The Precise Breakpoint Mapping in Paracentric Inversion 10q22.2q23.3 by Comprehensive Cytogenomic Analysis, Multicolor Banding, and Single-Copy Chromosome Sequencing.

Detection and precise genomic mapping of balanced chromosomal abnormalities in patients with impaired fertility or a clinical phenotype represent a challenge for current cytogenomics owing to difficulties with precise breakpoint localization in the regions enriched for DNA repeats and high genomic variation in such regions. Here, we present a comprehensive cytogenomic approach to breakpoint mapping in a rare paracentric inversion on 10q (in a patient with oligoasthenoteratozoospermia and necrozoospermia) that does not affect other phenotype traits. Multicolor banding, chromosomal microarray analysis, chromosome microdissection with reverse painting, and single-copy sequencing of the rearranged chromosome were performed to determine the length and position of the inverted region as well as to rule out a genetic imbalance at the breakpoints. As a result, a paracentric 19.251 Mbp inversion at 10q22.2q23.3 was described. The most probable location of the breakpoints was predicted using the hg38 assembly. The problems of genetic counseling associated with enrichment for repeats and high DNA variability of usual breakpoint regions were discussed. Possible approaches for cytogenomic assessment of couples with balanced chromosome rearrangements and problems like reproductive failures were considered and suggested as useful part of effective genetic counseling.

Prenatal Diagnosis of Small Supernumerary Marker Chromosome 10 by Array-Based Comparative Genomic Hybridization and Microdissected Chromosome Sequencing.

Interpreting the clinical significance of small supernumerary marker chromosomes (sSMCs) in prenatal diagnosis is still an urgent problem in genetic counselling regarding the fate of a pregnancy. We present a case of prenatal diagnosis of mosaic sSMC(10) in a foetus with a normal phenotype. Comprehensive cytogenomic analyses by array-based comparative genomic hybridization (aCGH), sSMC microdissection with next-generation sequencing (NGS) of microdissected library, fluorescence in situ hybridization (FISH) with locus-specific and telomere-specific DNA probes and quantitative real-time PCR revealed that sSMC(10) had a ring structure and was derived from the pericentromeric region of chromosome 10 with involvement of the 10p11.21-p11.1 and 10q11.21-q11.23 at 1.243 Mb and 7.173 Mb in size, respectively. We observed a difference in the length of sSMC(10) between NGS data of the DNA library derived from a single copy of sSMC(10), and aCGH results that may indicate instability and structural mosaicism for ring chromosomes in foetal cells. The presence of a 9 Mb euchromatin region in the analysed sSMC(10) did not lead to clinical manifestations, and a healthy girl was born at term. We suggest that the ring structure of sSMCs could influence sSMC manifestations and should be taken into account in genetic counselling during prenatal diagnosis.

Homogeneously Staining Regions (HSR) in Chromosome 1 of the House Mouse: Synapsis and Recombination at Meiosis.

Amplified sequences constitute a large part of mammalian genomes. A chromosome 1 containing 2 large (up to 50 Mb) homogeneously staining regions (HSRs) separated by a small inverted euchromatic region is present in many natural populations of the house mouse (Mus musculus musculus). The HSRs are composed of a long-range repeat cluster, Sp100-rs, with a repeat length of 100 kb. In order to understand the organization and function of HSRs in meiotic chromosomes, we examined synapsis and recombination in male mice hetero- and homozygous for the HSR-carrying chromosome using FISH with an HSR-specific DNA probe and immunolocalization of the key meiotic proteins. In all homozygous and heterozygous pachytene nuclei, we observed fully synapsed linear homomorphic bivalents 1 marked by the HSR FISH probe. The synaptic adjustment in the heterozygotes was bilateral: the HSR-carrying homolog was shortened and the wild-type homolog was elongated. The adjustment was reversible: desynapsis at diplotene was accompanied by elongation of the HSRs. Immunolocalization of H3K9me2/3 indicated that the HSRs in the meiotic chromosome retained the epigenetic modification typical for C-heterochromatin in somatic cells. MLH1 foci, marking mature recombination nodules, were detected in the proximal HSR band in heterozygotes and in both HSR bands of homozygotes. Unequal crossing over within the long-range repeat cluster can cause variation in size of the HSRs, which has been detected in the natural populations of the house mouse.

