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Biomedical subjects

G Novelli

Publications and source records attributed to G Novelli.

At least 19 recordsLinked to original sources
PubMed2006

Identification of a novel mutation in the SRY gene in a 46, XY female patient.

BACKGROUND: The SRY gene encodes for a testis-specific transcription factor (TDF, testis determining factor) that plays a key role in sexual differentiation and development in males. Several SRY mutations have been described in patients with gonadal dysgenesis, accounting for 10-15% of the sex reversal cases. The reported mutations are both point mutations and deletions, mostly involving the high mobility group (HMG) box domain of SRY, which is a conserved region through the evolution, suggesting that SRY function strictly depends on the HMG box. CASE PRESENTATION: Here we describe the clinical, endocrinological and molecular data of a patient with complete 46, XY gonadal dysgenesis caused by SRY mutation located within the conserved HMG box. Using DNA direct sequencing of the SRY coding region, we identified a single nucleotide insertion at codon 89 with subsequent frameshift of the reading frame sequence, which results in a truncated protein as consequence of an introduction of a stop codon at the position 103. CONCLUSION: A novel SRY mutation has been described in a female with a gonadal dysgenesis associated with a 46, XY karyotype. The described case is of importance for genetic counseling.

Adolescent↗
PubMed2006

Gene expression profile study in CFTR mutated bronchial cell lines.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis conductance transmembrane regulator (CFTR). Symptoms are pancreatic insufficiency, chronic obstructive lung disease, liver disease, chronic sinusitis and infertility in male patients. The phenotypic variability may be explained only in part by the more than 1200 CFTR mutations, which are grouped into six different classes, according to their effect on the protein ranging from a severe (no synthesis or blocked processing) to mild mutation (altered conductance or reduced synthesis). However, it is now accepted that other genes (CF modifiers) influence the phenotypic spectrum of the disease. In order to identify CF modifier genes, we built a low-density home-made oligoarray containing 144 genes selected according to biochemical criteria and evaluated their expression in two CF bronchial epithelial cell lines (CuFi1 F508del/F508del; CuFi3 F508del/R553X). If we consider both cell lines, 38 genes (26.3%) show an altered expression pattern with a threshold > +/-1.5. Of these 38 genes, 12 are altered in CuFi1, and 26 in CuFi3. Some of these genes share the same expression pattern in both cell lines, while others have a different behaviour. These results were validated by a QRT-PCR assay (R2 CuFi1 = 0.81 and R2 CuFi3 = 0.91). These data could suggest that the presence of a class I allele (R553X) determines a more profound alteration of gene expression pattern than the presence of a class II allele (F508del). The identification of the genes altered by a specific CF mutation could lead to the development of a pharmacological approach specific for different CFTR genotypes.

Alleles↗
PubMed2006

Intractable pruritus in patients with hepatitis C virus.

Intractable pruritus is one of the most common symptoms of chronic liver disease, especially experienced by patients with prolonged cholestasis. It can become the most distressing symptom in patients affected by chronic liver disease, causing a reduction in quality of life, interfering with daily activities, and leading to sleep deprivation or contributing to psychological disturbances up to suicide ideation. Therefore, pruritus that does not respond to medical therapy is an indication for liver transplantation. We treated nine patients with hepatitis C virus affected by intractable pruritus with the molecular adsorbent recirculating system. In each patient, liver function, renal function, and hemodynamic variables were evaluated before and after the treatment. Before undergoing the treatment each patient underwent abdominal ultrasound or computed tomography scan to exclude organic causes for pruritus. We observed a decrease in total bilirubin, creatinine, and bile acids together with a significant improvement in Visual Analog Scale for staging of pruritus in all the patients. Due to the small number of patients the results were not significant.

Adult↗
PubMed2006

Italian guidelines for molecular analysis in myotonic dystrophies.

