Le Basi biochimiche e molecolari delle talassemie e la diagnosi dell'Alfa-talassemia eterozigote

Recent aspects of molecular and biochemical basis of thalassemic syndromes are described together with laboratory methods suitable for diagnosis and screening of heterozygous α-thalassemia

Le Sindromi talassemiche

Recent developements of molecular biology and genetic of thalassemia syndromes are discussed with special regard to Sardinian situation. In the island heterozygous α and β thalassernias are present with incidences of 13,5 and 12,5% respectively. β+ and δβ thalassemias together with several types of HPFH, are also present. Screening programs at level of couples are suggested for the prevention of β thalassemic homozygosity

The Oxidative state of LDL is the major determinant of anti/prooxidant effect of coffee on Cu²⁺ catalysed peroxidation

Antioxidants exert contrasting effect on low density lipoprotein (LDL) oxidation catalysed by metals, acting as pro-oxidants under select in vitro conditions. Through our study on the effect of coffee on LDL oxidation, we identified the parameters governing this phenomenon, contributing to the comprehension of its mechanism and discovering significant implications for correct alimentary recommendations. By measuring conjugated diene formation, we have analysed the quantitative and qualitative effects exerted by an extract of roasted coffee on LDL oxidation triggered by copper sulphate. When the relative effects of different coffee concentrations were plotted against the lag time (LT) of control LDL (C-LDL), the apparently random experimental data arranged in sensible patterns: by increasing the LT the antioxidant activity of coffee decreased progressively to become prooxidant. The critical LT, at which coffee switches from antioxidant to prooxidant, increased by increasing coffee concentration. Also the contrasting results obtained following a delayed addition of coffee to the assay, arranged in a simple pattern when referred to the LT of C-LDL: the prooxidant effect decreased to become antioxidant as the LT of C-LDL increased. The dependence of coffee effect on the LT of C-LDL was influenced by LDL but not by metal catalyst concentration. These novel findings point to the oxidative state of LDL as a major parameter controlling the anti/prooxidant effect of coffee and suggest the LT of C-LDL as a potent analytical tool to express experimental data when studying the action exerted by a compound on LDL oxidation

Detection of the common Hb F Sardinia [^Aγ(E19)Ile→Thr]variant by isoelectric focusing in normal newborns and in adults affected by elevated fetal hemoglobin syndromes

A simple and rapid conventional isoelectric focusing technique for the detection of the silent Hb F Sardinia variant, containing the mutated AγT chain, is described. The method is based on thin-layer gels of shallow pH gradient (pH 6.7–7.7) and allows the direct detection of this rather common and widespread Hb variant at a screening level. 15–30 hemolysates from newborns and adults affected by elevated Hb F syndromes, both in the heterozygous and homozygous condition, could be examined simultaneously. The frequency of the AγT gene in Sardinian newborn (f = 0.175), in βo-thalassemia (f = 0.722), in β+-thalassemia (f = 0.346), and in the non-deletional type of Aγ-HPFH (f = 0), as evaluated with this method, is in accordance with that previously reported by means of other methodologies

A Simple approach to the determination of the γ chain composition of F in adult human blood samples

A procedure for the determination of the γ chain heterogeneity of adult human blood samples with low Hb F has been developed. It consists of the isoelectrofocusing of lysates at a rather high hemoglobin concentration, the isolation of the focused Hb F by elution from the gel, and the analysis of the isolated hemoglobin by reversed phase high performance liquid chromatography for the separation and quantitation of globin chains. Depending on the isoelectrofocusing apparatus, many samples with as little as 1% Hb F may be focused in a single run and the γ chain composition of the recovered hemoglobin (enriched from 15% to 95% in Hb F) easily analyzed by high performance liquid chromatography. The same procedure might be used for the isolation of low level hemoglobin variants for structural studies

High-performance liquid chromatography of globin chains in the identification of human globin gene abnormalities

