Hemoglobin Dallas (alpha 97(G4)Asn-->Lys): functional characterization of a high oxygen affinity natural mutant.

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.

Molecular analysis of atypical beta-thalassemia heterozygotes.

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.

Hb Matera [beta 55(D6)Met----Lys]: a new unstable hemoglobin variant in an Italian family.

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 silent carrier of beta thalassemia: interaction with the typical beta thalassemic trait.

The case of a girl affected by a Colley's disease of moderate severity is reported. A brother and a sister had levels of Hb F respectively of 18% and 45%. The father showed all the hematological signs of heterozygous thalassemia. The mother, however, was normal so far as osmotic fragility, red cell morphology, and Hb A2 level are concerned. In vitro hemoglobin chains biosynthesis was performed in all the subjects. Both the parents showed an alfa/non alfa ratio typical of beta thalassemia. Therefore, the mother has to be considered a "silent" carrier of the trait. The daughters and the son have a less severe Colley disease originating by such a double heterozygosity.

A drug-induced haemolytic anaemia due to Hb Torino (alpha43(CD1)Phe replaced by Val). second finding in an Italian family.

The unstable haemoglobin, Tb Torino (alpha43(CD1)Phe replaced by Val), has been found for the second time in a family from the Treviso region of Italy. The haemoglobin has a slightly lower oxygen affinity than normal. In both cases, the abnormal haemoglobin is associated with inclusion body anaemia but the course of the disorder in the present case is much less severe than that previously reported. The oxygen affinity of the haemolysate has been measured and been found lower than normal.

Alternate organization of alpha G-Philadelphia globin genes among U.S. black and Italian Caucasian heterozygotes.

Seven Hb G-Philadelphia (Hb G) heterozygotes from three Caucasian families from Northern Italy and Sardegna were found to have proportions of Hb G averaging 23%. This value is considerably lower than the 34% or 48% found in Blacks from the Southeastern U.S.A. in whom the alpha G gene is in linkage with alpha-thalassemia-2, i.e. the alpha o alpha G/alpha alpha or alpha o alpha G/alpha o alpha genotypes. Gene mapping identified tandem organization of the alpha G gene in cis with a normal alpha A gene, i.e. the alpha alpha G/alpha alpha genotype, among the Hb G heterozygotes from Italy. The data on the Italian heterozygotes are similar to those obtained by Bruzdzinski et al (14) on a Black family. These results indicate alternate organization of the alpha G genes probably across racial or ethnic boundaries. Comparison of the mean cellular globin amount of alpha G/alpha G gene/cell among Hb G heterozygotes with 4, 3, 2 or 1 alpha globin genes (i.e. alpha A + alpha G) revealed considerable reactivation of individual alpha genes in conditions of mild to severe alpha globin deficiencies.

A novel beta-globin structural mutant, Hb Brescia (beta 114 Leu-Pro), causing a severe beta-thalassemia intermedia phenotype.

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.

A beta-thalassaemia phenotype not linked to the beta-globin cluster in an Italian family.

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.

A nucleotide change at a splice junction in the human beta-globin gene is associated with beta 0-thalassemia.

beta 0-Thalassemia is a heterogeneous group of disorders associated with absence of beta-globin. In a survey of DNAs from patients with beta 0-thalassemia of diverse ethnic origins, a change at the splice junction at the 5' end of the large intervening sequence (IVS 2) of the human beta-globin gene has been found in one patient of Italian and another two of Iranian ethnic origins. The enzyme Hph I recognizes a change at this site and generates a large-than-normal fragment of DNA, which hybridizes specifically to a beta-globin IVS 2 probe. No other changes in beta-globin gene DNA structure or organization are detectable by extensive restriction endonuclease analysis. The enzyme HinfI which recognizes a sequence beginning three nucleotides from the 5' end of the IVS 2 splice junction, produces normal fragments and localizes the defect to a G-G-T sequence at the 5'-end IVS 2 splice junction. This sequence is known to be remarkably conserved in all globin genes from many species and in most other genes examined to date. Thus, in at least some beta 0-thalassemia patients, the beta 0-thalassemia defect is associated with a nucleotide change at a splice junction. These patients provide unique examples of naturally occurring defects in splice junctions of eukaryotic genes associated with absence of specific gene function.