Two Separate Cases: Complex Chromosomal Abnormality Involving Three Chromosomes and Small Supernumerary Marker Chromosome in Patients with Impaired Reproductive Function.

For medical genetic counseling, estimating the chance of a child being born with chromosome abnormality is crucially important. Cytogenetic diagnostics of parents with a balanced karyotype are a special case. Such chromosome rearrangements cannot be detected with comprehensive chromosome screening. In the current paper, we consider chromosome diagnostics in two cases of chromosome rearrangement in patients with balanced karyotype and provide the results of a detailed analysis of complex chromosomal rearrangement (CCR) involving three chromosomes and a small supernumerary marker chromosome (sSMC) in a patient with impaired reproductive function. The application of fluorescent in situ hybridization, microdissection, and multicolor banding allows for describing analyzed karyotypes in detail. In the case of a CCR, such as the one described here, the probability of gamete formation with a karyotype, showing a balance of chromosome regions, is extremely low. Recommendation for the family in genetic counseling should take into account the obtained result. In the case of an sSMC, it is critically important to identify the original chromosome from which the sSMC has been derived, even if the euchromatin material is absent. Finally, we present our view on the optimal strategy of identifying and describing sSMCs, namely the production of a microdissectional DNA probe from the sSMC combined with a consequent reverse painting.

Germline-restricted chromosome (GRC) in the sand martin and the pale martin (Hirundinidae, Aves): synapsis, recombination and copy number variation.

All songbirds studied to date have an additional Germline Restricted Chromosome (GRC), which is not present in somatic cells. GRCs show a wide variation in genetic content and little homology between species. To check how this divergence affected the meiotic behavior of the GRC, we examined synapsis, recombination and copy number variation for GRCs in the closely related sand and pale martins (Riparia riparia and R. diluta) in comparison with distantly related estrildid finches. Using immunolocalization of meiotic proteins and FISH with GRC-specific DNA probes, we found a striking similarity in the meiotic behavior of GRCs between martins and estrildid finches despite the millions of years of independent evolution. GRCs are usually present in two copies in female and in one copy in male pachytene cells. However, we detected polymorphism in female and mosaicism in male martins for the number of GRCs. In martin and zebra finch females, two GRCs synapse along their whole length, but recombine predominately at their ends. We suggest that the shared features of the meiotic behavior of GRCs have been supported by natural selection in favor of a preferential segregation of GRCs to the eggs.

Dysfunction telomeres in embryonic fibroblasts and cultured in vitro pluripotent stem cells of Rattus norvegicus (Rodentia, Muridae).

We studied the level of spontaneous telomere dysfunction in Rattus norvegicus (Berkenhout, 1769) (Rodentia, Muridae) embryonic fibroblasts (rEFs) and in cultured in vitro rat pluripotent stem cells (rPSCs), embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs), on early passages and after prolonged cultivation. Among studied cell lines, rESCs showed the lowest level of telomere dysfunction, while the riPSCs demonstrated an elevated level on early passages of cultivation. In cultivation, the frequency of dysfunctional telomeres has increased in all studied cell lines; this is particularly true for dysfunctional telomeres occurring in G1 stage in riPSCs. The obtained data are mainly discussed in the connection with the specific structure of the telomere regions and their influence on the differential DNA damage response in them.

Germline-restricted chromosome (GRC) is widespread among songbirds.

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages.

Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and Apodemus B chromosomes.

