Biomedical subjects

Giuseppe Novelli

Publications and source records attributed to Giuseppe Novelli.

At least 55 records · Page 3Linked to original sources

PubMed2004

A long PCR-based molecular protocol for detecting normal and expanded ZNF9 alleles in myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) lacks the expansion on chromosome 19q13 present in DM1 and is characterized by a mutation on 3q21. It has been shown that the DM2 mutation is a huge [CCTG]n repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. The longest normal allele observed has a approximately 30 CCTG repeat, whereas the range of expansion is extremely variable, starting from 75 up to 11,000 CCTGs. Direct analysis by Southern blot, after restriction enzyme digestion of genomic DNA, was the first method chosen for studying the DM2 mutation. However, the expansion size and the elevated grade of somatic instability have limited the sensitivity of the test to approximately 80% of known carriers. We developed a long PCR-formatted protocol, which involves a single genomic in vitro amplification, followed by agarose gel electrophoresis and oligospecific hybridization. We were able to detect normal alleles and expanded ZNF9 alleles, starting from low amounts of genomic DNA (>/= 1 ng) in virtually all the DM2 patients analyzed, obtaining a molecular detection rate of 100%. This method is quick, sensitive, and reproducible, and it reduces the cost of diagnostic laboratory processing for DM2 diagnosis.

Alleles↗

PubMed2003

Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.

An abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is found in approximately 10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals (fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% (21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% (10 of 107) of affected individuals. Conversely, 91% (97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% (6 of 27) of unaffected individuals (P<.00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats (odds ratio 34.0, 95% CI 11.1-103.7, P<.00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles.

Base Sequence↗

PubMed2003

Association of dopamine D4 receptor (DRD4) exon III repeat polymorphism with temperament in 3-year-old infants.

The long forms of the dopamine D4 receptor (DRD4) exon III repeat polymorphism (L-DRD4) have been linked in some studies to the adult personality trait of novelty seeking (NS), as well as to infant personality traits related to interest and activity. The current investigation extends the results of our previous longitudinal study on 1- to 5-month-old neonates assessed by the Early and Revised Infancy Temperament Questionnaire (EITQ/RITQ), in which we found a significant correlation between the DRD4 polymorphism and the adaptability trait at 1 month of age. In this study, we examined the relationship between children's behavior at 3 years of age, measured with the Toddler Temperament Scale (TTS), and DRD4 exon III repeat polymorphism. We found a significant association between the behavioral dimension of intensity of reaction and DRD4 genotypes. Current data failed to confirm the association with the adaptability trait. None of the extraversion and/or exploratory behavior measures was related to the L-DRD4 allele, as expected. In contrast, children with 4/7 genotypes showed worse response to new stimuli compared with 4/4 genotypes. This study corroborates only in part previous results on the link between the DRD4 gene and human temperament.

Child Behavior↗

PubMed2003

Association study between CAG trinucleotide repeats in the PCQAP gene (PC2 glutamine/Q-rich-associated protein) and schizophrenia.

Schizophrenia or schizoaffective disorders are quite common features in patients with DiGeorge/velocardiofacial syndrome (DGS/VCFS) as a result of hemizygosity of chromosome 22q11.2. We evaluated the PCQAP gene, which maps within the DGS/VCFS interval, as a potential candidate for schizophrenia susceptibility. PCQAP encodes for a subunit of the large multiprotein complex PC2, which exhibits a coactivator function in RNA polymerase II mediated transcription. Using a case-control study, we searched association between schizophrenia and the intragenic coding trinucleotide polymorphism. The distribution of the CAG repeat alleles was significantly different between patients and controls with the Mann-Whitney test (z = -2.5694, P = 0.0051; schizophrenics: n = 378, W = 161,002.5, Mean rank = 425.9325; controls: n = 444, W = 177,250.5, Mean rank = 399.2128). This result may indicate a possible involvement of the multiprotein complex PC2 in schizophrenia susceptibility.

Adult↗

PubMed2003

Analysis of intracellular distribution and apoptosis involvement of the Ufd1l gene product by over-expression studies.

UFD1L is the human homologue of the yeast ubiquitin fusion degradation 1 (Ufd1) gene and maps on chromosome 22q11.2 in the typically deleted region (TDR) for DiGeorge/velocardiofacial syndromes (DGS/VCFS). In yeast, Ufd1 protein is involved in a degradation pathway for ubiquitin fused products (UFD pathway). Several studies have demonstrated that Ufd1 is a component of the Cdc48-Ufd1-Npl4 multiprotein complex which is active in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for protein degradation or even more specific processing. The multiprotein complex Cdc48-Ufd-Npl4 is also active in mammalian cells. The biochemical role of UFD1L protein in human cells is unknown, even though the interaction between UFD1L and NPL4 proteins has been maintained. In order to clarify this issue, we examined the intracellular distribution of the protein in different mammalian cells and studied its involvement in the Fas and ceramide factors-mediated apoptotic pathways. We established that in mammalian cells, Ufd1l is localized around the nucleus and that it does not interfere with Fas-and ceramide-mediated apoptosis.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

The strange case of the "lumper" lamin A/C gene and human premature ageing.

Gary Rosenberg said of conchologists that they "are often characterized as lumpers or splitters. From one perspective, splitters recognize more species than really exist, and lumpers go around fixing the resulting damage." The recent explosion in the number of identified mutations within the LMNA gene, which encodes two protein products lamins A and C, has allowed the identification of an allelic series of disorders, all caused by mutations within this one gene. Hence, LMNA might be thought of as a "lumper" gene. These results have initiated analysis of the relationship between disease phenotype and gene, and have led to dramatic advances in our knowledge of the dynamic and complex organization of the nuclear architecture.

Aging, Premature↗

PubMed2003

Construction and purification of pSABR 01, a pUC19-derived vector optimized for cloning full-length cDNA.

A novel pUC19-derived vector, pSABR 01, was constructed by sub-cloning a fragment of the pSPORT1 polylinker into PUC19. The insertion of the polylinker generated two inactivating mutations in the LacZ open reading frame. These were then repaired by a PCR-based Site Directed Mutagenesis strategy. The pSABR 01 plasmid has four sites that are recognized by 'rare-cutter' restriction endonucleases that will optimize the cloning of full-length cDNA and five dual restriction sites that increase the versatility of subcloning the inserted cDNA. Protocols were also defined for purification of pSABR 01 from residual pSPORT1, following pSABR 01 construction, and from another contaminating plasmid.

Cloning, Molecular↗

PubMed2003

Role of genetics in prevention of coronary atherosclerosis.

Atherosclerosis is currently regarded as a complex disorder, triggered by gene-gene and gene-environment interactions. Despite the difficulties of multifactorial disease genetic analysis, significant progress has been achieved in the search for atherosclerosis susceptibility genes. Here, we review these advances with regard to genome scan results and candidate gene analysis.

Coronary Artery Disease↗

PubMed2003

Sequence-specific modification of genomic DNA by small DNA fragments.

Small DNA fragments have been used to modify endogenous genomic DNA in both human and mouse cells. This strategy for sequence-specific modification or genomic editing, known as small-fragment homologous replacement (SFHR), has yet to be characterized in terms of its underlying mechanisms. Genotypic and phenotypic analyses following SFHR have shown specific modification of disease-causing genetic loci associated with cystic fibrosis, beta-thalassemia, and Duchenne muscular dystrophy, suggesting that SFHR has potential as a therapeutic modality for the treatment of monogenic inherited disease.

Anemia, Sickle Cell↗

PubMed2002

Characterisation of mutations in 77 patients with X-linked myotubular myopathy, including a family with a very mild phenotype.

X-linked myotubular myopathy is characterised by neonatal hypotonia, muscle weakness and respiratory distress in affected males, leading often to early death, although prolonged survival is observed in milder forms, or as a result of prolongation of ventilation support. It is caused by mutations in the MTM1 gene, which encodes a phosphatase called myotubularin, which has been highly conserved during evolution, down to yeasts ( S. cerevisiae and S. pombe). To date, 251 mutations have been identified in unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene. We have found additional mutations in 77 patients, including 35 novel ones. We identified a missense mutation N180K in a 67-year-old grandfather (the oldest known patient with an MTM1 mutation), previously suspected to have autosomal centronuclear myopathy, and in his two grandsons also mildly affected. Mild and moderate phenotypes associated with novel missense mutations and with a translation initiation defect mutation are discussed, as well as severe phenotypes associated with particular novel mutations. With the present report, 192 different mutations in the MTM1 gene have been described in 328 families. The spectrum of mutations is now enlarged from the very severe classic neonatal phenotype to very mild phenotype allowing survival to the age of 67 years.

Adolescent↗

PubMed2002

In vitro correction of cystic fibrosis epithelial cell lines by small fragment homologous replacement (SFHR) technique.

BACKGROUND: 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. METHODS: In this study, small fragments of genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) DNA, that comprise the wild-type and DeltaF508 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). RESULTS: 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. CONCLUSIONS: 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.

Journal Article↗

PubMed2002

Association of the G289S single nucleotide polymorphism in the HSD17B3 gene with prostate cancer in Italian men.

BACKGROUND: Prostate cancer is a significant public health problem in this country. Substantial data support a plausible role for androgens in the etiology of this disease. The human HSD17B3 gene encodes the testicular (or type III) 17 beta-hydroxysteroid dehydrogenase enzyme, which catalyzes testosterone biosynthesis in men. METHODS: We have investigated the G289S (glycine at codon 289 replaced by serine) polymorphism at the HSD17B3 locus as a candidate single nucleotide polymorphism (SNP) for prostate cancer risk in constitutional DNA from 103 Italian prostate cancer patients and 109 Italian disease-free centenarians to assess the role of this SNP in susceptibility to prostate cancer. RESULTS: The G289S polymorphism confers a significant increase in risk for prostate cancer (odds ratio = 2.5; 95% confidence interval, 1.03-6.07) in our study population. CONCLUSION: Our data are consistent with a plausible role of the G289S SNP in prostate cancer susceptibility. Therefore, the HSD17B3 gene may be a plausible candidate gene for prostate cancer risk.

17-Hydroxysteroid Dehydrogenases↗

PubMed2002

Assignment of a locus for autosomal dominant idiopathic scoliosis (IS) to human chromosome 17p11.

Idiopathic scoliosis (IS) is a spine deformity of unknown etiology. Family studies have suggested that IS may be inherited as a mendelian autosomal dominant trait. We have performed linkage analysis on a three-generation IS Italian family. A positive LOD score value of 3.20 at theta=0.00 was detected with marker D17S799 after a genome-wide scanning. Analysis of six flanking microsatellites confirmed the linkage and haplotype inspection defined an interval of about 20 cM between D17S947 and D17S798. This is the first locus reported for IS. We scored genes mapping in this interval and studied the heparan sulfotransferase genes as candidates on the basis of their biochemical role. No causative mutation was detected in the affected patients.

Adult↗

PubMed2002

Evidence for differential S100 gene over-expression in psoriatic patients from genetically heterogeneous pedigrees.

Psoriasis is an inflammatory skin disorder characterised by keratinocyte hyper-proliferation and altered differentiation. To date, linkage analyses have identified at least seven distinct disease susceptibility regions (PSORS1-7). The PSORS4 locus was mapped by our group to chromosome 1q21, within the Epidermal Differentiation Complex. This cluster contains 13 genes encoding S100 calcium-binding proteins, some of which ( S100A7, S100A8 and S100A9) are known to be up-regulated in individual patient keratinocytes. In this study, we analysed S100 gene expression in psoriatic individuals from families characterised by linkage studies. We first selected individuals from two large pedigrees, one of which was linked to the 1q21 locus, whereas the other was unlinked to that region. We studied the expression of 12 S100 genes, by semi-quantitative RT-PCR and Northern blot. These analyses demonstrated up-regulation of S100A8, S100A9 and, to a lesser extent, S100A7 and S100A12, only in the 1q21 linked family. We subsequently analysed S100A7, S100A8, S100 A9 and S100 A12 in three additional samples and were able to confirm S100A8/ S100A9-specific over-expression in 1q-linked pedigrees. Thus, our data provide preliminary evidence for a locus-specific molecular mechanism underlying psoriasis susceptibility.

Base Sequence↗

PubMed2002

Mutational analysis of Peroxiredoxin IV: exclusion of a positional candidate for multinodular goitre.

