Identifizierung und Charakterisierung von "fragile sites" und Vergleich mit Neoplasie-assoziierten und evolutionär fixierten Chromosomenbruchpunkten der Hominidae

Ziel der vorliegenden Arbeit war die zytogenetisch bestehende chromosomale Kolokalisation von „fragile sites“ (FS), evolutionär konservierten Bruchpunkten und Neoplasie-assoziierten Bruchpunkten mit Hilfe molekularzytogenetischer Methoden zu überprüfen (hauptsächlich von „common fragile sites“ (cFS)), wobei eine FS-Kartierung m.H. von BACs Vorraussetzung war. Neben der Beantwortung der eigentlichen Fragestellung erfolgte die Beurteilung der FS-Expressionsrate in unterschiedlichen Individuen und Bandenstadien. In dem Bandenstadium 300-350 wurden nur wenige Chromosomenbrüche pro Metaphaseplatte identifiziert, in den höheren Bandenstadien signifikant mehr. Des Weiteren waren interindividuelle Unterschiede bezüglich der FS-Häufigkeiten feststellbar. Anhand der Beurteilung aller exprimierter FS konnten, neben den in der NCBI-Datenbank Gelisteten, 61 neue und 52 bereits in diversen Veröffentlichungen aufgeführte FS nachgewiesen werden. Dies ermöglichte erstmals die Erstellung einer „Fragilen Karte“ von Aphidicolin-induzierten FS in Lymphozyten des gesamten Genoms, einschließlich einer einheitlichen Benennung. Es erfolgte in dieser Arbeit eine vollständige Kartierung von 5 FS, von weiteren 15 war eine proximale oder distale Bruchpunkt-Bestimmung möglich und an 14 weiteren FS erfolgten Einzel-BAC-Hybridisierungen. Es konnte gezeigt werden, dass evolutionär konservierte Bruchpunkte zu einem hohen Anteil innerhalb von fragilen Bereichen des Genoms liegen (~74%), im Gegensatz zu Neoplasie-assoziierten chromosomalen Veränderungen (Translokationen). Eine spezifische Sequenzeigenschaft, welche für die Fragilität verantwortlich gemacht werden könnte, wurde nicht festgestellt

The hierarchically organized splitting of chromosomal bands for all human chromosomes

<p>Abstract</p> <p>Background</p> <p>Chromosome banding is widely used in cytogenetics. However, the biological nature of hierarchically organized splitting of chromosomal bands of human chromosomes is an enigma and has not been, as yet, studied.</p> <p>Results</p> <p>Here we present for the first time the hierarchically organized splitting of chromosomal bands in their sub-bands for all human chromosomes. To do this, array-proved multicolor banding (aMCB) probe-sets for all human chromosomes were applied to normal metaphase spreads of three different G-band levels. We confirmed for all chromosomes to be a general principle that only Giemsa-dark bands split into dark and light sub-bands, as we demonstrated previously by chromosome stretching. Thus, the biological band splitting is in > 50% of the sub-bands different than implemented by the ISCN nomenclature suggesting also a splitting of G-light bands. Locus-specific probes exemplary confirmed the results of MCB.</p> <p>Conclusion</p> <p>Overall, the present study enables a better understanding of chromosome architecture. The observed difference of biological and ISCN band-splitting may be an explanation why mapping data from human genome project do not always fit the cytogenetic mapping.</p

Position of chromosomes 18, 19, 21 and 22 in 3D-preserved interphase nuclei of human and gorilla and white hand gibbon

<p>Abstract</p> <p>Background</p> <p>Even though comparative nuclear architecture studies in hominoids are sparse, nuclear chromosome architecture was shown to be conserved during hominoid evolution. Thus, it is suspected that yet unknown biological mechanisms must underlie this observation.</p> <p>Results</p> <p>Here for the first time a combination of multicolor banding (MCB) and three-dimensional analysis of interphase cells was used to characterize the position and orientation of human chromosomes #18, #19, #21 and #22 and their homologues in primate B-lymphocytic cells. In general, our data is in concordance with previous studies. The position of the four studied human chromosomes and their homologues were conserved during primate evolution. However, comparison of interphase architecture in human B-lymphocytic cells and sperm revealed differences of localization of acrocentric chromosomes. The latter might be related to the fact that the nucleolus organizing region is not active in sperm.</p> <p>Conclusion</p> <p>Studies in different tissue types may characterize more – potentially biologically relevant differences in nuclear architecture.</p

