A novel beta-thalassemia mutation (G-->A) at the initiation codon of the beta-globin gene.
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Base Sequence↗
Biomedical subjects
M C Rosatelli
Publications and source records attributed to M C Rosatelli.
A simple electrophoretic procedure for fetal diagnosis of beta-thalassaemia due to short deletions.
This study describes three couples at risk for homozygous beta-thalassaemia in which one of the partners carried a short deletion beta-thalassaemia defect. Detection of short deletions in trophoblast DNA was accomplished by the very simple procedure of non-denaturing polyacrylamide gel electrophoresis. This method may be applied to detect beta-thalassaemia mutations due to deletion or addition of more than two nucleotides.
Base Sequence↗
Molecular screening and fetal diagnosis of beta-thalassemia in the Italian population.
This paper reports our experience of molecular screening and fetal diagnosis of beta-thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [beta zero 39 (C----T); beta zero 6 (-A); beta+ -87 (C----G); beta+ IVSI nt 110 (G----A); beta zero IVSI nt 1 (G----A); beta+ IVSI nt 6 (T----C); beta zero IVSII nt 1 (G----A); beta+ IVSII nt 745 (C----G)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [beta zero 76 (-C); beta+ IVSI nt 5 (G----A); beta+ IVSI nt 5 (G----C); beta+ IVSI -1 (cod 30) (G----C); beta+ -87 (C----T), beta zero -290 bp del.; beta+ -101 (C----T)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the beta-globin gene (beta IVSII nt 850 -1 bp). In the remaining four cases, the beta-globin gene showed entirely normal sequences and the beta-globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of beta-thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of beta-thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.
Base Sequence↗
Promoter mutations producing mild beta-thalassaemia in the Italian population.
In this study we have investigated the molecular basis for a mild form of beta-thalassaemia in three patients of Italian descent. In two, belonging to different families and affected by a mild and late-presenting form of thalassaemia major, direct sequencing of amplified DNA detected a C----T substitution at position -87 of the beta-globin gene in the compound heterozygous state either with codon 39 nonsense mutation or beta +IVSI, nt 110 mutation. The -87 (C----T) mutation has been previously described, in combination with the beta +IVSI, nt 110 mutation, in a single patient with thalassaemia intermedia. Both our patients showed a more severe phenotype as compared to that resulting from compound heterozygosity for a severe beta-thalassaemia mutation and another promoter mutation (-87, C----G) at the same position. In the third patient with the thalassaemia intermedia phenotype, we detected a novel promoter mutation, consisting in a C----A substitution at position -86, in combination with the codon 39 nonsense mutation. The results of this study indicate that different nucleotide substitutions affecting the proximal CACCC box of the beta-globin gene in combination with severe beta-thalassaemia, produce a mild form of thalassaemia ranging in severity from thalassaemia intermedia to late-presenting thalassaemia major.
Adolescent↗
Molecular characterization of beta-thalassemia in the Sardinian population.
This study reports the molecular characterization of beta-thalassemia in the Sardinian population. Three thousand beta-thalassemia chromosomes from prospective parents presenting at the genetic service were initially analyzed by dot blot analysis with oligonucleotide probes complementary to the most common beta-thalassemia mutations in the Mediterranean at-risk populations. the mutations which remained uncharacterized by this approach were defined by denaturing gradient gel electrophoresis (DGGE) followed by direct sequence analysis on amplified DNA. We reconfirmed that the predominant mutation in the Sardinian population is the codon 39 nonsense mutation, which accounts for 95.7% of the beta-thalassemia chromosomes. The other two relatively common mutations are frameshifts at codon 6 (2.1%) and at codon 76 (0.7%), relatively uncommon in other Mediterranean-origin populations. In this study we have detected a novel beta-thalassemia mutation, i.e., a frameshift at codon 1, in three beta-thalassemia chromosomes. The DGGE procedure followed by direct sequencing on amplified DNA is a powerful approach for the characterization of unknown mutations in this genetic system. The results herein presented allowed an expansion of the applicability of prenatal diagnosis by DNA analysis, to all couples at risk for beta-thalassemia in our population.
Base Sequence↗
Population-based genetic screening.
A preventive genetic programme aimed to control beta-thalassemia in the Sardinian population is based on a combination of increased awareness of the population, carrier screening, genetic counselling and prenatal diagnosis. As a result, the registry of thalassemia major demonstrated a profound decline in the incidence of this disease from 1 per 250 to 1 per 1200 live births, with 90% of cases effectively prevented.