Supernumerary chromosomes sporadically arise in many eukaryotic species as a result of genomic rearrangements. If present in a substantial part of species population, those are called B chromosomes, or Bs. This is the case for 70 mammalian species, most of which are rodents. In humans, the most common types of extra chromosomes, sSMCs (small supernumerary marker chromosomes), are diagnosed in approximately 1 of 2000 postnatal cases. Due to low frequency in population, human sSMCs are not considered B chromosomes. Genetic content of both B-chromosomes and sSMCs in most cases remains understudied. Here, we apply microdissection of single chromosomes with subsequent low-pass sequencing on Ion Torrent PGM and Illumina MiSeq to identify unique and repetitive DNA sequences present in a single human sSMC and several B chromosomes in mice Apodemus flavicollis and Apodemus peninsulae. The pipeline for sequencing data analysis was made available in Galaxy interface as an addition to previously published command-line version. Human sSMC was attributed to the proximal part of chromosome 15 long arm, and breakpoints leading to its formation were located into satellite DNA arrays. Genetic content of Apodemus B chromosomes was species-specific, and minor alterations were observed in both species. Common features of Bs in these Apodemus species were satellite DNA and ERV enrichment, as well as the presence of the vaccinia-related kinase gene Vrk1. Understanding of the non-essential genome elements content provides important insights into genome evolution in general.

Chromosome Synapsis and Recombination in Male Hybrids between Two Chromosome Races of the Common Shrew (Sorex araneus L., Soricidae, Eulipotyphla).

Hybrid zones between chromosome races of the common shrew (Sorex araneus) provide exceptional models to study the potential role of chromosome rearrangements in the initial steps of speciation. The Novosibirsk and Tomsk races differ by a series of Robertsonian fusions with monobrachial homology. They form a narrow hybrid zone and generate hybrids with both simple (chain of three chromosomes) and complex (chain of eight or nine) synaptic configurations. Using immunolocalisation of the meiotic proteins, we examined chromosome pairing and recombination in males from the hybrid zone. Homozygotes and simple heterozygotes for Robertsonian fusions showed a low frequency of synaptic aberrations (<10%). The carriers of complex synaptic configurations showed multiple pairing abnormalities, which might lead to reduced fertility. The recombination frequency in the proximal regions of most chromosomes of all karyotypes was much lower than in the other regions. The strong suppression of recombination in the pericentromeric regions and co-segregation of race specific chromosomes involved in the long chains would be expected to lead to linkage disequilibrium between genes located there. Genic differentiation, together with the high frequency of pairing aberrations in male carriers of the long chains, might contribute to maintenance of the narrow hybrid zone.

Spatial organization of fibroblast and spermatocyte nuclei with different B-chromosome content in Korean field mouse, Apodemus peninsulae (Rodentia, Muridae).

Korean field mouse (Apodemus peninsulae) shows a wide variation in the number of B chromosomes composed of constitutive heterochromatin. For this reason, it provides a good model to study the influence of the number of centromeres and amount of heterochromatin on spatial organization of interphase nuclei. We analyzed the three-dimensional organization of fibroblast and spermatocyte nuclei of the field mice carrying a different number of B chromosomes using laser scanning microscopy and 3D fluorescence in situ hybridization. We detected a co-localization of the B chromosomes with constitutive heterochromatin of the chromosomes of the basic set. We showed a non-random distribution of B chromosomes in the spermatocyte nuclei. Unpaired B chromosomes showed a tendency to occur in the compartment formed by the unpaired part of the XY bivalent.

Comprehensive Analyses of White-Handed Gibbon Chromosomes Enables Access to 92 Evolutionary Conserved Breakpoints Compared to the Human Genome.

Gibbon species (Hylobatidae) impress with an unusually high number of numerical and structural chromosomal changes within the family itself as well as compared to other Hominoidea including humans. In former studies applying molecular cytogenetic methods, 86 evolutionary conserved breakpoints (ECBs) were reported in the white-handed gibbon (Hylobates lar, HLA) with respect to the human genome. To analyze those ECBs in more detail and also to achieve a better understanding of the fast karyotype evolution in Hylobatidae, molecular data for these regions are indispensably necessary. In the present study, we obtained whole chromosome-specific probes by microdissection of all 21 HLA autosomes and prepared them for aCGH. Locus-specific DNA probes were also used for further molecular cytogenetic characterization of selected regions. Thus, we could map 6 yet unreported ECBs in HLA with respect to the human genome. Additionally, in 26 of the 86 previously reported ECBs, the present approach enabled a more precise breakpoint mapping. Interestingly, a preferred localization of ECBs within segmental duplications, copy number variant regions, and fragile sites was observed.

