Prenatal diagnosis of most common Mediterranean beta-thalassaemia mutants.
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Chorionic Villi↗
Biomedical subjects
M Pirastu
Publications and source records attributed to M Pirastu.
The prenatal diagnosis of thalassaemia.
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DNA↗
Molecular pathology of haemoglobin H disease in Sardinians.
We investigated the molecular basis for haemoglobin H disease in 50 Sardinian patients by restriction endonuclease analysis. We found that the majority (78% of the cases) are due to gene deletion (- -/- alpha). Among those with a combination of deletion and nondeletion defects (- -/alpha alpha th), the most prevalent nondeletion lesion (70% of the nondeletion defects) was the initiation codon mutation of the alpha 2 gene (alpha Nco alpha), previously discovered in this population. Of the remaining patients with the (- -/alpha alpha th) genotype, two showed the IVS-1 splice junction lesion and one a mutation in the alpha 1 gene, removing the Nco I site within the 5' part of the alpha 1 gene, which may arise from a process of gene conversion from the initiation codon mutant of the alpha 2 gene. A single patient had the homozygous state for the initiation codon mutant of the alpha 2 gene. Study of genotype-phenotype correlations indicates that the (alpha Nco alpha) haplotype is associated with a more severe defect in the alpha-globin chain output than that resulting from the (-alpha) haplotype. We may conclude that restriction endonuclease analysis is a powerful method for the definition of the molecular heterogeneity of haemoglobin H disease.
Adolescent↗
Prenatal diagnosis of inherited hemoglobinopathies.
In this paper we reviewed the different methods presently available for prenatal diagnosis of hemoglobin disorders and the impact of this technology in the control of beta-thalassemia in several Mediterranean populations. The vast majority of the inherited hemoglobinopathies can now be detected in the fetus by amniocyte or trophoblast DNA analysis. alpha-thalassemias, delta beta-thalassemias and gamma delta beta-thalassemias, which are usually caused by a gross structural rearrangement of the DNA, may be directly detected by Southern blot analysis. Only a few beta-thalassemia lesions are caused by gene deletion or affect a restriction recognition site and thus may be directly identified by this method. The major part of beta-thalassemia are due to single nucleotide substitution, small deletion or addition which do not alter a restriction recognition site. These mutations may be directly detected by complementary oligonucleotide probes. Alternatively, when normal or affected children are available, fetal diagnosis may be accomplished by linkage analysis with polymorphic restriction sites. Fetal blood analysis is used at present time for those cases presenting too late in the pregnancy for characterization of the molecular defect and in prospective parents in whom the defect is not known. Introduction of prenatal diagnosis in combination with carrier screening in several mediterranean populations led to a consistent reduction in the incidence of homozygous beta-thalassemia.
Base Sequence↗
Antenatal diagnosis of thalassemia major in Sardinia.
In this report, we summarized our experience, carried out in Sardinia, with antenatal diagnosis in one thousand pregnancies in which the fetus was at risk for homozygous beta-thalassemia. In the majority of these cases, the thalassemia lesion segregating in the family was the nonsense mutation at the codon corresponding to amino-acid 39. At the outset (976 cases) we used globin chain synthesis analysis by column chromatography on fetal blood obtained by placental aspiration, and recently (24 cases) we employed the synthetic oligonucleotide method on amniocyte DNA. Apart from 126 pregnancies still in progress, in all the other cases the diagnosis has been confirmed. In the majority of the cases (99%), we obtained sufficient fetal blood for the analysis. The fetal mortality associated with placental aspiration was 6.1%. The biochemical analysis gave reliable results. We had two misdiagnoses (0.2%): one due to a nonglobin protein comigrating with the beta chains and the other for a misclassification of the type of thalassemia segregating in the family. The oligonucleotide method gave clear-cut results in all the cases tested. The method was sensitive enough to detect the mutation directly in the DNA isolated from 20-25 ml of amniotic fluid in 75% of the pregnancies tested. In one case, we successfully employed this method for the analysis of the DNA isolated from chorionic villi. The oligonucleotide method seems to be the best procedure for monitoring the pregnancies at risk for beta-thalassemia in places where one or a few beta-thalassemia lesions are prevalent.
