beta-Thalassemia is a significant public health problem in Egypt where over 1000 of the annual 1.5 million newborns are expected to be affected with this disorder. A preventive program of the disease should be multifaceted with its technical component based on carrier screening and prenatal diagnosis through mutation detection. In addition, it should have an information and educational component with the aim of increasing public awareness of the disease. Proper selection of the technique(s) to be utilized in such a program is highly important. The appropriate technique to be used in screening should be reliable, simple and cost effective. It should also circumvent the problem of marked heterogeneity of the disease in Egypt. The reverse dot-blot technique has been used in the present study for the characterization of mutations in 138 Italian and 108 Egyptian thalassemia chromosomes, confirming its reliability as a screening method. The technique is now in routine use for thalassemia diagnosis in the Microcitemia Center of the Galliera Hospital in Genoa, Italy. Based on these results, we recommend the reverse dot-blot method as the technique of choice in the preventive program of this disease in Egypt.

Hemoglobin Dallas, an alpha-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity (P1/2 = 1 mmHg at pH 7.3 and 20-degrees-C), diminished cooperativity (n, the Hill coefficient = 1.7) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20-degrees-C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be attributed to a more `relaxed' T-state.

Human MutationVolume 1, Issue 2 p. 169-171 Mutation in Brief A novel δ-thalassemia mutation A G→C substitution at codon 30 of the δ-globin gene in a person of Southern Italian origin G. Loudianos, G. Loudianos Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorS. Murru, S. Murru Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorM. S. Ristaldi, M. S. Ristaldi Istituto di Ricerca sulle Talassemie ed Anemie, Mediterranee-CNR, Via Jenner, s/n 09100 Cagliari, ItalySearch for more papers by this authorP. Cossu, P. Cossu Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorG. Pilia, G. Pilia Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorS. Porcu, S. Porcu Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorG. V. Sciarratta, G. V. Sciarratta Centro per le Microcitemie, Ospedale Galliera, Genova, ItalySearch for more papers by this authorM. I. Parodi, M. I. Parodi Centro per le Microcitemie, Ospedale Galliera, Genova, ItalySearch for more papers by this authorA. Cao, A. Cao Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorM. Pirastu, Corresponding Author M. Pirastu Istituto di Ricerca sulle Talassemie ed Anemie, Mediterranee-CNR, Via Jenner, s/n 09100 Cagliari, ItalyIstituto di Ricerca sulle Talassemie ed Anemie, Mediterranee-CNR, Via Jenner, s/n 09100 Cagliari, ItalySearch for more papers by this author G. Loudianos, G. Loudianos Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorS. Murru, S. Murru Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorM. S. Ristaldi, M. S. Ristaldi Istituto di Ricerca sulle Talassemie ed Anemie, Mediterranee-CNR, Via Jenner, s/n 09100 Cagliari, ItalySearch for more papers by this authorP. Cossu, P. Cossu Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorG. Pilia, G. Pilia Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorS. Porcu, S. Porcu Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorG. V. Sciarratta, G. V. Sciarratta Centro per le Microcitemie, Ospedale Galliera, Genova, ItalySearch for more papers by this authorM. I. Parodi, M. I. Parodi Centro per le Microcitemie, Ospedale Galliera, Genova, ItalySearch for more papers by this authorA. Cao, A. Cao Istituto di Clinica e Biologia dell'Età Evolutiva, Università degli Studi di CagliariSearch for more papers by this authorM. Pirastu, Corresponding Author M. Pirastu Istituto di Ricerca sulle Talassemie ed Anemie, Mediterranee-CNR, Via Jenner, s/n 09100 Cagliari, ItalyIstituto di Ricerca sulle Talassemie ed Anemie, Mediterranee-CNR, Via Jenner, s/n 09100 Cagliari, ItalySearch for more papers by this author First published: 1992 https://doi.org/10.1002/humu.1380010215Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume1, Issue21992Pages 169-171 RelatedInformation

