Different hematological phenotypes caused by the interaction of triplicated α-globin genes and heterozygous β-thalassemia

The pathophysiology and clinical severity of β-thalassemia are related to the degree of α/non-α-chain imbalance. A triplicated α-globin gene locus can exacerbate effects of excess α-chains caused by a defective β-globin gene, although this is not observed in all cases. Extensive studies on this condition are lacking. We report a group of 17 patients who are heterozygous for both the ααα^anti-3.7 allele and a mutation in the β-globin gene cluster. Their clinical phenotypes varied: six had mild anemia with microcytosis and hypochromia, while 11 had more severe anemia with splenomegaly requiring splenectomy (three cases) and blood transfusions (four cases). Different phenotypes were also evident in the presence of the same β-thalassemia mutation: in one family, two individuals had the same α- and β-globin genotypes but presented with different hematologic phenotypes. In addition, the complex interaction involving a triplicated α-globin gene, β³⁹- and δ⁺²⁷-thalassemia mutations is studied in a family with two siblings presenting with hemolytic anemia, normal Hb A₂ and increased Hb F. Analysis of this series of patients suggests that additional genetic determinants play a role in modulating phenotypic expression in individuals with identical α- and β-globin genotypes. Interaction with a triplicated α-gene can play a role in the clinical presentation of patients with defective β-globin gene expression and should be considered in the diagnosis of atypical cases. Am. J. Hematol. 55:83-88, 1997. © 1997 Wiley-Liss, Inc.     

Detection of the (–SEA) double α-globin gene deletion by a simple immunologic assay for embryonic ζ-globin chains

Homozygous α-thalassemia [α-thal-1], with loss of all four α-globin genes, causes lethal hydrops fetalis. The most common mutation producing this syndrome is the Southeast Asian (–^SEA ) double α-globin gene deletion. Erythrocytes from adults heterozygous for the (–^SEA ) deletion have minute amounts of embryonic ζ-globin chains detectable by anti-ζ-globin monoclonal antibodies. Among 225 peripheral blood samples tested by a simple anti-ζ-immunobinding tetra-zolium dye test, 81 were positive and 144 were negative. The majority of subjects were of Filipino, Chinese, or Laotian ancestry. All 81 positive samples were confirmed by Bam HI digests and a ζ-cDNA probe to have the (–^SEA ) mutation. The (–^SEA ) double α-deletion was the only abnormality in 58. In the others, it was combined with α-globin or β-globin mutations, or coincidental iron deficiency. Four other samples from (–^SEA ) heterozygotes were negative by this immunologic assay. Anti-ζ negative samples included 78 deletions of the total α-globin region, (–^Tot), 23 single α-globin deletions, and a variety of β-globin mutations; 16 normocytic samples with normal α-genes were also negative. Ten anti-ζ positive and 25 anti-ζ negative samples had benign triplicated ζ-globin genes. In this population, the sensitivity of this test was 95%; and specificity for the (–^SEA ) mutation was 100%. Anti-ζ immunobinding testing provides rapid, simple, and reliable screening for the (–^SEA ) double α-globin deletion, although it does not detect the (–^Tot) total α-deletions. © 1993 Wiley-Liss, Inc.     

Nondeletional α-thalassemia: First description of αHphα and αNcoα mutations in a Spanish population

Several different deletions underlie the molecular basis of α-thalassemia. The most common α-thalassemia determinant in Spain is the rightward deletion (−α^3.7). To our knowledge, however, no cases of α-thalassemia due to nondeletional mutations have so far been described in this particular Mediterranean area. Here, we report the existence of nondeletional forms of α-thalassemia in ten Spanish families. The α₂-globin gene was characterized in ten unrelated patients and their relatives only when the presence of deletional α-thalassemia was ruled out. The α₂-globin gene analysis was performed using the polymerase chain reaction (PCR) followed by restriction enzyme analysis or by allele-specific priming. This allowed the identification of a 5-base pair (bp) deletion at the donor site of IVS I (α^Hphα) in 9 cases and the α₂ initiation codon mutation (α^Ncoα) in one case. Although these α₂-globin gene mutations are found in other Mediterranean areas, our results demonstrate their presence in the Spanish population and suggest that the α^Hphα/αα genotype is probably the most common nondeletional form of α-thalassemia in Spain. © 1996 Wiley-Liss, Inc.     

