Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia

α-Thalassemia commonly results from deletions or point mutations in one or both α-globin genes located on chromosome 16p13.3 giving rise to complex and variable genotypes and phenotypes. Rarely, unusual non-deletion defects or atypical deletions down-regulate the expression of the α-globin gene. In the last decade of the program for β-thalassemia carrier screening and genetic counseling in Sardinia, the association of new techniques of molecular biology such as gene sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) to conventional methods has allowed to better define several thalassemic genotypes and the complex variability of the α-cluster with its flanking regions, with a high frequency of different genotypes and compound heterozygosity for two α mutations even in the same family.The exact molecular definition of the genotypes resulting from the interactions among the large number of α-thalassemia determinants and with β-thalassemia, is important for a correct correlation of genotype–phenotype and to prevent underdiagnosis of carrier status which could hamper the effectiveness of a screening program particularly in those regions where a high frequency of hemoglobinopathies is present.     

beta 0 thalassemia, a nonsense mutation in man.

We determined the complete nucleotide sequence of the 5' noncoding region and the first 74 amino acids of the nonfunctional beta-globin mRNA in a patient with homozygous beta 0 thalassemia. We identified the molecular defect as a single nucleotide substitution in the coding region of the mRNA. At the position corresponding to amino acid 17, replacement of an adenine by a uracil changes the triplet AAG, which codes for lysine in the normal beta chain, to an amber termination codon, UAG. This type of beta 0 thalassemia represents an example of a nonsense mutation in man.     

A paucity of thalassemia trait in Italian men with myocardial infarction

We studied the prevalence of thalassemia trait in a group of Italian male myocardial infarction (MI) patients and an ethnically similar group of men admitted for other conditions. Italian men constituted approximately 13% of each group. Of 359 Italian men with a MI, only 2 had thalassemia trait. In contrast, of 330 adult Italian males in the non-MI group (mean age 59.6 years), 11 had thalassemia trait. Because the frequency of thalassemia trait was significantly (p less than 0.01) lower in the myocardial infarct group, we conclude that thalassemia trait may be a protective attribute as regards MI. A prospective study of individuals with thalassemia minor is indicated to elucidate the mechanism for the observed protective effect and to determine what risk factors may offset the protective effect in those that do experience MI.     

Rheological behaviour of red blood cells in beta and deltabeta thalassemia trait

In major and intermediate thalassemia a decrease in erythrocyte deformability and increased erythrocyte aggregability has been described, but few studies have dealt with the question of rheological red blood cell behaviour in minor beta and deltabeta thalassemia carriers, mostly in deltabeta, because it is a less common entity. To ascertain whether there are differences in red blood cell behaviour between minor thalassemia and controls and between both types of thalassemia trait beta and deltabeta, we determined erythrocyte deformability and aggregability in 30 beta and 30 deltabeta trait carriers diagnosed both with conventional methods and globin gene analysis, and in 40 age- and sex-matched controls. Erythrocyte deformability determined by means of the Rheodyn SSD showed a statistically significant lower Elongation Index (EI) at all the shear stresses tested in both thalassemic groups compared with controls (p<0.001). Minor beta thalassemia carriers showed lower EI than deltabeta carriers (p<0.001). Erythrocyte aggregability measured with the Myrenne aggregometer was significantly lower in both thalassemic groups than in controls (p<0.001), although no significant differences could be observed between both thalassemic groups. The rheological alterations found in thalassemia carriers are in part due to microcytosis, hypochromia and the morphological changes that characterize this kind of anaemia. The less altered deformability found in deltabeta carriers, is in agreement with the fact that it deals with a more benign trait.     

Demonstration of non-functional beta-globin mRNA in homozygous beta (0) thalassemia.