Molecular characterization of beta-thalassemia intermedia in patients of Italian descent and identification of three novel beta-thalassemia mutations.

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.

Promoter mutations producing mild beta-thalassaemia in the Italian population.

In this study we have investigated the molecular basis for a mild form of beta-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 beta-globin gene in the compound heterozygous state either with codon 39 nonsense mutation or beta +IVSI, nt 110 mutation. The -87 (C----T) mutation has been previously described, in combination with the beta +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 beta-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 beta-globin gene in combination with severe beta-thalassaemia, produce a mild form of thalassaemia ranging in severity from thalassaemia intermedia to late-presenting thalassaemia major.

Identification of Mediterranean beta-thalassemia mutations by reverse dot-blot in Italians and Egyptians.

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.

Hb Belfast (beta 15 Trp replaced by Arg) in an Italian family.

An uncommon abnormal hemoglobin, Hb Belfast (beta 15 Trp replaced by Arg) was discovered in a patient of Italian origin. The patient was a 42-year-old man who developed two episodes of jaundice after a prolonged administration of an antituberculous drugs. Family studies revealed that several members were asymptomatic carriers of Hb Belfast.

The C-T substitution in the distal CACCC box of the beta-globin gene promoter is a common cause of silent beta thalassaemia in the Italian population.

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.

The characterization of hemoglobin Manitoba or alpha (2)102(G9)Ser----Arg beta 2 and hemoglobin Contaldo or alpha (2)103(G10)His----Arg beta 2 by high performance liquid chromatography.

Hb Contaldo with a His----Arg substitution at position 103(G10) of the alpha chain is a newly discovered unstable Hb variant observed in an Italian child. Its instability is probably due to the disruption of the hydrogen bond between alpha 103(G10)His and beta 108(G10)Asn. The structural variation in the core segment was determined through analysis of tryptic peptides from digests of the alpha X and oxidized alpha X (with performic acid) chains, which were separated by HPLC. Similar analyses were made for the alpha X chain of the rare Hb Manitoba in which alpha 102(G9) Ser is replaced by Arg. This variant was observed for the first time in an Italian patient, and was also studied in a member of a previously described Canadian family.

The occurrence of different levels of G gamma chain and of the A gamma T variant of fetal hemoglobin in newborn babies from several countries.

The gamma chain compositions of the fetal hemoglobins of 2453 newborn babies from East Asian countries (1350 babies), from Italy, Yugoslavia, Bulgaria, and Georgia (417 Caucasian babies), and 686 black babies from Georgia were determined by high pressure liquid chromatography. Unusual results for a limited number of babies were confirmed by chemical analyses, and were evaluated further by family studies. Statistical analyses indicated high gene frequencies for the A gamma T chain in Italian (f = 0.237), Yugoslavian and Bulgarian (f = 0.238), and white Georgia babies (f = 0.224), a lower frequency in Japan (f = 0.178), and India (f = 0.173), and particularly in mainland China (f = 0.079). The A gamma T gene frequency in normal (AA) Black babies was 0.102. When a beta S or beta C mutation was also present this frequency was greatly decreased, particularly in babies with the AC condition (f = 0.036). These results suggest the near absence of the A gamma T mutation on the chromosome also carrying the beta C determinant. Most babies had the expected G gamma values which vary between 60 and 80%, but several (mainly black) babies had higher values (between 80 and 90%), while one normal black baby had a G gamma value of (nearly) 100%. This condition may be a form of A gamma +1-thalassemia and has been discussed in detail elsewhere (Blood 58:491-500, 1981). Thirty-five clinically normal (mainly Chinese, Indian, and Japanese) babies had G gamma values of about 40%. Twenty-six babies had A gamma I values of about 60%, while the remaining nine babies had A gamma T and A gamma I chains in a ratio of either 1 to 2 or 1 to 1. Two additional newborns did not produce any G gamma chains, but had only A gamma I chains or A gamma T chains. Family studies failed to indicate a specific hematological abnormality. These unusual ratios between the G gamma and A gamma (either A gamma I or A gamma T) chains have led to speculations regarding possible genetic abnormalities present in these infants.