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Improvements in microarray-based comparative genomic hybridization technology have allowed for high-resolution detection of genome wide copy number alterations, leading to a better definition of rearrangements and supporting the study of pathogenesis mechanisms. In this study, we focused our attention on chromosome 8p. We report 12 cases of 8p rearrangements, analyzed by molecular karyotype, evidencing a continuum of fragility that involves the entire short arm. The breakpoints seem more concentrated in three intervals: one at the telomeric end, the others at 8p23.1, close to the beta-defensin gene cluster and olfactory receptor low-copy repeats. Hypothetical mechanisms for all cases are described. Our data extend the cohort of published patients with 8p aberrations and highlight the need to pay special attention to these sequences due to the risk of formation of new chromosomal aberrations with pathological effects.

The presence of thousands of repetitive sequences makes the centromere a fragile region subject to breakage. In this study we collected 31 cases of rearrangements of chromosome 18, of which 16 involved an acrocentric chromosome, during genetic screening done in three centers. We noticed a significant enrichment of reciprocal translocations between the centromere of chromosome 18 and the centromeric or pericentromeric regions of the acrocentrics. We describe five cases with translocation between chromosome 18 and an acrocentric chromosome, and one case involving the common telomere regions of chromosomes 18p and 22p. In addition, we bring evidence to support the hypothesis that chromosome 18 preferentially recombines with acrocentrics: (i) the presence on 18p11.21 of segmental duplications highly homologous to acrocentrics, that can justify a NAHR mechanism; (ii) the observation by 2D-FISH of the behavior of the centromeric regions of 18 respect to the centromeric regions of acrocentrics in the nuclei of normal subjects; (iii) the contact analysis among these regions on published Hi-C data from the human lymphoblastoid cell line (GM12878).

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.

Satellited non-acrocentric autosomal chromosomes (ps–qs-chromosomes) are the result of an interchange between sub-or telomeric regions of autosomes and the p arm of acrocentrics. The sequence homology at the rearrangement breakpoints appears to be, among others, the most frequent mechanism generating these variant chromosomes. The unbalanced carriers of this type of translocation may or may not display phenotypic abnormalities. With the aim to understand the causative mechanism, we revised all the ps–qs-chromosomes identified in five medical genetics laboratories, which used the same procedures for karyotype analysis, reporting 24 unrelated cases involving eight chromosomes. In conclusion, we observed three different scenarios: true translocation, benign variant and complex rearrangement. The detection of translocation partners is essential to evaluate possible euchromatic unbalances and to infer their effect on phenotype. Moreover, we emphasize the importance to perform both, molecular and conventional cytogenetics methods, to better understand the behavior of our genome.

Uterine smooth muscle tumors are the most common female genital tract neoplasms. While leiomyosarcoma has been studied at length, smooth muscle tumors of uncertain malignant potential (STUMPs) still have ambiguous and unresolved issues, with a risk of relapse and evolution largely undefined. We performed an array comparative genomic hybridization analysis on a primitive STUMP and its local recurrence, histologically diagnosed as undifferentiated sarcoma. To the best of our knowledge, our report is the first genomic study on primitive STUMPs and the different relapsed tumors. The results showed few copy number alterations shared between both samples and the high heterogeneity in the STUMP was apparently lost in the sarcoma. Surprisingly the STUMP presented an amplification of the BCL2 gene, not observed in the relapsed tumor. Additionally, fluorescence in situ hybridization and immunohistochemical staining were performed to confirm BCL2 amplification and expression in these samples and in two other cases of primitive STUMPs and their corresponding relapsed tumors. The presence of BCL2 in multiple copies and expression in the two primitive STUMPs and two relapsed tumors was confirmed. The marked amplification of the BCL2 gene present in the primitive STUMP and the multiple copies also observed in other cases, suggest its potential role as a marker of STUMP malignant potential and recurrence.

