Informativity of intragenic microsatellites for carrier detection and prenatal diagnosis of cystic fibrosis in the Italian population
Clinical Genetics, Jun 28, 2008
Molecular diagnosis of cystic fibrosis (CF) in the Italian population, based on the detection of ... more Molecular diagnosis of cystic fibrosis (CF) in the Italian population, based on the detection of the deltaF508 mutation (51.2% of CF chromosomes), provides full informativity for prenatal diagnosis (PDN) in about 28% of families at risk. Identification of the predominant non-deltaF508 mutations allows the characterization of about 70% of CF chromosomes, making approximately 48% of couples fully informative. In families where at least one chromosome remains uncharacterized, allele segregation is still determined using RFLPs closely linked to the CF gene. The recent identification of three polymorphic clusters of dinucleotide repeats (IVS8/GT, IVS17b/TA and IVS17b/CA) led us to evaluate whether their analysis might improve feasibility studies for prenatal diagnosis or heterozygote identification. One hundred nuclear families with a CF child, reflecting the general Italian deltaF508 mutation distribution, were genotyped for the three microsatellites. In this study microsatellite analysis using IVS8/GT and IVS17b/TA allowed the identification of both parental CF chromosomes in 94% of couples; inclusion in the study of the less polymorphic repeat locus, IVS17b/CA, slightly improved this percentage (97%). Hence, a strategy involving primarily the detection of the deltaF508 mutation and secondarily microsatellite analysis makes possible PDN of CF in virtually all Italian CF families.
A novel mutation (M1V) in the translation initiation codon of the cystic fibrosis transmembrane conductance regulator gene, in three CF chromosomes of Italian origin
Human Molecular Genetics, 1994
Cystic Fibrosis (CF) is the most common severe autosomal recessive disorder of the Caucasian popu... more Cystic Fibrosis (CF) is the most common severe autosomal recessive disorder of the Caucasian population, with an estimated birth incidence of 1 in 2,500. Since the identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene ...
Background: The recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP),... more Background: The recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required. Results: In this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-ras v12 transgenic mice or from human adeno-or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II). Conclusions: Epigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples.
Lung cancer is the leading cause of tumor-related death worldwide and more efforts are needed to ... more Lung cancer is the leading cause of tumor-related death worldwide and more efforts are needed to elucidate lung carcinogenesis. Here we investigated the expression of 641 miRNAs in lung tumorigenesis in a K-Ras (+/LSLG12Vgeo);RERTn(ert/ert) mouse model and 113 human tumors. The conserved miRNA cluster on chromosome 12qF1 was significantly and progressively upregulated during murine lung carcinogenesis. In particular, miR-494-3p expression was correlated with lung cancer progression in mice and with worse survival in lung cancer patients. Mechanistically, ectopic expression of miR-494-3p in A549 lung cancer cells boosted the tumor-initiating population, enhanced cancer cell motility, and increased the expression of stem cellrelated genes. Importantly, miR-494-3p improved the ability of A549 cells to grow and metastasize in vivo, modulating NOTCH1 and PTEN/PI3K/AKT signaling. Overall, these data identify miR-494-3p as a key factor in lung cancer onset and progression and possible therapeutic target.
Novel cancer biomarkers like microRNA (miRNA) are promising tools to gain a better understanding ... more Novel cancer biomarkers like microRNA (miRNA) are promising tools to gain a better understanding of lung cancer pathology and yield important information to guide therapy. In recent years, new less invasive methods for the diagnosis and staging of NSCLC have become key tools in thoracic oncology and the worldwide spread of endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA). However, appropriate specimen handling is mandatory to achieve adequate results and reproducibility. The aim of this single centre prospective study was to evaluate the feasibility of a complete miRNA expression profile in fresh NSCLC cell lines obtained by EBUS-TBNA. Patients with proven NSCLC underwent EBUS-TBNA for the diagnosis of suspect lymph node metastasis, and cytological specimens were collected for epithelial cell culture and miRNA expression analysis. To validate the miRNA expression profile, we compared the results from EBUS-TBNA NSCLC specimens with those obtained from formalin-fi...
Background: CT screening of lung cancer allows the detection of very small tumors. The objective ... more Background: CT screening of lung cancer allows the detection of very small tumors. The objective of our study was to verify whether a selected population of initial asymptomatic lung cancers show genetic abnormalities able to mark the early events of lung carcinogenesis. Methods: A pilot study was performed for the standardization of the technique, enrolling heavy smokers with asymptomatic early stage lung cancers non-screening detected. Since 2004, a low dose CT screening study enrolled 5201 asymptomatic heavy smokers volunteers. Tissue samples were collected from patients of these two groups who underwent surgical resection in our institute and short-term cultures were started from tumoral tissues. Cultures were suitably treated for the analysis of metaphase spreads. Samples were classified according to the normal (N) or aneuploid (A) karyotype. N samples were further analyzed by the Affymetrix HuSNP 50K technology. Results: 101 tumor samples were analyzed (33 collected during a pilot project and 78 as part of the screening program). Metaphase spreads were obtained in 70% (71/101) of the selected samples: 77% (55/71) showed an aneuploid karyotype, while 20% (14/71) resulted as normal, 3% (2/71) were uncharacterized. The subgroup of tumors with p-stage IA showed aneuploidy in 70% (24/34) of cases with a mean size of 15 mm. A correlation between tumor size and chromosome number was shown, having small size tumors a higher probability to show a normal karyotype. 8/14 of the N samples were found suitable for SNPs analysis. Subtle genomic alterations were found in 4 of the analyzed patients, the remaining 4 lacking to date of an evidence for the presence of chromosomal aberrations throughout the genome. A common region of amplification was identified at 8p11 in 2 samples and is currently under investigation. Conclusions: i) The majority of screening detected tumors presented aneuploidy despite very initial stage. ii) A correlation between tumor size and ploidy status exists in early lung cancers. ii) Genomic scanning is able to detect subtle region of chromosomal alterations in early lung cancer patients. No significant financial relationships to disclose.
of intragenic microsatellites for carrier detection and prenatal diagnosis of cystic fibrosis in ... more of intragenic microsatellites for carrier detection and prenatal diagnosis of cystic fibrosis in the Italian population.
