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M Pirastu

Publications and source records attributed to M Pirastu.

At least 73 records · Page 4Linked to original sources
PubMed1989

Prenatal diagnosis of beta-thalassaemia in Mediterranean populations by dot blot analysis with DNA amplification and allele specific oligonucleotide probes.

In this study, we describe a simple strategy to detect beta-thalassaemia mutations in prospective parents and to make prenatal diagnosis in pregnancies at risk in the Mediterranean population. Screening of prospective parents is carried out by dot blot analysis on enzymatically amplified DNA with a set of oligonucleotide probes complementary to the most common mutations in this population. Prenatal diagnosis is accomplished by the same procedure on enzymatically amplified amniocyte or trophoblast DNA. The main advantages of this procedure are the simplicity, sensitivity (0.05 micrograms of DNA), and rapidity (12-24 h). Further simplification is obtained by amplification of the DNA from crude amniotic cell lysate. The very low amount of fetal material necessary for this analysis eliminates the need to culture amniotic fluid cells and may decrease the fetal loss rate associated with trophoblast sampling. The number of specific DNA sequences obtained by the amplification procedure allowed us to use non-radioactive labelled oligonucleotide probes, which have several advantages compared to radioactive probes.

Alleles↗
PubMed1989

Prenatal diagnosis of beta thalassaemia based on restriction endonuclease analysis of amplified fetal DNA.

In the Mediterranean area, 50% of the beta thalassaemia mutations abolish or create a restriction endonuclease site in the beta globin gene. This study describes a new procedure for prenatal detection of these beta thalassaemia defects based on the direct visualisation, on an ethidium bromide stained polyacrylamide gel, of the discrete DNA fragments produced by restriction endonuclease digestion of fetal DNA, enzymatically amplified using the DNA polymerase from the thermophilus bacterium Thermus aquaticus. We applied this procedure to the Sardinian population to detect the nonsense mutation at codon 39 and the frameshift at codon 6 of the beta globin gene; these are the most frequent beta thalassaemia mutations in this population, accounting for 95% and 2.2% of the beta thalassaemia chromosomes. The main advantages of this procedure are simplicity (no radioactivity), sensitivity (0.2 microgram of DNA), and rapidity (12 hours). The very small amount of fetal material required makes amniotic fluid cell culture unnecessary and may decrease the fetal loss rate associated with trophoblast sampling. By circumventing the use of radioactive and non-radioactive probes, the spread of this technology to the high risk areas will be facilitated.

Amniocentesis↗
PubMed1988

The peculiar spectrum of beta-thalassemia genes in Tunisia.

We have determined the spectrum of mutations producing beta-thalassemia (beta-thal) in Tunisia by direct DNA analysis using hybridization with allele-specific oligonucleotide probes and restriction endonuclease assay. In all, 34 unrelated beta-thal patients originating from different parts of the country were available for study. The beta-globin gene cluster of each was subjected to haplotype analysis, and on the basis of this analysis, we tested each patient's DNA for one or more mutations previously shown to be linked to that haplotype. We identified four previously unreported haplotypes and found that this population differs from others in Mediterranean areas in the frequency of the beta-thal haplotypes, the unexpected observation being the high frequency of haplotype IX. Six different point mutations were found, accounting for 62% of beta-thal genes in this Tunisian population. The molecular defects known to be the most frequent in Mediterraneans (nonsense codon 39, IVS1 nt 110, IVS1 nt 6) only make up 37% of the mutant genes. One as yet undescribed mutation (IVS1 nt 2 T----G) was identified by molecular cloning and sequencing. Our results should help the implementation of a prenatal diagnosis program for beta-thal in Tunisia.

Genetic Markers↗
PubMed1988

Beta-thalassemia mutations in the Portuguese population.

In this study we have carried out haplotype analysis on the beta-globin gene cluster and characterized the beta-thalassemia mutation by oligonucleotide hybridization in 14 patients with thalassemia major and 5 with sickle cell/beta-thalassemia originating from southern Portugal. We found that three mutations, namely the beta(0)-39, beta(0) IVS-1 nt 1 and beta(+) IVS-1 nt 110 are prevalent accounting for 53%, 32% and 10% of the beta-thalassemia chromosomes respectively. In general each mutation was associated with a specific chromosomal haplotype; the beta(0)-39 mutation, however, was linked to three different haplotypes. These results indicate that three oligo-probes complementary to the most common mutations allow prenatal diagnosis by oligonucleotide analysis in 96% of the couples at risk of having offspring with thalassemia major in southern Portugal.