Background: Neocentromeres are rare and considered chromosomal aberrations, because a non-centromeric region evolves in an active centromere by mutation. The literature reported several structural anomalies of X chromosome and they influence the female reproductive capacity or are associated to Turner syndrome in the presence of monosomy X cell line. Case presentation: We report a case of chromosome X complex rearrangement found in a prenatal diagnosis. The fetal karyotype showed a mosaicism with a 45,X cell line and a 46 chromosomes second line with a big marker, instead of a sex chromosome. The marker morphology and fluorescence in situ hybridization (FISH) characterization allowed us to identify a tricentric X chromosome constituted by two complete X chromosome fused at the p arms telomere and an active neocentromere in the middle, at the union of the two Xp arms, where usually are the telomeric regions. FISH also showed the presence of a paracentric inversion of both Xp arms. Furthermore, fragility figures were found in 56% of metaphases from peripheral blood lymphocytes culture at birth: a shorter marker chromosome and an apparently acentric fragment frequently lost. Conclusions: At our knowledge, this is the first isochromosome of an entire non-acrocentric chromosome. The neocentromere is constituted by canonical sequences but localized in an unusual position and the original centromeres are inactivated. We speculated that marker chromosome was the result of a double rearrangement: firstly, a paracentric inversion which involved the Xp arm, shifting a part of the centromere at the p end and subsequently a duplication of the entire X chromosome, which gave rise to an isochromosome. It is possible to suppose that the first event could be a result of a non-allelic homologous recombination mediated by inverted low-copy repeats. As expected, our case shows a Turner phenotype with mild facial features and no major skeletal deformity, normal psychomotor development a

Background: Segmental duplication of the long arm of chromosome 14 (14q) has commonly been reported to affect the proximal segment of 14q, while distal duplication is a rare condition and often associated with segmental monosomy of other chromosomes. Case presentation: We report the clinical and genetic characterization of a 4-year-old male patient with 14q32.3-qter trisomy resulting from an adjacent segregation of a paternal reciprocal translocation (14;21)(q32.1;p12). The child shows minor facial anomalies, severe developmental delay, growth retardation, and a history of congenital hypothyroidism and neonatal transitory hyperglycemic crises. Conclusions: To the best of our knowledge, only 15 other cases of segmental 14q trisomy were documented. We compared molecularly defined cases to identify a minimal common duplicated region and to find genes with a hypothetical role in the phenotype. The presented case supports the previous suggestion of a pure "distal 14q partial duplication" and underlines the clinical variability.

As shown by genomic studies, colorectal cancer (CRC) is a highly heterogeneous disease, where copy number alterations (CNAs) may greatly vary among different patients. To explore whether CNAs may be present also in histologically normal tissues from patients affected by CRC, we performed CGH + SNP Microarray on 15 paired tumoral and normal samples. Here, we report for the first time the occurrence of CNAs as a common feature of the histologically normal tissue from CRC patients, particularly CNAs affecting different oncogenes and tumor-suppressor genes, including some not previously reported in CRC and others known as being involved in tumor progression. Moreover, from the comparison of normal vs paired tumoral tissue, we were able to identify three groups: samples with an increased number of CNAs in tumoral vs normal tissue, samples with a similar number of CNAs in both tissues, and samples with a decrease of CNAs in tumoral vs normal tissue, which may be likely due to a selection of the cell population within the tumor. In conclusion, our approach allowed us to uncover for the first time an unexpected frequency of genetic alteration in normal tissue, suggesting that tumorigenic genetic lesions are already present in histologically normal colonic tissue and that the use in array comparative genomic hybridization (CGH) studies of normal samples as reference for the paired tumors can lead to misrepresented genomic data, which may be incomplete or limited, especially if used for the research of target molecules for personalized therapy and for the possible correlation with clinical outcome.

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.