Amnion↗
beta (+)-Thalassaemia in the Po river delta region (northern Italy): genotype and beta globin synthesis.
Six beta(+)-thalassaemic patients from the Po river delta region have been studied. Using synthetic oligonucleotides as specific hybridisation probes, the beta(+) IVS I mutation (G----A at position 108) was demonstrated. This lesion and the enzyme polymorphism pattern in the subjects examined are the same as have been described for other Mediterranean beta(+)-thalassaemias. Antenatal diagnosis through DNA analysis of beta(+)-thalassaemia is therefore possible. The production of beta globin in a beta(+), homozygote and in a beta (+), beta(0) 39 (nonsense mutation at codon 39) double heterozygote is approximately 20% and 10% respectively of total non-alpha globin synthesis. Despite some overlapping of the results, similar beta globin synthesis levels have been obtained in 43 beta(+)-thalassaemia patients. This suggests that in the Po river delta region the most common thalassaemic genes are beta(0) 39 and beta(+) IVS I.
Autoradiography↗
A distant gene deletion affects beta-globin gene function in an atypical gamma delta beta-thalassemia.
We describe an English family with an atypical gamma delta beta-thalassemia syndrome. Heterozygosity results in a beta-thalassemia phenotype with normal hemoglobin A2. However, unlike previously described cases, no history of neonatal hemolytic anemia requiring blood transfusion was obtained. Gene mapping showed a deletion that extended from the third exon of the G gamma-globin gene upstream for approximately 100 kilobases (kb). The A gamma-globin, psi beta-, delta-, and beta-globin genes in cis remained intact. The malfunction of the beta-globin gene on a chromosome in which the deletion is located 25 kb away suggests that chromatin structure and conformation are important for globin gene expression.
Adult↗
Initiation codon mutation as a cause of alpha thalassemia.
Cloning and sequence analysis of the alpha-globin genes from a Sardinian patient with the nondeletion type of hemoglobin-H disease revealed a new type of thalassemia lesion. A mutation in the alpha 2-globin gene changes the initiation codon ATG to ACG and abolishes the function of this gene. Globin mRNA output from the affected alpha 2 locus is decreased relative to the alpha 1 locus. The mutation is detectable in genomic DNA by restriction analysis with the enzyme NcoI. Of the seven Sardinian patients with nondeletion alpha thalassemia screened with this enzyme, six had the initiation codon lesion.
Base Sequence↗
Multiple mutations produce delta beta 0 thalassemia in Sardinia.
In Sardinia the common form of beta thalassemia is a beta 0 thalassemia due to a nonsense mutation at codon 39. delta beta 0 Thalassemia is rare in Sardinia and is associated with increased production of hemoglobin F of the A gamma type. In this study we used a synthetic oligomer assay and detected the beta 39 nonsense mutation on the delta beta 0 thalassemia chromosome. Hence at least two different mutations have occurred on this chromosome; one that increases A gamma globin synthesis and another that silences the beta globin gene.
Chromosomes, Human↗
Longitudinal study of a newborn with a combination of deletion and nondeletion alpha-thalassemia-2.
In this study we have characterized by DNA analysis the molecular basis of an alpha-thalassemia condition found in an infant, with 16% Hb Bart's at birth, who developed an hematologic picture similar to the alpha-thalassemia carrier state. Restriction endonuclease analysis and hybridization with alpha and zeta specific probes have provided strong evidence that this patient carries a genetic compound of deletion alpha-thalassemia-2 lesion (-alpha) and a non-deletion defect [(alpha alpha)th] with both alpha-structural genes intact on chromosome 16. He inherited the deletion alpha-thalassemia-2 chromosome (-alpha) from the father and the chromosome with non-deletion alpha-thalassemia defect from the mother. Because the deletion of one, two, or three alpha-globin structural genes is associated with 1-2%, 5-6%, or 25%, Hb Bart's respectively, these findings suggest that the non-deletion chromosome [(alpha alpha)th] contains two alpha-globin structural genes that are less active than a single alpha gene (-alpha).
