Biomedical subjects

Maria Cristina Rosatelli

Publications and source records attributed to Maria Cristina Rosatelli.

10 recordsLinked to original sources

PubMed2006

Spinal muscular atrophy genotyping by gene dosage using multiple ligation-dependent probe amplification.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction-restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.

Adult↗

PubMed2006

Thalassaemia-like carriers not linked to the beta-globin gene cluster.

This study describes the largest series reported to date, of individuals belonging to unrelated families carrying a beta-thalassaemia-like phenotype in whom the beta-globin gene was found to be structurally intact by sequence analysis. This genetic determinant appears haematologically heterogeneous, displaying either a silent beta-thalassaemia-like phenotype or a typical beta-thalassaemia carrier-like phenotype in different families. Compound heterozygosity for both beta-thalassaemia-like determinant and typical beta-thalassaemia allele resulted either in thalassaemia intermedia or thalassaemia major. By linkage analysis both the silent and the typical beta-like determinants were found not to be linked to the beta-globin cluster. Sequence analysis of the hypersensitive site cores of locus control region and of the genes coding for the transcription factors erythroid Kruppel-like factor and nuclear factor (erythroid-derived 2) were normal. beta-globin mRNA levels determined by real-time polymerase chain reaction were reduced in both types of beta-like carriers. These results indicate the existence of causative genetic determinants not yet molecularly defined, but most likely, resulting from either the reduction or loss of function of a gene coding for unknown transcriptional regulator(s) of the beta-globin gene. The knowledge of these rare beta-thalassaemia-like determinants have implications for clinical and, especially, prenatal diagnosis of beta-thalassaemia.

Alleles↗

PubMed2006

A new insertion/deletion of the cystic fibrosis transmembrane conductance regulator gene accounts for 3.4% of cystic fibrosis mutations in Sardinia: implications for population screening.

Previous studies performed on Sardinian patients affected by cystic fibrosis (CF) have led to the identification of molecular defects in 87 of 88 patients. Two mutations, the F508del and T338I, were quite prevalent and accounted for 50% and 20% of the molecular defects, respectively. T338I has been detected rarely in other populations, most likely because of the genetic isolation of Sardinians. In the present study, we have performed a molecular analysis of the CF gene in eight Sardinian patients in whom only a single mutation has been defined. Using DNA analyses (Southern blot, single nucleotide polymorphisms, microsatellite analyses, and Extra-Long polymerase chain reaction) selected to detect gross gene rearrangement and by using mRNA studies, we detected a novel mutation c.54-5811_164 + 2186del8108ins182 in six of the eight patients investigated. This mutation consists of a gross deletion of 8108 bp spanning exon 2 with an insertion of 182 bp at the deletion junction, between nucleotide 54-5811 of intron 1 (IVS1 nt16864) and nucleotide 164 + 2186 of intron 2 (IVS2 nt 2186). By including the novel mutation in our mutation panel we are now able to reach a 95% detection rate, thereby improving the process of carrier detection and genetic counseling in Sardinia.

Alleles↗

PubMed2005

Immunophenotypic characterisation of peripheral blood lymphocytes in autoimmune polyglandular syndrome type 1: clinical study and review of the literature.

Autoimmune endocrinopathies are characterised by an increased number of peripheral blood lymphocytes (PBL) expressing activation/ memory markers on their surface. The aim of this study was to determine whether a similar finding could be detected in a group of 11 paediatric and young adult patients suffering from autoimmune polyglandular syndrome type 1 (APS1), also called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), as very few data have previously been reported in this field. The control group was made up of 11 sex- and age-matched healthy subjects. Fifteen lymphocyte subsets were compared, in terms of percentage and absolute number, and statistical analysis was performed by the Mann-Whitney test. Measurement of T (CD3+), B (CD19+), natural killer (NK, CD3-CD16/56+), CD4+ and CD8+ T lymphocytes showed that patients with APS1 had a higher percentage and absolute count of T lymphocytes: this was entirely due to the statistically larger CD3+CD4+ fraction. Patients with APS1 also had slightly fewer B and NK lymphocytes, but the difference was negligible. Comparison of CD4+ subpopulations bearing activation and naive/memory antigens (marked by CD69, CD25, anti-HLA-DR, CD45RA and CD45RO) showed that patients with APS1 had generally larger percentages and absolute counts of these subsets: however, only the percentage and absolute size of the CD4+CD25+ subset (p = 0.0354 and p = 0.0151, respectively), and the absolute number of the CD4+ anti-HLA-DR+ and CD4+ CD45RO+ subsets (p = 0.0193 and p = 0.0209, respectively) were significantly higher. Interestingly, patients with APS1 also had significantly fewer CD8+CD11b+ and CD3-CD8+ cells. In conclusion, PBL distribution in APS1 resembles that of other autoimmune diseases. Further studies are needed to confirm and possibly extend these data.

Adolescent↗

PubMed2004

Preimplantation genetic diagnosis for beta-thalassaemia: the Sardinian experience.