Genotype↗
PubMed1988

Beta thalassaemia mutations in Turkish Cypriots.

Using oligonucleotide hybridisation or restriction endonuclease analysis, we have characterised the molecular defect in 94 patients with thalassaemia major and four with thalassaemia intermedia of Turkish Cypriot descent. We found that four mutations, namely beta+ IVS-1 nt 110, beta zero IVS-1 nt, beta+ IVS-1 nt 6, and beta+ IVS-2 nt 745 were prevalent, accounting for 69.9%, 11.7%, 8.7%, and 5.6% respectively of the beta thalassaemia chromosomes. This information may help in the organisation of a large scale prevention programme based on fetal diagnosis of beta thalassaemia by DNA analysis in the Turkish population.

Cyprus↗
PubMed1988

Sardinian multiple sclerosis is associated with HLA-DR4: a serologic and molecular analysis.

HLA haplotypes in 45 unrelated Sardinian multiple sclerosis patients and in six multiplex families were defined, using both serologic and restriction fragment length polymorphism (RFLP) analysis. In unrelated MS patients, we found an association with HLA-DR4 (p less than 0.01, relative risk = 2.5) and DQw3 (p less than 0.04, relative risk = 2.2). Using a beta-DR cDNA probe, we observed no variation of the DR4 RFLP profile in sporadic or related MS patients compared with DR4-specific pattern in controls. Using a beta-DQ cDNA probe, we identified two DQw3 patterns (DQw3.1 and DQw3.2) with similar frequency in patients and in controls. No specific RFLPs were observed in association with different disease courses. The frequency of haplotype sharing in affected members of multiplex families was not different from that expected by chance. This study shows that Sardinian MS patients carry predominantly the HLA-DR4 allele, in contrast to the DR2 prevalence reported in Caucasian populations. The lack of association with HLA haplotypes in affected members of multiplex families may indicate that genetic factors outside the HLA system play a substantial role in families with MS.

Adult↗
PubMed1988

Homozygous beta zero-39 mutation with thalassemia intermedia in northern Sardinia: clinical, hematological and molecular analysis.

In this study, we investigated the clinical and hematological features and carried out alpha- and beta-globin gene analyses in 11 Sardinian adult beta zero-thalassemia homozygotes from Northern Sardinia who were not transfusion-dependent. Oligonucleotide analysis revealed in nine out of 11 patients the nonsense mutation at codon 39, which was associated either with haplotype II or IX (14/16 and 2/16 chromosomes, respectively). Haplotype II was linked to the A gamma T mutation. The G gamma globin level ranged from 50 to 70%. Four out of nine patients (44%) were heterozygous and 3/9 (33%) homozygous for the rightward deletional type of alpha-thalassemia; two (22%) had the normal alpha-gene complement. Patients who were alpha-thalassemia homozygotes (-alpha/-alpha) showed a more balanced globin chain synthesis ratio. This study confirms that alpha-thalassemia may ameliorate the clinical picture of homozygous beta zero-thalassemia.

Adult↗
PubMed1988

Delineation of specific beta-thalassemia mutations in high-risk areas of Italy: a prerequisite for prenatal diagnosis.

In this study, we defined by haplotype characterization combined with oligonucleotide hybridization or direct restriction endonuclease analysis the specific beta-thalassemia mutations in a representative sample of beta-thalassemia chromosomes from patients with homozygous beta-thalassemia originating from different parts of Italy. We characterized the mutations in 90% of the thalassemia chromosomes and found that three mutations, namely the beta(+)IVS 1-110, beta (0)-39 and beta(+)IVS 1-6 are prevalent in the Italian population. Most of the patients investigated were compound heterozygotes for two beta-thalassemia mutations, and only a few were homozygotes for one mutant. On the basis of these findings, we predict that prenatal diagnosis in this population would be feasible in most cases by fetal DNA analysis with the oligonucleotide method using a limited number of oligonucleotide probes selected after screening parents for the most common beta-thalassemia mutations. We have also devised a method based on hybridization with a mixture of two oligonucleotides that allows rapid and simultaneous screening of prospective parents for the two most frequent mutations in Italians, the beta(+)IVS 1-110 and beta(0)-39 mutants. This method may be applicable to prenatal diagnosis in cases at risk for the genetic compound of these mutations.

