A novel mutation (Leu817Pro) causing type 2A von Willebrand disease.

We studied a patient affected by von Willebrand disease type 2A who experienced several mild bleeding episodes and was characterized by markedly reduced haemostatic parameters. In the exon 28 of von Willebrand factor (vWF) gene a T to C transition at nucleotide 8680, resulting in the missense mutation Leu817Pro, was found in the heterozygous form in the patient and in two affected relatives. As suggested by the presence in platelets of a complete spectrum of vWF multimers as well as by the increased vWF antigen levels and improved haemostasis after DDAVP treatment, the mutation is compatible with normal multimerization, and could be responsible for a reduced stability or an impaired physiological secretion of vWF.

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.

Pitfalls in genetic counselling for beta-thalassemia: an individual with 4 different thalassemia mutations.

This paper describes a complex combination of four thalassemia genes (delta(+), beta(0), nondeletion and deletion alpha-thalassemia) in the spouse of a typical high Hb A2 beta-thalassemia carrier presenting for genetic counselling. This complex gene combination resulted in a hematological phenotype, characterized by thalassemia-like red cell indices, normal Hb A2 and Hb F levels and slightly reduced alpha/beta globin chain synthesis ratio, and therefore not indicative for the presence of beta-thalassemia trait. Family studies in combination with alpha-globin gene mapping, haplotype analysis at the beta-globin gene cluster and definition of the beta-thalassemia mutation by oligonucleotide hybridization led us to identify a beta-thalassemia mutation, to define the molecular basis for this phenotype and give the appropriate genetic counselling.

[Determination of HbA2 by a microchromatographic method. Reliability and simplification of the use of prepared columns]. / Dosaggio della Hb A2 con metodo microcromatografico. Affidabilità e semplificazione d'uso di colonne preconfezionate.

A modified procedure for hemoglobin A2 determination by microchromatography was developed simplifying an easily commercially available column kit method. Blood samples were analyzed simultaneously by means of DE-52 microchromatography described by Huisman et al. (reference 12) to check the reliability of the new modified method. In the Pediatric Clinic of Cagliari University where the original Huisman method was used the values of HbA2 obtained were as follows: Normal subjects (35 cases) 2.470 +/- 0.351, beta-Thalassemia heterozygous subjects (38 cases) 5.142 +/- 0.342. The modified method gave respectively the following results: Normal subjects 2.578 +/- 0.303, beta-Thalassemia heterozygous subjects 5.465 +/- 0.400. No overlap was found between normal and beta-thalassemia trait samples. It is suggested that the simplified method is very reliable and very simple to organize even in laboratories with few facilities and when needs for only few tests per week exist.

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.

Globin gene mapping studies in Sardinian patients homozygous for beta zero Thalassaemia.

The genetic factors responsible for the relatively mild clinical phenotypes of some cases of homozygous beta zero thalassaemia (thalassaemia intermedia) in Sardinia have been evaluated. The frequency of deletion forms of alpha thalassaemia was higher in patients with thalassaemia intermedia (6/8) than in those with thalassaemia major (6/17). The beta globin gene clusters were also studied, first to determine whether there were any rearrangements of the gamma genes, and second to see whether the restriction fragment length polymorphism patterns (haplotypes) of the two groups of patients were similar. The structure of the gamma genes was normal in all the patients with the single exception of a thalassaemia major patient with a triplicated gamma gene arrangement. The beta globin gene cluster haplotypes of the two groups of patients were not significantly different. However, the frequency of the various haplotypes in the thalassaemic as compared to the normal (beta A) chromosomes was different. This finding is of potential value in the antenatal diagnosis of homozygous beta thalassaemia in this population.

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.

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.

The interaction of alpha thalassaemia with heterozygous beta thalassaemia.

The alpha globin genotypes of 55 beta thalassaemia heterozygotes have been determined by restriction endonuclease analysis to identify those with interacting alpha thalassaemia genes. A comparison of the haematological and haemoglobin synthesis findings of individuals with normal alpha genotypes (alpha alpha/alpha alpha) with those with one (-alpha/alpha alpha) or two (-alpha/-alpha) alpha genes deleted shows that the latter two groups have more balanced globin chain synthesis ratios, higher haemoglobin levels, and larger, better haemoglobinized red cells. This suggests that the degree of globin chain imbalance is a significant factor in determining the red cell characteristics in heterozygous beta thalassaemia. Screening programmes for thalassaemia, based on the detection of low MCVs, could miss cases of the interaction of alpha and beta thalassaemia.

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.

Delta beta (F)-thalassaemia in Sardinia.

A population survey carried out in southern sardinia on more than 5000 people has shown that delta beta (F)-thalassaemia, with a gene frequency of 0-00088, is a rare trait in this population. We examined the members of three families segregating for both delta beta- and beta(0)-thalassemia and a number of delta beta carriers identified during the screening. The doubly heterozygous children suffer from a mild form of Cooley's disease with non-alpha/alpha biosynthetic ratios within the range of values observed in beta (0)-thalassaemia homozygotes. Three of them have been transfusion dependent for some time. The delta beta carriers, although in many respects showing the usual picture of delta beta-thalassaemia, such as abnormal red cell indices, normal Hb A2, Hb F heterogeneously distributed in the erythrocytes, and low beta/alpha synthetic ratios, have unusually high levels of Hb F (range 10 to 20%) and particularly low glycine content (range 0.02 to 0.14 residues) in the isolated gamma CB3 peptide. These results have led us to the conclusion that the delta beta-thalassaemia found in Sardinia is different from the similar kind of delta beta defect found in Negroes and in other Mediterranean populations, including continental Italians.

Haematological and obstetric aspects of antenatal diagnosis of beta-thalassaemia: experience with 200 cases.