Small supernumerary marker chromosomes (sSMC) in humans; are there B chromosomes hidden among them

<p>Abstract</p> <p>Background</p> <p>Small supernumerary marker chromosomes (sSMC) and B-chromosomes represent a heterogeneous collection of chromosomes added to the typical karyotype, and which are both small in size. They may consist of heterochromatic and/or euchromatic material. Also a predominance of maternal transmission was reported for both groups. Even though sSMC and B-chromosomes show some similarity it is still an open question if B-chromosomes are present among the heterogeneous group of sSMC. According to current theories, sSMC would need drive, drift or beneficial effects to increase in frequency in order to become B chromosome. However, up to now no B-chromosomes were described in human.</p> <p>Results</p> <p>Here we provide first evidence and discuss, that among sSMC B-chromosomes might be hidden. We present two potential candidates which may already be, or may in future evolve into B chromosomes in human: (i) sSMC cases where the marker is stainable only by DNA derived from itself; and (ii) acrocentric-derived inverted duplication sSMC without associated clinical phenotype. Here we report on the second sSMC stainable exclusively by its own DNA and show that for acrocentric derived sSMC 3.9× more are familial cases than reported for other sSMC.</p> <p>Conclusion</p> <p>The majority of sSMC are not to be considered as B-chromosomes. Nonetheless, a minority of sSMC show similarities to B-chromosomes. Further studies are necessary to come to final conclusions for that problem.</p

Chromosome distribution in human sperm – a 3D multicolor banding-study

<p>Abstract</p> <p>Background</p> <p>Nuclear architecture studies in human sperm are sparse. By now performed ones were practically all done on flattened nuclei. Thus, studies close at the <it>in vivo </it>state of sperm, i.e. on three-dimensionally conserved interphase cells, are lacking by now. Only the position of 14 chromosomes in human sperm was studied.</p> <p>Results</p> <p>Here for the first time a combination of multicolor banding (MCB) and three-dimensional analysis of interphase cells was used to characterize the position and orientation of all human chromosomes in sperm cells of a healthy donor. The interphase nuclei of human sperm are organized in a non-random way, driven by the gene density and chromosome size.</p> <p>Conclusion</p> <p>Here we present the first comprehensive results on the nuclear architecture of normal human sperm. Future studies in this tissue type, e.g. also in male patients with unexplained fertility problems, may characterize yet unknown mechanisms of infertility.</p

Clinically abnormal case with paternally derived partial trisomy 8p23.3 to 8p12 including maternal isodisomy of 8p23.3: a case report

<p>Abstract</p> <p>Background</p> <p>Because of low copy repeats (LCRs) and common inversion polymorphisms, the human chromosome 8p is prone to a number of recurrent rearrangements. Each of these rearrangements is associated with several phenotypic features. We report on a patient with various clinical malformations and developmental delay in connection with an inverted duplication event, involving chromosome 8p.</p> <p>Methods</p> <p>Chromosome analysis, multicolor banding analysis (MCB), extensive fluorescence in situ hybridization (FISH) analysis and microsatellite analysis were performed.</p> <p>Results</p> <p>The karyotype was characterized in detail by multicolor banding (MCB), subtelomeric and centromere-near probes as 46,XY,dup(8)(pter->p23.3::p12->p23.3::p23.3->qter). Additionally, microsatellite analysis revealed the paternal origin of the duplication and gave hints for a mitotic recombination involving about 6 MB in 8p23.3.</p> <p>Conclusion</p> <p>A comprehensive analysis of the derivative chromosome 8 suggested a previously unreported mechanism of formation, which included an early mitotic aberration leading to maternal isodisomy, followed by an inverted duplication of the 8p12p23.3 region.</p