DNA Mutational Analysis↗
PubMed1988

Delineation of the molecular basis of delta- and normal HbA2 beta-thalassemia.

In this study, we used cloning and sequence analysis to define the molecular defect in two delta-thalassemia genes, one associated with reduced output of delta-globin chains (delta +thal) from a Sardinian and the other with a complete suppression of delta-chain production from the affected locus (delta zerp thal) from a Southern Italian. Sequence analysis of the delta +thal gene showed a G----T substitution at the first nucleotide of codon 27 (delta +27) which produces an amino acid change (Ala----Ser) and presumably activates a cryptic splice site located at this position. Therefore, only a fraction of the transcript is processed from this site, as indicated by the clinical phenotype of delta +thal. DNA sequencing of the delta zero thal gene revealed a T----C substitution at position 1 of IVS-1, which abolishes the splicing at this site and thus leads to complete deficiency of normal mRNA explaining the clinical phenotype of delta zero thal. Oligonucleotide analysis was used to confirm the coinheritance of the delta +27 mutation in a group of Sardinians with thalassemia like phenotype and normal HbA2 level who, on the basis of genetic criteria, were supposed to be double heterozygous for delta-thalassemia and beta-thalassemia. The definition of delta-thalassemia defects in each high-risk area facilitates identification of double heterozygotes for delta- and beta-thalassemia by DNA analysis and may thus improve genetic counseling.

Base Sequence↗
PubMed1987

The same beta-globin gene mutation is present on nine different beta-thalassemia chromosomes in a Sardinian population.

The predominant beta-thalassemia in Sardinia is the beta 0 type in which no beta-globin chains are synthesized in the homozygous state. We determined the beta-thalassemia mutations in this population by the oligonucleotide-probe method and defined the chromosome haplotypes on which the mutation resides. The same beta 39(CAG----TAG) nonsense mutation was found on nine different chromosome haplotypes. Although this mutation may have arisen more than once, the multiple haplotypes could also be generated by crossing over and gene conversion events. These findings underscore the frequency of mutational events in the beta-globin gene region.

Chromosome Mapping↗
PubMed1987

Molecular characterization of a normal Hb A2 beta-thalassaemia determinant in a Sardinian family.

In this study we have carried out haplotype analysis at the beta-globin gene cluster and defined the beta-thalassaemia mutations in a large Sardinian family, ascertained through a proband with thalassaemia major, in which several members were carriers of a beta-thalassaemia allele characterized by microcytosis, hypochromia and normal Hb A2 levels (type 2 normal Hb A2 heterozygous beta-thalassemia). The proband was a compound heterozygote for the beta zero 39 and the beta + IVS-2, nt 745 mutations and all the beta-thalassaemia heterozygotes with normal Hb A2 showed the beta + IVS-2, nt 745 mutation, always associated with haplotype VII. Because of the consistent association of a specific beta-thalassaemia mutation and normal Hb A2 levels, we postulate that this beta-thalassaemia chromosome carries a delta gene (delta-thalassaemia) which is unable to increase the delta-globin output in response to beta-thalassaemia.

Child, Preschool↗
PubMed1987

An initiation codon mutation (AUG----GUG) of the human alpha 1-globin gene. Structural characterization and evidence for a mild thalassemic phenotype.

alpha-globin is encoded by two adjacent genes, alpha 1 and alpha 2. Recent evidence suggests that these genes are not equally expressed and that the alpha 2-globin gene encodes the majority of alpha-globin. This finding would predict that a thalassemic mutation of the alpha 2-globin gene would result in a more severe loss of alpha-chain synthesis than a similar mutation in the alpha 1-globin gene. In a previous study we described a nondeletion alpha-thalassemia defect in the alpha 2-globin gene resulting from an AUG----ACG initiation codon mutation. In the present study we describe a different initiation codon mutation, AUG----GUG, present in the alpha 1-globin gene. The alpha 1- and alpha 2-globin gene initiation codon mutations result in similarly lowered levels of encoded mRNA. Despite the similarity of these two mutations, the alpha 2 mutant results in a more severe loss of alpha-globin synthesis and a more severe clinical alpha-thalassemia phenotype than the corresponding alpha 1-globin gene mutation. This difference reflects the dominant role of alpha 2-globin gene in overall alpha-globin synthesis.

Base Sequence↗