Characterization of nondeletion α-thalassemia mutations in the Greek population

α-Thalassemia is usually due to deletions within the α-globin gene cluster, leading to loss of function of one (-α) or both [-(α) or –] α-globin genes. Nondeletion mutations (denoted αα^T or α^Tα) are less frequent and in Greece are not well defined. We report the analysis of 16 nondeletion α-thalassemia chromosomes using a polymerase chain reaction method to amplify specifically the α2-globin gene, which was subsequently screened using ASO hybridization or restriction enzyme analysis for four mutations already characterized in other Mediterranean and Middle Eastern populations. Of the 16 nondeletion chromosomes, nine had the polyadenylation signal mutation (α^PolyAα), two the IVSI 5′ pentanucleotide deletion (α^Hphα), two the Hb Icaria mutation (α^icα), and one the initiation codon mutation (α^Ncoα). In two, the defects are still undefined. These findings show that nondeletion α-thalassemia in Greece is heterogeneous and that the most frequent mutation (accounting for >50%) is the polyadenylation signal mutation, which to date was most commonly found in the Saudi Arabian population. © 1993 Wiley-Liss, Inc.     

Quantities of αQ chain variants in heterozygotes with and without a concomitant β-thalassemia trait

We have analyzed the quantities of α^x chain-containing hemoglobins (α^xβ₂ and α₂^xβ₂,) in 14 heterozygotes for Hb Q-India [α64(E13)Asp→His] or Hb Q-Thalland [α74(EF3)Asp→Hls]; both amino acid replacements are the result of mutations in the α1-globin gene. Five of these persons (three with Hb Q-India and two with Hb Q-Thailand) had an additional β°-thalassemia heterozygoslty. The average quantities for Hb Q + Hb Q₂ in the four groups were 17.2% (^Q/αα; β^A/β^A), 9.5% (αα^Q/αα; β^A/β°^Th), 26.8% (-α^Q/αα;β^A/β^A), and 16.95% (-α^Q/αα; β^A/β°^Th). These variations can best be explained by a posttranslational control mechanism; an imbalance in the α^A, α^Q, and β^A chain ratio will favor the α_z^Qβ_z formation when an α-thalassemia is present and will reduce its formation in the presence of a β-thalassemia heterozygosity. © 1994 Wiley-Liss, Inc.     

α-thalassemia carrier identification by DNA analysis in the screening for thalassemia

Differentiation between heterozygous α-thalassemia and several phenotypically resembling alleles at the β-globin gene cluster such as coinherited δ- and β-thalassemia or γδβ-thalassemia is a critical step in genetic counseling. In this paper we report our experience in the identification of the α-thalassemia carrier state using polymerase chain reaction (PCR)-based methods, and the feasibility and simplification of screening for thalassemia using this approach. α-Globin genotype was determined by PCR-based method in 526 adult subjects with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), normal hemoglobin A₂ and F, and normal serum iron. To verify the reliability of the protocol used, in 68 of these subjects we performed globin chain synthesis analysis and in 101 we determined α-globin genotype by Southern blot analysis. Five hundred twenty-one (99%) of 526 subjects examined were identified as carriers of one or two α-thalassemia alleles. The identification of the α-thalassemia carrier state may be fast and accurate by PCR-based method, avoiding other cumbersome and expensive methods such as globin chain synthesis and Southern blot analysis. Am. J. Hematol. 59:273–278, 1998. © 1998 Wiley-Liss, Inc.     

