Chromosome territories, X;Y translocation and Premature Ovarian Failure: is there a relationship?

<p>Abstract</p> <p>Background</p> <p>Premature ovarian failure (POF) is a secondary hypergonadotrophic amenorrhea occurring before the age of 40 and affecting 1-3% of females. Chromosome anomalies account for 6-8% of POF cases, but only few cases are associated with translocations involving X and Y chromosomes.</p> <p>This study shows the cytogenetic and molecular analysis of a POF patient came to our attention as she developed a left ovary choriocarcinoma at the age of 10 and at 14 years of age she presented secondary amenorrhea with elevated levels of gonadotropins.</p> <p>Results</p> <p>Breakpoint position on X and Y chromosomes was investigated using Fluorescent In Situ Hybridisation (FISH) with a panel of specific BAC probes, microsatellite analysis and evaluation of copy number changes and loss of heterozigosity by Affymetrix^®GeneChip platform (Santa Clara, CA, USA). Patient's karyotype resulted 46, X, der(Y)t(X;Y)(q13.1;q11.223). X inactivation study was assessed by RBA banding and showed preferential inactivation of derivative chromosome. The reciprocal spatial disposition of sexual chromosome territories was investigated using whole chromosome painting and centromeres probes: patient's results didn't show a significant difference in comparison to normal controls.</p> <p>Conclusion</p> <p>The peculiar clinical case come to our attention highlighted the complexity of POF aetiology and of the translocation event, even if our results seem to exclude any effect on nuclear organisation. POF phenotype could be partially explained by skewed X chromosome inactivation that influences gene expression.</p

Mutations in MAPT give rise to aneuploidy in animal models of tauopathy

Tau is a major microtubule-associated protein in brain neurons. Its misfolding and accumulation cause neurodegenerative diseases characterized by brain atrophy and dementia, named tauopathies. Genetic forms are caused by mutations of microtubule-associated protein tau gene (MAPT). Tau is expressed also in nonneural tissues such as lymphocytes. Tau has been recently recognized as a multifunctional protein, and in particular, some findings supported a role in genome stability. In fact, peripheral cells of patients affected by frontotemporal dementia carrying different MAPT mutations showed structural and numerical chromosome aberrations. The aim of this study was to assess chromosome stability in peripheral cell from two animal models of genetic tauopathy, JNPL3 and PS19 mouse strains expressing the human tau carrying the P301L and P301S mutations, respectively, to confirm the previous data on humans. After demonstrating the presence of mutated tau in spleen, we performed standard cytogenetic analysis of splenic lymphocytes from homozygous and hemizygous JNPL3, hemizygous PS19, and relevant controls. Losses and gains of chromosomes (aneuploidy) were evaluated. We detected a significantly higher level of aneuploidy in JNPL3 and PS19 than in control mice. Moreover, in JNPL3, the aneuploidy was higher in homozygotes than in hemizygotes, demonstrating a gene dose effect, which appeared also to be age independent. Our results show that mutated tau is associated with chromosome instability. It is conceivable to hypothesize that in genetic tauopathies the aneuploidy may be present also in central nervous system, possibly contributing to neurodegeneration

Biological heterogeneity of putative bladder cancer stem-like cell populations from human bladder transitional cell carcinoma samples.

Transitional cell carcinoma (TCC) is the most common type of bladder cancer. Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells, the cancer stem-like cells (CSCs) or tumor initiating cells. We report on the isolation and biological characterization of putative bladder CSC populations from primary TCCs. Isolated cells were induced to proliferate in stem cell culture conditions (serum-free medium containing mitogenic growth factors). The proliferating cells formed spheroids (urospheres) and their abilities for extensive proliferation and self-renewal were assayed. Their positivity for several stem cell markers (CD133, Oct-3/4, nestin, and cytokeratins) was also assessed by immunofluorescence tests and they could have the potential to differentiate in the presence of serum. In stem cell culture conditions they gradually showed loss of proliferation, adherence to the substrate, and morphological changes, which might reflect their progressive acquisition of differentiative capacity and loss of self-renewal ability. To evaluate if effective cell selection occurred after isolation, conventional cytogenetic studies on fresh chromosome spreads immediately after isolation and after culture were carried out. In addition, a molecular cytogenetic study by UroVysion assay was carried out on paraffin-embedded tissue sections and on fresh and after culture nuclei preparations. The data collected indicated important karyotype changes and a positive selection for hypo- or near-diploid cells, losing the complexity present in fresh tumors

