Since being established in 1963, the murine fibroblast cell line NIH 3T3 has been used in thousands of studies. NIH 3T3 immortalized spontaneously and became tetraploid shortly after its establishment. Here we report the first molecular cytogenetic characterization of NIH 3T3 using fluorescence in situ hybridization based multicolor banding (mcb). Overall, a complex rearranged karyotype presenting 16 breakpoints was characterized. Also it was possible to deduce the resulting gains and losses of copy numbers in NIH 3T3. Overall, only 1.8% of the NIH 3T3 genome is disome, 26.2% tri-, 60% tetra-, 10.8% quinta-, and 1.2% hexasome. Strikingly, the cell line gained only 4 derivative chromosomes since its first cytogenetic description in 1989. An attempt to align the observed imbalances of the studied cell line with their homologous regions in humans gave the following surprising result: NIH 3T3 shows imbalances as typically seen in human solid cancers of ectodermal origin.

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.

BackgroundHeterochromatic variants of pericentromere of chromosome 9 are reported and discussed since decades concerning their detailed structure and clinical meaning. However, detailed studies are scarce. Thus, here we provide the largest ever done molecular cytogenetic research based on >300 chromosome 9 heteromorphism carriers.ResultsIn this study, 334 carriers of heterochromatic variants of chromosome 9 were included, being 192 patients from Western Europe and the remainder from Easter-European origin. A 3-color-fluorescence in situ hybridization (FISH) probe-set directed against for 9p12 to 9q13~21.1 (9het-mix) and 8 different locus-specific probes were applied for their characterization. The 9het-mix enables the characterization of 21 of the yet known 24 chromosome 9 heteromorphic patterns. In this study, 17 different variants were detected including five yet unreported; the most frequent were pericentric inversions (49.4%) followed by 9qh-variants (23.9%), variants of 9ph (11.4%), cenh (8.2%), and dicentric- (3.8%) and duplication-variants (3.3%). For reasons of simplicity, a new short nomenclature for the yet reported 24 heteromorphic patterns of chromosome 9 is suggested. Six breakpoints involved in four of the 24 variants could be narrowed down using locus-specific probes.ConclusionsBased on this largest study ever done in carriers of chromosome 9 heteromorphisms, three of the 24 detailed variants were more frequently observed in Western than in Eastern Europe. Besides, there is no clear evidence that infertility is linked to any of the 24 chromosome 9 heteromorphic variants.

Abstract. The differentiation of homologous chromosomes as well as their parental origin can presently be conducted and determined exclusively by molecular genetic methods using microsatellite or SNP analysis. Only in exceptional cases is a distinction on a single-cell level possible, e.g. due to variations within the heterochromatic regions of chromosomes 1, 9, 16 and Y or the p-arms of the acrocentric chromosomes. In the absence of such polymorphisms, an individual distinction of the homologous chromosomes is not currently possible. Consequently, various questions of scientific and diagnostic relevance are unable to be answered. Based on the recently detected large-scale copy-number variations (LCV) or copynumber polymorphisms (CNP) spanning up to several megabase pairs of DNA, in this study, a molecular cytogenetic technique for the inter-individual differentiation of homologous chromosomes called parental-origin-determination fluorescence in situ hybridization (pod-FISH) is presented. All human chromosomes were covered with 225 LCVand/or CNP-specific BAC probes, and one-to five-color chromosome-specific pod-FISH sets were created, evaluated and optimized. We demonstrated that pod-FISH is suitable for single-cell analysis of uniparental disomy (UDP) in clinical cases such as Prader-Willi syndrome caused by maternal UPD. A rare clinical case with a mosaic form of a genome-wide isodisomy was used to determine the detection limits of pod-FISH. Additionally we analyzed the informativeness of conventional microsatellite analysis for the first time and compared the results to pod-FISH. With this new possibility to study the parental origin of individual human chromosomes on a single-cell level, new doors for diagnostic and basic research are opened.

Background: Small supernumerary marker chromosomes (sSMC) are present ~2.6 × 10 6 human worldwide. sSMC are a heterogeneous group of derivative chromosomes concerning their clinical consequences as well as their chromosomal origin and shape. Besides the sSMC present in Emanuel syndrome, i.e. der(22)t(11;22)(q23;q11), only few so-called complex sSMC are reported.

A prenatally ascertained case with a de novo small supernumerary marker chromosome (sSMC) derived from chromosome 1 is reported. Due to a fetal heart defect the parents decided in favour of an induced abortion. Postmortem, a molecular cytogenetic study on eleven formalin fixed, paraffin-embedded tissues of the fetus was performed, to further characterize the levels of mosaicism of the sSMC(1). sSMC presence varied between 13 and 62% within different tissues of sSMC carriers. This finding is something common in sSMC carriers and could explain why up to the present no clinical correlations for sSMC mosaicism and clinical outcome in the corresponding carriers could be established.

The discovery of copy number variations (CNV) in the human genome opened new perspectives in the study of the genetic causes of inherited disorders and the etiology of common diseases. Differently patterned instances of somatic mosaicism in CNV regions have been shown to be present in monozygotic twins and throughout different tissues within an individual. A single-cell-level investigation of CNV in different human cell types led us to uncover mitotically derived genomic mosaicism, which is stable in different cell types of one individual. A unique study of immortalized B-lymphoblastoid cell lines obtained with 20 year interval from the same two subjects shows that mitotic changes in CNV regions may happen early during embryonic development and seem to occur only once, as levels of mosaicism remained stable. This finding has the potential to change our concept of dynamic human genome variation. We propose that further genomic studies should focus on the single-cell level, to understand better the etiology and physiology of aging and diseases mediated by somatic variations.

BackgroundThe chromosomal homologies of human (Homo sapiens = HSA) and silvered leaf monkey (Trachypithecus cristatus = TCR) have been previously studied by classical chromosome staining and by fluorescence in situ hybridization (FISH) applying chromosome-specific DNA probes of all human chromosomes in the 1980s and 1990s, respectively.ResultsHowever, as the resolution of these techniques is limited we used multicolor banding (MCB) at an ~250-band level, and other selected human DNA probes to establish a detailed chromosomal map of TCR. Therefore it was possible to precisely determine evolutionary conserved breakpoints, orientation of segments and distribution of specific regions in TCR compared to HSA. Overall, 69 evolutionary conserved breakpoints including chromosomal segments, which failed to be resolved in previous reports, were exactly identified and characterized.ConclusionsThis work also represents the first molecular cytogenetic one characterizing a multiple sex chromosome system with a male karyotype 44,XY1Y2. The obtained results are compared to other available data for old world monkeys and drawbacks in hominoid evolution are discussed.