Child, Preschool↗
Prenatal diagnosis of beta-thalassemia. Detection of a single nucleotide mutation in DNA.
We investigated a method employing synthetic oligonucleotides for the prenatal diagnosis of beta-thalassemia due to a single nucleotide mutation. The beta 0 thalassemia we tested is produced by a nonsense mutation and is commonly found in Sardinia and other parts of the Mediterranean. In this DNA lesion, the glutamine codon CAG at the beta 39 position is mutated to TAG, which results in a stop codon and premature termination of the beta-globin chain. We synthesized two oligonucleotides: one homologous to the normal beta A gene and the other to the beta 0 thalassemia gene at the beta 39 location. The oligonucleotides were labeled with 32P and used as hybridization probes for normal and thalassemic DNA. The beta A probe hybridized only to the normal DNA, and the beta-thalassemia probe only to thalassemic DNA, thus providing a technique for direct demonstration of the mutation. The method is sensitive enough to be applied directly to DNA that is isolated from uncultured cells obtained from only 20 ml of amniotic fluid as early as the 16th gestational week.
Amniocentesis↗
Phenotype-genotype correlation in haemoglobin H disease in childhood.
In this study we used restriction endonuclease mapping to characterise the molecular defect responsible for haemoglobin H disease in 14 Sardinian children. The resulting genotypes were then correlated with the respective clinical and haematological phenotypes. We found that patients with the combination of non-deletion alpha(+)-thalassaemia [(alpha alpha)th] and deletion alpha(0)-thalassaemia (-Med) have a more severe phenotype than that resulting from the interaction of deletion alpha(0)-thalassaemia (-Med) and alpha(+)-thalassaemia (-alpha) determinants. Clinically, presentation was earlier and with moderate anaemia or haemolytic crisis, enlargement of the liver and spleen, and thalassaemic bone changes. Haematologically, the anaemia was more severe and there were higher bilirubin levels, reticulocyte counts, Hb H levels, and percentage of red blood cells with inclusion bodies. These results suggest that in those Hb H disease patients with the non-deletion [(alpha alpha)th] determinant, two alpha globin genes produce fewer alpha globin chains than a single alpha globin locus.
Adolescent↗
Hemolytic disease of the newborn caused by a new deletion of the entire beta-globin cluster.
We describe a new type of gamma delta beta-thalassemia in four generations of a family of Scotch-Irish descent. The proposita presented with hemolytic disease of the newborn, which was characterized by a microcytic anemia. Initial restriction endonuclease analysis of the DNA showed no grossly abnormal patterns, but studies of polymorphic restriction sites and gene dosage revealed an extensive deletion that removed all the beta- and beta-like globin genes from the affected chromosome. In situ hybridization of chromosome preparations with radioactive beta-globin gene probes showed that only one 11p homolog contained the beta-globin gene cluster in the affected family members.
Adult↗
Phenotypic effect of heterozygous alpha and beta 0-thalassemia interaction.
In this study, we carried out restriction endonuclease mapping in order to characterize the alpha-globin genotype of 10 Sardinian beta 0-thalassemia heterozygotes, all of whom presented with normal red blood cell indices and increased HbA2 levels. In 8 of these subjects, we found the deletion of two alpha-globin genes (-alpha/-alpha), and in the remaining two the deletion of a single alpha-globin gene (-alpha/alpha alpha). In three of these carriers with the (-alpha/-alpha) alpha-globin genotype and in one with the (-alpha/alpha alpha) genotype, we also found the glucose-6-phosphate dehydrogenase (G6PD) defect of the Mediterranean type. On the basis of these findings, we may conclude that the interaction of heterozygous beta 0-thalassemia with alpha-thalassemia, due to the deletion of either one or two alpha-globin genes, may lead to the production of red blood cells with normal indices. The association of the G6PD defect with this thalassemia gene complex may eventually contribute to this effect. We suggest, therefore, that screening programs for heterozygous beta-thalassemia in populations where alpha-thalassemia is also prevalent, should incorporate the determination of HbA2 in the first set of tests.