Myotonic dystrophies, the most common form of adult muscular dystrophy, comprise at least two forms, clinically and genetically heterogeneous. Myotonic dystrophy type 1 and type 2 are both caused by unstable repetitions in untranslated gene regions: a [CTG]n expansion in the 3' region of the DMPK gene on chromosome 19q13 (DM1) and [CCTG]n tetranucleotide repeat located in the first intron of the ZNF9 gene on chromosome 3q21 (DM2). DM clinical features are caused by a gain of functions RNA mechanism in which the CUG and CCUG repeats alter nuclear functions, including alternative splicing of shared genes. Southern blot and/or polymerase chain reaction PCR-based approaches allow the detection of DM mutations in almost 100% of cases, however, the expansion size and the elevated grade of somatic instability make molecular testing for DM a diagnostic challenge. The increased use of DNA testing for DM generates many questions regarding the indications and interpretations of the test which require standardized methods, routinely available in molecular genetic laboratories. Here, we propose Guidelines for the molecular diagnosis of DM1 and DM2 approved by the Italian Ministry of Health in 2005 (Piano Nazionale Linee Guida, PNLG). Best practice for DM molecular analysis in diagnostic application, presymptomatic and prenatal testing, using direct and indirect approaches are described, with particular attention focused on ethical, legal and social issues. Overviews of materials used in the molecular diagnosis, as well as internet resources, are also included.

Genetic Testing↗
PubMed2006

[Liver transplantation in swine: a model for tolerance induction].

AIM: The liver as a solid graft has a known immunological privilege. Its tolerogenic property has been demonstrated in rodents. In humans the onset of chronic rejection and the severity of such complication is less frequent after liver transplantation compared to other organs. The underlying events whose effect is graft acceptance instead of rejection should be further investigated. Their control could open new ways to decrease the need for long-term immunosuppression after transplantation of other organs. Aim of this study is to evaluate a model of liver transplantation in swine as a preliminary step for immunological studies. METHODS: Ten outbred Landrace/Large White mismatched swine underwent to liver transplantation with a simple passive portocaval jugular bypass. The onset of rejection was monitored daily by liver function test. After death or sacrifice the liver parenchyma was studied to evaluate tissue damage and inflammatory infiltrate. RESULTS: The postoperative liver function showed a critical period for organ rejection about postoperative day 5. The animals that survived longer were sacrificed with a normal biochemical hepatic function. However, histology consistently showed a pattern of mild rejection in a still preserved architecture. CONCLUSIONS: The evidence of a prolonged liver function in a rejecting model of liver transplantation makes this model suitable for studies of tolerance induction.

Animals↗
PubMed2005

Transmission ratio distortion in the spinal muscular atrophy locus: data from 314 prenatal tests.

BACKGROUND: Spinal muscular atrophy (SMA) is a recessive neurodegenerative disorder characterized by the loss of alpha-motor neurons in the spinal cord and subsequent death of motor neuron cells. SMA occurs with a frequency of 1 in 6,000 live births, with a carrier frequency of 1 in 40, and is a leading genetic cause of infant mortality. SMA is caused by loss or mutation of the telomeric survival motor neuron gene (SMN1), which is deleted in almost 94% of SMA patients OBJECTIVE: To analyze the transmission ratio at the SMA locus, examining the segregation of the SMN1-deleted alleles in 314 fetuses from carrier parents who requested prenatal testing for the disease. METHODS: Prenatal diagnosis of SMA in families at 25% risk of the disease has been performed on chorionic villous sampling specimens, through direct detection of the SMN1 gene mutation and linkage analysis using microsatellite markers from the 5q13 region. Analysis of the genotypic/allelic frequencies of the SMN1 gene was performed using the chi2 test, assuming a recessive mendelian inheritance. RESULTS: Of 314 fetuses analyzed, 95 were homozygous for the wild-type allele (30.3%), 154 were carriers (49.0%), and the remaining 65 were homozygous for the mutated allele (20.7%). Statistical analysis demonstrated that proportion of fetuses predicted with SMA is lower than 25% expected for a recessive disorder, resulting in a transmission rate of the SMN1-deleted allele deviant from the 50% expected in a random the segregation of a mendelian tract (p = 0.016) CONCLUSIONS: This is the first study to evaluate the genotypic frequencies at the spinal muscular atrophy (SMA) locus based on data derived from prenatal analysis, which are not subject to ascertainment bias. The analysis showed a transmission ratio distortion at the SMA locus in favor of the SMN1 wild-type alleles.