In two Chinese patients with homozygous beta(0)-thalassemia, messenger RNAs from peripheral blood reticulocytes and the bone marrow failed to direct beta-chain synthesis in vivo and in vitro in a cell-free system. Molecular hybridization showed that the beta cDNA annealed to the RNAs at almost the same rate as the alpha and gamma cDNA. The beta cDNA-RNA hydrid formed efficiently and was thermally stable, whereas hybrids between gamma and beta sequences formed slowly and denatured at a significantly lower temperature. Thus, we conclude that the beta cDNA was annealing to beta-globin sequences in these two patients, and that nonfunctional beta-globin mRNA was present. Similar results were obtained in the reticulocyte RNA from an Italian patient with homozygous beta(0)-thalassemia.     

Myocardial infarction and thalassemia trait: An example of heterozygote advantage

This study was designed to test the hypothesis that thalassemia trait diminishes the likelihood of myocardial infarction. If the hypothesis is true the prevalence of thalassemia trait should be less in myocardial infarct patients than in a control population. Blood counts and blood chemistry data from 500 men in Taiwan with a discharge diagnosis of myocardial infarction were recorded, and similar data were gathered on 500 men admitted to the same hospital for a routine check-up. Thalassemia trait was diagnosed via a discriminant function applied to the red blood cell count and indices. The study was done in a large modern hospital in Taipei; 14 of the 500 infarct patients and 33 of the 500 non-infarct subjects had thalassemia trait as defined here. The prevalence of thalassemia trait in myocardial infarct patients is significantly less than in the control subjects. This case control study supports the hypothesis that thalassemia trait lessens the risk of myocardial infarction. © 193s Wiley-Liss, Inc.     

Beta thalassemia and energy consumption in hemoglobin A1C formation: a model

Hemoglobin (Hb) A1C is the nonenzymatic glycated product of the Hb beta chain at the valine terminal residue. Recently, the nature of energy-consuming reaction in HbA1C formation was reported, and this was proposed as an underlying pathophysiology for poor nutritional status, muscle loss, and functional impairment in poor-control diabetic patients. Here, the author focuses on energy change in HbA1C formation in the case of beta thalassemia. According to this study, the energy range required for any type of beta thalassemia is greater than that required for normal Hb. Hence, it might be expressed that beta thalassemia did significantly increase complications due to energy consumption during HbA1C formation in poor-control diabetic cases.     

Beta thalassemia minor is a beneficial determinant of red blood cell storage lesion

Blood donor genetics and lifestyle affect the quality of red blood cell (RBC) storage. Heterozygotes for beta thalassemia (bThal⁺) constitute a non-negligible proportion of blood donors in the Mediterranean and other geographical areas. The unique hematological profile of bThal⁺ could affect the capacity of enduring storage stress, however, the storability of bThal⁺ RBC is largely unknown. In this study, RBC from 18 bThal⁺ donors were stored in the cold and profiled for primary (hemolysis) and secondary (phosphatidylserine exposure, potassium leakage, oxidative stress) quality measures, and metabolomics, versus sex- and age-matched controls. The bThal⁺ units exhibited better levels of storage hemolysis and susceptibility to lysis following osmotic, oxidative and mechanical insults. Moreover, bThal⁺ RBC had a lower percentage of surface removal signaling, reactive oxygen species and oxidative defects to membrane components at late stages of storage. Lower potassium accumulation and higher uratedependent antioxidant capacity were noted in the bThal⁺ supernatant. Full metabolomics analyses revealed alterations in purine and arginine pathways at baseline, along with activation of the pentose phosphate pathway and glycolysis upstream to pyruvate kinase in bThal⁺ RBC. Upon storage, substantial changes were observed in arginine, purine and vitamin B6 metabolism, as well as in the hexosamine pathway. A high degree of glutamate generation in bThal⁺ RBC was accompanied by low levels of purine oxidation products (IMP, hypoxanthine, allantoin). The bThal mutations impact the metabolism and the susceptibility to hemolysis of stored RBC, suggesting good post-transfusion recovery. However, hemoglobin increment and other clinical outcomes of bThal⁺ RBC transfusion deserve elucidation by future studies.     

