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Biomedical subjects

L Zelante

Publications and source records attributed to L Zelante.

At least 109 records · Page 6Linked to original sources
PubMed1996

Scanning the first part of the neurofibromatosis type 1 gene by RNA-SSCP: identification of three novel mutations and of two new polymorphisms.

Neurofibromatosis type 1 (NF1) of von Recklinghausen is a common autosomal dominant disorder, characterized by peripheral neurofibromas, café-au-lait spots and Lisch nodules of the iris. The high mutation rate at the NF1 locus results in a wide range of molecular abnormalities. We have scanned 14 different exons from the first part of the NF1 gene using the RNA-single strand conformation polymorphism (RNA-SSCP) method in a series of 40 NF1 patients. Three novel mutations, two nonsense and one missense, and two polymorphisms have been detected in familial cases. Genotype-phenotype correlations have been investigated, but no particular association has been detected. After this screening, the majority of NF1 chromosomes has not yet been characterized, confirming the difficulty in detecting the defect underlying NF1 in most families, even following extensive DNA analysis.

Adolescent↗
PubMed1996

Molecular analysis of the cystinuria disease gene: identification of four new mutations, one large deletion, and one polymorphism.

A cystinuria disease gene (rBAT) has recently been identified, but evidence strongly suggests that only Type-I cystinuria is due to mutations in this gene. Sixteen point mutations and a large deletion causing the disease have so far been described in the rBAT gene sequence. To identify new mutated alleles, genomic DNA was analyzed, after the determination of the entire genomic structure of the rBAT gene, by RNA-single strand conformation polymorphism analysis, an accurate and sensitive method able to detect nucleotide changes. Four new point mutations, a large deletion, and a common intragenic polymorphism were detected. These new mutations increase to 22 the number of mutated alleles so far characterized in rBAT. In addition, the frequency of 21 mutations was assessed in a sample of accurately defined Type-I cystinuria chromosomes. They account for about 58% of all Type-I chromosomes, mutation M467T being the most common (0.26).

Alleles↗
PubMed1996

The Williams syndrome: an Italian collaborative study.

Williams syndrome (WS) is a multiple congenital anomalies/mental retardation syndrome caused by a microdeletion on the long arm of chromoome 7 including the elastin gene. Possibly it is a contiguous gene syndrome with autosomal dominant transmission. Seventy-seven WS patients from 11 Italian Pediatric-Dysmorphology-Genetics Units were collected by means of a questionnaire designed to draw a comprehensive clinical picture, to define the frequency of different traits and associations thereof, to better understand the clinical evolution, to improve the prognosis and to ameliorate the follow-up. The most important signs for diagnosis, based on their relative frequencies, are: mental retardation with characteristic outgoing behaviour and hoarse voice; facial findings like stellate iris, periorbital fullness and thick lips; congenital heart disease. The frequency of the clinical signs reported in our patients are on the whole concordant with those found in the literature; the only significant differences concern low stature, hallus valgus, hypoplastic nails, joint contractures and ear infections. The multisystemic nature of this syndrome requires a coordinated and integrated approach in order to avoid fragmentary interventions.

Abnormalities, Multiple↗
PubMed1995

Molecular basis for the recently described hereditary hyperferritinemia-cataract syndrome: a mutation in the iron-responsive element of ferritin L-subunit gene (the "Verona mutation")

Recently, we described a new genetic disorder (the "hereditary hyperferritinemia-cataract syndrome") clinically characterized by the combination of elevated serum ferritin and congenital bilateral nuclear cataract, both cotransmitted as an autosomal dominant trait. In affected subjects, hyperferritinemia (ranging from 950 to 2,259 micrograms/L) is typically not related to iron overload. Differently from subjects with hereditary hemochromatosis, they have normal to low levels of serum iron and percent of transferrin saturation and absence of iron overload in parenchymal organs. When unnecessary phlebotomies are performed, they rapidly develop iron-deficient anemia, with persistently elevated levels of serum ferritin. By RNA-single-strand conformation polymorphism screening of the L-subunit ferritin gene on chromosome 19, we were able to identify in affected subjects a mutation in the 5' untranslated region. This mutation involves the five nucleotides sequence [CAGUG] of the iron-responsive element (IRE), which is critical for the posttranscriptional regulation of ferritin synthesis by means of IRE-binding protein (IRE-BP). Thus, it is very likely to provide the molecular basis for the iron-insensitive upregulation of ferritin synthesis in affected subjects.

