- To improve the level of understanding of fundamental biochemical mechanisms that regulate the in vitro expression of globin genes during different phases of postnatal development;
- To contribute to the control and prevention of thalassemia.

- Collection of data on the occurrence of different DNA-mutations in DNA causing thalassemia and related hemoglobinopathies in the three countries from where participants are included (Italy, Malta & Cyprus);
- Testing of new-born babies and premarital couples in addition to antenatal maternal and fetal testing;
- Follow up of probands with a variety of haemoglobin structure abnormalities (including sickle cell disease and fetal Hb variants) or biosynthesis (i.e. the different types of thalassemia) employing state of the art haematology, protein chemistry and molecular biology techniques;
- Precise documentation of the incidence of different hemoglobinopathies and thalassemias in the populations of Malta, Sardinia and Cyprus including identification of mutations;
- Genotype-phenotype correlation in the most frequent beta-thalassemia mutations (i.e. beta39 and betaIVSI-110) in the Maltese, Sardinian and Cypriot populations to evaluate possible expression differences due to the ethnic origin;
- Identification of couples at risk of having progeny with clinically significant hemoglobinopathy or thalassemia;
- Evaluation of findings in experimental systems (in vitro transfections and in vivo footprinting).

Results

- In our studies we have been able to define the spectrum of beta- and alpha-thalassemia mutations in the three countries participating in this project. This information has been used to set up a strategy for carrier identification and fetal testing of all Mediterranean origin populations. We have also set up the technique to establish the genotype of the oocytes in the mouse, as necessary step to carry out preimplantation diagnosis.
- In this part of the study we have identified and described a new beta-chain variant and a number of mild beta-thalassemia mutations (frameshift at codon 59, IVSII nt 844 (C -> G), -92 (C -> T). One of these mutations, IVSII nt844 (C -> G), is completely silent in the heterozygous state and results in the carrier state phenotype when in homozygosity. The -92 mutation is also a silent mutation, while frameshift at codon 59 is obviously a beta-thalassemia mutation.
- We have expanded also our knowledge on the correlation between genotype and phenotype in the field of beta-thalassemia mutations. On this topic, we have been able to quantify the residual output of beta-thalassemia gene affected by a beta-thalassemia mutation by quantifying the HbA level in compound heterozygosity for the beta-thalassemia mutation and a Hb variant.
- We have studied the in vitro expression of the different beta-thalassemia mutations to date described affecting the proximal and distal CAAC box and found a defined correlation with their phenotype effect in vivo.
- Finally, we have, independently from others, cloned the Erythroid Kruppel like Factor (EKLF) gene, which codes for an essential transcription factor for the expression of the beta-globin gene and mapped to chromosome 19p13.2-p13.3. We have also identified two polymorphisms of the EKLF gene, which may be useful for further linkage analysis between this gene and different beta-thalassemia carriers unlinked to the beta-globin cluster.

Follow-up

Our planning for the next year is as follows :
- Start in our countries preimplantation diagnosis in those couples who already had a number of abortion and who, after counselling, are willing to carry out this approach in order to have healthy children without the risk of pregnancy interruption;
- Try to set up on large scale the technique recently developed for making fetal diagnosis by analysis of fetal cells in maternal circulation. This technique is based on PCR amplification of the DNA from single fetal cell recovered after identification by epsilon or gamma globin chain staining in slides prepared from maternal blood;
- Definition of the reasons for the occurrence in some cases of a mild phenotype in patients homozygous for different beta-thalassemia. In this project we are planning to carry out whole genome analysis with polymorphic microsatellites spanning the human chromosomes in patients with mild different beta-thalassemia versus patients with thalassemia major carrying the same mutation and originating from the same region;
- Definition of the molecular defect in cases of different beta-thalassemia not linked to the beta-globin gene. In case of negative results we plan whole genome linkage analysis as before.

These studies need a large number of patients from different countries, that will be available by a collaborative Mediterranean project.