Background and Objectives. The ETV6 gene undergoes rearrangements with tyrosine kinases in hemato... more Background and Objectives. The ETV6 gene undergoes rearrangements with tyrosine kinases in hematologic malignancies and solid tumors. ETV6/ABL1 chimeric proteins have been detected both in lymphoid and myeloid disorders. Our objective was to study two new cases of ETV6/ABL1-positive acute myeloid leukemia (AML) and to focus on bone marrow morphology and on molecular cytogenetics of eosinophilic cells. Design and Methods. Fluorescence in situ hybridization (FISH) was performed in two AML cases with different translocations, i.e. t(8;12)(p21;p13) and t(9;12) (q34; p13). We used probes for the short arm of chromosome 12, for ABL1 and BCR, for centromeric regions, and for whole chromosome arms. Polymerase chain reaction (PCR) was carried out by applying primers selected for the ETV6 gene. Results. In both cases, bone marrow morphology was characterized by trilineage dysplasia and increased abnormal eosinophils. FISH showed the 5'ETV6 translocated to chromosome 8 in patient #1, and to chromosome 9 in patient #2. A 3' PCR identified chimeric products resulting from fusion between ETV6 exon 4 or exon 5, and ABL1 exon 2. Accordingly, an ETV6/ABL1 fusion signal was detected on der(8) in patient #1, and on der(9) in patient #2. Using interphase FISH abnormal bone marrow eosinophils were proved to belong to the neoplastic clone, carrying the ETV6 rearrangement. Interpretation and Conclusions. Our findings provide new information on the heterogeneity of conventional cytogenetics in ETV6/ABL1 positive leukemias, and indicate the putative target cell in this AML is an immature precursor capable of terminally differentiating towards eosinophils.
A role for bone-marrow-derived cells (BMDCs) in tissue repair and malignancy onset has been propo... more A role for bone-marrow-derived cells (BMDCs) in tissue repair and malignancy onset has been proposed, but their contribution is still debated. We tested the ability of BMDCs containing the inducible kras V12 oncogene to initiate lung adenocarcinoma. For our experimental strategy, we reconstituted lethally irradiated wild type mice with BMDCs carrying inducible kras V12 and subsequently induced oncogene expression by 4-OHT administration. Epithelial lung lesions, from adenoma to adenocarcinomas, appeared at successive time points. These results show that lung tumors were derived from donor BMDCs and indicate a direct involvement of bone marrow cells in the development of epithelial cancers.
A role for bone-marrow-derived cells (BMDCs) in tissue repair and malignancy onset has been propo... more A role for bone-marrow-derived cells (BMDCs) in tissue repair and malignancy onset has been proposed, but their contribution is still debated. We tested the ability of BMDCs containing the inducible kras V12 oncogene to initiate lung adenocarcinoma. For our experimental strategy, we reconstituted lethally irradiated wild type mice with BMDCs carrying inducible kras V12 and subsequently induced oncogene expression by 4-OHT administration. Epithelial lung lesions, from adenoma to adenocarcinomas, appeared at successive time points. These results show that lung tumors were derived from donor BMDCs and indicate a direct involvement of bone marrow cells in the development of epithelial cancers.
A homeobox gene, HLXB9, is the major locus for dominantly inherited sacral agenesis
Nature genetics, 1998
Partial absence of the sacrum is a rare congenital defect which also occurs as an autosomal domin... more Partial absence of the sacrum is a rare congenital defect which also occurs as an autosomal dominant trait; association with anterior meningocoele, presacral teratoma and anorectal abnormalities constitutes the Currarino triad (MIM 176450). Malformation at the caudal end of the developing notochord at approximately Carnegie stage 7 (16 post-ovulatory days), which results in aberrant secondary neurulation, can explain the observed pattern of anomalies. We previously reported linkage to 7q36 markers in two dominantly inherited sacral agenesis families. We now present data refining the initial subchromosomal localization in several additional hereditary sacral agenesis (HSA) families. We excluded several candidate genes before identifying patient-specific mutations in a homeobox gene, HLXB9, which was previously reported to map to 1q41-q42.1 and to be expressed in lymphoid and pancreatic tissues.
The ETV6 gene undergoes rearrangements with tyrosine kinases in hematologic malignancies and soli... more The ETV6 gene undergoes rearrangements with tyrosine kinases in hematologic malignancies and solid tumors. ETV6/ABL1 chimeric proteins have been detected both in lymphoid and myeloid disorders. Our objective was to study two new cases of ETV6/ABL1-positive acute myeloid leukemia (AML) and to focus on bone marrow morphology and on molecular cytogenetics of eosinophilic cells. Fluorescence in situ hybridization (FISH) was performed in two AML cases with different translocations, i.e. t(8;12)(p21;p13) and t(9;12) (q34; p13). We used probes for the short arm of chromosome 12, for ABL1 and BCR, for centromeric regions, and for whole chromosome arms. Polymerase chain reaction (PCR) was carried out by applying primers selected for the ETV6 gene. In both cases, bone marrow morphology was characterized by trilineage dysplasia and increased abnormal eosinophils. FISH showed the 5'ETV6 translocated to chromosome 8 in patient #1, and to chromosome 9 in patient #2. A 3' PCR identified ch...
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