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.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system. GBM is a fatal tumor, incurable by conventional therapies. One of the factors underlying tumor recurrence and poor long-term survival is the presence of a cancer stem-like cell population, termed glioma stem cells (GSCs), which is particularly resistant to chemotherapy and radiotherapy and supports tumor self-renewal. The aim of the present study was to evaluate the impact and difference in effects of short-term and long-term treatments with valproic acid (VPA), a histone deacetylase inhibitor, on seven GSC lines. We investigated for the first time the changes in the genome-wide DNA methylation profile and the differentiation behavior of GSCs induced by short-term and long-term VPA treatments. Moreover, we verified VPA sensitivity after long-term VPA pretreatment and, notably, the results provide evidence of a subpopulation more resistant to further VPA treatments. Finally, since short-term VPA treatment induced a reversal of the MGMT methylation status, we aimed to sensitize GSCs to temozolomide, the drug commonly used for this tumor, using this regimen. The overall data highlighted the heterogeneous behavior of GSC lines that is representative of tumor heterogeneity in GBM. The VPA effects were variable among these cell lines in terms of pro-differentiating ability and DNA methylation switch. Here, we attempted to identify a suitable therapy for the eradication of the stem cell subpopulation, which is mandatory to achieve an effective treatment for this tumor. Differentiation-inducing and epigenetic therapies are the most promising approaches to affect the multiple properties of GSCs and, finally, defeat GBM.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma and the most common malignant brain tumor. Current therapies provide a median survival of 12–15 months after diagnosis, due to the high recurrence rate. The failure of current therapies may be due to the presence, within the tumor, of cells characterized by enhanced self-renewal capacity, multilineage differentiation potential and elevated invasive behavior, called glioma stem cells (GSCs). To evaluate the pharmacological efficacy of selected drugs on six GSC lines, we set up a multiple drug responsivity assay based on the combined evaluation of cytomorphological and functional parameters, including the analysis of polymorphic nuclei, mitotic index and cell viability. In order to understand the real pharmacological efficacy of the tested drugs, we assigned a specific drug responsivity score to each GSC line, integrating the data produced by multiple assays. In this work we explored the antineoplastic effects of paclitaxel (PTX), an inhibitor of microtubule depolymerization, utilized as standard treatment in several cancers, and of valproic acid (VPA), an inhibitor of histone deacetylases (HDACs) with multiple anticancer properties. We classified the six GSC lines as responsive or resistant to these drugs, on the basis of their responsivity scores. This method can also be useful to identify the best way to combine two or more drugs. In particular, we utilized the pro-differentiating effect of VPA to improve the PTX effectiveness and we observed a significant reduction of cell viability compared to single treatments.

Background: The existence of two distinct groups of tumors with different clinical characteristic is a remarkable feature of transitional cell carcinomas (TCCs) of the bladder. More than 70% are low-grade (LG) non-infiltrating (NI) cancers at diagnosis, but 60-80% of them recur at least one time and 10-20% progress in stage and grade. On the other hand, about 20% of tumors show muscle invasion (IN) and have a poor prognosis with <50% survival after 5 years. This study focuses on the complexity of the bladder cancer genome, and for the first time to our knowledge, on the possibility to compare genomic alterations of in vitro selected cancer stem-like cells (CSCs), and their original biopsy in order to identify different genomic signature already present in the early stages of tumorigenesis of LG and HG tumors.Methods: We initially used conventional chromosome analysis on TCC biopsies with different histotypes (LG vs HG) in order to detect rough differences between them. Then, we performed array comparative genomic hybridization (aCGH) on 10 HG and 10 LG tumors providing an overview of copy number alterations (CNAs). Finally, we made a comparison of the overall CNAs in 16 biopsies and their respective CSCs isolated from them.Results: Our findings indicate that LG and HG bladder cancer differ with regard to their genomic profile even in the early stage of tumorigenesis; moreover, we identified a subgroup of LG samples with a higher tendency to lose genomic regions which could represent a more aggressive phenotype.Conclusions: The outcomes not only provide valuable information to deeper studying TCC carcinogenesis, but also could help in the clinic for diagnosis and prognosis of patients who will benefit from a more aggressive therapy.

Background. Glioblastoma multiforme (GBM) is one of the deadliest cancers since it shows a very limited sensitivity to chemo- and/or radiotherapy. Experimental evidence suggests that the presence in the tumour environment of GBM stem-like cells might be particularly relevant to confer this extraordinary resistance to treatment. Aim. In this study, we aimed at providing a proof-of-concept of the efficacy of Photo Activation Therapy (PAT) using monochromatic X-rays, issued from a synchrotron radiation (SR) source, in killing human glioma stem cells pre-treated with cisplatin. We have also investigated if lethality of this treatment was superior to that achievable with conventional X-ray irradiation. Material and Methods. Cisplatin was chosen as the carrier of platinum atoms in the cells based on previous experience in glioma and non-glioma cancer cells; the irradiation was performed by delivering 1-8 Grays using beams of energies just above or below the platinum K-shell edge (78.39 keV) or with a conventional X-ray source. Cells were exposed to low drug concentrations allowing 90% or more of GBM stem-like cells survival thus mimicking the unfavourable tissue distribution generally achieved in GMB patients. Results. Under these experimental conditions a significant enhancement in cell lethality was observed using SR with respect to conventional X-ray irradiation. Conclusions. Our results provide additional support to the suggestion that PAT deserves to be further explored in relevant in vivo models based on GBM stem-like cells.