Adult↗
PubMed1995

Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'.

Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of two complementary, 35 nt long, oligonucleotides. The accessibility of DNA modifying enzymes to this molecule was significantly decreased: T4-ligase and kinase activity were respectively reduced up to 1/2 and to 1/6, as compared to the normal phosphodiester molecule. Nucleolytic stability was increased either to purified enzymes (DNase I and Bal31) or to incubations in fresh serum, cell culture medium or in muscle protein extract. Phosphorothioate end-capped complete eukaryotic transcription units (obtained by Taq polymerase amplification with PS-primers) were not significantly protected from nucleolytic attack. On the contrary, synthetic transcription units, 'mini genes', obtained by Taq amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant to DNase I and Bal31 nucleolytic activity. Transcription efficiency, driven by the T7 promoter, was 96.5, 95 and 33.5% (respectively with 1, 2 or 3 substitutions), as compared to the normal phosphodiester molecules.

Base Sequence↗
PubMed1995

Genetic heterogeneity in cystinuria: the SLC3A1 gene is linked to type I but not to type III cystinuria.

Cystinuria is an autosomal recessive amino-aciduria where three urinary phenotypes have been described (I, II, and III). An amino acid transporter gene, SLC3A1 (formerly rBAT), was found to be responsible for this disorder. To assess whether mutations in SLC3A1 are involved in different cystinuria phenotypes, linkage with this gene and its nearest marker (D2S119) was analyzed in 22 families with type I and/or type III cystinuria. Linkage with heterogeneity was proved (alpha = 0.45; P < 0.008). Type I/I families showed homogeneous linkage to SLC3A1 (Zmax > 3.0 at theta = 0.00; alpha = 1), whereas types I/III and III/III were not linked. Our data suggest that type I cystinuria is due to mutations in the SLC3A1 gene, whereas another locus is responsible for type III. This result establishes genetic heterogeneity for cystinuria, classically considered as a multiallelic monogenic disease.

Adolescent↗
PubMed1995

Analysis of the complete coding region of the CFTR gene in a cohort of CF patients from north-eastern Italy: identification of 90% of the mutations.

A complete coding-region analysis on 225 cystic fibrosis (CF) chromosomes from a cohort that includes all the affected subjects born in two North-Eastern Italian regions over eight years was performed. In a previous study, we identified mutations on 166/225 (73.8%) CF chromosomes after screening for 62 mutations. To characterise the remaining 59 CF chromosomes, we carried out automated direct DNA sequencing (exons 9 and 13), RNA single-strand conformation polymorphism (exons 1-8 and 10-12) and denaturing gradient gel electrophoresis (exons 14a-24) of the 27 exons and flanking regions of the CF transmembrane conductance regulator gene. We identified 22 mutations, four of which are novel, viz. 711 + 5G-->A, R709X, 3132delTG and 2790-2A-->G, and we characterised 90.2% (203/225) of the CF chromosomes. Taking advantage of the homogeneity of the sample, an evaluation of the most important clinical parameters, assessed at the age of 12 years, is presented. We confirm some previously reported genotype-phenotype correlations and we report a new nonsense mutation (R709X) associated with a pancreatic sufficient phenotype.

Adolescent↗
PubMed1995

New polymorphisms and markers in the HLA class I region: relevance to hereditary hemochromatosis (HFE).