Chorionic Villi Sampling↗
PubMed2005

Rescue of heterochromatin organization in Hutchinson-Gilford progeria by drug treatment.

Hutchinson-Gilford progeria (HGPS) is a premature aging syndrome associated with LMNA mutations. Progeria cells bearing the G608G LMNA mutation are characterized by accumulation of a mutated lamin A precursor (progerin), nuclear dysmorphism and chromatin disorganization. In cultured HGPS fibroblasts, we found worsening of the cellular phenotype with patient age, mainly consisting of increased nuclear-shape abnormalities, progerin accumulation and heterochromatin loss. Moreover, transcript distribution was altered in HGPS nuclei, as determined by different techniques. In the attempt to improve the cellular phenotype, we applied treatment with drugs either affecting protein farnesylation or chromatin arrangement. Our results show that the combined treatment with mevinolin and the histone deacetylase inhibitor trichostatin A dramatically lowers progerin levels, leading to rescue of heterochromatin organization and reorganization of transcripts in HGPS fibroblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectified by drug treatment.

Cell Nucleus↗
PubMed2004

Characterization of a single nucleotide polymorphism in the ZNF9 gene and analysis of association with myotonic dystrophy type II (DM2) in the Italian population.

Myotonic dystrophy type 2 (DM2) is a dominant inherited disorder clinically similar to myotonic dystrophy type 1 (DM1) with a peculiar pattern of multisystemic phenotypic features. The mutation responsible for DM1 is a CTG repeat in the 3' UTR of the dystrophia myotonica protein kinase gene (DMPK) on chromosome 19q13.3, while DM2 is caused by an unstable CCTG expansion in intron 1 of the zinc finger protein 9 gene (ZNF9) on chromosome 3q21.3. Southern blotting analysis is the conventional test used to determinate the size of the repeats in the molecular diagnosis of DM2. However, the large number of CCTG repeats and their somatic instability complicates this diagnostic protocol. In order to improve the DM2 test, we have recently characterised a single nucleotide polymorphism located in the first intron of the ZNF9 gene. This SNP consists in a C to A nucleotide change, which creates or disrupts and ApaI enzyme restriction site, easily detectable by PCR amplification followed by restriction analysis. We genotyped this SNP in 30 unrelated DM2 patients and 70 unrelated Italians healthy individuals. Our results show that this polymorphism is in linkage disequilibrium with the DM2 mutation.

Case-Control Studies↗
PubMed2004

Survival in kidney transplantation from living donors: a single-center experience.

BACKGROUND: Transplantation from living donors, in Italy, is still not accepted, in particular those from unrelated donors. The aim of this paper was to present the experience of one transplant center. MATERIALS AND METHODS: Since 1982, 608 transplants were performed from living donors using cyclosporine as the main component of immunosuppressive therapy. Among those, 402 transplants were from related living donors (338 one haplotype pairs and 25 zero haplotypes pairs) and 206 from unrelated living donors (171 spouses and 35 emotionally related subjects). RESULTS: Graft survival at 1, 5, and 10 years showed no statistically meaningful difference between the two groups. A group of 19 transplants performed in predialytic phase patients was compared with a contemporaneous group of 167 transplants performed in patients who were already receiving dialysis. These two groups did not show any statistically meaningful difference in graft survival at 1, 5, or 10 years. DISCUSSION AND CONCLUSIONS: We think that transplants from living donors, whether related or unrelated, must always be proposed as a therapeutic option for end-stage renal disease patients, since they show an higher graft survival than that from cadaveric donors, independent of the compatibility between donor and recipient and independent of the degree of relationship of the pair. Transplantation from living donors definitely is a complementary, not substitutive, program to that from cadaveric donors, which should always be encouraged with awareness campaigns among the population and targeted programs for healthy personnel.

