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Alessia Colosimo

Publications and source records attributed to Alessia Colosimo.

14 recordsLinked to original sources
PubMed2007

Sequence-specific modification of a beta-thalassemia locus by small DNA fragments in human erythroid progenitor cells.

Gene therapy has been proposed as a definitive cure of beta-thalassemia. We applied a gene targeting approach, based on the introduction of small DNA fragments (SDF) into erythroid progenitor cells, to specifically modify the beta-globin gene sequence at codon 39. The strategy was first tested in normal individuals by delivering mutant SDF that were able to produce the beta-39 (C->T) mutation. Secondly, wild-type SDF were electroporated into target cells of beta-3i9/beta-39 b-thalassemic patients to correct the endogenous mutation. In both cases, gene modification was assayed by allele-specific polymerase chain reaction of DNA and mRNA, by restriction fragment length polymorphism analysis and by direct sequencing.

Alleles↗
PubMed2006

Hematologic and molecular characterization of a Sicilian cohort of alpha thalassemia carriers.

Detailed hematologic and molecular analyses were carried out on a cohort of Sicilian individuals with suspected or asymptomatic beta-thalassemia. Iron deficiency, mild beta-thalassemia alleles and most common Mediterranean alpha-globin deletional mutations were excluded. All negative individuals were then tested for alpha-thalassemia point mutations by a denaturing high-performance liquid chromatography (DHPLC)-based assay. Four rare alpha-globin variants (Hb Interlaken, Hb Chesapeake, Hb Lombard, Hb Sun Prairie) and one point mutation (polyA: AATAAA-G in alpha2) were identified in 15 out of 80 carriers. Direct sequence analysis carried out in the remaining 65 negative individuals revealed no further sequence variants.

Cohort Studies↗
PubMed2006

Detection of a rare beta-globin nonsense mutation [codon 59 (AAG-->TAG)] in an Italian family.

In this study we report on the hematological and molecular findings of a family from Central Italy, whose 33-year-old male proband presented with a beta0-thalassemia (thal) trait associated to a relevant Hb F level. The proband and his family (parents and a sister) were investigated by hematological analysis. The two beta-thal carriers of the beta-globin nonsense mutation [codon 59 (AAG-->TAG)] (the proband and his father) showed the hematological picture of a beta0-thal trait: the only hematological difference between the two beta-thal carriers was in the Hb F level (3.3% in the proband and 1% in his father).

Adult↗
PubMed2006

Influence of Ggamma-158C --> and beta- (AT)x(T)y globin gene polymorphisms on HbF levels in Italian beta-thalassemia carriers and wild-type subjects.

Clinical manifestations of beta-thalassemia (beta-thal) intermedia phenotypes are influenced by the persistence of fetal hemoglobin (HbF) and by several polymorphisms located in the promoters of A- and beta-globin genes. The aim of this study was to evaluate the distribution of the -158Ggamma (C-->T) polymorphism and of the (AT)x(T)y configuration, as well as their eventual association with elevated levels of HbF in 188 beta-thal carriers and 229 wild-type individuals of Italian descent. The -158GgammaT and the (AT)9(T)5alleles were found to be associated with increased levels of HbF in beta-thal carriers, but not in wild-type subjects.

Fetal Hemoglobin↗
PubMed2005

Screening of mutations in the CFTR gene in 1195 couples entering assisted reproduction technique programs.

Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.

Alleles↗
PubMed2004

Identification of 14 rare marker chromosomes and derivatives by spectral karyotyping in prenatal and postnatal diagnosis.

Extra structurally abnormal chromosomes (ESACs) and cryptic rearrangements are often associated with mental retardation and phenotypic abnormalities. In some cases their characterisation, using standard cytogenetic techniques and fluorescence in situ hybridization (FISH), is difficult and time consuming, where a fast and accurate identification is essential, especially when such chromosomal aberrations are found in prenatal diagnosis. A recent molecular technique, spectral karyotyping (SKY), based on the spectral signature of 24 chromosome-specific painting probes labelled with different combinations of five fluorochromes, allows the simultaneous visualisation of all human chromosomes in different colours. We used SKY analysis on 14 cases with rare ESACs or cryptic unbalanced rearrangements found at pre- or postnatal diagnosis. SKY analysis permitted the classification of chromosome rearrangements in all 14 cases analysed in combination with FISH analysis.

Chromosome Aberrations↗
PubMed2004

Mutational analysis of parkin gene by denaturing high-performance liquid chromatography (DHPLC) in essential tremor.