This paper describes a family of Central Italian origin in which three patients in two generations had either thalassaemia intermedia or a late presenting form of thalassaemia major. Sequence analysis of the patients' DNA revealed that only one of the beta-globin genes was affected by a beta-thalassaemia mutation (the codon 39 nonsense mutation), the other being completely normal, apart from the complex rearrangement (-T +ATA) at position -530 5' to the CAP site of the beta-globin gene, which has uncertain clinical significance. Haematologically, all these patients were characterized by unusually low HbF levels (1.8-7.3%) for a beta-thalassaemia major or intermedia phenotype. The mother of the two patients with thalassaemia intermedia was heterozygous for beta-thalassaemia (codon 39 nonsense mutation), while the father had thalassaemia-like red cell indices, an increased alpha/non alpha chain synthesis ratio, a slight increase of HbF and a low HbA2 level, but showed entirely normal beta-globin gene sequences, apart from the complex rearrangement (-T +ATA) at position -530 5' to the CAP site. One of the thalassaemia intermedia patients married a normal woman and they had a child with thalassaemia major who inherited only the codon 39 nonsense mutation but not the complex rearrangement at position -530. The clinical phenotype of thalassaemia-intermedia or major in the patients from this family may be explained by postulating the inheritance of the double heterozygous state for beta-thalassaemia and for a mutation in a gene coding for an erythroid-specific DNA binding protein which may impair the function of the normal beta-globin gene. Heterozygosity for this postulated mutation (father of the patients with thalassaemia intermedia) may result in the production of a beta-thalassaemia carrier state with normal HbA2 level.

This study describes a patient with a thalassemia intermedia-like phenotype in whom beta-globin gene sequencing detected a novel abnormal hemoglobin (Hb) due to a T-C substitution at codon 114 of the beta-globin gene arising as a de novo mutation. The abnormal variant was designated Hb Brescia after the place of birth of the propositus. Normal sequences were detected at the in trans beta-globin locus. In addition, alpha-globin gene analysis detected a triple alpha-globin locus which was inherited from the father. The T-C change at position 114 of the beta-globin gene results in a leucine to proline substitution (Leu-Pro) in the G-helix. The resulting Hb tetramer is highly unstable and precipitates forming inclusion bodies in the peripheral red blood cells. Moreover, the Leu-Pro substitution interferes negatively with the four alpha 1 beta 1 contact points of the G-helix most likely adversely affecting the alpha beta dimer formation. The very severe phenotype presented by our patient is unusual in a heterozygote for an unstable Hb variant and may be explained by the coinheritance of the triple alpha-globin locus.

Summary. In this study we have investigated the molecular basis for a mild form of β‐thalassaemia in three patients of Italian descent. In two, belonging to different families and affected by a mild and late‐presenting form of thalassaemia major, direct sequencing of amplified DNA detected a C→T substitution at position −87 of the β‐globin gene in the compound heterozygous state either with codon 39 nonsense mutation or β+IVSI, nt 110 mutation. The −87 (C→T) mutation has been previously described, in combination with the β+IVSI, nt 110 mutation, in a single patient with thalassaemia intermedia. Both our patients showed a more severe phenotype as compared to that resulting from compound heterozygosity for a severe β‐thalassaemia mutation and another promoter mutation (−87, C→G) at the same position. In the third patient with the thalassaemia intermedia phenotype, we detected a novel promoter mutation, consisting in a C→A substitution at position −86, in combination with the codon 39 nonsense mutation. The results of this study indicate that different nucleotide substitutions affecting the proximal CACCC box of the β‐globin gene in combination with severe β‐thalassaemia, produce a mild form of thalassaemia ranging in severity from thalassaemia intermedia to late‐presenting thalassaemia major.

In this study, we have defined by dot-blot analysis with allelic specific oligonucleotide probes or direct sequencing on amplified DNA the beta-thalassemia mutations in a large group of patients (23) of Italian descent with thalassemia intermedia. These patients had one parent with either the silent beta-thalassemia carrier phenotype or borderline-normal hemoglobin A2 (HbA2) levels (2.5% to 3.5%). Nearly all were genetic compounds for a severe beta-thalassemia mutation and a beta-thalassemia mutation associated with high residual output of beta- globin chains (beta + intervening sequence [IVS]-I-nt6, beta -87, beta - 101), indicating that inheritance of a mild beta-thalassemia allele, even in a single dose, is the most common molecular mechanism producing thalassemia intermedia in the Italian population. In three cases, in whom we failed to define by dot-blot analysis the mutations, we sequenced the beta + globin gene and found three novel beta-thalassemia mutations, which are certainly very rare because they have been hitherto detected solely in a single patient. These mutations consist of: (1) a T-A substitution at position 2 of IVS-I, in a patient compound heterozygote for this mutation and the -87 promoter mutation; (2) a G-C substitution at position 844 of IVS-II, in a patient heterozygous for this mutation who showed normal sequences at the in trans beta-globin gene (The reason for the presence of clinical manifestations in a beta-thalassemia heterozygote has not been defined.); and (3) a deletion of one nucleotide (-T) at codon 126, resulting in a frameshift and readthrough of the 5′ untranslated region and most likely producing an elongated Hb molecule of 156 amino acid residues, in a patient heterozygous for this mutation with normal beta- globin gene sequences at the other locus.

