Teresa Tuveri

PRENATAL DIAGNOSIS OF BETA-THALASSAEMIA WITH THE SYNTHETIC-OLIGOMER TECHNIQUE

103 couples attending the antenatal clinic in Sardinia were screened for the beta o-39 (nonsense) mutation, which codes for beta-thalassaemia, with the oligonucleotide method. In 94 couples both members had the beta o-39 mutant and thus were eligible for antenatal testing with this method. These pregnancies were monitored with amniocentesis (61) or trophoblast biopsy (33). Prenatal diagnosis in those monitored with amniocentesis was carried out with DNA analysis of uncultivated amniocytes (19) or cultivated cells (38). In 4 pregnancies results were unsatisfactory, and prenatal diagnosis was repeated with fetal-blood analysis. Trophoblast biopsy was unsuccessful in 1 pregnancy and gave a misdiagnosis in another because of maternal contamination. In the latter case the genotype of the fetus was established with amniocyte DNA analysis and globin-chain-synthesis studies.

Molecular screening and fetal diagnosis of β-thalassemia in the Italian population

SummaryThis paper reports our experience of molecular screening and fetal diagnosis of β-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 [β∘39 (C→T); ∘6 (-A); β+-87 (C→G); β+ IVSI nt 110 (G→A); β∘IVSI nt 1 (G→A); β+ IVSI nt 6 (T→C); β∘ IVSII nt 1 (G→A); β+ 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 [β∘76 (-C); β+ IVSI nt 5 (G→A); β+ IVSI nt 5 (G→C); β+ IVSI -1 (cod 30) (G→C); β+-87 (C→T), β∘-290 bp del.; β+-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 β-globin gene (β IVSII nt 850-1 bp). In the remaining four cases, the β-globin gene showed entirely normal sequences and the β-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 β-thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of β-thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.

Beta thalassaemia mutations in Sardinians: implications for prenatal diagnosis.

In this study we have characterised by oligonucleotide hybridisation and direct restriction endonuclease analysis the beta thalassaemia mutation in 494 Sardinian beta thalassaemia heterozygotes. The most prevalent mutation, accounting for 95.4% of the cases, was the nonsense mutation at codon 39. The remainder, in decreasing order of frequency, were a frameshift at codon 6 (2.2%), beta + IVS-1, nt 110 (0.4%), and beta + IVS-2, nt 745 (0.4%). This information allows prenatal diagnosis by DNA analysis to be made in the great majority of Sardinian couples at risk for beta thalassaemia.

Hematological phenotype of the double heterozygous state for alpha and beta thalassemia.

In this study, we have correlated the hematological phenotype of 56 Sardinian beta o-thalassemia heterozygotes with their alpha-globin genotype as defined by restriction endonuclease mapping. We found that the coinheritance of the deletion of one alpha-globin and, more obviously, two alpha-globin genes tend to normalize the thalassemia-like hematological phenotype commonly associated with the beta o-thalassemia carrier state. On the other hand, the association of the deletion of three alpha-globin genes caused a more severe phenotype. By globin chain synthesis analysis, those beta o-thalassemia heterozygotes with the (-alpha/alpha alpha) alpha-globin genotype had less deficiency of beta-chain synthesis than did those with the normal alpha-globin genotype (alpha alpha/alpha alpha). In heterozygotes with the (-alpha/-alpha) and in those with the (--/-alpha) alpha-globin genotype the imbalance was actually reversed with a mild or marked alpha-chain synthesis excess respectively.

Antenatal Diagnosis of β-Thalassemia in Sardiniaa

: 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.

Fetal HLA typing in β 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.

β° Thalassemia Trait in Sardinia

The red cell indices and results of globin chain synthesis in peripheral blood of obligate β° thalassemia (β° thal) carriers (parents of hamozygous β° thal children) and β thalassemia (β thal) carriers identified during mass screening are reported. Red cell indices were similar in obligate β° carriers and in carriers diagnosed during mass screening. However there was a higher incidence of anemia in female obligate β° thal carriers. In Sardinia the β° thal carrier showed the usual hematological characteristics of the high Hb A2 β thal carrier with microcytosis, hypochromia, reduced osmotic fragility: Hb F > 1% was found in 30% of the carriers. With MCV, MCH, osmotic fragility test (OFT) and Shine and Lal discriminant function we found 3.5%, 1.5%, 3.5% and 4.0% respectively false negatives in carrier identification. A part from one subject, all obligate carriers had elevated Hb A2 levels. The α/β ratio in obligate carriers (mean±SD) was 1.83±0.26 (N=30).

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.

Prenatal Diagnosis of β Thalassaemia by Fetal Red Cell Enrichment with NH4Cl-NH4HCO3 Differential Lysis of Maternal Cells

Summary. Prenatal diagnosis with globin chain synthesis analysis on fetal red blood cells concentrated by NH4Cl‐NH2HCO3 differential lysis of maternal cells (Ørskov lysis) was carried out in 27 pregnancies at risk for β thalassaemia and one at risk for sickle cell β0 thalassaemia. The β/γ globin chain synthesis ratio was also determined after anti‐i differential agglutination (12 cases), in almost pure fetal samples (six cases) and by extrapolation (one case). Differential lysis permitted the study of samples drawn by placental aspiration containing as little as 3.2% fetal red blood cells. There was no consistent difference between the β/γ ratios observed after differential lysis and those determined after the use of the other approaches. A presumptive diagnosis of homozygous β thalassaemia was made in nine cases. All but one of these pregnancies was terminated. The absence of β chain synthesis was confirmed by the study of fetal blood after abortion in four cases with suitable samples. Of the remaining pregnancies, six proceeded to term and non‐homozygous infants were delivered. The others are still in progress. No fetal loss occurred. ørskov lysis seems to be a very reliable method for prenatal diagnosis of β chain abnormalities. Moreover it can minimize the number and duration of placental aspirations required and thus the risk to the fetus.

Molecular mechanism accounting for milder types of thalassemia major

We carried out alpha-globin gene analysis by restriction endonuclease mapping in 91 Sardinians with homozygous transfusion-dependent beta 0-thalassemia and correlated the clinical findings with the alpha-globin genotype. In patients (n = 6) with deletion of two alpha-globin structural genes, disease onset and transfusion dependence occur later than in those (n = 50) with a full complement of alpha-globin genes. There was no statistically significant difference in the group of patients (n = 35) with deletion of only one alpha-globin gene. Patients with deletion of two alpha-globin genes had significantly higher Hb A2 levels than those with a full complement of alpha-structural genes and those with deletion of a single alpha-globin gene. From this and other studies, it seems that the deletion of two alpha-globin structural genes may convert the common severe clinical picture associated with homozygous beta 0-thalassemia to milder forms, ranging from a later occurring but still transfusion-dependent type to a non-transfusion-dependent form.