Molecular characterization of β-thalassemia mutations in Guadeloupe

In order to perform genetic counselling and prenatal diagnosis of Hb-S-β-thalassemia disease and β-thalassemia, we have delineated the spectrum of β-thalassemia alleles in the Guadeloupean population. A sample of 63 unrelated families was analyzed including 70 β-thalassemia carriers, 52 Hb-S-β-thalassemia, and 8 patients with different β-thalassemic hemoglobinopathies. Among the eleven mutations identified, four of them [-29 (A → G), IVS-I-5 (G → A), IVS-II-1 (G → A), and IVS-I-5 (G → C)] account for 77.6% of the β-thalassemia chromosomes present in the studied families. The seven other variants, CD 24 (T → A), IVS-I-2 (T → C), Poly A (T → C), -88 (C → T), IVS-II-849 (A → G), Hb E, and Hb Lepore are less frequent. As a result, Hb S-β -thalassemia type 1 (low Hb A values: 5–15%) together with Hb S-β°-thalassemia phenotypes are as frequent as Hb S-β⁺-thalassemia type 2 (high Hb A values: 20–30%) in the Guadeloupean population. Patients with Hb S-β -thalassemia type 2 have milder hematological manifestations of the disease compared to patients with Hb S-β°-thalassemia and Hb S-β⁺-thalassemia type 1. This first report on the type and nature of β-thalassemia mutations in Guadeloupe shows that prenatal diagnosis of Hb S-β-thalassemia and β-thalassemia should be feasible by direct detection of point mutation in most cases. © 1996 Wiley-Liss, Inc.     

Mild β+(−87)-thalassemia CACCC box mutation is associated with elevated fetal hemoglobin expression in cis

The β°-thalassemia codon 39 nonsense mutation predominant in Sardinia is severe, and homozygotes are transfusion dependent. Two-thirds of β° 39 alleles are linked to ^Aγ^T (haplotype II). Onefourth are linked to ^Aγ^I (haplotypes I and IX), as is the mild β⁺-thalassemia ?87 C→G mutation (haplotype VIII). β⁺/β°-Thalassemla VIII/II compound heterozygotes have Significantly higher ^Aγ^I:^Aγ^T (23:7) than β°-thalassemia I/II (24:20) or IX/II (16:17) cases. This suggests that the β⁺ ?87 mutation is associated with elevated γ expression in cis, which may contribute to the lack of transfusion-dependence in β⁺/β° Cases. © 1994 Wiley-Liss, Inc.     

Phenotypic variability of Filipino β°-thalassemia/HbE patients in Indonesia

Three Indonesian patients with identical genotypes, each compound heterozygotes for Filipino β°-thalassemia/HbE, expressed different clinical severities. One patient has mild disease and is transfusion independent, while the other two are severely affected and transfusion dependent. The size of the Filipino β°-globin gene deletion was confirmed to be 45 kb, resolving conflicting values given in the literature. Neither ameliorating genetic factors such as α-globin gene deletions or the XmnI restriction site polymorphism at position -158 upstream of the ^Gγ-globin gene, nor differences in β-globin gene haplotype, explain the phenotypic variation. These observations have implications for the development of antenatal diagnosis in Indonesia, as at present it is not possible to give an accurate prediction of severity of phenotype for this common genotype. Am. J. Hematol. 62:7–12, 1999. © 1999 Wiley-Liss, Inc.     

Rapid detection of the common mediterranean α-globin deletions/rearrangements using PCR

The most frequent molecular lesions causing α-thalassemia are deletions of one or more α-globin genes. Detection of these deletions generally requires genomic Southern analysis, which is cumbersome and time consuming. We have designed new sets of primers for PCR identification of the common Mediterranean α-globin gene rearrangements, including the -α^3.7 deletion and the ααα^anti3.7 triplication, the -α^4.2 deletion, and the --^Med allele. We have established reaction conditions that provide easily interpretable, unambiguous diagnoses. Some of the PCR reactions are multiplex, simultaneously identifying several genotypes, thus reducing the time and cost of screening and prenatal testing. The use of these methods should facilitate carrier screening and identification of couples at risk for α-thalassemia. Am. J. Hematol. 58:306–310, 1998. © 1998 Wiley-Liss, Inc.     

α-thalassemia-1 (— −-CAL mutation) in a Spanish family

We have detected a second family (five members affected) with a large (32 kb) deletion involving the α₁, α₂, ψα₁, ψα₂,ψζ₁, and ζ-globin genes. This mutation has been previously described in Calabria, Italy, in a child and his mother and has been named α-thalassemia ?- ?-^CAL . © 1994 Wiley-Liss, Inc.     

