenetics has advanced significantly over the past 10 years, spurred on largely by the surge of interest in the dog as a biomedical model for genetic disease and the availability of advanced genomics resources. As such the role of canine cytogenetics has moved rapidly from one that served initially to define the gross genomic organization of the canine genome and provide a reliable means to determine the chromosomal location of individual genes, to one that enabled the assembled sequence of the canine genome to be anchored to the karyotype. Canine cytogenetics now presents the biomedical research community with a means to assist in our search for a greater understanding of how genome architectures altered during speciation and in our search for genes associated with cancers that affect both dogs and humans. The cytogenetics 'toolbox' for the dog is now loaded. This review aims to provide a summary of some of the recent advancements in canine cytogenetics.     

Chromosome landmarks as tools to study the genome of Arabidopsis thaliana

The model plant Arabidopsis thaliana has long been used for genetic, cellular and molecular studies. Whereas this plant was used as a model of genetics in the 1940's, the first cytogenetic observation of A. thaliana chromosomes was published in the beginning of the 20th century. Although Arabidopsis was not originally considered to be a good plant model for cytogenetics due to smallness of its genome, the number of published chromosome studies has expanded enormously in recent years. The advent of fluorescence in situ hybridization techniques on meiotic chromosomes together with indirect immuno-fluorescence localization of key chromosomal and nuclear proteins and wide accessibility of Arabidopsis mutants have resulted in a synergistic boost in Arabidopsis cytogenetics. In comparison to other plant species, the small genome with under-represented DNA repeats together with a small number of chromosomes makes this model plant easy to comprehend for a cytologist.