Somatic Mosaicism in Cases with Small Supernumerary Marker Chromosomes

Somatic mosaicism is something that is observed in everyday lives of cytogeneticists. Chromosome instability is one of the leading causes of large-scale genome variation analyzable since the correct human chromosome number was established in 1956. Somatic mosaicism is also a well-known fact to be present in cases with small supernumerary marker chromosomes (sSMC), i.e. karyotypes of 47,+mar/46. In this study, the data available in the literature were collected concerning the frequency mosaicism in different subgroups of patients with sSMC. Of 3124 cases with sSMC 1626 (52%) present with somatic mosaicism. Some groups like patients with Emanuel-, cat-eye- or i(18p)- syndrome only tend rarely to develop mosaicism, while in Pallister-Killian syndrome every patient is mosaic. In general, acrocentric and non-acrocentric derived sSMCs are differently susceptible to mosaicism; non-acrocentric derived ones are hereby the less stable ones. Even though, in the overwhelming majority of the cases, somatic mosaicism does not have any detectable clinical effects, there are rare cases with altered clinical outcomes due to mosaicism. This is extremely important for prenatal genetic counseling. Overall, as mosaicism is something to be considered in at least every second sSMC case, array-CGH studies cannot be offered as a screening test to reliably detect this kind of chromosomal aberration, as low level mosaic cases and cryptic mosaics are missed by that

Derivative chromosome 1 and GLUT1 deficiency syndrome in a sibling pair

<p>Abstract</p> <p>Background</p> <p>Genomic imbalances constitute a major cause of congenital and developmental abnormalities. GLUT1 deficiency syndrome is caused by various de novo mutations in the facilitated human glucose transporter 1 gene (1p34.2) and patients with this syndrome have been diagnosed with hypoglycorrhachia, mental and developmental delay, microcephaly and seizures. Furthermore, 1q terminal deletions have been submitted in the recent reports and the absence of corpus callosum has been related to the deletion between <it>C1orf100 </it>and <it>C1orf121 </it>in 1q44.</p> <p>Results</p> <p>This study reports on a sibling pair with developmental delay, mental retardation, microcephaly, hypotonia, epilepsy, facial dysmorphism, ataxia and impaired speech. Chromosome analysis revealed a derivative chromosome 1 in both patients. FISH and MCB analysis showed two interstitial deletions at 1p34.2 and 1q44. SNP array and array-CGH analysis also determined the sizes of deletions detailed. The deleted region on 1p34.2 encompasses 33 genes, among which is <it>GLUT1 </it>gene (<it>SLC2A1</it>). However, the deleted region on 1q44 includes 59 genes and distal-proximal breakpoints were located in the ZNF672 gene and SMYD3 gene, respectively.</p> <p>Conclusion</p> <p>Haploinsufficiency of <it>GLUT1 </it>leads to GLUT1 deficiency syndrome, consistent with the phenotype in patients of this study. Conversely, in the deleted region on 1q44, none of the genes are related to findings in these patients. Additionally, the results confirm previous reports on that corpus callosal development may depend on the critical gene(s) lying in 1q44 proximal to the <it>SMYD3 </it>gene.</p

First molecular-cytogenetic characterization of Fanconi anemia fragile sites in primary lymphocytes of FA-D2 patients in different stages of the disease

Background: Fanconi anemia (FA) is a chromosomal instability syndrome characterized by increased frequency of chromosomal breakages, chromosomal radial figures and accelerated telomere shortening. In this work we performed detailed molecular-cytogenetic characterization of breakpoints in primary lymphocytes of FA-D2 patients in different stages of the disease using fluorescent in situ hybridization. Results: We found that chromosomal breakpoints co-localize on the molecular level with common fragile sites, whereas their distribution pattern depends on the severity of the disease. Telomere quantitative fluorescent in situ hybridization revealed that telomere fusions and radial figures, especially radials which involve telomere sequences are the consequence of critically shortened telomeres that increase with the disease progression and could be considered as a predictive parameter during the course of the disease. Sex chromosomes in FA cells are also involved in radial formation indicating that specific X chromosome regions share homology with autosomes and also could serve as repair templates in resolving DNA damage. Conclusions: FA-D2 chromosomal breakpoints co-localize with common fragile sites, but their distribution pattern depends on the disease stage. Telomere fusions and radials figures which involve telomere sequences are the consequence of shortened telomeres, increase with disease progression and could be of predictive value

Early Embryonic Chromosome Instability Results in Stable Mosaic Pattern in Human Tissues

The discovery of copy number variations (CNV) in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations