Biomedical subjects

Roberto Ravazzolo

Publications and source records attributed to Roberto Ravazzolo.

At least 37 records · Page 2Linked to original sources

PubMed2005

A common haplotype at the 5' end of the RET proto-oncogene, overrepresented in Hirschsprung patients, is associated with reduced gene expression.

Hirschsprung disease (HSCR) is a complex genetic defect of intestinal innervation mainly ascribed to loss of function mutations of the RET gene. Although RETcoding mutations account for only 15% of HSCR sporadic cases, several linkage and association studies still indicate RET as a major HSCR gene, suggesting the existence of noncoding RET variants or common polymorphisms which can act in HSCR pathogenesis. We previously described a predisposing RET haplotype (A-C-A) composed of alleles at three SNPs (-1 bp and -5 bp from the RET transcription start site, NT_033985.6:g.975824G>A and NT_033985.6:g.975820C>A, respectively, and silent polymorphism c.135G>A), which was present in 62% of chromosomes from HSCR patients but only in 22% of control chromosomes. Here we address the question of how this 5' ACA haplotype may functionally act as a predisposing factor in HSCR pathogenesis by performing functional analysis of the same three SNPs. We demonstrate that neither the two promoter variants nor the exon 2 SNP interfere with reporter gene transcription or RET mRNA splicing, respectively. However, real-time RT-PCR, performed in RNA obtained from lymphoblasts of selected individuals, has shown that homozygosity for the whole ACA haplotype is associated with reduced RET gene expression. We propose that a yet unidentified variant in linkage disequilibrium with the ACA haplotype, rather than the single characterizing SNPs, acts as a HSCR susceptibility allele by affecting the normal amount of RET receptor on the cell surface.

5' Flanking Region↗

PubMed2005

Molecular characterization of a t(2;6) balanced translocation that is associated with a complex phenotype and leads to truncation of the TCBA1 gene.

The molecular characterization of balanced chromosomal rearrangements has often been a powerful tool for the positional identification of genes associated with specific diseases. In some instances, these rearrangements may be associated with a variety of different phenotypes, and thus establishing a genotype-phenotype correlation may be a complex process. However, molecular characterization of the rearrangement remains a useful tool for diagnoses or prognoses, or for identifying new genes and establishing a gene-to-function relationship. In this work we describe the characterization of a de novo balanced translocation t(2;6)(q24.3;q22.31) found in a patient with a complex phenotype. The major clinical finding was a severe neurological involvement. Thanks to the molecular characterization of this translocation we found that the rearrangement led to the truncation of the TCBA1 gene on chromosome 6q. We found that the gene is transcribed in different splice variants and is highly specific for the central nervous system. TCBA1 does not show any similarity with other known genes, and no information is available about its function. However, the gene appears to be well conserved among species, and we were able to infer the sequence of a putative mouse homolog of TCBA1. This allowed us to perform a more detailed expression study in mice, thus confirming its specificity for the nervous system. This finding is of particular interest because it suggests that TCBA1 may be correlated with the neurological phenotype of our patient, and possibly mutated in genetic diseases with a neurological phenotype.

Alternative Splicing↗

PubMed2005

MVK mutations and associated clinical features in Italian patients affected with autoinflammatory disorders and recurrent fever.

Autosomal recessive autoinflammatory disorder caused by mutations of the mevalonate kinase gene (MVK), leading to mild, incomplete MK enzyme deficiency (MKD), has been known so far as Hyper-IgD and periodic fever syndrome (HIDS) and regarded as mostly occurring in Northern Europe. Here we report the results of the molecular characterization of the first Italian series of patients affected with autoinflammatory disorders and periodic fever. A total of 13 different mutations, scattered throughout the MVK coding region, were identified in either homozygous or compound heterozygous state in 15 patients. The mutation leading to the V377I amino-acid change, already described also in other series, resulted the most common with a frequency of 50% of all MKD alleles. Among the other mutations, eight had never been described before, including an interstitial deletion of 19 nucleotides in exon 2. In addition to these nucleotide changes, private and polymorphic MVK variants have been detected in the patients under analysis and checked also in a set of control individuals. Clinical features are reported for each of the 15 MKD patients, and life-threatening infections and systemic amyloidosis presented as unexpected MKD-related complications. Our study demonstrates that MKD is a common cause of recurrent fever also in the Italian population, where it is associated with both a wide spectrum of previously unreported MVK mutations and peculiar phenotypic features.

Adolescent↗

PubMed2005

Interaction of the LMX1B and PAX2 gene products suggests possible molecular basis of differential phenotypes in Nail-Patella syndrome.

The LMX1B gene, encoding a protein involved in limb, kidney and eye development, is mutated in patients affected by Nail-Patella syndrome. Inter- and intrafamilial variability is common in this disorder for skeletal abnormalities, presence and severity of nephropathy and ocular anomalies. Phenotypic variability might depend on interactions of the LMX1B causative gene with other genes during development of both kidney and eye, which might act as modifier genes. Results are presented on the interaction between LMX1B and PAX2 proteins, obtained by both direct yeast two-hybrid assay and coimmunoprecipitation. Such interaction provides support to further studies on pathways underlying important developmental processes.

Animals↗

PubMed2005

An in vitro approach to test the possible role of candidate factors in the transcriptional regulation of the RET proto-oncogene.

Neural crest cells arise from the epithelium of the dorsal neural tube and migrate to various districts giving origin, among others, to sympathetic, parasympathetic, and enteric ganglia. It has been shown that the transcription factors HOX11L1, HOX11L2, MASH1, PHOX2A, and PHOX2B are all necessary, to various extents, to the correct development of the autonomic nervous system. To investigate their possible role in the transcriptional regulation of the RET proto-oncogene, a gene playing a crucial role in correct intestinal innervation, we undertook a specific in vitro experimental strategy. Two neuroblastoma cell lines (SK-N-MC and SK-N-BE) were cotransfected with each transcription factor expressing plasmids and sequential deletion constructs of the 5' c-RET flanking region cloned upstream of the Luciferase reporter gene. Here we show that HOX11L1 enhances the activity of the c-RET promoter in SK-N-MC cell line by stimulating a region between -166 bp and -35 bp. Gel shift assays performed with oligonucleotides spanning this promoter sequence showed a change of the SP1 interaction with its binding sites, consequent to transfection with HOX11L1. While HOX11L2 showed no effect in both the cell lines, we have observed PHOX2A, PHOX2B, and MASH1 triggering a reproducible increase in the Luciferase activity in SK-N-BE cell line. A sequence responsible of the PHOX2A-dependent activation has been identified, while PHOX2B seems to act indirectly, as no physical binding has been demonstrated on c-RET promoter.

Animals↗

PubMed2005

A very short segment of the murine Ret promoter contains elements sensitive to in vitro neural cell differentiation.

Regulation of the murine Ret gene has only been partially studied, specifically cis-acting promoter elements and related transcription factors, although in vivo experiments have shown the ability of Ret 5' flanking sequences to drive expression of transgenic reporter genes. This work describes the characterization of a 387-bp fragment of the Ret 5' flanking sequence with the ability to activate expression of the LacZ gene in transfected cells, in which SP1 recognition elements played a fundamental role. Using P19 teratocarcinoma cells differentiated by retinoic acid treatment, we observed an induction of Ret mRNA accompanied by a decrease of SP1 binding due to its proteolytic cleavage.

5' Flanking Region↗

PubMed2004

Polymorphisms in the osteopontin promoter affect its transcriptional activity.

Understanding the molecular mechanisms that underlie regulation of transcription of the human osteopontin encoding gene (OPN) may help to clarify several processes, such as fibrotic evolution of organ damage, tumorigenesis and metastasis, and immune response, in which OPN overexpression is observed. With the aim to evaluate variants with functional effect on transcription, we have analyzed the promoter region and focused our investigation on three common variants present in the first 500 bp upstream of the transcription start site. Transfection of constructs carrying the four most frequent haplotypes relative to variants at -66, -156, and -443 fused to the luciferase reporter gene in a panel of different cell lines showed that one haplotype conferred a significantly reduced level of reporter gene expression in all tested cell lines. We describe that the -66 polymorphism modifies the binding affinity for the SP1/SP3 transcription factors, the -156 polymorphism is included in a yet uncharacterized RUNX2 binding site, and the -443 polymorphism causes differential binding of an unknown factor. The finding of differential effects of various combination of variants in haplotypes may contribute to explain data of association studies reported in several already published articles. Future association studies using haplotypes instead of single OPN variants will allow to achieve more accurate results referable to differential expression of OPN in several common diseases, in which OPN is considered a candidate susceptibility gene.

Alleles↗

PubMed2004

Double mechanism for apical tryptophan depletion in polarized human bronchial epithelium.

Indoleamine 2,3-dioxygenase is an enzyme that catabolizes tryptophan to kynurenine. We investigated the consequences of IDO induction by IFN-gamma in polarized human bronchial epithelium. IDO mRNA expression was undetectable in resting conditions, but strongly induced by IFN-gamma. We determined the concentration of tryptophan and kynurenine in the extracellular medium, and we found that apical tryptophan concentration was lower than the basolateral in resting cells. IFN-gamma caused a decrease in tryptophan concentration on both sides of the epithelium. Kynurenine was absent in control conditions, but increased in the basolateral medium after IFN-gamma treatment. The asymmetric distribution of tryptophan and kynurenine suggested the presence of a transepithelial amino acid transport. Uptake experiments with radiolabeled amino acids demonstrated the presence of a Na(+)-dependent amino acid transporter with broad specificity that was responsible for the tryptophan/kynurenine transport. We confirmed these data by measuring the short-circuit currents elicited by direct application of tryptophan or kynurenine to the apical surface. The rate of amino acid transport was dependent on the transepithelial potential, and we established that in cystic fibrosis epithelia, in which the transepithelial potential is significantly more negative than in noncystic fibrosis epithelia, amino acid uptake was reduced. This work suggests that human airway epithelial cells maintain low apical tryptophan concentrations by two mechanisms, a removal through a Na(+)-dependent amino acid transporter and an IFN-gamma-inducible degradation by IDO.

Amino Acid Transport Systems↗

PubMed2004

Infantile steroid-resistant nephrotic syndrome associated with double homozygous mutations of podocin.

Mutations of NPHS2, ie, the gene coding for podocin, are associated with nephrotic syndrome (NS) in children, with a clinical phenotype characterized by variable age at onset (from 1 to 10 years) and steroid/cyclosporine resistance. The authors describe an infantile variant in 2 families (3 patients) from Turkey, characterized by homozygosity of a complex haplotype, in which 2 podocin mutations (P20L-R168H) are present in cis. It results from the insertion of a new mutation (R168H), only found in Turkey, on a more ancient haplotype containing the P20L mutation observed in the European population. All patients described had presented with NS within the first 6 months of life with strict resistance to drugs and a histologic background of focal segmental glomerulosclerosis. This is the first description of double homozygous mutations in an autosomal recessive renal disease reported in the literature. The association with infantile NS widens the panel of clinical presentation related to NPHS2 mutations.

Child↗

PubMed2004

Effect of inflammatory stimuli on airway ion transport.

The airway epithelium controls the chemical and physical properties of airway surface fluid and consequently mucociliary clearance. The treatment for 24-48 hours of human bronchial epithelial cells with interferon-gamma or interleukin-4 leads to marked changes in transepithelial ion transport properties. Both cytokines downregulate the activity of the epithelial Na+ channel and, at the same time, upregulate Ca2+-dependent Cl- secretion. Interleukin-4 also increases the expression and function of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results suggest that some inflammatory stimuli may change the balance between fluid absorption and secretion to favor hydration of the airway surface and consequently mucus clearance.

Animals↗

PubMed2004

Sex chromosome rearrangements leading to partial aneuploidies and mosaicisms: use of QF-PCR for detection and quantification of the involved cell lines.

Chromosome rearrangements can lead to aneuploidies of specific chromosome regions and could be present in the entire individual or limited to some tissues (mosaicism) depending on the developmental stage of the embryo when the rearrangement occurs. We report 6 cases with sex chromosome rearrangements identified by conventional cytogenetics and tested by quantitative fluorescent polymerase chain reaction (QF-PCR). QF-PCR has been largely employed for rapid detection of common aneuploidies in pre-natal and post-natal diagnosis and consists in DNA amplification by polymerase chain reaction (PCR) using fluorescent labelled primers and the analysis of chromosome specific small tandem repeats (STR). We tested 5 sex chromosome specific STR markers in multiplex PCR amplifications together with other chromosome specific STR markers as control amplifications. The PCR products were analysed by capillary electrophoresis. The results from QF-PCR analysis were obtained within one day and confirmed our cytogenetic observations. This study shows that QF-PCR analysis can detect sex chromosome imbalance and also suspect mosaicism or chromosome rearrangement.

Aneuploidy↗

PubMed2003

Myotubularin-related 2 protein phosphatase and neurofilament light chain protein, both mutated in CMT neuropathies, interact in peripheral nerve.

Charcot-Marie-Tooth disease type 4B1, CMT4B1, is a severe, autosomal-recessive, demyelinating peripheral neuropathy, due to mutations in the Myotubularin-related 2 gene, MTMR2. MTMR2 is widely expressed and encodes a phosphatase whose substrates include phosphoinositides. However, this does not explain how MTMR2 mutants specifically produce demyelination in the peripheral nerve. Therefore, we analysed the cellular and subcellular distribution of Mtmr2 in nerve. Mtmr2 was detected in all cytoplasmic compartments of myelin-forming Schwann cells, as well as in the cytoplasm of non-myelin-forming Schwann cells and both sensory and motorneurons. In contrast, Mtmr2 was detected in the nucleus of Schwann cells and motorneurons, but not in the nucleus of sensory neurons. As Mtmr2 is diffusely present also within the nerve, a specific function could derive instead from nerve-specific interacting proteins. Therefore, we performed two yeast two-hybrid screenings, using either fetal brain or peripheral nerve cDNA libraries. The neurofilament light chain protein, NF-L, was identified repeatedly in both screenings, and found to interact with MTMR2 in both Schwann cells and neurons. Interestingly, NF-L, encoding NF-L, is mutated in CMT2E. These data may provide a basis for the nerve-specific pathogenesis of CMT4B1, and further support for the notion that hereditary demyelinating and axonal neuropathies may represent different clinical manifestations of a common pathological mechanism.

Animals↗

PubMed2003

Genetic heterogeneity in inherited spastic paraplegia associated with epilepsy.

We have recently mapped a new rare form of spastic paraplegia complicated by bilateral cataracts, gastroesophageal reflux with persistent vomiting, and amyotrophy to chromosome 10q23.3-q24.2. This locus, named SPG9, is located in an interval spanning about 12 cM of genomic DNA, between markers D10S536 and D10S603, where different neurological disorders have been mapped. In particular, a gene for partial epilepsy has been assigned to a 3 cM interval between markers D10S185 and D10S577, which is completely included in the SPG9 critical region. A few families affected with spastic paraplegia and epilepsy have been reported; in the present study, we tested a pedigree with concurrence of spastic paraplegia, epilepsy, and mental retardation inherited as an autosomal dominant trait, using markers located in the SPG9 interval. Haplotype reconstruction excluded the linkage to 10q23.3-q24.2. In addition, the seven different loci so far reported to be associated with autosomal dominant pure forms of spastic paraplegia have been tested and excluded by linkage analysis and haplotype reconstruction, including SPG4 on chromosome 2p22-p21, where a familial form of spastic paraplegia associated with dementia and epilepsy has been mapped. These data confirm genetic heterogeneity in familial spastic paraplegia with epilepsy and suggest a specific locus for the family here analyzed.

DNA↗

PubMed2003

Previously undescribed nonsense mutation in SHH caused autosomal dominant holoprosencephaly with wide intrafamilial variability.

Holoprosencephaly (HPE) is the most common developmental defect of the forebrain and midface in humans, with a frequency of 1/16,000 live births. Different genes are implicated in the pathogenesis of HPE; these include SHH, ZIC2, SIX3, TGIF, and human DKK1. We describe here a family with recurrence of autosomal dominant HPE in different members showing a wide clinical variability. The mother presents a single central maxillary incisor and mild hypotelorism as signs of the diseases, while three of her sons were affected by HPE. By direct sequencing and restriction analysis of exon 2 of the SHH gene, we have identified a previously undescribed nonsense mutation at codon 128 (W128X). The identification of this mutation allowed us to give a prenatal diagnosis in this family and confirms a wide intrafamilial variability in the phenotypic spectrum.

Abortion, Spontaneous↗

PubMed2003

Genetics, clinical and pathological features of glomerulonephritis associated with mutations of nonmuscle myosin IIA (Fechtner syndrome).

BACKGROUND: Fechtner syndrome (FTNS), also known as Alport-like syndrome, is a rare inherited condition characterized by progressive nephritis, macrothrombocytopenia, Döhle-like leukocyte inclusions, deafness, and cataract. Although it recently was shown that FTNS derives from mutation of MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA), its pathophysiological characteristics remain unknown. METHODS: We studied a large FTNS family in which 10 components carried a missense mutation of MYH9 determining the D1424H substitution. RESULTS: All affected subjects presented with macrothrombocytopenia and leukocyte Döhle-like bodies consisting of macroaggregates of NMMHC-IIA, but only two subjects had major renal problems characterized by proteinuria and renal failure. Electron microscopy showed focal and segmental effacement of podocytes and loss of the interpodocyte slit diaphragm. Immunohistochemistry showed apical localization of NMMHC-IIA in tubular epithelia and less podocyte staining in the two patients, whereas it was diffuse in normal epithelia. Three patients presented with stable microhematuria, and another five patients had no renal lesions, although they carried the same mutation of MYH9. Therefore, MYH9 mutation per se was responsible for platelet and leukocyte abnormalities, whereas additional predisposing conditions and/or environmental factors are necessary for nephropathy, cataract, and deafness. Looking at podocyte components conferring permselectivity properties to the kidney, we characterized the haplotype of podocin and found cosegregation of one specific allele in the two patients with nephrotic syndrome, suggesting a relationship between podocin features and proteinuria. CONCLUSION: Our study indicates a major role for the NMMHC-IIA abnormality in the pathogenesis of leukocyte, platelet, and kidney defects in FTNS. The basic feature in all cases is aggregation and compartmentation of NMMHC-IIA. However, proteinuria and podocyte lesions are the hallmark of nephropathy in patients who develop renal failure, and podocin may have some function in this setting.

Adolescent↗

PubMed2003

MYH9-related disease: May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but represent a variable expression of a single illness.

May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are autosomal dominant macrothrombocytopenias distinguished by different combinations of clinical and laboratory signs, such as sensorineural hearing loss, cataract, nephritis, and polymorphonuclear Döhle-like bodies. Mutations in the MYH9 gene encoding for the nonmuscle myosin heavy chain IIA (NMMHC-IIA) have been identified in all these syndromes. To understand the role of the MYH9 mutations, we report the molecular defects in 12 new cases, which together with our previous works represent a cohort of 19 families. Since no genotype-phenotype correlation was established, we performed an accurate clinical and biochemical re-evaluation of patients. In addition to macrothrombocytopenia, an abnormal distribution of NMMHC-IIA within leukocytes was observed in all individuals, including those without Döhle-like bodies. Selective, high-tone hearing deficiency and cataract was diagnosed in 83% and 23%, respectively, of patients initially referred as having May-Hegglin anomaly or Sebastian syndrome. Kidney abnormalities, such as hematuria and proteinuria, affected not only patients referred as Fechtner syndrome and Epstein syndrome but also those referred as May-Hegglin anomaly and Sebastian syndrome. These findings allowed us to conclude that May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but rather a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to a severe form complicated by hearing loss, cataracts, and renal failure. For this new nosologic entity, we propose the term "MHY9-related disease," which better interprets the recent knowledge in this field and identifies all patients at risk of developing renal, hearing, or visual defects.

Adolescent↗

PubMed2003

The first intron of the human osteopontin gene contains a C/EBP-beta-responsive enhancer.

The osteopontin (OPN) protein is found expressed at high level in several processes including fibrotic evolution of organ injuries, tumorigenesis, and immune response. The molecular mechanisms that underly overexpression, especially at the transcriptional level, have been only partially clarified. Therefore, this study was undertaken in search for additional DNA elements in the regulatory regions of the OPN gene and cognate transcription factors. Our results on the region upstream of the transcription start site confirmed that essential elements are located within the first 100 bp. Analysis of the sequence that includes the first untranslated exon and first intron revealed that it could enhance the promoter activity. Experiments of transfection of constructs containing different fragments of this sequence showed that most of the enhancer activity was confined in the terminal 30-bp tract of the first intron, although it was not functioning in a myofibroblast cell line. DNA/protein binding assays and cotransfection experiments showed that the C/EBP-beta transcription factor was able to bind a recognition sequence in this 30-bp segment. We found a bi-allelic sequence polymorphism at +245 in the first intron, which did not show a significant functional effect, but is a useful tool for future association studies.

Base Sequence↗

PubMed2003

Confirmation of CLIM2/LMX1B interaction by yeast two-hybrid screening and analysis of its involvement in nail-patella syndrome.

Nail-patella syndrome (NPS), an autosomal dominant disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns, and often associated with nephropathy and, less frequently, with open angle glaucoma, is caused by mutations in the LMX1B gene. Inter-familial and intra-familial phenotypic variability raises the question whether modifier genes can be identified to explain differences in the expression and severity of clinical features of NPS. Genes encoding proteins that interact with the LMX1B protein are good candidates and, therefore, methods to search for interactions can be used to this purpose. By the yeast two-hybrid screening we detected the CLIM2 gene as a LMX1B interactor, confirming previous reports which described the same interaction by biochemical methods. Sequencing of the CLIM2 coding region in seven NPS cases in which no LMX1B mutation had been found, did not detect any molecular variant in these patients. Moreover, by genotyping a polymorphic dinucleotide repeat close to the CLIM2 gene in affected members of a large Dutch NPS family with high incidence of nephropathy, we were unable to find a correlation between the presence of a specific allele and the expression of nephropathy. In conclusion, although the results of this study could not provide any proof of CLIM2 involvement in the pathogenesis of NPS or in determination of the clinical phenotype, we suggest that the CLIM2 gene can be considered as a good candidate for further studies on normal and disturbed kidney development associated with NPS or other hereditary glomerulopathies.

DNA Mutational Analysis↗