The results of 200 antenatal diagnoses in pregnancies at risk for homozygous beta-thalassaemia, carried out on fetal blood samples obtained by placental aspiration in the second trimester, are described. Globin chain synthesis in the fetuses was measured by means of 3H-leucine incorporation and separation of the chains on carboxy-methyl-cellulose columns. Fetal red cell enrichment was performed by NH4Cl-NH4HCO3 differential lysis of maternal cells or anti-i differential agglutination. Sufficient fetal blood for analysis was obtained in 97.5% of the cases. The overall fetal loss rate was 6.5%, but it declined from 10% in the first consecutive 100 cases to 3% in the last 100 cases. Fetal loss was the result of early or late intrauterine death or spontaneous abortion. Forty-two homozygous fetuses had no beta-chain synthesis and one had a very low beta/gamma ratio (0.005). Of the pregnancies, 37 were terminated at parental request and four aborted spontaneously. Absence of beta-chain radioactivity was confirmed in 12 abortuses with suitable cord blood samples for analysis. Two pregnancies with homozygous fetuses were not terminated, as one member of each couple was a devout Catholic. As expected, both infants developed Cooley's anaemia. Follow-up of the 146 infants, diagnosed in utero as non-homozygotes, showed cerebral palsy in one and a small cutaneous needle injury in three. None of these developed homozygous beta-thalassaemia. Even beta-thalassaemia trait with a beta/gamma ratio of 0.046 +/- 0.012 can be distinguished from normal, showing a beta/gamma ratio of 0.086 +/- 0.019 with a high degree of certainty.

Prenatal diagnosis of thalassaemia major resulting from Lepore/ beta-thalassaemia genotype.

Antenatal diagnosis was carried out in a pregnancy at risk for beta-thalassaemia major/intermedia, resulting from the Lepore/ beta-thalassaemia genotype, by globin chain synthesis analysis on fetal blood obtained by placentocentesis at 19 weeks' gestation. As there was no radioactive incorporation in the beta-region, the fetus was considered to be a Lepore/ beta-thalassaemia genetic compound and aborted on parental request. After abortion, cord blood analysis confirmed the absence of beta-chain radioactivity.

beta zero thalassemia in Sardinia is caused by a nonsense mutation.

We report the characterization of a molecular lesion of beta thalassemia in Sardinia. Beta thalassemia in this area is predominantly the beta zero type with low levels of beta-globin mRNA. Translation assay of this messenger RNA in a cell-free system showed beta-globin chain synthesis only with the addition of an amber (UAG) suppressor transfer RNA. Double-stranded complementary DNA prepared from reticulocyte mRNA from a Sardinian patient was cloned in a bacterial plasmid and a beta-globin complementary DNA containing clone was isolated and sequenced. At the position corresponding to amino acid number 39, a single nucleotide mutation converted a glutamine codon (CAG) to an amber termination codon (UAG). We previously reported an amber nonsense mutation at amino acid 17 as a cause of Chinese beta zero thalassemia. Thus, beta zero thalassemia in Sardinia represents the second example of a nonsense mutation, and we predict that other beta zero thalassemias with mutations at various points along the beta-globin chain will be found to form a discrete subgroup of beta zero thalassemia. These experiments further illustrate the heterogeneity of lesions that lead to defective globin chain synthesis in beta thalassemia.

Prevention of homozygous beta-thalassemia by carrier screening and prenatal diagnosis in Sardinia.

We report here results of a 3-year pilot voluntary screening program coupled with prenatal diagnosis directed to the prospective prevention of homozygous beta-thalassemia (beta-thal) in Sardinia. The screening program took two approaches: outreach community testing and hospital testing on request after a period of sensibilization. The outreach testing was very effective as, taking into account the already known number of couples at risk with an affected proband (20), 74% of the couple at risk expected (61) on the basis of the carrier rate were identified. Less effective was the hospital testing in which half of the couples at risk expected were detected (502 with the 199 without an affected proband). After nondirective genetic counseling, approximately 85% of the couples at risk, which had a pregnancy, with no statistically significant difference between those with and those without a proband, requested prenatal testing. This figure showed a steadily increase from the beginning in 1977 to 1980. All the pregnancies (42), but two carrying homozygous fetuses, were terminated on parental request. A continuous hospital survey of thal-major admissions in the different hospitals of the counties showed a steady decline in the incidence figure at birth from 1976 (1:213) to 1978 (1:290). These results showed that even in a medium-developed, rural, Catholic population screening coupled with prenatal diagnosis can be successful in the control of a fatal, recessively inherited disorder.

Beta-Thalassaemia types in southern Sardinia.

In this study the prevalence of the different beta-thalassaemia types in southern Sardinia was investigated by cellulose acetate and agar gel electrophoresis or globin chain synthesis analysis on column chromatography or both in (1) all the patients (347) presenting with thalassaemia major or intermedia at our haematology service from 1976 to 1979, and (2) a group of 82 patients with transfusion-dependent thalassaemia major randomly chosen from 236 under our care. Apart from six subjects with delta(beta)0/beta+-thalassaemia genotype and eight with beta0/beta+ or less probably beta+/beta/-thalassaemia, all thalassaemia major and intermedia patients studied were beta0-thalassaemia homozygotes. Globin chain synthesis on peripheral blood cells from these patients, performed at different intervals from blood transfusion, showed no incorporation of radioactive leucine into beta-globin peak, the same as before the transfusion. No correlation between kappa/gamma ratios and clinical severity or hypersplenism was found. Globin chain synthesis analysis carried out at birth in three infants later found to have homozygous beta0-thalassaemia demonstrated imbalanced or borderline kappa/gamma ratios.