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.

Glioblastoma multiforme (GBM) is one of the deadliest cancers characterized by very limited sensitivity to chemo- and/or radiotherapy. The presence of GBM stem-like cells in the tumor might be relevant for GBM treatment resistance

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments

Glioblastoma multiforme (GBM), the most common and malignant type of glioma, is characterized by a poor prognosis and the lack of an effective treatment, which are due to a small sub-population of cells with stem-like properties, termed glioma stem cells (GSCs). The term “multiforme” describes the histological features of this tumor, that is, the cellular and morphological heterogeneity. At the molecular level multiple layers of alterations may reflect this heterogeneity providing together the driving force for tumor initiation and development. In order to decipher the common “signature” of the ancestral GSC population, we examined six already characterized GSC lines evaluating their cytogenomic and epigenomic profiles through a multilevel approach (conventional cytogenetic, FISH, aCGH, MeDIP-Chip and functional bioinformatic analysis). We found several canonical cytogenetic alterations associated with GBM and a common minimal deleted region (MDR) at 1p36.31, including CAMTA1 gene, a putative tumor suppressor gene, specific for the GSC population. Therefore, on one hand our data confirm a role of driver mutations for copy number alterations (CNAs) included in the GBM genomic-signature (gain of chromosome 7- EGFR gene, loss of chromosome 13- RB1 gene, loss of chromosome 10-PTEN gene); on the other, it is not obvious that the new identified CNAs are passenger mutations, as they may be necessary for tumor progression specific for the individual patient. Through our approach, we were able to demonstrate that not only individual genes into a pathway can be perturbed through multiple mechanisms and at different levels, but also that different combinations of perturbed genes can incapacitate functional modules within a cellular networks. Therefore, beyond the differences that can create apparent heterogeneity of alterations among GSC lines, there’s a sort of selective force acting on them in order to converge towards the impairment of cell development and differentiation p

Bladder cancer (BC) is the seventh most common cancer worldwide. Over 90 % are transitional cell carcinoma (TCC) and at least 70 % of TCC are superficial with unpredictable clinical outcome. It has been suggested that the heterogeneity of BC originates from underlying genetic alterations that lead to different pathways of tumor development and progression. In the last decade a subset of neoplastic cells, known as cancer stem cells (CSCs) has been isolated and characterized from different kind of tumors, including BC. Since the CSC subpopulation seems to be the driving force for tumor initiation, growth and relapse, the identification of a specific CSC genomic signature is a crucial step in order to find new markers for tumor recurrence and progression and new targeted therapies. In this study we compare the genomic profiles of whole biopsies of 16 TCCs with the corresponding CSCs isolated from them, in order to identify any shared copy number alterations (CNAs). The molecular karyotype was evaluated by means of Human Genome kit 8×60 K (Agilent Technologies). The comparison between the genomic profiles evidenced 5 CNAs shared by at least three samples: 3p, two at 9p, 9q and 19q. In particular we observed the loss of tumor suppressor genes (CDKN2A, CDKN2B, PTPRD, TSC1, PTCH1, ABL1 and NOTCH1) while the gain of PPARG, a member of the peroxisome proliferator-activated receptor (PPAR) subfamily of nuclear receptors. Gene Ontology analysis showed that the most represented classes in the CNAs of biopsies and CSCs are apoptosis and transcription regulation. The identification of a specific genomic signature that characterize CSCs is an outstanding endpoint and the identification of commonly shared alterations could provide a first clue in deciphering putative genomic markers useful for diagnostic, prognostic or therapeutic purpose

