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A Pizzuti

Publications and source records attributed to A Pizzuti.

At least 91 records · Page 5Linked to original sources
PubMed1993

Failure in detecting mRNA transcripts from the mutated allele in myotonic dystrophy muscle.

The expression of myotonin-protein kinase (MT-PK) gene was studied in myotonic dystrophy (DM) muscle and normal controls using a polymerase chain reaction protocol to analyse the 3' intragenic p(CTG) polymorphism. Unaffected individuals show bi-allelic expression, while the sole wild-type allele was transcribed in DM muscle. Our findings support a gene dosage effect in the pathogenesis of the disease.

Adult↗
PubMed1992

Triplet repeat mutations in human disease.

Triplet repeats are the sites of mutation in three human heritable disorders, spinal and bulbar muscular atrophy (SBMA), fragile X syndrome, and myotonic dystrophy (DM). These repeats are GC-rich and highly polymorphic in the normal population. Fragile X syndrome and DM are examples of diseases in which premutation alleles cause little or no disease in the individual, but give rise to significantly amplified repeats in affected progeny. This newly identified mechanism of mutation has, so far, been identified in two of the most common heritable disorders, fragile X syndrome and DM, and one rare disease, SBMA.

Female↗
PubMed1992

An unstable triplet repeat in a gene related to myotonic muscular dystrophy.

Synthetic oligonucleotides containing GC-rich triplet sequences were used in a scanning strategy to identify unstable genetic sequences at the myotonic dystrophy (DM) locus. A highly polymorphic GCT repeat was identified and found to be unstable, with an increased number of repeats occurring in DM patients. In the case of severe congenital DM, the paternal triplet allele was inherited unaltered while the maternal, DM-associated allele was unstable. These studies suggest that the mutational mechanism leading to DM is triplet amplification, similar to that occurring in the fragile X syndrome. The triplet repeat sequence is within a gene (to be referred to as myotonin-protein kinase), which has a sequence similar to protein kinases.

Amino Acid Sequence↗
PubMed1992

Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR.

About one third of Duchenne muscular dystrophy (DMD) patients have no gross DNA rearrangements in the dystrophin gene detectable by Southern blot analysis or multiplex exon amplification. Presumably, in these cases, the deficiency is caused by minor structural lesions of the dystrophin gene. However, to date, only a single human DMD case has been described where a point mutation, producing a stop codon, accounts for the DMD phenotype. To screen for microheterogeneities in the dystrophin gene, we applied analysis by chemical mismatch cleavage to thirteen exons amplified in multiplex sets by the polymerase chain reaction. This analysis covers approximately 20% of the dystrophin-coding sequence. Sixty DMD patients without detectable deletions or duplications were investigated, leading to the identification of two point mutations and four polymorphisms with a frequency higher than 5%. Both point mutations are frameshift mutations in exons 12 and 48, respectively, and are closely followed by stop codons, thus explaining the functional deficiency of the dystrophin gene products in both patients.

Base Sequence↗
PubMed1992

A transposon-like element in the deletion-prone region of the dystrophin gene.

The central portion of the dystrophin gene locus is a preferential site for deletions causing progressive muscular dystrophy of the Duchenne type (DMD). The nucleotide sequence of a deletion junction fragment from a DMD patient was determined, revealing that the proximal breakpoint of the deletion in intron 43 fell within the sequence of a transposon-like element. This segment, belonging to the THE-1 family of human transposable elements, is normally present in a complete form in intron 43 of the dystrophin gene. The deletion mutation was maternally transmitted and eliminated two-thirds of the THE-1 element. Analysis of DNA from additional DMD patients revealed a second deletion with the proximal breakpoint mapping within the same THE-1 element.

Base Sequence↗
PubMed1992

Anticipation in myotonic dystrophy. II. Complex relationships between clinical findings and structure of the GCT repeat.

We studied the expansion of the GCT repeats within the myotonic dystrophy protein kinase gene in nine myotonic dystrophy (DM) kindreds. Southern blot and polymerase chain reaction analyses of the repeat region demonstrated the expansion in all 62 patients with the diagnosis of DM. Among 43 DM parent-child pairs, age of onset in the child was earlier than in the parent in 36 pairs, in the same decade as the parent in five, and undetermined in two. The clinical anticipation observed in the 36 pairs accompanied an increase in the fragment size in 32, a decrease in two, and no apparent change in two pairs. In the remaining pairs without documented clinical anticipation, the fragment size increased in four, decreased in two, and was apparently unchanged in one. Overall, the size of expansion showed an inverse correlation with the age of onset (p < 0.001). In all seven pairs in which the fragment did not increase in size, the affected parent was male. Two congenital DM children born to affected mothers had expanded DNA greater than 4.5 kb. The differences between parent and child in age of onset significantly correlated with the differences in the expansion size among father-child pairs (p < 0.001) but not mother-child pairs (p > 0.5). Our data suggest that the expansion of the GCT repeats plays an important role in anticipation although other factors, including the sex of the affected parent, may have significant effects on molecular mechanisms of anticipation.

Base Sequence↗
PubMed1991

Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.

Fragile X syndrome results from mutations in a (CGG)n repeat found in the coding sequence of the FMR-1 gene. Analysis of length variation in this region in normal individuals shows a range of allele sizes varying from a low of 6 to a high of 54 repeats. Premutations showing no phenotypic effect in fragile X families range in size from 52 to over 200 repeats. All alleles with greater than 52 repeats, including those identified in a normal family, are meiotically unstable with a mutation frequency of one, while 75 meioses of alleles of 46 repeats and below have shown no mutation. Premutation alleles are also mitotically unstable as mosaicism is observed. The risk of expansion during oogenesis to the full mutation associated with mental retardation increases with the number of repeats, and this variation in risk accounts for the Sherman paradox.