The mitochondria-targeted antioxidant SkQ1 restores αB-crystallin expression and protects against AMD-like retinopathy in OXYS rats.

Age-related macular degeneration (AMD), a neurodegenerative and vascular retinal disease, is the leading cause of blindness in the developed world. Accumulating evidence suggests that alterations in the expression of a small heat shock protein (αB-crystallin) are involved in the pathogeneses of AMD. Here we demonstrate that senescence-accelerated OXYS rats-an animal model of the dry form of AMD-develop spontaneous retinopathy against the background of reduced expression of αB-crystallin in the retina at the early preclinical stages of retinopathy (age 20 days) as well as at 4 and 24 months of age, during the progressive stage of the disease. The level of αA-crystallin expression in the retina of OXYS rats at all the ages examined was no different from that in disease-free Wistar rats. Treatment with the mitochondria-targeted antioxidant SkQ1 (plastoquinonyl-decyltriphenylphosphonium) from 1.5 to 4 months of age, 250 nmol/kg, increased the level of αB-crystallin expression in the retina of OXYS rats. SkQ1 slowed the development of retinopathy and reduced histological aberrations in retinal pigment epithelium cells. SkQ1 also attenuated neurodegenerative changes in the photoreceptors and facilitated circulation in choroid blood vessels in the retina of OXYS rats; this improvement was probably linked with the restoration of αB-crystallin expression.

A comparative study of genome organization and inferences for the systematics of two large bushcricket genera of the tribe Barbitistini (Orthoptera: Tettigoniidae: Phaneropterinae).

BACKGROUND: Poecilimon and Isophya are the largest genera of the tribe Barbitistini and among the most systematically complicated and evolutionarily intriguing groups of Palearctic tettigoniids. We examined the genomic organization of 79 taxa with a stable chromosome number using classical (C-banding, silver and fluorochrome staining) and molecular (fluorescence in situ hybridization with 18S rDNA and (TTAGG)n telomeric probes) cytogenetic techniques. These tools were employed to establish genetic organization and differences or similarities between genera or species within the same genus and determine if cytogenetic markers can be used for identifying some taxonomic groups of species. RESULTS: Differences between the karyotypes of the studied genera include some general changes in the morphology of the X chromosome in Isophya (in contrast to Poecilimon). The number of major rDNA clusters per haploid genome divided Poecilimon into two main almost equal groups (with either one or two clusters), while two rDNA clusters predominated in Isophya. In both genera, rDNA loci were preferentially located in the paracentromeric region of the autosomes and rarely in the sex chromosomes. Our results demonstrate a coincidence between the location of rDNA loci and active NORs and GC-rich heterochromatin regions. The C/DAPI/CMA3 bands observed in most Poecilimon chromosomes suggest the presence of more families of repetitive DNA sequences as compared to the heterochromatin patterns in Isophya. CONCLUSIONS: The results show both differences and similarities in genome organization among species of the same genus and between genera. Previous views on the systematics and phylogenetic grouping of certain lineages are discussed in light of the present cytogenetic results. In some cases, variation of chromosome markers was observed to correspond with variation in other evolutionary traits, which is related to the processes of ongoing speciation and hybridization in zones of secondary contact. It was concluded that the physical mapping of rDNA sequences and heterochromatin may be used as an additional marker for understanding interspecific relationships in these groups and their routes of speciation.

DNA probes for FISH analysis of C-negative regions in human chromosomes.