Cytogenetics of Premature Ovarian Failure: An Investigation on 269 Affected Women

The importance of X chromosome in the aetiology of premature ovarian failure (POF) is well-known but in many cases POF still remains idiopathic. Chromosome aneuploidy increase is a physiological phenomenon related to aging, but the role of low-level sex chromosome mosaicism in ovarian function is still undiscovered. Standard cytogenetic analysis was carried out in a total of 269 patients affected by POF: 27 chromosomal abnormalities were identified, including X chromosome and autosomal structural and numerical abnormalities. In 47 patients with 46,XX karyotype we performed interphase FISH using X alpha-satellite probe in order to identify X chromosome mosaicism rate. Aneuploidy rate in the patient group was significantly higher than the general population group. These findings underline the importance of X chromosome in the aetiology of POF and highlight the potential role of low-level sex chromosome mosaicism in ovarian aging that may lead to a premature onset of menopause

Cytogenetics of Premature Ovarian Failure: An Investigation on 269 Affected Women

The importance of X chromosome in the aetiology of premature ovarian failure (POF) is well-known but in many cases POF still remains idiopathic. Chromosome aneuploidy increase is a physiological phenomenon related to aging, but the role of low-level sex chromosome mosaicism in ovarian function is still undiscovered. Standard cytogenetic analysis was carried out in a total of 269 patients affected by POF: 27 chromosomal abnormalities were identified, including X chromosome and autosomal structural and numerical abnormalities. In 47 patients with 46,XX karyotype we performed interphase FISH using X alpha-satellite probe in order to identify X chromosome mosaicism rate. Aneuploidy rate in the patient group was significantly higher than the general population group. These findings underline the importance of X chromosome in the aetiology of POF and highlight the potential role of low-level sex chromosome mosaicism in ovarian aging that may lead to a premature onset of menopause

Genomic and epigenomic profile of uterine smooth muscle tumors of uncertain malignant potential (Stumps) revealed similarities and differences with leiomyomas and leiomyosarcomas

Uterine smooth muscle tumors of uncertain malignant potential (STUMPs) represent a heterogeneous group of tumors that cannot be histologically diagnosed as unequivocally benign or malignant. For this reason, many authors are working to obtain a better definition of diagnostic and prognostic criteria. In this work, we analyzed the genomic and epigenomic profile of uterine smooth muscle tumors (USMTs) in order to find similarities and differences between STUMPs, leiomyosarcomas (LMSs) and leiomyomas (LMs), and possibly identify prognostic factors in this group of tumors. Array-CGH data on 23 USMTs demonstrated the presence of a more similar genomic profile between STUMPs and LMSs. Some genes, such as PRKDC and PUM2, with a potential prognostic value, were never previously associated with STUMP. The methylation data appears to be very promising, especially with regards to the divergent profile found in the sample that relapsed, characterized by an overall CGI hypomethylation. Finally, the Gene Ontology analysis highlighted some cancer genes that could play a pivotal role in the unexpected aggressive behavior that can be found in some of these tumors. These genes could prove to be prognostic markers in the future

Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells

Bone-marrow-derived mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and differentiation into multiple cell types. Accumulating preclinical and clinical evidence indicates that MSCs are good candidates to use as cell therapy in many degenerative diseases. For MSC clinical applications, an adequate number of cells are necessary so an extensive expansion is required. However, spontaneous immortalization and malignant transformation of MSCs after culture expansion have been reported in human and mouse, while very few data are present for rat MSCs (rMSCs). In this study, we monitored the chromosomal status of rMSCs at several passages in vitro, also testing the influence of four different cell culture conditions. We first used the conventional traditional cytogenetic techniques, in order to have the opportunity to observe even minor structural abnormalities and to identify low-degree mosaic conditions. Then, a more detailed genomic analysis was conducted by array comparative genomic hybridization. We demonstrated that, irrespective of culture conditions, rMSCs manifested a markedly aneuploid karyotype and a progressive chromosomal instability in all the passages we analyzed and that they are anything but stable during in vitro culture. Despite the fact that the risk of neoplastic transformation associated with this genomic instability needs to be further addressed and considering the apparent genomic stability reported for in vitro cultured human MSCs (hMSCs), our findings underline the fact that rMSCs may not in fact be a good model for effectively exploring the full clinical therapeutic potential of hMSCs

Chromosomal Aberrations in Bladder Cancer: Fresh versus Formalin Fixed Paraffin Embedded Tissue and Targeted FISH versus Wide Microarray-Based CGH Analysis

Bladder carcinogenesis is believed to follow two alternative pathways driven by the loss of chromosome 9 and the gain of chromosome 7, albeit other nonrandom copy number alterations (CNAs) were identified. However, confirmation studies are needed since many aspects of this model remain unclear and considerable heterogeneity among cases has emerged. One of the purposes of this study was to evaluate the performance of a targeted test (UroVysion assay) widely used for the detection of Transitional Cell Carcinoma (TCC) of the bladder, in two different types of material derived from the same tumor. We compared the results of UroVysion test performed on Freshly Isolated interphasic Nuclei (FIN) and on Formalin Fixed Paraffin Embedded (FFPE) tissues from 22 TCCs and we didn't find substantial differences. A second goal was to assess the concordance between array-CGH profiles and the targeted chromosomal profiles of UroVysion assay on an additional set of 10 TCCs, in order to evaluate whether UroVysion is an adequately sensitive method for the identification of selected aneuploidies and nonrandom CNAs in TCCs. Our results confirmed the importance of global genomic screening methods, that is array based CGH, to comprehensively determine the genomic profiles of large series of TCCs tumors. However, this technique has yet some limitations, such as not being able to detect low level mosaicism, or not detecting any change in the number of copies for a kind of compensatory effect due to the presence of high cellular heterogeneity. Thus, it is still advisable to use complementary techniques such as array-CGH and FISH, as the former is able to detect alterations at the genome level not excluding any chromosome, but the latter is able to maintain the individual data at the level of single cells, even if it focuses on few genomic regions

Using Copy Number Alterations to Identify New Therapeutic Targets for Bladder Carcinoma

Bladder cancer represents the ninth most widespread malignancy throughout the world. It is characterized by the presence of two different clinical and prognostic subtypes: non-muscle-invasive bladder cancers (NMIBCs) and muscle-invasive bladder cancers (MIBCs). MIBCs have a poor outcome with a common progression to metastasis. Despite improvements in knowledge, treatment has not advanced significantly in recent years, with the absence of new therapeutic targets. Because of the limitations of current therapeutic options, the greater challenge will be to identify biomarkers for clinical application. For this reason, we compared our array comparative genomic hybridization (array-CGH) results with those reported in literature for invasive bladder tumors and, in particular, we focused on the evaluation of copy number alterations (CNAs) present in biopsies and retained in the corresponding cancer stem cell (CSC) subpopulations that should be the main target of therapy. According to our data, CCNE1, MYC, MDM2 and PPARG genes could be interesting therapeutic targets for bladder CSC subpopulations. Surprisingly, HER2 copy number gains are not retained in bladder CSCs, making the gene-targeted therapy less interesting than the others. These results provide precious advice for further study on bladder therapy; however, the clinical importance of these results should be explored

The Effect of Culture on Human Bone Marrow Mesenchymal Stem Cells: Focus on DNA Methylation Profiles

Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-term in vitro culture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions for in vitro expansion in order to minimize these epigenetic changes and to standardize safer procedures