Rosamaria Silipigni

Beckwith–Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder that can be prenatally suspected or diagnosed based on established clinical guidelines. Molecular confirmation is commonly performed on amniocytes. The possibility to use fresh (CVF) and cultured (CVC) chorionic villi has never been investigated. To verify whether CVF and CVC are reliable sources of DNA to study fetal methylation, we used pyrosequencing to test the methylation level of a number of differentially methylated regions (DMRs) at several imprinted loci (ICR1, ICR2, H19, PWS/AS-ICR, GNASXL, GNAS1A, ZAC/PLAGL1, and MEST) and at non-imprinted MGMT and RASSF1A promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, GNASXL, PWS/AS-ICR, and MEST. Conversely, the methylation levels at H19 promoter, GNAS1A and ZAC/PLAGL1 were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level of CVF/CVC of 2 BWS-suspected fetuses (P1 and P2). P1 showed ICR2 hypomethylation, P2 showed normal methylation at both ICR1 and ICR2. Our findings, although limited to one case of BWS fetus with an imprinting defect, can suggest that ICR1 and ICR2, but not H19, could be reliable targets for prenatal BWS diagnosis by methylation test in CVF and CVC. In addition, PWS/AS-ICR, GNASXL, and MEST, but not GNAS1A and ZAC/PLAGL1, are steadily hemimethylated in CV from healthy pregnancies, independently from culture. Thus, prenatal investigation of genomic imprinting in CV needs to be validated in a locus-specific manner.

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

MGMT-methylated alleles are distributed heterogeneously within glioma samples irrespective of IDH Status and chromosome 10q Deletion

Several molecular markers drive diagnostic classification, prognostic stratification, and/or prediction of response to therapy in patients with gliomas. Among them, IDH gene mutations are valuable markers for defining subtypes and are strongly associated with epigenetic silencing of the methylguanine DNA methyltransferase (MGMT) gene. However, little is known about the percentage of MGMT-methylated alleles in IDH-mutated cells or the potential association between MGMT methylation and deletion of chromosome 10q, which encompasses the MGMT locus. Here, we quantitatively assessed MGMT methylation and IDH1 mutation in 208 primary glioma samples to explore possible differences associated with the IDH genotype. We also explored a potential association between MGMT methylation and loss of chromosome 10q. We observed that MGMT methylation was heterogeneously distributed within glioma samples irrespective of IDH status suggesting an incomplete overlap between IDH1-mutated and MGMT-methylated alleles and indicating a partial association between these 2 events. Moreover, loss of one MGMT allele did not affect the methylation level of the remaining allele. MGMT was methylated in about half of gliomas harboring a 10q deletion; in those cases, loss of heterozygosity might be considered a second hit leading to complete inactivation of MGMT and further contributing to tumor progression.

Delineating the Mosaic Trisomy 15 Phenotype Using a Serendipitous Mechanism as a Clue

Parental balanced translocation is one of the traditional indications for invasive prenatal diagnosis. Usually, the diagnostic process is straightforward. Sometimes, however, results are not entirely clear and may reveal unexpected biological processes. We performed chorionic villi sampling for a paternal 8;15 reciprocal translocation in the sixth pregnancy of a Caucasian woman. Cytogenetic analysis of chorionic villi, after both short- and long-term cultures, revealed the presence of the same rearrangement found in the father as well as a trisomy 15. Surprisingly, the trisomy, which was initially expected to derive from aberrant segregation during paternal meiosis, resulted instead from maternal nondisjunction. Although a sonogram of the fetus appeared to be normal, follow-up amniocentesis demonstrated a low-level mosaic trisomy 15 in cells extracted from the amniotic fluid, while 10% of cells from fetal tissues sampled after termination of the pregnancy were also found to be trisomic. Fetal autopsy showed dysmorphic features, confirming the diagnosis of mosaic trisomy 15 and enabled deeper insight into the prenatal phenotype of this rare condition.

Molecular cytogenetic characterization of a 2q35-q37 duplication and a 4q35.1-q35.2 deletion in two cousins: A genotype–phenotype analysis