This article summarizes experience and data obtained using a previously developed reverse-phase high-performance liquid chromatography method (J.B. Shelton, J.R. Shelton and W.A. Schroeder, J. Liq. Chromatogr. 7 (1984) 1969) in the study of a number of hemoglobinopathies in the Sardinian population. The occurrence and incidence of several abnormal hemoglobins are described, as well as aspects of the expression of abnormal γ-globin gene arrangements and thalassemic genes

Disorders of the synthesis of human fetal hemoglobin

Fetal hemoglobin (HbF), the predominant hemoglobin in the fetus, is a mixture of two molecular species (α2Gγ2 and α2Aγ2) that differ only at position 136 reflecting the products of two nonallelic γ-globin genes. At the time of birth, HbF accounts for ~70% of the total Hb. The Gγ:Aγ globin ratio in the HbF of normal newborn is 70:30 whereas in the trace amounts of HbF that is found in the adult it reverses to 40:60 because of a γ- to β-globin gene switch. Alterations of these ratios are indicative of a molecular defect at the level of the HbF synthesis. Qualitative hemoglobinopathies due to Gγ and Aγ chain structural variants, and quantitative hemoglobinopathies affecting the synthesis of HbF such as γ-thalassemias, duplications, triplications, and even sextuplications of the γ-globin genes, which may be detected in newborn blood lysates, have been described. Moreover, several pathological and nonpathological conditions affecting the β-globin gene cluster, such as β-thalassemia, sickle cell disease, δβ-thalassemia, and hereditary persistence of HbF syndromes, are characterized by the continued synthesis of γ-globin chains in the adult life. Studies of these natural mutants associated with increased synthesis of HbF in adult life have provided considerable insight into the understanding of the control of globin gene expression and Hb switching

Characterization of four novel variants of goat β^A-globin gene

Four novel alleles of the adult β-globin gene of Capra hircus were observed in an extended study on hemoglobin polymorphism in goat breeds living in the island of Sardinia. Nucleotide sequencing showed that one of these alleles is due to a 2 bp substitution at codon 125 (CT→GAG, G, "Leu→Glu). Two substitutions, the silent CT T→CTC for Leu at codon 78 and the conservative A AG→AG G (Lys→Arg) at codon 104, are shared by the other three alleles, two of them having additional mutations, which suggests a common origin. The allele we provisionally called the βY shares four out of five amino acid substitutions, together with the same polymorphisms in the IVSII, we observed previously in the rather common βE gene. This evidence allowed the origin of the βE gene to be better characterized. The data increase to seven the number of alleles at the goat βA-globin locus characterized thus far at the molecular level. A simplified nomenclature for the increasing number of goat β-globin alleles is presented

Hb G-Philadelphia, or [α68(E17)Asn→Lys], in North Sardinia: detection by isoelectric focusing and identification by HPLC of tryptic peptides

11129 individuals of the northern area of the island of Sardinia (7717 newborns and 3412 adults) were examined for the Hb G-Philadelphia variant [α68(E17)Asn→Lys]. Hemolysates were analysed by isoelectric focusing, and the variant identified by reversed phase high performance liquid chromatography of tryptic peptides. A total of seven heterozygotes (1 in 1589) were identified. This is probably the highest prevalence of this mutant so far described. Percentages of the variant (average 35.1 ± 6.2%) were trimodally distributed with modes centering on 28, 36, and 42%, respectively. These values suggest that the G-Philadelphia allele occurs in Sardinians both on a single and on a double locus chromosome. The linkage with α-thalassemia may be the reason for the high frequency of the variant

The Levels of adenine nucleotides and pyridine coenzymes in red blood cells from the newborn, determined simultaneously by HPLC

The concentrations of ATP, ADP, AMP; NADP and NADPH; NAD and NADH were determined in erythrocytes from healthy newborns and compared with those obtained in healthy adults. No significant differences were found for the adenine nucleotide concentrations, but NADH levels were reduced in newborn erythrocytes, with a consequent increase in the NAD/NADH ratio. Moreover, in newborn erythrocytes increased levels of NADP were observed, with a consequent increase in the NADP/NADPH ratio and a decrease in the NAD/NADP ratio