Two different mutations in codon 68 are observed in Hb G-Philadelphia heterozygotes

Through sequencing of amplified DNA containing the appropriate α-giobin genes we have identified the base substitution leading to the formation of Hb G-Philadelphia [α6B(El7)Asn Lys]. Three subjects (～25% Hb G) had an AAC AAA change at codon 68 of the α2-globin gene; the chromosome with this mutation carried two α genes (αα). Six subjects (～33% Hb G) had an AAC AAG change at the same codon of the α2α1 hybrid gene on a chromosome with the 3.7 kb deletion (-α^3.7). These results indicate two inde pendent mutations which likely occurred in different populations; Increase In the level of Hb G is primarily dependent upon the loss of one or more α-globin genes. © 1994 Wiley-Liss, Inc.     

Maximal γ-globin expression in the compound heterozygous state for –175 Gγ HPFH and β°39 nonsense thalassaemia: a case study

The –175 (T→C) ^Gγ hereditary persistence of fetal haemoglobin is a very rare promoter mutation occurring in Caucasians as well as in African-Americans. Heterozygotes for this non-deletional HPFH show 20% HbF, mostly of ^Gγ type. We describe here a healthy Sardinian man who coinherited –175 (T→C) ^Gγ HPFH with the β-thalassaemia codon 39 nonsense mutation in trans; he showed 64% HbF, 100% of ^Gγ type. Although the β-globin haplotype pattern (II/II) was indicative of the presence of the ^Aγ^T allele on both chromosomes, the ^Aγ^T expression was undetectable by HPLC even in red cell populations separated by age. The proband was, moreover, homozygous for the –4 bp deletion at position -225 to -222 of ^Aγ promoter which has recently been associated with decreased ^Aγ^T globin expression. These findings suggest that this maximal overexpression of ^Gγ-globin probably reflects intensified stimulation of the mutated ^Gγ promoter in this hitherto undescribed genetic condition.     

Hemoglobin E diseases: Hematological,analytical, and biosynthetic studies in homozygotes and double heterozygotes for α-thalassemia

Two families with Hb E diseases are described. In the Laotian family S, three homozygous Hb E were found. In the Vietnamese family H, double heterozygous Hb E-α-thalassemia-2 and Hb E-Hb H diseases were found. Anemia or hemolysis was absent in Hb E carriers, unless complicated by iron deficiency, the presence of severe α-thalassemia gene (Hb H disease), or oxidative drug (paraaminosalicylic acid). Moreover, iron deficiency or concurrent α-thalassemia genes resulted in a decreased amount of Hb E in its heterozygous carriers. Mild microcytosis and hypochromia were observed in Hb E heterozygotes, whereas the microcytosis and hypochromia were more pronounced in Hb E homozygotes. Globin chain synthesis studies yielded unbalanced α/non-α ratios in both heterozygotes and homozygotes (average ratios were 1.13 and 1.56, respectively). The unbalanced biosynthetic ratios with microcytosis and hypochromia in Hb E carriers represented a β-thalassemia phenotype, which could be a result of reduced synthesis of β^E-globin mRNA, as suggested by recent hybridization studies.     

β-thalassemia and hemoglobin types in Argentina: Determination of most frequent mutations

In order to know the spectrum of β-thalassemia alleles and other mutations affecting the β-globin gene, we analyzed the hemoglobin abnormalities in 24 patients from the Province of Córdoba in Argentina. Molecular screening of samples was performed by the polymerase chain reaction (PCR), using six sets of oligonucleotides to amplify fragments encompassing the whole β-globin coding region and splice junctions, as well as the promoter and 3′ untranslated regions. The altered fragments were determined by denaturing gradient gel electrophoresis (DGGE), and the corresponding mutations were identified by restriction enzyme analysis or by direct sequencing of PCR products. Using this approach, three different β-thalassemia mutations were detected, codon 39 (C→T), IVS-1-110 (G→A), and IVS-1-1 (G→A), and also the hemoglobin S trait. This is the first report of β-thalassemia mutations described in Argentina. Our results show that these mutations are similar to those found in Spain and Italy, possibly due to the important Mediterranean migratory stream received in our country, and could be important for prenatal diagnosis of these diseases in Córdoba, Argentina. Am. J. Hematol. 54:160–163, 1997 © 1997 Wiley-Liss, Inc.     