Hereditary hemochromatosis (HFE) is an inherited disorder whose gene lies in the proximity of the histocompatibility antigen (HLA) class I region, on 6p21.3. Despite efforts in refining the HFE region, a number of informative DNA markers, linked to the disease locus and amenable to use in an assay based on the polymerase chain reaction (PCR) is available. The gene content of this region is high, and the HFE gene has not so far been identified. We have used a strategy based on PCR protocols potentially able to detect both polymorphisms and expressed sequences. This approach has been applied to a 700-kb stretch (approximately) of DNA corresponding to the insert of a Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (225 B1) of the possible candidate region. Five new polymorphisms have been detected among 20 specific fragments isolated. Four of them are tightly linked to the HFE locus. Because of the strong linkage disequilibrium with the disease demonstrated by these markers, they could represent starting points for the identification and characterization of the HFE gene. The remaining non-polymorphic fragments, being amplifiable and in most cases linked to NotI sites, may be useful starting points for the generation of a genomic contig of band 6p21.3 and for gene identification.

Base Sequence↗
PubMed1995

Assignment of the gene responsible for cystinuria (rBAT) and of markers D2S119 and D2S177 to 2p16 by fluorescence in situ hybridization.

We have established rBAT (named as SLC3A1 in the Genome Data Base) as a gene responsible for cystinuria, a heritable disorder of amino acid transport. The cystinuria locus has been mapped by linkage between microsatellite markers D2S119 and D2S177. Fluorescence in situ hybridization (FISH) either with Alu-polymerase-chain-reaction (PCR)-amplified sequences of a yeast artificial chromosome (YAC) containing the rBAT gene or with rBAT-specific PCR-amplified genomic fragments, and chromosome G-banding have cytogenetically mapped rBAT to 2p16.3. In order to correlate the physical and genetic information on cystinuria, we have performed FISH with combinations of Alu-PCR-amplified sequences from YACs containing rBAT or the D2S119 and D2S177 loci. In all cases, a fused signal is obtained that demonstrates their close physical location; this allows the assignment of rBAT, cystinuria and their linked markers, D2S119 and D2S177, to 2p16.

Amino Acid Transport Systems, Basic↗
PubMed1995

Homozygosity for a novel splice site mutation (2790-2 A--->G) preceding exon 15 of the CFTR gene in a cystic fibrosis patient of North-East Italian descent.

We have been screening a cohort of 225 chromosomes from cystic fibrosis patients for mutations in the cystic fibrosis transmembrane regulator gene using a combination of DGGE,RNA-SSCP and DNA sequencing. A novel splice site mutation was detected by multiplex DGGE in a homozygous patient. Restriction-site generating PCR (RG-PCR) analysis demonstrated that both parents carried the same mutation. The molecular haplotype was the same. All the known ancestors came from the same (Veneto) region, and no consanguinity was documented up to the sixth generation.

Alternative Splicing↗
PubMed1995

Characterization of the 5' region of the Fanconi anaemia group C (FACC) gene.

Fanconi anaemia (FA) is an autosomal recessive disease characterised by progressive pancytopenia, chromosome instability and an increased risk of cancer. The Fanconi Anaemia Complementation Group C (FACC) gene is mutated in patients of complementation group C. Several different forms of FACC mRNA that share the same coding region have been isolated. At least two species result from the use of alternative exons at the 5' end and three result from the use of distinct polyadenylation signals. As a first step toward the characterization of this gene we have isolated the genomic clones corresponding to the 5' region, including a putative promoter and two alternate 5' exons. These exons, named -1 and -1a, were found to be separated by a small intron, with exon -1 located 5' to exon -1a. Further, these exons are flanked by consensus sequences of donor sites at the 5' ends of introns. An acceptor splice site was not evident 5' of exon -1a, suggesting that exon -1 is not spliced onto exon -1a. The sequences upstream of exons -1 and -1a have no obvious TATA or CAAT boxes but include CG-rich sequences. Functional analysis of the sequence upstream of the putative transcription start site of both alternative exons indicates that the region upstream exon -1 is sufficient to drive the expression of the luciferase reporter gene in CaCo-2 cells and that the transcriptional regulation of this gene is complex.