OBJECTIVES: To evaluate the relationship between point mutations within the parkin gene and essential tremor (ET). BACKGROUND: Essential tremor, the most common movement disorder, has long been recognised as an autosomal dominant disease. To date the genes involved in ET pathogenesis are still unknown. Several authors reported the association of ET with Parkinson's disease (PD). PATIENTS AND METHODS: One hundred and ten unrelated ET patients were analysed for point mutations within the parkin gene. Experimental conditions for DHPLC mutational analysis of the coding region of the parkin gene were set up. RESULTS: Neither obvious disruptive mutations, nor mutations previously described in patients with Parkinson's disease were identified in the cohort of patients analysed. DHPLC analysis detected two already reported polymorphisms [c.1138G>C (V380L) and c.1180G>A (D394N)], and four novel rare variants (frequency <1%) [c.645C>A (H215Q); c.847C>T (H279H); c.1393G>A (V465M) and c.2695A>G] located within exonic regions. Four new polymorphisms [c.413-20T>C; c.872-35G>A; c.872-68C>G; c.1286-117A>G], and one rare variant (c.934-3C>T) were also found within intronic regions. CONCLUSION: Causative sequence variants in the parkin gene have not been identified in this cohort of Italian ET patients.

Aged↗
PubMed2003

Molecular detection of novel WFS1 mutations in patients with Wolfram syndrome by a DHPLC-based assay.

Wolfram syndrome (WS) is a recessively inherited mendelian form of diabetes and neurodegeneration also known by the acronym DIDMOAD from the major clinical features, including diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Affected individuals may also show renal tract abnormalities as well as multiple neurological and psychiatric symptoms. The causative gene for WS (WFS1) encoding wolframin maps to chromosome 4p16.1 and consists of eight exons, spanning 33.44 Kb of genomic DNA. In this study we report on the mutational analysis of the WFS1 coding region in 19 Italian WS patients and 25 relatives, using a DHPLC-based protocol. A total of 19 different mutations in WFS1 were found in 18 of 19 patients (95%). All these mutations, except one, are novel, preferentially located in WFS1 exon 8, and include deletions, insertions, duplications, and nonsense and missense changes. In particular, a 16 base-pair deletion in WFS1 codon 454 was detected in five different unrelated nuclear families, being the most prevalent alteration in this Italian group. Nine neutral changes and polymorphisms were also identified. Overall, this study represents the molecular characterization of the largest cohort of Italian WS patients and carriers studied so far, and increases the number of identified WFS1 allelic variants worldwide.

Adolescent↗
PubMed2003

Validation of dHPLC for molecular diagnosis of beta-thalassemia in Southern Italy.

Beta-thalassemia, the most common hereditary anemia in the Mediterranean area, results from over 200 causative mutations in the beta-globin locus. The aim of this study was to validate a denaturing high-performance liquid chromatography (dHPLC)-based assay for postnatal and prenatal molecular diagnosis of beta-thalassemia in Southern Italy. Sixty beta-thalassemic patients, affected either by thalassemia intermedia or thalassemia major, were analyzed in a blind study. We also carried out prenatal molecular diagnosis in 12 couples at-risk for having affected offspring. Chorionic villi samples were subjected to dHPLC analysis upon molecular characterization of the parental beta-globin alleles. Direct sequence analysis was used to validate each result, showing an accuracy rate of 100% for dHPLC. Overall, our protocol was able to identify the responsible mutations in all 96 analyzed subjects (including 12 prenatals in at-risk pregnancies), detecting the eight most common mutations in Southern Italy. Three rare mutations (one of which, reported here for the first time) that standard mutation detection methods failed to reveal, were also identified. dHPLC assay proved to be a reliable, rapid, and sensitive method for detecting both common and rare mutations within the beta-globin gene. Because of this property our protocol has the potential to be implemented for mutational screening in different areas of high prevalence for beta-thalassemia.

Chorionic Villi Sampling↗
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

Reliability of DHPLC in mutational screening of beta-globin (HBB) alleles.

The inherited disorders of hemoglobin represent the most common Mendelian disease worldwide, with a higher prevalence among Mediterraneans, Asians, Africans, and Indians. Altered beta-globin sequences, causing either hemoglobinopathies or beta-thalassemia syndromes, are due to more than 200 different mutations in the beta-globin gene. Prevention programs based on postnatal and prenatal molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations. We have developed a rapid and highly specific mutation screening test based on the denaturing high-performance liquid chromatography (DHPLC) system. The sensitivity and specificity of the method were tested on the full genomic region of the beta-globin gene in 30 normal Italian subjects and 40 heterozygous carriers in which 25 different beta-globin mutations had been previously characterized by multiplex-ARMS technique. The results showed DHPLC to be 100% sensitive and specific. All the 25 sequence alterations and two previously undetected polymorphisms were precisely identified with neither false positive nor false negative results. In addition, 12 compound heterozygous and four homozygous patients were successfully subjected to DHPLC. Overall, the method was able to rapidly identify the most common beta-globin mutations, accounting for more than 97% of beta-globin alleles in the Italian population. Compared to classical approaches of mutation screening, this method allows a rapid, highly sensitive, cost-effective, and semi-automated simultaneous mutational scanning of a large number of samples.

Alleles↗