OBJECTIVES: To report the experiences on preimplantation genetic diagnosis (PGD) in couples at risk for beta-thalassaemia in Sardinia. METHODS: 23 couples at risk for beta-thalassaemia were included in the PGD programme with a total of 42 cycles performed. Among these, 11 couples were fertile, while the remaining 12 had associated fertility problems. In vitro Fertilization (IVF), PGD and prenatal genetic molecular confirmation protocols and results are reported. RESULTS: All the patients followed the protocol of ovarian stimulation, oocyte retrieval, intracytoplasmic sperm injection (ICSI), embryo biopsy and genetic analysis. A total of 272 oocytes were fertilized in the regular way, and embryo biopsy was performed on 202 embryos. Out of these 202 embryos, 192 (95%) were successful. The genetic diagnosis was performed on 150 embryos (78.1%). Ninety-eight were identified as unaffected and 75 were transferred in 31 cycles. In the infertile patient group, two biochemical pregnancies (11.1% per transfer), in the fertile patient group, four clinical pregnancies, two twin and two singleton pregnancies (30.8% per transfer), were obtained. The genetic molecular results were confirmed in all pregnancies by first-trimester chorionic villus sampling (CVS). CONCLUSION: Our study shows that PGD for beta-thalassaemia is an available procedure for couples who wish to avoid termination of pregnancy, except in cases where the IVF cycle efficiency is very poor.

Biopsy↗

PubMed2004

A novel silent beta-thalassemia mutation in the distal CACCC box affects the binding and responsiveness to EKLF.

The silent beta-thalassemia mutation, beta(+)-101C-->T, is the only mutation currently described in the distal beta-globin CACCC box. We present a novel mutation, a C-->G transversion, in the same position. Expression analysis in heterozygous subjects demonstrated that the mutation determines a 20% reduction in the output of the beta-globin gene. DNA-protein interaction and transactivation analysis correlated the decrease in the beta-globin synthesis with the reduced binding and transactivation of EKLF to the mutant promoter. These data predict that the beta-101C-->G mutation will display a silent thalassemia phenotype similar to that of the beta-101C-->T mutation.

DNA-Binding Proteins↗

PubMed2004

Beta-thalassemia microelectronic chip: a fast and accurate method for mutation detection.

BACKGROUND: beta-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the beta-thalassemia alleles in the Mediterranean area. METHODS: We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, -87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes. RESULTS: Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip. CONCLUSIONS: The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing beta-thalassemia in other regions of the world.

Fluorometry↗

PubMed2003

Fetal HLA typing in beta thalassaemia: implications for haemopoietic stem-cell transplantation.

Stem-cell transplantation can cure beta thalassaemia. We aimed to assess whether fetal HLA typing done early in the pregnancy of couples who were at risk of beta thalassaemia could provide an alternative to pregnancy termination if the prospect of a bone-marrow transplantation from a family member was available. In our clinic in Sardinia, we did fetal HLA typing for 49 couples at risk of having a baby with beta thalassaemia. Two affected children were born and successfully received a transplantation from a family donor. Five non-affected fetuses were HLA compatible with an affected sibling and their cord blood was harvested for a future transplantation.

Fetal Diseases↗

PubMed2002

Screening for thalassemia: a model of success.

Programs of prospective carrier screening and genetic counseling for beta-thalassemia among couples planning marriage, preconception, or during early pregnancy are ongoing in several at-risk populations in the Mediterranean area, including Greeks, Greek Cypriots and Continental Italians. Carrier detection is carried out by haematological analysis followed by mutation detection by DNA analysis. Once carrier couples are identified, prenatal diagnosis is accomplished by mutation analysis on PCR amplified DNA from chorionic villi. These programs have been very effective, due to education programs and subsequent acceptance of screening. Future prospects include automation of the process of mutation detection by microchips analysis, introduction of preconception and preimplantation diagnosis and hopefully fetal diagnosis by analysis of fetal cells in maternal circulation.

DNA Mutational Analysis↗

PubMed2002

Delineation of the molecular defects in the AIRE gene in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients from Southern Italy.

In this study, we have carried out molecular analysis of the AIRE (autoimmune regulator) gene in 11 patients (from 8 families) affected by autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, originating from a restricted area of Southern Italy (the Salento peninsula in Puglia). Of the 16 mutant AIRE alleles from the 8 probands studied, 12 carried a missense mutation (W78R in 9, P539L in 2, and P252L in 1), 2 carried the Q358X nonsense mutation, and 2 carried the 1058delT frameshift mutation. All these mutations except the 1058delT are novel. Each of the detected mutations either predicts a premature termination of the protein or results in a nonconservative amino acid change, most likely adversely affecting the function of the protein. The W78R missense mutation is relatively common in these patients, having been detected (in homozygosity or compound heterozygosity) in 6 of the 8 probands tested, indicating the presence of a founder effect. The results of this study contribute to the delineation of the molecular pathology of the AIRE gene and enhance our ability to perform a molecular diagnosis in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients from Southern Italy.

Adolescent↗