Mesenchymal stemcells (MSCs) hold great promise for the treatment of numerous diseases. A major problem for MSC therapeutic use is represented by the very low amount of MSCs which can be isolated from different tissues; thus ex vivo expansion is indispensable. Long-term culture, however, is associated with extensive morphological and functional changes of MSCs. In addition, the concern that they may accumulate stochastic mutations which lead the risk of malignant transformation still remains. Overall, the genome of human MSCs (hMSCs) appears to be apparently stable throughout culture, though transient clonal aneuploidies have been detected. Particular attention should be given to the use of low-oxygen environment in order to increase the proliferative capacity of hMSCs, since data on the effect of hypoxic culture conditions on genomic stability are few and contradictory. Furthermore, specific and reproducible epigenetic changes were acquired by hMSCs during ex vivo expansion, which may be connected and trigger all the biological changes observed. In this review we address current issues on long-term culture of hMSCs with a 360-degree view, starting from the genomic profiles and back, looking for an epigenetic interpretation of their genetic stability

In addition to the main function of promoting polymerization and stabilization of microtubules, other roles are being attributed to tau, now considered a multifunctional protein. In particular, previous studies suggest that tau is involved in chromosome stability and genome protection. We performed cytogenetic analysis, including molecular karyotyping, on lymphocytes and fibroblasts from patients affected by frontotemporal lobar degeneration carrying different mutations in the microtubule-associated protein tau gene, to investigate the effects of these mutations on genome stability. Furthermore, we analyzed the response of mutated lymphoblastoid cell lines to genotoxic agents to evaluate the participation of tau to DNA repair systems. We found a significantly higher level of chromosome aberrations in mutated than in control cells. Mutated lymphocytes showed higher percentages of stable lesions, clonal and total aneuploidy (medians: 2 versus 0, p $\ll$ 0.01; 1.5 versus 0, p $\ll$ 0.01; 16.5 versus 0, p $\ll$ 0.01, respectively). Fibroblasts of patients showed higher percentages of stable lesions, structural aberrations and total aneuploidy (medians: 0 versus 0, p = 0.03; 5.8 versus 0, p = 0.02; 26.5 versus 12.6, p $\ll$ 0.01, respectively). In addition, the in depth analysis of DNA copy number variations showed a higher tendency to non-allelic homologous recombination in mutated cells. Finally, while our analysis did not support an involvement of tau in DNA repair systems, it revealed its role in stabilization of chromatin. In summary, our findings indicate a role of tau in genome and chromosome stability that can be ascribed to its function as a microtubule-associated protein as well as a protein protecting chromatin integrity through interaction with DNA

The importance of the genetic factor in the aetiology of premature ovarian failure (POF) is emphasized by the high percentage of familial cases and X chromosome abnormalities account for 10% of chromosomal aberrations. In this study, we report the detailed analysis of 4 chromosomal abnormalities involving the X chromosome and associated with POF that were detected during a screening of 269 affected women. Conventional and molecular cytogenetics were valuable tools for locating the breakpoint regions and thus the following karyotypes were defined: 46,X,der(X)t(X;19)(p21.1;q13.42)mat, 46,X,t(X;2)(q21.33;q14.3)dn, 46,X,der(X)t(X;Y)(q26.2;q11.223)mat and 46,X,t(X;13)(q13.3;q31)dn. A bioinformatic analysis of the breakpoint regions identified putative candidate genes for ovarian failure near the breakpoint regions on the X chromosome or on autosomes that were involved in the translocation event. HS6ST1, HS6ST2 and MATER genes were identified and their functions and a literature review revealed an interesting connection to the POF phenotype. Moreover, the 19q13.32 locus is associated with the age of onset of the natural menopause. These results support the position effect of the breakpoint on flanking genes, and cytogenetic techniques, in combination with bioinformatic analysis, may help to improve what is known about this puzzling disorder and its diagnostic potential.