BACKGROUND: Multinodular goitre (MNG) is a common disorder characterised by an enlargement of the thyroid, occurring as a compensatory response to hormonogenesis impairment. The incidence of MNG is dependent on sex (female:male ratio 5:1) and several reports have documented a genetic basis for the disease. Last year we mapped a MNG locus to chromosome Xp22 in a region containing the peroxiredoxin IV (Prx-IV) gene. Since Prx-IV is involved in the removal of H2O2 in thyroid cells, we hypothesize that mutations in Prx-IV gene are involved in pathogenesis of MNG. METHODS: Four individuals (2 affected, 2 unrelated unaffected) were sequenced using automated methods. All individuals were originated from the original three-generation Italian family described in previous studies. A Southern blot analysis using a Prx-IV full-length cDNA as a probe was performed in order to exclude genomic rearrangements and/or intronic mutations. In addition a RT-PCR of PRX-IV was performed in order to investigate expression alterations. RESULTS: No causative mutations were found. Two adjacent nucleotide substitutions were detected within introns 1 and 4. These changes were also detected in unaffected individuals, suggesting that they were innocuous polymorphisms. No gross genomic rearrangements and/or restriction fragment alterations were observed on Southern analysis. Finally, using RT-PCR from tissue-specific RNA, no differences of PRX-IV expression-levels were detected between affected and unaffected samples. CONCLUSIONS: Based on sequence and genomic analysis, Prx-IV is very unlikely to be the MNG2 gene.

Journal Article↗

PubMed2002

Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C.

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder, characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, and mottled cutaneous pigmentation. The LMNA gene encoding two nuclear envelope proteins (lamins A and C [lamin A/C]) maps to chromosome 1q21 and has been associated with five distinct pathologies, including Dunnigan-type familial partial lipodystrophy, a condition that is characterized by subcutaneous fat loss and is invariably associated with insulin resistance and diabetes. Since patients with MAD frequently have partial lipodystrophy and insulin resistance, we hypothesized that the disease may be caused by mutations in the LMNA gene. We analyzed five consanguineous Italian families and demonstrated linkage of MAD to chromosome 1q21, by use of homozygosity mapping. We then sequenced the LMNA gene and identified a homozygous missense mutation (R527H) that was shared by all affected patients. Patient skin fibroblasts showed nuclei that presented abnormal lamin A/C distribution and a dysmorphic envelope, thus demonstrating the pathogenic effect of the R527H LMNA mutation.

Abnormalities, Multiple↗

PubMed2002

Coding haplotype analysis supports HCR as the putative susceptibility gene for psoriasis at the MHC PSORS1 locus.

PSORS1, near HLA-C, is the major genetic determinant of psoriasis. We present genetic and structural evidence suggesting a major role for the HCR gene at the PSORS1 locus. Genotyping of 419 families from six populations revealed that coding single-nucleotide polymorphisms of HCR formed a conserved allele HCR*WWCC that associated highly significantly with psoriasis and with the HLA-Cw6 allele in all populations. Because of strong linkage disequilibrium between HLA-Cw6 and HCR*WWCC, the two genes could not be genetically distinguished by this sample size. However, the variant HCR allele was predicted to differ in secondary structure from the wild-type protein. HCR protein expression in lesional psoriatic skin differed considerably from that observed in normal skin. These results provide strong evidence for the HCR*WWCC allele as a major genetic determinant for psoriasis, probably by a mechanism impacting on keratinocyte proliferation.

Age of Onset↗

PubMed2002

Functional characterization of the 5' flanking region of human ubiquitin fusion degradation 1 like gene (UFD1L).

UFD1L (Ubiquitin Fusion Degradation 1 Like) gene encodes for a component of a multi-complex involved in the degradation of ubiquitin fusion proteins. The gene maps on chromosome 22q11, in a region commonly deleted in severe congenital disorders such as DiGeorge (DGS) and velo-cardio-facial (VCFS) syndromes. UFD1L is a single copy gene ubiquitously expressed in high levels in the pharyngeal pouches and fourth branchial arch artery during development. To understand the regulation of UFD1L expression we performed a functional analysis of its 5' regulatory region. 5'-RACE and primer extension analyses revealed the presence of different transcription start sites in adult and fetal tissues. UFD1L 5' flanking region contains a TATA-box motif and is also very GC-rich with a CpG island encompassing exon 1. Transcriptional activity of this region was examined by transfection experiments of promoter-GFP reporter gene constructs in a human epithelial cell line. These experiments revealed the importance of the region between -17 and -463 nt which contains the TATA-box. EMSA assay resulted in the detection of five functional consensus sequences respectively for the transcription complex TFIID and for the transcription factors AP-1 (one site), AP-2 (one) and Sp1 (two).

5' Flanking Region↗