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1.  De Novo 13q13.3-21.31 deletion involving RB1 gene in a patient with hemangioendothelioma of the liver 
Interstitial deletions of the long arm of chromosome 13 (13q) are related with variable phenotypes, according to the size and the location of the deleted region. The main clinical features are moderate/severe mental and growth retardation, cranio-facial dysmorphism, variable congenital defects and increased susceptibility to tumors. Here we report a 3-year-old girl carrying a de novo 13q13.3-21.32 interstitial deletion. She showed developmental delay, growth retardation and mild dysmorphism including curly hair, high forehead, short nose, thin upper lip and long philtrum. An abnormal mass was surgically removed from her liver resulting in a hemangioendothelioma. Array analysis allowed us to define a deleted region of about 27.87 Mb, which includes the RB1 gene. This is the first report of a 13q deletion associated with infantile hemangioendothelioma of the liver.
PMCID: PMC3896849  PMID: 24433316
RB1; Tumor; Hemangioendothelioma; Liver; Chromosome 13q; Deletion; Syndrome
3.  Prenatal diagnosis of genomic disorders and chromosome abnormalities using array-based comparative genomic hybridization 
Journal of Prenatal Medicine  2007;1(1):16-22.
Cytogenetic analysis is a crucial tool of prenatal diagnosis. The ability to rapidly detect aneuploidy and identify small structural abnormalities of foetal chromosomes has been greatly improved by the use of molecular cytogenetic technologies. Microarray-based Comparative Genomic Hybridization (aCGH) has been recently employed in postnatal diagnosis of cryptic chromosomal aberrations, but use in prenatal diagnosis is still limited.
We set-up a diagnostic protocol which uses aCGH technology on genomic DNA isolated from uncultured chorionic villus sampled at 11–12 week’s gestation. We used a commercially targeted microarray (MDTelArray, Technogenetics Srl – Bouty Group, Sesto S. Giovanni, Milan, Italy) constituted by 167 genomic clones corresponding to 34 critical regions frequently involved in microdeletions and microduplications and 126 subtelomeric clones. Array validation has been carried-out via retrospective analysis of DNA isolated from a series of cytogenetically normal chorionic villus samples (CVS) and of DNA isolated from cytogenetically abnormal cultured amniocytes, CVS or peripheral blood. A pilot prospective study was undertaken analyzing 25 CVS obtained from foetuses at risk for chromosomal aberrations. aCGH results both for retrospective and prospective studies were in agreement with data obtained using “classical” cytogenetic analysis, and/or FISH analysis or DNA testing. Although these preliminary data support the usefulness of aCGH in prenatal diagnosis, further prospective studies are required to verify the feasibility of introducing this technique as part of the diagnostic armamentarium for identify affected foetuses.
PMCID: PMC3309345  PMID: 22470819
prenatal diagnosis; array-based comparative genomic hybridization (aCGH); aneuploidy; cryptic chromosomal aberrations; chorionic villus samples (CVS)
4.  Molecular analysis using DHPLC of cystic fibrosis: increase of the mutation detection rate among the affected population in Central Italy 
Cystic fibrosis (CF) is a multisystem disorder characterised by mutations of the CFTR gene, which encodes for an important component in the coordination of electrolyte movement across of epithelial cell membranes. Symptoms are pulmonary disease, pancreatic exocrine insufficiency, male infertility and elevated sweat concentrations. The CFTR gene has numerous mutations (>1000) and functionally important polymorphisms (>200). Early identification is important to provide appropriate therapeutic interventions, prognostic and genetic counselling and to ensure access to specialised medical services. However, molecular diagnosis by direct mutation screening has proved difficult in certain ethnic groups due to allelic heterogeneity and variable frequency of causative mutations.
We applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons and characterise sequence variations in a subgroup of CF Italian patients from the Lazio region (Central Italy) characterised by an extensive allelic heterogeneity.
We have identified a total of 36 different mutations representing 88% of the CF chromosomes. Among these are two novel CFTR mutations, including one missense (H199R) and one microdeletion (4167delCTAAGCC).
Using this approach, we were able to increase our standard power rate of mutation detection of about 11% (77% vs. 88%).
PMCID: PMC419352  PMID: 15084222
Cystic fibrosis; CFTR mutation screening; DHPLC
5.  In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique 
SFHR (small fragment homologous replacement)-mediated targeting is a process that has been used to correct specific mutations in mammalian cells. This process involves both chemical and cellular factors that are not yet defined. To evaluate potential of this technique for gene therapy it is necessary to characterize gene transfer efficacy in terms of the transfection vehicle, the genetic target, and the cellular processing of the DNA and DNA-vehicle complex.
In this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and ΔF508 sequences, were transfected into immortalized CF and normal airway epithelial cells, respectively. Homologous replacement was evaluated using PCR and sequence-based analyses of cellular DNA and RNA. Individual stages of cationic lipid-facilitated SFHR in cultured cell lines were also examined using transmission electron microscopy (TEM).
We demonstrated that the lipid/DNA (+/-) ratio influences the mode of entry into the cell and therefore affects the efficacy of SFHR-mediated gene targeting. Lipid/DNA complexes with more negative ratios entered the cell via a plasma membrane fusion pathway. Transfer of the DNA that relies on an endocytic pathway appeared more effective at mediating SFHR. In addition, it was also clear that there is a correlation between the specific cell line transfected and the optimal lipid/DNA ratio.
These studies provide new insights into factors that underlie SFHR-mediated gene targeting efficacy and into the parameters that can be modulated for its optimization.
PMCID: PMC130050  PMID: 12243649
gene therapy; cystic fibrosis transmembrane conductance regulator (CFTR); gene targeting; transmission electron microscopy (TEM); transfection

Results 1-5 (5)