Adult↗
Antenatal diagnosis of beta-thalassemia in Sardinia.
This paper reviews the characteristics and the results of 15 years of experience with a preventive program, based on carrier screening and prenatal diagnosis, designed to control thalassemia major in the Sardinian population. The education of the population about thalassemia and the modalities for its prevention was accomplished via the mass media. Carrier screening was carried out voluntarily on couples of child-bearing age. Prenatal diagnosis was initially carried out by fetal blood analysis; since 1983, it has been done by DNA analysis on non-amplified or amplified DNA. Different chorionic villous sampling procedures have been used. Nowadays, we have adopted the transabdominal approach because, in our experience, it seems to be associated with a low risk (2%) of fetal mortality. At the present time, the beta-thalassemia mutations are detected directly by dot-blot analysis of amplified DNA with 32P- or horseradish peroxidase-labeled allele-specific oligonucleotide probes. Two oligonucleotide probes, one complementary to the codon-39 nonsense mutation, which accounts for 95.7% of the beta-thalassemia chromosomes in the Sardinian population, and the other complementary to the frameshift at codon 6, which is the second most common mutation in our population (2.1%), allow us to make prenatal diagnosis in the large majority of cases. Notwithstanding a careful dissection of maternal decidua from chorionic villi, co-amplification of maternal sequence was detected in 4 out of 425 cases tested by this procedure. In order to avoid this pitfall, the simultaneous amplification of highly polymorphic VNTR (variable number of tandem repeats) segments could be used. On the whole we have so far carried out 2711 prenatal tests: 1130 by fetal blood analysis, 1156 by oligonucleotide hybridization on electrophoretically separated DNA fragments, and 425 by dot-blot analysis on amplified DNA with allele-specific oligonucleotide probes. Two errors occurred by fetal blood analysis and none by DNA analysis. The incidence of thalassemia major declined from 1:250 live births in the absence of prevention to 1:1000 after the establishment of this program, indicating that carrier screening and prenatal diagnosis are effective means for preventing thalassemia major at the population level.
Female↗
Reliability of prenatal diagnosis of genetic diseases by analysis of amplified trophoblast DNA.
Dot blot analysis on enzymatically amplified trophoblast DNA with allele specific oligonucleotide probes is currently used for the prenatal diagnosis of single gene disorders characterised at the molecular level, such as the beta thalassaemias, phenylketonuria, sickle cell anaemia, and alpha 1-anti-trypsin deficiency. A potential problem with the use of this procedure is the co-amplification of maternal sequences, which may obscure the diagnosis in the fetus. To address this question, we carried out prenatal diagnosis of beta thalassaemia in 300 couples at risk by dot blot analysis on enzymatically amplified DNA with 32P or horseradish peroxidase labelled allele specific oligonucleotide probes. We verified the diagnosis obtained by this procedure with oligonucleotide hybridisation on electrophoretically separated non-amplified trophoblast DNA fragments. We detected no co-amplified maternal sequences, even with a faint signal, in the dot blot of trophoblast DNA from those fetuses diagnosed as normal or homozygotes, nor in those diagnosed as heterozygotes, who were born to parents carrying different mutations and had inherited the paternal mutation. These results indicate that, when careful dissection of trophoblast tissue from maternal decidua is carried out, amplification of chorionic villi DNA is not associated with amplification of maternal DNA sequences. We may thus conclude that dot blot analysis of trophoblast DNA is a very reliable procedure for prenatal diagnosis.
Chorionic Villi Sampling↗
Erythropoiesis following bone marrow transplantation from donors heterozygous for beta-thalassaemia.
This study shows a marked and protracted activation of HbF synthesis in homozygous beta.-thalassaemia patients transplanted from HLA identical siblings heterozygous for beta-thalassaemia, as compared to patients transplanted from normal donors. HbF synthesis in recipients was much higher in relation to the corresponding bone marrow donor values either normal or heterozygous for beta thalassaemia. gamma-chain synthesis and G gamma/A gamma ratio were also studied in peripheral blood BFU-E from recipients and their donors. BFU-E from donors heterozygous for beta-thalassaemia showed higher gamma chain synthesis as compared to normal donors. Peripheral blood BFU-E gamma/beta + gamma ratios and G gamma percentage were higher in recipients than in their corresponding donors both normal or heterozygotes. The marked and protracted reactivation of HbF synthesis in recipients of heterozygous beta-thalassaemia bone marrow most likely results from an increased erythropoietic stress on erythroid progenitors. In order to obtain adequate Hb levels heterozygous beta-thalassaemia bone marrow should produce more red blood cells to compensate for the low MCH. The magnitude of activation of HbF synthesis was very variable. This variability may result from inherited differences in the capacity of reactivation of HbF synthesis of red cell progenitors from heterozygous beta-thalassaemia under stressed erythropoiesis.