Alleles↗
PubMed1991

Human fetal brain beta-nerve growth factor cDNA: molecular cloning of 5' and 3' untranslated regions.

The mRNA of beta-nerve growth factor (beta-NGF) has been demonstrated to be present in the human brain, both in adult and fetal stages of development. However, its complete nucleotide sequence is unknown since a full-length cDNA has yet to be isolated. We report here the cloning and complete analysis of the human fetal brain beta-NGF transcript. cDNA synthesis was performed from total brain RNA and overlapping regions of beta-NGF cDNA were enzymatically amplified and sequenced. The central portion of the cDNA was amplified using primers designed on the known genomic DNA sequence. The 5' and 3' unknown regions were amplified by the anchored polymerase chain reaction (PCR) and by complementary DNA Ends-PCR respectively. The latter method is an original variation of Inverted PCR. The sequenced transcript is very similar to the most common form of beta-NGF mRNA present in the mouse central nervous system. In addition, both the 5' and 3' untranslated regions show a high degree of homology to the corresponding murine sequences.

Base Sequence↗
PubMed1991

Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.

Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27.3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal of the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome.

Alleles↗
PubMed1991

Characterization of a murine gene expressed from the inactive X chromosome.

In mammals, equal dosage of gene products encoded by the X chromosome in male and female cells is achieved by X inactivation. Although X-chromosome inactivation represents the most extensive example known of long range cis gene regulation, the mechanism by which thousands of genes on only one of a pair of identical chromosomes are turned off is poorly understood. We have recently identified a human gene (XIST) exclusively expressed from the inactive X chromosome. Here we report the isolation and characterization of its murine homologue (Xist) which localizes to the mouse X inactivation centre region and is the first murine gene found to be expressed from the inactive X chromosome. Nucleotide sequence analysis indicates that Xist may be associated with a protein product. The similar map positions and expression patterns for Xist in mouse and man suggest that this gene may have a role in X inactivation.

Amino Acid Sequence↗
PubMed1991

Beta-nerve growth factor (beta-NGF) mRNA expression in the parkinsonian adrenal gland.

The adrenal gland is a well-demonstrated source for different neurotrophic factors. The presence of the beta-nerve growth factor (beta-NGF) mRNA in the adrenal tissue used for grafting in a Parkinsonian patient is reported here. Adrenal samples were obtained on the day of implantation, and a specific cDNA was synthesized after the extraction of total RNA using a synthetic oligonucleotide as a reverse transcription primer. A 168-bp portion of the cDNA was amplified using two other oligonucleotides as Taq polymerase primers in a polymerase chain reaction. Thirty-two cycles of amplification were performed. The amplification products were identified by agarose gel electrophoresis and Southern blot analysis as a single DNA band hybridizing with a third beta-NGF specific oligonucleotide. The identity of the fragment was confirmed by DNA sequencing. Quantitative analysis demonstrated a beta-NGF mRNA concentration exceeding 5 fg/micrograms of total adrenal RNA. These findings add NGF to the other neurotrophic factors produced by the gland (i.e., basic fibroblast growth factor) and demonstrate the retained functional capacity of the Parkinsonian adrenal to express the beta-NGF mRNA. All these data may assume relevant meaning for neurotransplantation research.

Adrenal Glands↗
PubMed1990

Detection of beta-nerve growth factor mRNA in the human fetal brain.

Nerve growth factor (NGF) is a trophic molecule recently demonstrated to interact with different structures in the central nervous system. The expression of the beta-NGF mRNA from human fetal cortices at the 15-16th week of gestational age has been demonstrated and quantitated by polymerase chain reaction amplification of the specific cDNA. beta-NGF mRNA expression in the human brain coincides with the period of active differentiation and synaptogenesis, suggesting that the trophic agent plays a role in the cerebral development.

Base Sequence↗
PubMed1990

Human neuronal cells in culture: from concepts to basic methodology.

The paper reviews some conceptual and methodological aspects of the tissue culture models which, during the past three decades, demonstrated a remarkable mimicry of many important structures and functions of the mammalian Central Nervous System (CNS) and related peripheral sensory and motor elements. Emphasis is placed on an original human neuronal tissue culture model obtained from selective CNS areas. The different cell types were identified and the neurotrophic interactions preliminary characterized. Neuropathological findings suggest hypothesis that can be fully tested using in vitro human models of affected cerebral specific areas.

Brain↗
PubMed1988

Human neuronal cell viability demonstrated in culture after cryopreservation.

A human neuronal cell freezing technique has been developed. The results indicate that human fetal neuronal cells can be frozen with 7%-10% dimethyl sulfoxide (DMSO) as cryoprotectant. The survival of isolated thawed cells has been evaluated in culture. Cells have been frozen at -196 degrees C for 370 days without loss of viability. Average recovery rate of frozen cells was 62% of the recovery rate of cultured unfrozen controls. Thawed cells show neurite outgrowth and maintain both cellular markers such as neuron specific enolase (NSE) and neurochemical characteristics (GABA synthesis). Morphological integrity of cryopreserved neurons has been confirmed at ultrastructural level.

Cell Survival↗