The 2q3 duplication and 4q3 deletion are two distinct conditions with variable phenotypes including developmental delay, intellectual disability, Pierre Robin sequence (PRS), and cardiovascular, craniofacial, digital and skeletal anomalies. We describe two cousins, a 37‐year‐old man (Patient 1) and a 17‐year‐old girl (Patient 2), with a derivative chromosome leading to a 4q35 deletion–2q35q37 duplication. Conventional karyotype showed in both patients the same rearrangement derived from unbalanced segregation of a parental reciprocal translocation involving the long arms of chromosome 2 and 4. Patient 1s father and Patient 2s mother were identified as the carriers of a balanced translocation t(2;4)(q35;q35). Array‐CGH analysis, performed to characterize the rearrangement, documented in both patients the presence of a 26 Mb duplication of the 2q35‐q37.3 region of chromosome 2 and a 6.3 Mb deletion of the 4q35.1‐q35.2 region of chromosome 4. Both patients showed intellectual disability, minor facial, and digital anomalies, hearing, ocular, and genitourinary abnormalities. The comparison of their features with those of published cases of 2q3 duplication and 4q3 deletion allowed us to further delineate the genotype–phenotype correlation as well as the combined effect of partial 2q duplication and 4q deletion syndromes in adulthood.

Mitochondrial Dysregulation and Impaired Autophagy in iPSC-Derived Dopaminergic Neurons of Multiple System Atrophy

Summary Multiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets.

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A) from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene

Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table.

Derivation of human induced pluripotent stem cell line EURACi004-A from skin fibroblasts of a patient with Arrhythmogenic Cardiomyopathy carrying the heterozygous PKP2 mutation c.2569_3018del50

Arrhythmogenic Cardiomyopathy (ACM) is an inherited cardiac disease characterized by arrhythmias and fibro-fatty replacement in the ventricular myocardium. Causative mutations are mainly reported in desmosomal genes, especially in plakophilin2 (PKP2). Here, using a virus-free reprogramming approach, we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of one ACM patient carrying the frameshift heterozygous PKP2 mutation c.2569_3018del50. The iPSC line (EURACi004-A) showed the typical morphology of pluripotent cells, possessed normal karyotype and exhibited pluripotency markers and trilineage differentiation potential, including cardiomyogenic capability. Thus, this line can represent a human in vitro model to study the molecular basis of ACM.

Expression of C19MC miRNAs in HCC associates with stem-cell features and the cancer-testis genes signature

BACKGROUND Intratumor heterogeneity of hepatocellular carcinoma (HCC) and, among HCC cell subsets, the cancer stem cell population (hCSC), is responsible for therapeutic resistance and disease relapse. AIMS To characterize hCSC-enriched HCCs at the molecular level. METHODS Side population (SP) was used to identify the hCSCs in multiple tumor sampling from different patients and primary HCCs cultures. FACS was used to immunoprofile cultures. miRNAs were profiled in samples and correlated to SP. The Cancer Genome Atlas (TCGA) HCC dataset was analyzed to search for signatures associated with C19MC miRNAs expression. Results were confirmed by immunohistochemistry. RESULTS The miRNA cluster on chromosome 19 (C19MC) was enriched in SP and in HCCs with a high SP fraction. At the molecular level, an elevated C19MC was correlated with expression of precursor transcripts. In TCGA-HCC series, high C19MC expression identified a subset of patients with poorer prognosis, advanced disease and overexpression of the cancer-testis (CT) antigens. These data were confirmed in an independent cohort of HCCs and at the protein level. CONCLUSION C19MC miRNAs and CT antigens overexpression represents a novel oncogenic pathway in a subset of hCSC-enriched HCCs with dismal prognosis. CT antigens are promising immunotherapy targets. Therefore, these molecular signatures could identify HCCs who could benefit from immunotherapy.

Mass spectrometry-based assay for the molecular diagnosis of glioma: concomitant detection of chromosome 1p/19q codeletion, and IDH1 , IDH2 , and TERT mutation status

The World Health Organization recently revised the diagnosis of glioma, to integrate molecular parameters, including IDH mutations and codeletion (loss of heterozygosity; LOH) of chromosome arms 1p/19q, into the definitions of adult glioma histological subtypes. Mutations in the TERT promoter may also be useful for glioma diagnosis and prognosis. The integration of molecular markers into routine diagnosis requires their rapid and reliable assessment. We propose a MassARRAY (MS)-based test that can identify 1p/19q codeletion using quantitative SNP genotyping and, simultaneously, characterize hotspot mutations in the IDH1, IDH2, and TERT genes in tumor DNA. We determined the reliability of the MS approach testing 50 gliomas and comparing the MS results with those obtained by standard methods, such as short tandem repeat genotyping, array comparative genomic hybridization (array-CGH) and Fluorescence In Situ Hybridization (FISH) for 1p/19q codeletion and Sanger sequencing for hotspots mutations. The results indicate that MS is suitable for the accurate, rapid, and cost-effective evaluation of chromosome deletions combined with hotspot mutation detection. This MS approach could be similarly exploited in evaluation of LOH in other situations of clinical and/or research importance.