Blood Donors↗
Thalassemia intermedia resulting from a mild beta-thalassemia mutation.
We investigated the molecular basis for a mild phenotype in a group of patients with beta(+) thalassemia originating from Northern Sardinia by definition of the beta-thalassemia mutation, alpha-globin mapping and beta-globin haplotype determination. In nine patients, we detected the compound heterozygous state for the -87 promoter mutation and the codon 39 nonsense mutation; in one patient, we detected the combination of the codon 39 nonsense mutation and beta(+) IVS-1 nt 6 mutation. These patients were either nontransfusion dependent for survival or became transfusion dependent later. We did not detect the -87 promoter mutation in any of 115 thalassemia major patients originating from the same part of Sardinia, investigated as controls. Heterozygotes for the -87 promoter mutation showed statistically higher hemoglobin (Hb) levels and larger and better hemoglobinized RBCs as compared with heterozygotes for the codon 39 nonsense mutation. From these data, we conclude that the -87 promoter mutation is a mild thalassemia allele, able to produce a phenotype of intermediate severity even in combination with a beta(0)-thalassemia mutant. The coinheritance of alpha-thalassemia or the -++-- 5' subhaplotype in several cases may have contributed to development of the mild clinical picture. Characterization of the beta-thalassemia mutation in combination with alpha-globin mapping and haplotype analysis may allow a better estimate of the probability of a given clinical phenotype, thus permitting more accurate counseling.
Adolescent↗
Prenatal diagnosis of beta thalassaemia by oligonucleotide analysis in Mediterranean populations.
We have used four oligonucleotide probes and two restriction enzymes to detect the beta thalassaemia mutation in a group of 61 couples of Italian descent who were prospective parents. We have been able to define the beta thalassaemia mutation in both parents in 47 couples and in only one parent in 12 couples. Prenatal diagnosis was accomplished successfully either by amniocyte (two) or trophoblast (26) DNA analysis in 28 couples in which the pregnancy was in progress. These results indicate that direct identification of the mutation by oligonucleotide or restriction endonuclease analysis is a practical and useful method for prenatal diagnosis of beta thalassaemia in childless couples.
Amniocentesis↗
Chorionic villus sampling and acceptance rate of prenatal diagnosis.
In this paper, we compared the acceptance rate of fetal diagnosis for beta-thalassemia in three group of couples of Sardinian descent; the first counselled before DNA analysis was available, the second presenting after DNA analysis was introduced but too late for chorionic villus sampling and thus monitored by amniocyte DNA analysis and the third presenting within the first trimester after DNA analysis was introduced and thus in time for trophoblast DNA analysis. A higher proportion of couples from the latter group opted for fetal testing as compared to the 1st and 2nd group. These results indicate that in this population, introduction of 1st trimester diagnosis made prenatal testing acceptable to practically all counselled couples at risk.
Adult↗
Beta-globin haplotype and XmnI polymorphism at position G (gamma)-158 and HbF production in Fanconi's anemia.
BACKGROUND: Patients with aplastic anemia show to a variable degree an increase of the red blood cell volume and percentage of HbF. The extent of HbF reactivation in sickle cell anemia and thalassemia major is related to the presence of XmnI polymorphism at -158 G (gamma). In this study, we have investigated whether in Fanconi's anemia the increase of the HbF is also related to the XmnI polymorphism. METHODS: Restriction site polymorphisms in the beta-globin gene cluster were analyzed to define the beta-globin haplotype. The presence of a C --> T substitution at position -158 G (gamma) was investigated by XmnI digestion. RESULTS: We found that patients with the XmnI site at -158 G (gamma), which was contained either in the 5' -+-++ or in the rare -+--- sub-haplotype, tend to have higher HbF and MCV values. The differences between XmnI positive and XmnI negative patients were highly significative (p less than 0.0025) for the MCV values, but barely significant for HbF levels (p less than 0.05). CONCLUSIONS: Our results suggest that in Fanconi's anemia both the extent of HbF reactivation and the fetal-like erythropoiesis, which is responsible for high MCV, are at least partially related to the beta-globin haplotype.
Erythropoiesis↗
Prenatal diagnosis of beta-thalassemia in Italian population.
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Female↗