Mediterranean Biochemical Network

Molecular analysis of beta-thalassemia carriers with intact beta-globin gene

This is a collaborative research involving researchers from University of Cagliari, ASL N°8 of Cagliari and IRTAM, CNR of Cagliari.
The research laboratories of this University are carrying out prevalently studies on beta-globin gene disease (Thalassemia), and hemoglobinopathies. The beta-thalassemias are very heterogeneous at the molecular level. At the present time approximately more than 100 different molecular defects have been characterized. These defects consist mostly in single nucleotide substitutions, small addition or deletions. Less frequently beta-thalassemia results from a gross structural rearrangement of the DNA.

On the contrary, alfa-thalassemia are usually caused by the mechanism of gene deletion. Restriction endonuclease digestion, non-denaturing gradient gel electrophoresis, and allele specific oligonucleotide probes or allele specific oligonucleotide primers may define known mutations.
Unknown mutations are detected by denaturing gradient gel electrophoresis followed by direct sequencing.

Other potentially useful methods for unknown mutations are single strand conformation polymorphism analysis and chemical mismatch cleavage analysis. Beta-thalassemia genes are concentrated in peoples residing in regions of the world endemic of malaria, including Mediterraneans, North Africans and Middle Easterners, Asian Indians, Chinese and Southeast Asians, and black Africans. The large majority of heterozygous for beta-thalassemia, showing the classical phenotype, carry a mutation in the globin gene.
A few exceptional cases have been described, in whom in spite of the presence of beta-thalassemia-like hematological features, neither defects in the beta-globin gene nor deletions in the beta-globin gene cluster were found.

It was postulated that the erythroid Kruppel-like factor (EKLF) could play an important role in determining this phenotype. EKLF is a zinc finger, which binds the CACCC consensus sequence in the beta-globin gene promoter.
Mutations in the CACCC box abolish the EKLF binding. With the aim of investigating the involvement of EKLF in a fraction of thalassemia syndromes in which the defect appeared to be inherited unlinked to the beta-globin gene cluster, the group involved in this research sought up to clone and characterize the human EKLF gene.
Both complementary DNA and genomic DNA were cloned and entirely sequenced. No nucleotide variation was detected in those patients with normal beta-globin gene sequences.
These results give evidence that neither the entire beta-globin cluster nor the EKLF gene are involved in the pathogenesis of these determinants.

Universities involved in this project:

University of Cagliari - Italy

Prof. Maria Cristina Rosatelli
Prof. Antonio Cao

Dr. Paolo Moi - ASL N° 8-Cagliari (Local Sanitary Company N°8- Cagliari)
Dr. Valeria Faa - IRTAM, CNR Cagliari (Regional Institute Thalassemia and Mediterranean Anemias, National Research Council Cagliari)

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