The diagnosis of thalassemia trait by starch block electrophoresis of the hemoglobin

Starch block electrophoresis of the hemoglobin has been performed for a groupof adults who are the parents of children with thalassemia major. The hemoglobin electrophoretic pattern was found to be constantly abnormal in this group,in that the minor component with E-like mobility (designsated the A₂ component)constituted a greater than normal proportion of the total hemoglobin. Reducedmean cell (erythrocyte) volume was likewise found to be present in all membersof the patient group.

If it is assumed that typical childhood thalassemia major represents thehomozygous state for the thalassemia gene, then the patient group studied constitutes a populations of adults heterozygous for thalassemia. Since microcytosisand an increase in the A₂ content were constantly present in this group, they aresuggested as suitable minimum diagnostic criteria for thalassemia trait.

The degree of elevation of the A₂ fraction was noted to have a discontinuousdistribution. Preliminary studies have demonstrated that the degree of elevationappears to be identical in affected members of single pedigrees. The discontinuityin distribution observed is thus apparently under genetic control.

The rather small difference in A₂ content between some normal and somethalassemia trait adults implies that the alteration of hemoglobin synthesis is asecondary phenomenon, since such "low-valued" thalassemia traits were notdifferent in their clinical expression. Despite the smallness of this difference,quantitation of the A₂ fraction sufficed to distinguish these "low-valued" thalassemia heterozygotes from normal individuals.

Submitted on August 7, 1957 Accepted on September 15, 1957     

Erythrocyte aggregability and disaggregability in thalassemia trait carriers analyzed by a laser backscattering technique

Erythrocyte aggregability was determined by a laser backscattering light technique in 23 beta minor thalassemia carriers and in 36 age and sex matched controls. The aggregation time (Ta) was statistically higher in cases than in controls (2.8 +/- 1.0 vs 2.3 +/- 0.4, p < 0.05) and the aggregation index at 10 sec (AI10) was statistically lower (25.1 +/- 5.7 vs 28.2 +/- 3.8, p < 0.05), suggesting both parameters a statistically lower erythrocyte aggregability tendency. However, the total disaggregation threshold (gammaD) was statistically higher in cases than in controls (134.4 +/- 34.1 vs 105.1 +/- 33.1, p < 0.05), indicating that once aggregates are formed a higher shear rate is needed to break them up. No differences were observed in plasmatic factors, i.e., fibrinogen, total cholesterol and triglycerides, that could have influenced erythrocyte aggregation. A negative statistically significant correlation was found between erythrocyte indexes and the total disaggregation threshold. The lower erythrocyte aggregation found in minor thalassemia carriers could be attributed in part to the morphological alterations, although others mechanisms such as modifications in the membrane structure of the RBC can not be ruled out.     

Homozygous beta zero-39 mutation with thalassemia intermedia in northern Sardinia: clinical, hematological and molecular analysis

In this study, we investigated the clinical and hematological features and carried out alpha- and beta-globin gene analyses in 11 Sardinian adult beta zero-thalassemia homozygotes from Northern Sardinia who were not transfusion-dependent. Oligonucleotide analysis revealed in nine out of 11 patients the nonsense mutation at codon 39, which was associated either with haplotype II or IX (14/16 and 2/16 chromosomes, respectively). Haplotype II was linked to the A gamma T mutation. The G gamma globin level ranged from 50 to 70%. Four out of nine patients (44%) were heterozygous and 3/9 (33%) homozygous for the rightward deletional type of alpha-thalassemia; two (22%) had the normal alpha-gene complement. Patients who were alpha-thalassemia homozygotes (-alpha/-alpha) showed a more balanced globin chain synthesis ratio. This study confirms that alpha-thalassemia may ameliorate the clinical picture of homozygous beta zero-thalassemia.