Base Sequence↗
PubMed1995

Screening of neurofibromatosis type 1 gene: identification of a large deletion and of an intronic variant.

Neurofibromatosis type 1 of von Recklinghausen is a common autosomal dominant disorder, characterized by peripheral neurofibromas, café-au-lait spots and Lisch nodules of the iris. The high mutation rate at the neurofibromatosis type 1 locus results in a wide range of molecular abnormalities. We have screened seven different exons of the neurofibromatosis type 1 gene, including those codifying for the GAP-related domain, using the RNA-Single Strand Conformation Polymorphism (RNA-SSCP) method in a series of 59 neurofibromatosis type 1 patients. We have also analyzed four intragenic repeats and one RFLP to detect hemizygosity and evaluate informativeness in at-risk families. One deletion and a new intronic normal variant have been detected. Thus the majority of Neurofibromatosis type 1 chromosomes have not been characterized, confirming difficulty in providing proper genetic counselling in neurofibromatosis type 1 families, even following extensive DNA analysis.

Alleles↗
PubMed1994

Development of RNA-SSCP protocols for the identification and screening of CFTR mutations: identification of two new mutations.

A strategy is described that allows a rapid and accurate identification and screening of cystic fibrosis gene mutations. It consists of setting up and developing RNA single strand conformation polymorphism (rSSCP) protocols, a technique based on the large repertoire of secondary structure of single-stranded RNA. By incorporating the T7 phage promoter sequence into PCR primers, it is possible to carry out rSSCP and compare it to standard single-strand conformation polymorphism (SSCP). Several parallel tests indicate that rSSCP detects a higher fraction of single base changes, and is less time consuming than SSCP since it requires only one fairly short electrophoretic run. Using this technique we were able to identify two new splicing mutations in introns 5 (711 + 5G-->A) and 10 (1717-8G-->A) of the CFTR gene.

Cystic Fibrosis↗
PubMed1994

The motilin gene: subregional localisation, tissue expression, DNA polymorphisms and exclusion as a candidate gene for the HLA-associated immotile cilia syndrome.

The product of the human motilin gene (MLN) has an important role in regulating gastrointestinal motility. The precise chromosomal localisation and expression of this gene are still unresolved. Here, we report a detailed study assigning MLN to 6p21.3; MLN is tightly linked to the HLA-DQalpha locus. Moreover, MLN expression has been evaluated in a large series of tissues. Positive signals have been obtained for brain, bronchi and a gastrointestinal malignancy. Direct sequencing exon by exon of the codifying region, intron/exon boundaries and promoter has allowed the identification of three DNA polymorphisms, one of which corresponds to a common protein variant. The chromosomal localisation of MLN, and its expression in broncoepithelial cells suggests that this gene is involved in immotile-cilia syndrome (ICS) disease. Sequence and segregation analysis of the MLN gene carried out in two families, in which the disease locus was previously assigned to 6p21.3, exclude MLN as a candidate gene for the HLA-associated form of ICS.

Base Sequence↗
PubMed1994

A new complex polymorphic repeat close to the HLA-A and HLA-E loci.

We describe a complex polymorphic repeat discovered analyzing YAC 225B1, which spans the region between the HLA-A and HLA-E loci. The repeat consists of a variable number of the nucleotide A followed by a variable number of the trinucleotide AAG [(A)n(AAG)n]. Three different alleles were detected with an observed heterozygosity of 0.30.

Alleles↗
PubMed1994

Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine.

Cystinuria is a classic heritable aminoaciduria that involves the defective transepithelial transport of cystine and dibasic amino acids in the kidney and intestine. Six missense mutations in the human rBAT gene, which is involved in high-affinity transport of cystine and dibasic amino acids in kidney and intestine, segregate with cystinuria. These mutations account for 30% of the cystinuria chromosomes studied. Homozygosity for the most common mutation (M467T) was detected in three cystinuric siblings. Mutation M467T nearly abolished the amino acid transport activity induced by rBAT in Xenopus oocytes. These results establish rBAT as a cystinuria gene.

Adolescent↗