BACKGROUND: Bladder cancer is the seventh most common cancer worldwide and over 90% are transitional cell carcinoma (TCC). At the first time of diagnosis at least 70% of TCC present as superficial bladder cancer. Because the clinical outcome of superficial bladder tumors is relatively unpredictable, there is a pressing need to identify markers that may predict tumor recurrence and progression and new treatment strategies. CASE PRESENTATION: We present a unique case of a 67-years old male who underwent total cystectomy after repeated trans-urethral resections of the bladder for multifocal non-muscle invasive bladder cancer. The first and the third tumor were diagnosed as high grade non-infiltrating (HGNI), while the second as carcinoma in situ (CIS). We performed both array comparative genomic hybridization and a targeted chromosomal profile by UroVysion in order to detect copy number variations (CNVs) that may be involved with tumor recurrence and progression. The overall data from this study provide new evidence for the monoclonal origin of urothelial tumor multifocality as several genetic changes were found in different tumors of the same patient. From the analysis of shared CNVs two gained regions emerged at 3p25.2 and 12q23.2, including PPARG and ASCL1 genes, respectively. The copy number level of these genes would seem inversely mutually correlated and highly dependent on histological grade, because the highest level of amplification at 3p25.2 was evidenced in the two HGNI samples, while the highest level of copy number gain at 12q23.2 was reported in the CIS. CONCLUSION: We provide new evidence on the role of PPARG in initiation and maintenance of bladder cancer. For the first time we also suggest a possible explanation for the elevated expression of PPARG in this type of tumor through a focal high level amplification at 3p25.2. Furthermore, a new gene, ASCL1, emerged as a potential candidate to assist PPARG in bladder carcinogenesis.

Introduction. Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells that can differentiate into different cell lineages and have emerged as a promising tool for cell-targeted therapies and tissue engineering. Their use in a therapeutic context requires large-scale in vitro expansion, increasing the probability of genetic and epigenetic instabilities. Some evidence shows that an organized program of replicative senescence is triggered in human BM-MSCs (hBM-MSCs) on prolonged in vitro expansion that includes alterations in phenotype, differentiation potential, telomere length, proliferation rates, global gene-expression patterns, and DNA methylation profiles. Methods. In this study, we monitored the chromosomal status, the biologic behavior, and the senescence state of hBM-MSCs derived from eight healthy donors at different passages during in vitro propagation. For a more complete picture, the telomere length was also monitored in five of eight donors, whereas the genomic profile was evaluated in three of eight donors by array-comparative genomic hybridization (array-CGH). Finally, an epigenomic profile was delineated and compared between early and late passages, by pooling DNA of hBM-MSCs from four donors. Results: Our data indicate that long-term culture severely affects the characteristics of hBM-MSCs. All the observed changes (that is, enlarged morphology, decreased number of cell divisions, random loss of genomic regions, telomere shortening) might be regulated by epigenetic modifications. Gene Ontology analysis revealed that specific biologic processes of hBM-MSCs are affected by variations in DNA methylation from early to late passages. Conclusions: Because we revealed a significant decrease in DNA methylation levels in hBM-MSCs during long-term culture, it is very important to unravel how these modifications can influence the biologic features of hBM-MSCs to keep track of this organized program and also to clarify the conflicting observations on h

A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0-4%, obtained using targeted arrays, to 9-12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes

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

Transitional cell carcinoma (TCC) comprises the majority of bladder cancers accounting for more than 90%. In general, TCC is grouped in high- or low-grade (HG or LG), noninvasive (NI) or invasive (IN) lesions. The biological differences between these groups probably reflect underlying genetic heterogeneity leading to specific pathways of tumor development and progression. Twenty-two TCCs (nine HGIN, three HGNI, one LGIN, nine LGNI) were tested by UroVysion® Bladder Cancer Kit, a fluorescence in situ hybridization assay designed to detect aneuploidy for chromosomes 3, 7, 17 and loss of the 9p21 locus; the test was performed in duplicate on formalin-fixed paraffin-embedded (FFPE) samples and on freshly isolated nuclei (FIN) in order to evaluate the performance of this targeted test in two different materials derived from the same tumor. Although FFPE specimens offer the certainty of histological diagnosis and allow retrospective studies of a large number of sample, conversely fresh tissues are the most reliable for molecular genetic analysis and also provide a comprehensive analysis of the biopsy. At least 100 cells for each preparation were scored and the signals divided according to loss, disomy and gain (number of signals/cell <2; = 2; ≥ 3). Statistical analysis was conducted by means of a Poisson model. The data generated fromFIN material and from FFPE counterpart were generally comparable. However, in HGNI, significant difference was evidenced, except for chromosome 3; conversely, in HGIN, significant difference emerged only for this signal. A second level of comparison was applied on ten TCC (six HGIN, one HGNI, three LGNI) between data derived from array comparative genomic hybridization and UroVysion analysis on different areas of matched FFPE tissues. Our results reflect the high intra-tumor heterogeneity and also showed some shared aberrations that could be interesting for the therapy with monoclonal antibodies