Biomedical subjects
Publications and source records attributed to M S Ristaldi.
Adenylate kinase (AK), phosphoglucomutase (PGM1) and 6-phosphogluconate dehydrogenase (6PGD) polymorphisms were investigated in a sample of individuals from Central Sardinia. The gene frequencies were: AK1 = 0.973, PGM1(1) = 0.842 and PGDA = 0.969. The frequencies were compared with those of other Italian populations.
The distribution of phenotypes and gene frequencies of the group-specific component (Gc) and C3 complement were studied in South Sardinia. The gene frequencies were: Gc1 = 0.7346; C3F = 0.1963.
A sample of the population from Central Sardinia was studied with respect to acid phosphatase (AcP) and esterase D (EsD) enzymes. The gene frequencies were: AcPA = 0.322, AcPB = 0.617, AcPC = 0.061 and EsD1 = 0.892. The results were compared with those of other Italian populations.
The promoter region of adult beta globin genes in humans and other mammals contains conserved regions of pivotal importance for their regulated tissue specific expression. These include the CACCC and CAAT motifs. The CACCC motif is duplicated in humans and other mammals. The human delta-globin gene lacks these conserved regions and its expression in normal individuals is about 3% that of the beta globin gene. Previous studies have shown that the introduction of the beta-globin CACCC or CAAT can activate the delta-globin gene promoter, but the effect of the distal CACCC element has not yet been tested. In the present study, using site-specific mutagenesis, we have introduced the consensus sequence for the distal and proximal CACCC motif and the CAAT box alone or in combination in the wild-type delta-globin gene promoter. The resulting mutants, as well as the wild type (wt) delta- and beta-globin gene promoters, have been analyzed in a transient expression assay in Cos7, K562, and MEL cell lines. The results show that the CACCC boxes can increase the transcription efficiency of the delta-globin gene promoter in both erythroid and non-erythroid cell systems. The contribution of the two CACCC elements is almost equal in the non-erythroid (Cos7) and erythroid embryonic-fetal cell lines (K562), while the proximal CACCC element is more active in adult erythroid cells (MEL). Nonetheless, duplication of this element does not appear to affect the efficiency of the promoter synergistically. Furthermore, to assess the competitive ability of the delta globin promoter containing the proximal or distal CACCC consensus sequences over the wt beta globin gene promoter, we have carried out transient expression experiments using DNA constructs in which the delta and beta globin gene promoters are linked in cis and are sharing a single enhancer (competitive transient expression). The results show that both CACCC elements are able to activate the delta globin gene promoter in Cos7 and K562 cells, although to a different extent, whereas only the proximal CACCC element is effective in increasing the transcription efficiency in MEL cells. These findings are in agreement with the more severe clinical phenotype produced by the beta-thalassemia mutations affecting the proximal CACCC box as compared with those within the distal CACCC box. The Erythroid Kruppel Like Factor (EKLF) is a nuclear protein restricted to erythroid cells which specifically bind the CACCC box sequence and activate the beta-globin gene. In the present study we carried out transactivation experiments of the mutagenized delta-globin gene promoter by introducing an EKLF expressing construct in erythroid cells. Constructs containing the proximal but not those bearing the distal CACCC element are transactivated. Our results indicate that the proximal CACCC box and, to a lesser extent, also the distal box have a role in the regulated stage specific expression of a beta-like globin gene, and show that the insertion of a single CACCC motif in the delta-globin gene promoter is sufficient to increase its activity. Nevertheless only the delta globin gene promoter containing the proximal CACCC element is able to compete with the wt beta globin gene promoter in the adult erythroid environment. These findings have potential relevance for the future prospective treatment of inherited hemoglobinopathies based on the conversion of the low functioning delta-globin gene into a high functioning beta-like globin gene.
This paper reviews the methodology available to make prenatal diagnosis of inherited hemoglobinopathies by DNA analysis and the strategy to be used for the large scale application of this procedure to high-risk populations. The most straightforward approach for prenatal diagnosis is nowadays based on the analysis of DNA enzymatically amplified by the polymerase chain reaction (PCR). The mutations, produced by gross structural rearrangement of the DNA and those affecting a restriction recognition site, are directly detected by visualization following ethidium bromide staining of the electrophoretic pattern resulting from enzymatic digestion of amplified DNA. The remaining ones are detected by dot blot analysis with allelic specific oligonucleotide probes. Because in each population a limited number of specific beta-thalassemia mutations are prevalent, prenatal diagnosis by DNA analysis may be carried out by a population-specific strategy based on the amplification of those regions of the beta-globin genes containing the mutations most frequently occurring in each population followed by dot blot analysis with allelic specific oligonucleotide probes. This approach has the great advantage of being very simple, because radioactive probes are not necessary, very rapid, the results being obtained within 24 hours from sampling and very sensitive, only a limited amount of DNA in the order of 50 ng being necessary.
In this study, we have defined the beta thalassemia mutation and characterized the beta globin haplotype and the alpha globin gene arrangement in a group of patients of Sicilian descent with beta (s)/beta thalassemia. We found that those patients carrying a beta(+) thalassemia mutation associated with a moderate reduction of beta chain synthesis (beta(+) IVS-1 nt 6) have normal or reduced Hb levels and mild to moderate clinical manifestations, as defined by the number of hospital admission and sickle cell crises per year. Those patients carrying a beta(+) thalassemia mutation associated with a severe reduction of beta chain synthesis (beta(+) IVS-1 nt 110) have a disease of moderate severity. In those carrying a beta(0) thalassemia gene the disease was clinically very heterogeneous, ranging in severity from mild to severe with no difference related to the type of mutation [beta(0) 39, beta(0) IVS-1 nt 1, beta(0) IVS-2 nt 1, beta(0) 6 (-1bp)]. In this last group of patients part of the clinical variability may be attributed to the HbF levels, which were higher in those with mild to moderate clinical severity.
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