Actuarial Analysis↗
PubMed2004

Characterization of the loricrin (LOR) gene as a positional candidate for the PSORS4 psoriasis susceptibility locus.

Psoriasis is a chronic inflammatory disease of the skin with both genetic and environmental risk factors. Non-parametric linkage analyses have mapped many susceptibility loci on different chromosomes. We mapped one of these loci, PSORS4, on human chromosome 1q21. Using the linkage disequilibrium approach, we refined the critical region to a specific genomic interval of about 100 Kb which contains only the loricrin (LOR) gene. Here we report a genetic and functional study of this gene to verify its involvement in psoriasis pathogenesis. We document low expression of LOR in psoriatic skin of patients selected from families in which the disease was segregating with the PSORS4 locus. Re-sequencing of the entire gene in a subset of patients revealed the existence of novel polymorphisms able to influence the protein structure, as shown by molecular modelling studies. However, no evidence for genetic association was detected in a large cohort of Italian nuclear families. This rules out the LOR gene as a candidate for the PSORS4 locus.

Chromosomes, Human, Pair 1↗
PubMed2004

Sequence-specific modification of mouse genomic DNA mediated by gene targeting techniques.

The major impact of the human genome sequence is the understanding of disease etiology with deduced therapy. The completion of this project has shifted the interest from the sequencing and identification of genes to the exploration of gene function, signalling the beginning of the post-genomic era. Contrasting with the spectacular progress in the identification of many morbid genes, today therapeutic progress is still lagging behind. The goal of all gene therapy protocols is to repair the precise genetic defect without additional modification of the genome. The main strategy has traditionally been focused on the introduction of an expression system designed to express a specific protein, defective in the transfected cell. But the numerous deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders. Among these one is represented by gene repair based on homologous recombination (HR). Simply stated, the process involves targeting the mutation in situ for gene correction and for restoration of a normal gene function. Homologous recombination is an efficient means for genomic manipulation of prokaryotes, yeast and some lower eukaryotes. By contrast, in higher eukaryotes it is less efficient than in the prokaryotic system, with non-homologous recombination being 10-50 fold higher. However, recent advances in gene targeting and novel strategies have led to the suggestion that gene correction based on HR might be used as clinical therapy for genetic disease. This site-specific gene repair approach could represent an alternative gene therapy strategy in respect to those involving the use of retroviral or lentiviral vectors to introduce therapeutic genes and linked regulatory sequences into random sites within the target cell genome. In fact, gene therapy approaches involving addition of a gene by viral or nonviral vectors often give a short duration of gene expression and are difficult to target to specific populations of cells. The purpose of this paper is to review oligonucleotide-based gene targeting technologies and their applications on modifying the mouse genome.

Animals↗
PubMed2003

Biased T-cell receptor repertoires in patients with chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome).

Chromosome 22q11.2 deletion (del22q11.2) syndrome (DiGeorge syndrome/velocardiofacial syndrome) is a common syndrome typically consisting of congenital heart disease, hypoparathyroidism, developmental delay and immunodeficiency. Although a broad range of immunologic defects have been described in these patients, limited information is currently available on the diversity of the T-cell receptor (TCR) variable beta (BV) chain repertoire. The TCRBV repertoires of nine patients with del22q11.2 syndrome were determined by flow cytometry, fragment size analysis of the third complementarity determining region (CDR3 spectratyping) and sequencing of V(D)J regions. The rate of thymic output and the phenotype and function of peripheral T cells were also studied. Expanded TCRBV families were detected by flow cytometry in both CD4+ and CD8+ T cells. A decreased diversity of TCR repertoires was also demonstrated by CDR3 spectratyping, showing altered CDR3 profiles in the majority of TCRBV families investigated. The oligoclonal nature of abnormal peaks detected by CDR3 spectratyping was confirmed by the sequence analysis of the V(D)J regions. Thymic output, evaluated by measuring TCR rearrangement excision circles (TRECs), was significantly decreased in comparison with age-matched controls. Finally, a significant up-regulation in the percentage, but not in the absolute count, of activated CD4+ T cells (CD95+, CCR5+, HLA-DR+), IFN-gamma - and IL-2-expressing T cells was detected. These findings suggest that the diversity of CD4 and CD8 TCRBV repertoires is decreased in patients with del22q11.2 syndrome, possibly as a result of either impaired thymic function and/or increased T-cell activation.