Hb Matera is a newly discovered, slow-moving, beta chain variant observed in four members of an Italian family. The variant had a decreased stability in heat and isopropanol tests and numerous red cells contained inclusion bodies. Structural analysis revealed that methionine at position beta 55 was replaced by lysine. Hb Matera's instability is due to an interference in the alpha 1-beta 1 contact of the hemoglobin molecule.

the manifestations typical of @-thalassemia. have k e n described.'" Herein we a novel abnormal elongated hemoglobin (Hb) variant detected in a patient with a thalassemia intermedia-like phenotype born from normal parents. This variant eluded identification by conventional protein analysis and was detected solely by DNA sequencing. Hematologic analysis. DNA amplification. and dot blot analysis were carried out as previously described.' DNA sequencing was performed by the didaoxy chain termination method' on amplified single strand DNA' using the enzyme T7 DNA polymerase (Sequenasc-USB). The proband (Fig IA, 11-1). a girl of Southern Italian descent. presented at 3 years of age with anemia (Hb levels. 6.6 g/dl.) and spleen enlargement. Clinical examination showed moderate thalassemia-like bone manifestations. splenomegaly (3 cm below the costal margin), and hepatomegaly (3 cm below thecatal margin). She received sporadic blood transfusions until 6 years of age. when a regular transfusion program with packed red blood cells given every 2 to 3 months was initiated. She was splenectomized at 14 years of age and died at 27 years due to hepatic coma. Her Hb values. before starting a trans- fusion therapy. were in the range of6 g/dL. Blood film examination showed marked thalassemia-like red cell abnormalities. On incuba- tion with brilliant cresyl blue, large inclusion bodies were detected in peripheral red blood cells. The HbF and HbA, levels w m 10% and 2.24. respectively. Then/non-u-globin chain synthesis ratio was 1 5 Hb Hb Geneva, Hb Manhattan) result from mutations in exon 3 of the @-globin We to analyze the composition of the red blood cell inclusions, but, in to Hb Geneva, they most composed of a-chains in excess. Accumulation of a-chains that precipitate and damage the red cell membrane is most likely the result of the exhaustion of the proteolytic system, because of the continuous enzymatic breakdown of the abnormal @-chains. From this and previous studies, we can conclude that the presence of abnormal, very unstable Hb should be suspected in any sporadic patient showing the clinical phenotype of thalassemia intermedia and having both parents hematologically normal, or in families in which this phenotype shows a Mendelian dominant transmission pattern. Molecular diagnosis may be rapidly accomplished by direct sequencing of amplified @-globin gene sequences.

This paper reviews the molecular pathology of a heterogeneous group of beta-thalassemia heterozygotes which may be referred to as atypical beta-thalassemia. This group includes four different categories of heterozygous beta-thalassemia, which are characterized, respectively, by (1) normal MCV and MCH; (2) normal Hb A2; (3) normal MCV, MCH, and Hb A2 and imbalanced globin chain synthesis only or, (4) the presence of clinical manifestations. The first group is represented by a limited proportion of double heterozygotes for alpha- and beta-thalassemia. The second group includes two categories. One category is double heterozygotes for delta- and beta-thalassemia with the delta-thalassemia mutation in cis or in trans to beta-thalassemia. A number of delta-thalassemia mutations which produce this phenotype by interacting with beta-thalassemia have been described. The other category within the second group is heterozygotes for some mild beta(+)-thalassemia mutations. Within the third group, conclusive evidence for a mutation within the beta-globin gene cluster producing the silent beta-thalassemia phenotype has been obtained solely for a C----T substitution at -101 within the CACCC box of the beta-globin gene. Possible candidates are the complex rearrangements (-T, +ATA; -T, +ATATA) found at position -530 from the cap site. In the group of thalassemic hemoglobinopathies, a series of mutations mostly located in the third exon and producing elongated or truncated molecules have been recently reported. Most of the mutations are silent at the protein level, produce inclusion bodies in peripheral erythrocytes, and show a dominant transmission pattern or occur sporadically.