Fluorescent in situ hybridization (FISH) is a powerful technology for studying the chromosome organization and aberrations as well as for searching the homology between chromosomal regions in mammals. Currently, FISH is used as a simple, rapid, and reliable technique for analyzing chromosomal rearrangements and assigning chromosomal breakpoints in modern diagnosing of chromosomal pathology. In addition to cloned DNA fragments, the DNA probes produced by sequence-independent polymerase chain reaction are widely used in FISH assays. As a rule, the DNA probes generated from a genomic or chromosomal DNA by whole genome amplification are enriched for repetitive elements and, consequently, efficient FISH analysis requires that repetitive DNA hybridization is suppressed. The linker-adapter polymerase chain reaction (LA-PCR) using the genomic DNA hydrolyzed with HaeIII and RsaI restriction endonucleases allows the repetitive DNA fraction in DNA probe to be decreased and gene-rich DNA to be predominantly amplified. The protocol described here was proposed for production of the DNA probes for enhanced analysis of the C-negative regions in human chromosomes.

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae).

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.

Recombination map of the common shrew, Sorex araneus (Eulipotyphla, Mammalia).

The Eurasian common shrew (Sorex araneus L.) is characterized by spectacular chromosomal variation, both autosomal variation of the Robertsonian type and an XX/XY(1)Y(2) system of sex determination. It is an important mammalian model of chromosomal and genome evolution as it is one of the few species with a complete genome sequence. Here we generate a high-precision cytological recombination map for the species, the third such map produced in mammals, following those for humans and house mice. We prepared synaptonemal complex (SC) spreads of meiotic chromosomes from 638 spermatocytes of 22 males of nine different Robertsonian karyotypes, identifying each autosome arm by differential DAPI staining. Altogether we mapped 13,983 recombination sites along 7095 individual autosomes, using immunolocalization of MLH1, a mismatch repair protein marking recombination sites. We estimated the total recombination length of the shrew genome as 1145 cM. The majority of bivalents showed a high recombination frequency near the telomeres and a low frequency near the centromeres. The distances between MLH1 foci were consistent with crossover interference both within chromosome arms and across the centromere in metacentric bivalents. The pattern of recombination along a chromosome arm was a function of its length, interference, and centromere and telomere effects. The specific DNA sequence must also be important because chromosome arms of the same length differed substantially in their recombination pattern. These features of recombination show great similarity with humans and mice and suggest generality among mammals. However, contrary to a widespread perception, the metacentric bivalent tu usually lacked an MLH1 focus on one of its chromosome arms, arguing against a minimum requirement of one chiasma per chromosome arm for correct segregation. With regard to autosomal chromosomal variation, the chromosomes showing Robertsonian polymorphism display MLH1 foci that become increasingly distal when comparing acrocentric homozygotes, heterozygotes, and metacentric homozygotes. Within the sex trivalent XY(1)Y(2), the autosomal part of the complex behaves similarly to other autosomes.

Highly complex karyotypic changes in acute myelogenous leukemia: a case report.

We report on a patient with a clinically diagnosed acute myelogenous leukemia (AML) with so far unrecorded complex translocations. As GTG-banding was not able to resolve all karyotypic changes, spectral karyotyping (SKY) and multiplex-fluorescence in situ hybridization (M-FISH) were performed for comparison. Both methods gave nearly identical results, however, they were unable to characterize all involved chromosomal breakpoints in detail. Thus, multicolor banding (MCB) technique was applied and its results were confirmed for two large derivative chromosomes by microdissection and reverse painting. Using this battery of molecular cytogenetic approaches the karyotype of this AML case could be described as 40 approximately 44,XY,der(1)t(1;5;8;20) (1qter-->1p12::5q14.3-->5q15 or 5q15-->5q14.3::8p11.2-->8p23.? 3::20p11.1-->20p13),del(2)(q12),der(3)t(3;6),der(5)t(5;18) (5p15.33-->5q11::18q21.3-->18q23),del(6),-8,der(9)t(9;17;15),der(10)t(3;10),del(11)(q24),-15,-16,del(17),der(18) t(8;18;5;2;20)(8q24.3-->8q24.2 or 8q24.2-->8q24.3::18p11.22-->18q21.3::5q14.3-->5q11::2q32-->2q12::20q13.2-->20q13.33), der(20)t(1;20;18)(1p36.33-->1p31.3-22.3::20p11.1-->20q11.2 or 20q11.2-->20p11.1::18p11.22-->18p11.32).