DNA↗
Delta +-thalassemia in Sardinia.
We have defined a new type of delta-thalassemia in which beta-globin chain synthesis is incompletely suppressed. Homozygotes have unusually low HbA2 levels, and double heterozygosity for this delta-thalassemia gene and beta-thalassemia normalizes the HbA2 level. The delta-thalassemia occurs on a chromosome that is identifiable using polymorphic restriction endonuclease sites. We call this condition delta +-thalassemia, to distinguish it from the previously described delta 0-thalassemia syndromes in which no delta-globin chain synthesis occurs.
Chromosome Mapping↗
[Clinical and immunological study on 4 pediatric patients with chronic mucocandidiasis associated with hypoparathyroidism].
Four paediatrics patients affected by Chronic Mucocutaneous Candidiasis associated with hypoparathyroidism have been studied. In vivo and in vitro responses to Candida albicans antigens and to other common antigens (Dermatophytin T, Varidase), the percentage of T and B cells and the response to the selective polyclonal phytomitogens (PHA, ConA, PWM) have been investigated in order to quantitate and functionally characterize the B and T lymphocyte classes. The HLA A an B typing has been also performed. Our results show a remarkable and most likely primitive impairment of the T cells, unresponsive either to the C. albicans and the two other skin test antigens or to the selective polyclonal mitogens in vitro. The infectious disease (Mucocandidiasis) is most likely secondary to the T-cell failure. The association between the Mucocandidiasis, hypoparathyroidism and the T-cell deficiency is probably due to a common primitive disembryogenetic defect.
Antigens, Fungal↗
Alpha-thalassemia in two Mediterranean populations.
We used restriction endonuclease analysis to determine the incidence of alpha-thalassemia in two Mediterranean islands. In a random population sample, the gene frequency of deletion-type alpha-thalassemia-2 (-alpha) was 0.18 in Sardinians and 0.07 in Greek Cypriots. All cases were the rightward crossover type. From these frequencies and the known incidence of hemoglobin-H disease in these populations, we calculated the frequency of the alpha-thalassemia-1 genotype (--) and determined that it was low. We also found that beta-thalassemia homozygotes in sardinia have a higher incidence of alpha-thalassemia than normals and beta thalassemia heterozygotes because a significantly greater number of these homozygotes are also homozygous for the alpha-thalassemia-2 lesion. These findings support the theory that coinheritance of alpha-thalassemia mitigates the severity of beta-thalassemia and suggest that the protection is most pronounced when two alpha-globin genes are deleted.
Cyprus↗
Clinical presentation of thalassemia major due to homozygous beta (0)-thalassemia.
This study concerns the clinical presentation of all thalassemia major or intermedia in children admitted to out genetics department from January 1978 to December 1979. At electrophoresis and/or globin chain synthesis analysis on column chromatography. All these patients were shown to have homozygous beta(0)-thalassemia. The clinical severity was highly variable, dependent on age at presentation and when the patient first became dependent on transfusions. On the main reasons for this variability may be associated with thalassemia 1 or alpha-thalassemia 2 which have an incidence figure of 13 % in our population and hence also in these patients. Remarkable findings of the clinical and hematological picture were: (a) early spleen enlargement and growth deficiency; (b) MCV and MCH values intermediate between normals and beta-thalassemia heterozygotes; (c) normal mean hemoglobin A2 percentage and corpuscular concentration; (d) frequently increased transferrin saturation and ferritin levels; (e) presentation at age greater than 2 with hemoglobin levels about 9 g/dl usually associated with a milder course.
Adolescent↗