Adolescent↗
PubMed2002

Genome medicine: gene therapy for the millennium, 30 September-3 October 2001, Rome, Italy.

The recent surge of DNA sequence information resulting from the efforts of agencies interested in deciphering the human genetic code has facilitated technological developments that have been critical in the identification of genes associated with numerous disease pathologies. In addition, these efforts have opened the door to the opportunity to develop novel genetic therapies to treat a broad range of inherited disorders. Through a joint effort by the University of Vermont, the University of Rome, Tor Vergata, University of Rome, La Sapienza, and the CSS Mendel Institute, Rome, an international meeting, 'Genome Medicine: Gene Therapy for the Millennium' was organized. This meeting provided a forum for the discussion of scientific and clinical advances stimulated by the explosion of sequence information generated by the Human Genome Project and the implications these advances have for gene therapy. The meeting had six sessions that focused on the functional evaluation of specific genes via biochemical analysis and through animal models, the development of novel therapeutic strategies involving gene targeting, artificial chromsomes, DNA delivery systems and non-embryonic stem cells, and on the ethical and social implications of these advances.

Forecasting↗
PubMed2002

Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting.

Cystic fibrosis is the most common inherited disease in the Caucasian population. About 70% of all CF chromosomes carry the DeltaF508 mutation, a 3-bp deletion that results in the loss of a phenylalanine at amino acid 508 in the CF transmembrane conductance regulator (CFTR) protein. Direct modification of the DeltaF508 locus of endogenous CFTR was achieved by small fragment homologous replacement (SFHR). Transformed human airway epithelial cells (CFBE41o(-)), homozygous for DeltaF508 mutation, were transfected with small fragments (491-bp) of wild-type (WT) CFTR DNA comprising exon 10 and the flanking introns. The DNA fragments were in a liposome-DNA complex at a charge ratio of 6:1 (+:-), respectively). The population of transfected cells was subcloned by limiting dilution at approximately 1 cell/well in 96-well plates. Individual colonies were isolated and analyzed. The DNA from several colonies was characterized by radiolabeled, nonallele-specific and radiolabeled, allele-specific PCR amplification, as well as by genomic DNA fingerprinting. The CFTR-WT allele was detected in five of these colonies by allele-specific PCR amplification thus indicating that the cell lines carried both WT and DeltaF alleles. DNA fingerprint analysis confirmed that the colonies were isogenic and derived from the parental CFBE41o(-) cell line. Although, the WT allele was detected by allele-specific PCR, it was not detected initially when the same samples were analyzed by non allele-specific PCR. A sensitivity assay, mixing the genomic DNA of wild-type (16HBE14o(-)) and mutant (CFBE41o(-)) cell lines, indicated that the allele-specific PCR was at least 25-fold more sensitive than non allele-specific PCR. These results suggest that the colony is not yet clonal, but still contains a population of parental, CFBE41o(-) cells that have not been modified. Based on the mixing analysis, the proportion of corrected cells appears to be between 1 and 10% of the total population. Nonallele-specific reverse transcriptase PCR (RT-PCR) analysis of the CFTR mRNA indicated that two of the colonies expressed both WT and DeltaF508 CFTR mRNA, while one colony appeared to express only the WT mRNA. The mRNA results were confirmed by sequence analysis of 3' end primer extension products from the mRNA of CFTR exon 10 showing that the mRNA containing exon 10. Furthermore, a survey of primer extension products indicated no random insertion of the fragment in an expressed gene. This study demonstrates SFHR-mediated modification of the DeltaF508 allele in DeltaF508 homozygote human airway epithelial cells over multiple generations. The resultant cells express WT-CFTR mRNA and can be subcloned further to isolate isogenic clonal populations of cells.

Cell Line↗