This paper reviews the molecular pathology of a heterogeneous group of beta-thalassemia heterozygotes which may be referred to as atypical beta-thalassemia. This group includes four different categories of heterozygous beta-thalassemia, which are characterized, respectively, by (1) normal MCV and MCH; (2) normal Hb A2; (3) normal MCV, MCH, and Hb A2 and imbalanced globin chain synthesis only or, (4) the presence of clinical manifestations. The first group is represented by a limited proportion of double heterozygotes for alpha- and beta-thalassemia. The second group includes two categories. One category is double heterozygotes for delta- and beta-thalassemia with the delta-thalassemia mutation in cis or in trans to beta-thalassemia. A number of delta-thalassemia mutations which produce this phenotype by interacting with beta-thalassemia have been described. The other category within the second group is heterozygotes for some mild beta(+)-thalassemia mutations. Within the third group, conclusive evidence for a mutation within the beta-globin gene cluster producing the silent beta-thalassemia phenotype has been obtained solely for a C----T substitution at -101 within the CACCC box of the beta-globin gene. Possible candidates are the complex rearrangements (-T, +ATA; -T, +ATATA) found at position -530 from the cap site. In the group of thalassemic hemoglobinopathies, a series of mutations mostly located in the third exon and producing elongated or truncated molecules have been recently reported. Most of the mutations are silent at the protein level, produce inclusion bodies in peripheral erythrocytes, and show a dominant transmission pattern or occur sporadically.

This paper describes four families of Italian descent in each of which the propositus had the clinical phenotype of thalassaemia intermedia, resulting from the compound heterozygous state for high HbA2 beta thalassaemia and type I silent beta thalassaemia. Direct sequencing on amplified DNA and/or oligonucleotide analysis detected, in all families but one, the compound heterozygous state for codon 39 nonsense mutation and the C-T substitution at position -101 in the distal CACCC box of the beta-globin gene promoter (beta th-101). Members of these families who are heterozygous for high HbA2 beta thalassaemia showed the codon 39 nonsense mutation, while those with the clinical phenotype of silent beta thalassaemia had the beta th-101 mutation. In the remaining family, the propositus and one of his siblings had the compound heterozygous state for a molecularly undefined high HbA2 beta thalassaemia and the beta th-101 mutation in combination with the triple alpha globin gene arrangement. These patients showed a more severe thalassaemia intermedia like clinical phenotype as compared to those with the same beta-globin genotype and a normal alpha-globin gene arrangement. In the families investigated the beta th-101 was always associated with haplotype I. A group of patients with thalassaemia intermedia from Southern Italy, either homozygous or heterozygous for haplotype I and in whom previous studies had failed to define the mutation in one of the beta thalassaemia globin genes, were screened by oligonucleotide analysis for the presence of the beta th-101. Three out of nine were positive. These results indicate that the beta th-101 mutation is a common cause of the type I silent beta thalassaemia phenotype in the Southern Italian population.

An abnormal, slightly unstable, hemoglobin (Hb) was found in the red cell hemolysate of a 4-year-old female of Chilean ancestry, Living in Italy. Structural analysis of this variant identified it as Hb J-Guantanamo or α 2 β 2 128(H6)Ala→Asp

Hb Abruzzo was discovered in 1972 by Tentori et al. (1,2) in two brothers from a village in Abruzzo. Both had the compound heterozygosity of this variant and an apparent β o -thalassemia, and suffered from a mild hemolytic anemia, splenomegaly, and erythrocytosis. We recently observed the same variant in a second Italian family, also in association with a β o -thal. The family comes from the village of Troia (Foggia) which is located in the region known as Puglia, and was first studied at the Transfusional Center of Monza Hospital (Milan)