De novo initiation codon mutation (ATG→ACG) of the β-globin gene causing β-thalassemia in a swiss family

Investigation of microcytic anemia with normal ferrous status in two members (father and daughter) of a Swiss family originating from Bern revealed high levels of HbA₂ (4%, 7.3%) and HbF (3.2%, 3.1%). Direct sequence analysis of asymmetrically amplified DNA showed the ATGǎG mutation in the initiation codon of the β-globin gene. Heterozygous β-thalassemia was not found in either of the propositus's parents or in any of his brothers and sisters. Extended restriction fragment length polymorphism haplotyping of the β chromosomes led us to the conclusion of a recent spontaneous mutation in the paternal germ cell. The results of routine HLA and blood group testing supported the stated paternity. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the Bam HI polymorphism downstream from the β-globin gene as previously observed. This is the second family found to carry this initiation codon mutation in the β-globin gene. Unlike the first reported family, of Yugoslavian origin, our patients have high HbF levels and this in the absence of a C→T substitution at-158 site 5′to Gγ. © 1993 Wiley-Liss, Inc.     

Heterozygous β-thalassemia with thalassemia intermedia phenotype

In this study we investigated the molecular bases of the β-thalassemia intermedia phenotype in six patients belonging to two unrelated families of Sardinian descent. Sequence analysis of the β globin gene from these patients detected, as the sole abnormality, the heterozygosity for the codon 39 nonsense mutation. The Aγ and Gγ promoters as well as the HS2 and HS3 core sequences of the β globin LCR from these patients, did not show any non-polymorphic nucleotide variation from the consensus sequence. One of the parents was heterozygous for codon 39 nonsense mutation but showed the β-thalassemia carrier phenotype; the other was hematologically normal and had an entirely normal β globin gene sequence. In both families, other members showed the typical hematological phenotype, clinically silent, of heterozygous β thalassemia. To explain the thalassemia intermedia phenotype, we postulated the presence of an unknown molecular defect interacting with the β globin gene mutation. Haplotype analysis excluded that this postulated defect lies in the β globin gene cluster. Am. J. Hematol. 57:43–47, 1998. © 1998 Wiley-Liss, Inc.     

Sickle cell disorder, β-globin gene cluster haplotypes and α-thalassemia in neonates and adults from Guadeloupe

We have studied haplotype of β^s chromosome and α-globin gene status in 534 patients (255 adults and 279 children of whom 159 neonates) from Guadeloupe with various sickle cell-related conditions, namely SS (n = 298), SC (n = 170), S-β -thal (n = 56), and other rare forms (n = 10). Haplotype data on β^s chromosomes confirm our previous observation that Benin type is the most prevalent (75%) β^s chromosome in Guadeloupe, in disagreement with the historical records. Comparison of the frequency of distribution of various β^s haplotypes between neonates and adults on the one hand and between SS and SC cases on the other shows that the current β^s haplotype distribution in this island is not distorted by haplotype-related differential survival. We also show that the frequency of α-thalassemia (-3.7 kb) in Guadeloupe is one of the highest recorded in this region involved in Atlantic slave trade and also failed to reveal any age-dependent increase in frequency. We conclude that the African component of Guadeloupe is distinct from that of Brazil and Cuba but is close to that of Jamaica. Am. J. Hematol. 55:24-27, 1997. © 1997 Wiley-Liss, Inc.     

Severity of β-thalassemia due to genotypes involving the IVS-I-6 (T→C) mutation

Among individuals of Mediterranean or Middle Eastern descent, the IVS-I-6 (T→C) mutation is one of the most common causes of β-thalassemia. In this report, we describe the clinical phenotypes of a group of β-thalassemia patients who are compound heterozygotes for the relatively mild IVS-I-6 (T→C) β-thalassemia mutation and more severe β⁺- or β°-thalassemia mutations. Although most of these patients are transfusion-dependent, the requirement for regular transfusions generally occurred late in childhood. A correlation between concomitant α-thalassemia and a mild transfusion-independent phenotype is not apparent, indicating the involvement of other ameliorating determinants.     

Initiation codon mutation (ATG → ATA) of the β-globin gene causing β-thalassemia in a Swedish family

An initiation codon mutation ATG → ATA of the β-globin gene was found in seven members of three generations of a family living in northern Sweden. This mutation, which has not previously been described, changes the initiation codon for methionine into a codon for isoleucine and will then result in a β°-thalassemic phenotype. The affected family members all present hematological findings typical for β-thalassemic trait, with slight anemia, marked microcytosis, and increased levels of Hb A₂. ©1995 Wiley-Liss, Inc.