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Early prenatal diagnosis of thalassaemia and sickle cell disease using recent development in DNA technology


This Concerted Action was settled in order to carry out the appropriate studies and actions to fulfil the following requisites for the large application of prenatal diagnosis of inherited haemoglobinopathies and for improving the counselling in a high-risk population:
- Extensive knowledge on the frequency and distribution of the different beta and delta-thalassaemia mutations in each at-risk population;
- Delineation of the molecular bases of the attenuated forms of beta-thalassaemia in the same populations;
- Production of a protocol to define the known and unknown beta-thalassaemia mutations by a critical evaluation of the procedures nowadays available;
- Dissemination of the technologies to directly detect the beta-thalassaemia mutations in all countries of the European community in order to expand and improve the diagnostic capability of every state member;
- Organization of a registry for prenatal diagnosis;
- Evaluation of procedures for preimplantation diagnosis and analysis of fetal cells present in maternal blood.
A detailed protocol has been prepared to define the known and unknown beta-thalassaemia mutations by a critical evaluation of the procedures now available in prenatal diagnostic laboratories throughout Europe. Mutations may be detected by a number of different procedures on deoxyribonucleic acid (DNA) enzymatically amplified by polymerase chain reaction (PCR). The methodologies in use may be divided into 2 groups for detection of known and unknown mutations. For known mutations, the simplest and most suitable methods for large scale application are primer specific amplification and reverse dot blot analysis. Both methods may be amenable to complete automation. For the former method, specific primers for each common beta-thalassaemic mutation were synthesized and for the latter, a series of oligonucleotide probes complementary to the common Mediterranean beta-thalassaemic mutations were prepared. These were made available to all participating members. The most widely used method to detect unknown mutations is denaturing gradient gel electrophoresis (DGGE), which was found to be a very powerful method for detection of mutations in the beta-globin gene. Dissemination of the technologies to directly detect the beta-thalassaemic mutations to all countries of the European Community has improved the capacity to make prenatal diagnoses by corionic villi analysis.

Detection of delta-thalassaemia mutations has been carried out by using a limited number of allele specific primers or probes. The clinical relevance of delta-thalassaemia is that double heterozygotes for delta- and beta-thalassaemia have thalassemia like red cell indices but normal haemoglobin A2 (HbA2) levels and they may thus be confused with heterozygous alpha-thalassaemia. A map of the frequency and distribution of the beta-thalassaemia and delta-thalassaemia mutations in Mediterranean at risk populations has been produced. Detailed analysis of the phenotype genotype correlation of those mutations occurring in Mediterranean populations indicates that the factors able to ameliorate the clinical picture of homozygous beta-thalassaemia are: homozygosity or double heterozygosity for a mild beta-thalassaemia mutation; coinheritance of alpha-thalassaemia; coinheritance of a genetic determinant able to increase the synthesis of gamma chains in adult life. Among these factors, however, only homozygosity for a mild mutation produces a consistent effect. The information has improved the capacity to predict the clinical phenotype in offspring and has aided genetic counselling.
Beta-thalassaemia is a very common autosomal recessive disorder in Southern Europe. Countries with the highest prevalence are Italy, Greece and Portugal. However, because of South-to-North emigration, beta-thalassaemia has spread in Northern Europe and nowadays it is frequently detected in United Kingdom, France, Germany, Holland, Belgium and Luxembourg. Beta-thalassaemia is also brought to Northern countries (in addition to this Southern to Northern Europe Emigration) by emigration from the African and Asian countries. The distribution described above indicates that beta-thalassaemia nowadays affects a large part of the European Community, particularly the less developed countries and the less privileged subpopulations.

Homozygosity for beta-thalassaemia is a very severe and potentially fatal disorder for which the only available cure, at the present time, is bone marrow transplantation, that however, can be carried out in a limited proportion of individuals affected, i.e. those who have an HLA identical siblings. The traditional management, based on a continuous transfusion program in combination with iron chelators, may provide for an extended survival but is associated with the risks of transfusion-related infections (Hepatitis B and C and HIV) and iron-mediated organ damage. Bone marrow transplantation and traditional management are very costly procedures which drain a substantial amount of health resources of the population involved.

Immigration from Northern and Western Africa, and the Caribbeans have also introduced in Continental Europe, especially in the more industrializes countries, the sickle cell disorders. The sickle cell disorders require a continuous medical supervision and, at the present time, may be cured only by bone marrow transplantation.

Based on this premises, every effort able to prevent and control homozygous beta-thalassaemia and the sickle disorders should have a high priority in the management of these disorders.

Prenatal diagnosis for these disorders started at the beginning of the 70's and was carried out, at that time, by the analysis of fetal blood obtained by fetoscopy, a procedure associated with a substantial risk of fetal mortality. Since then a tremendous progress has been made in this field, so that nowadays fetal detection of inherited haemoglobinopathies is usually carried out by the direct definition of the mutation on fetal DNA obtained by chorionic villous biopsy. Beta-thalassaemia, being very heterogeneous at the molecular level (more than 120 different molecular defects have been defined), the use of this technology requires the knowledge of the molecular bases of beta-thalassaemia mutations in each at-risk population.

Homozygous beta-thalassaemia may result either in the production of transfusion dependent thalassaemia major or in more attenuated forms requiring transfusions for survival, which are referred to as thalassaemia intermedia.

The molecular basis for thalassaemia intermedia are only partially defined. Among the best known are the presence of beta-thalassaemic mutations associated with high residual output of beta-globin chains, the coinheritance of alpha-thalassaemia or hereditary persistence of fetal hemoglobin. Likewise the sickle cell disorders may be ameliorated by the coinheritance of alpha-thalassaemia or particular beta-globin haplotype associated with a high production of gamma chains in adult life. The capability to predict the clinical phenotype of the offsprings in a mating of two beta-thalassaemia heteozygotes could be a significative advance in the genetic counselling of these disorders.


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Università degli Studi di Cagliari
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Via Jenner s/n
09100 Cagliari

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