Phenotypic and molecular diversity of haemoglobin H disease: a Greek experience.

Haemoglobin H (Hb H) disease is the severest form of alpha-thalassaemia compatible with post-natal life and occurs when alpha-thalassaemia mutations interact to reduce alpha-globin synthesis to levels approximately equivalent to the output of a single alpha-globin gene. Hb H disease has variable clinical expression, mainly related to underlying genotypes. The spectrum of alpha-thalassaemia determinants in Greece appears greater than in any other population studied and, in 75 Greek Hb H disease patients, we found 12 alpha-thalassaemia mutations interacting to produce 15 Hb H disease genotypes. Evaluation of haematological, biochemical and clinical findings, and correlation with genotypes, defined genetic predictors of disease severity and factors involved in disease progression. In accordance with previous reports, patients with non-deletion alpha-thalassaemia mutations had more severe clinical expression. Additionally, we found that all patients with the most severe phenotypes had alpha-thalassaemic globin variants. Phenotypic severity was not simply related to the degree of alpha-globin deficiency: high Hb H levels were found to exacerbate anaemia by negatively influencing tissue oxygenation, and both Hb H and alpha-thalassaemic haemoglobin variants appear to reduce red cell survival within the bone marrow and circulation. Together with the long-term follow-up in many patients, this report provides comprehensive information for management of Hb H disease and appropriate family counselling.

Distinct phenotypic expression associated with a new hyperunstable alpha globin variant (Hb heraklion, alpha1cd37(C2)Pro>0): comparison to other alpha-thalassemic hemoglobinopathies.

Clinical phenotypes associated with abnormal globin chain biosynthesis may result in thalassemia (deficient quantity) or hemolytic anemia (abnormal hemoglobins). However, the phenotypic expression of hyperunstable hemoglobin variants often includes features of thalassemia, along with variable peripheral hemolysis. Hemoglobinopathies caused by highly unstable beta-chain variants have a dominant thalassemia-like phenotype, in which carriers have a clinical expression of thalassemia intermedia, but highly unstable alpha-globin variants are usually only phenotypically apparent when they interact with other alpha-thalassemia mutations. In a child with clinical and hematological features consistent with beta-thalassemia intermedia, DNA analysis excluded any beta-globin gene mutations but characterized a novel deletion cd37(C2)Pro>0 (Hb Heraklion) in the alpha1 globin gene, in trans to a common Mediterranean nondeletion alpha-thalassemia mutation (alpha(Hph)alpha). The deletion of proline at alpha37(C2) is predicted to result in severe instability of the variant hemoglobin, which on interaction with a synthesis-deficient alpha-thalassemia mutation causes a relatively severe dyserythropoietic anemia, representing an alternative phenotype associated with highly unstable alpha-chain variants. Hb Heraklion is the fourth highly unstable alpha-globin variant that we have observed in patients from Greece and Albania. Two variants involve the alpha2-globin gene: Hb Agrinio (alpha29(B10)Leu>Pro) and Hb Adana (alpha59(E8)Gly>Asp), and two the alpha1-gene: Hb Aghia Sophia (alpha62(E11)Val>0) and (Hb Heraklion a37(C2)Pro>0). Each has been observed on interaction with a different alpha-thalassemia mutation and the phenotypes associated with these highly unstable alpha-variants are presented.

Molecular studies of beta-thalassemia heterozygotes with raised Hb F levels.

Hb F levels in beta-thalassemia heterozygotes are usually less than 2%, but amongst 1,059 patients studied, 73 (7%) had Hb F levels above 2.5% (2.6-14.0%). To investigate factors that may influence the increase of Hb F levels in these heterozygotes, we characterized the beta-thalassemia mutations and their chromosomal background, gamma-globin gene promoter variations, and alpha-globin genotypes. All 73 beta-thalassemia heterozygotes carried beta-thalassemia point mutations previously observed in the Greek population; gene mapping excluded b gene cluster deletions; only two cases had an additional gamma-globin gene (gammagammagamma/gammagamma). Five alpha-globin genes (alphaalphaalpha/alphaalpha) were detected in 17/73 cases (23%) as compared to a carrier rate of 1.76% in the general population. Molecular, hematological, and biosynthetic findings in these compound heterozygotes indicated that the raised Hb F levels were caused by cell selection due to ineffective erythropoiesis. In the remaining 56 simple beta-thalassemia heterozygotes, 11 beta-thalassemia mutations were observed, each on the expected haplotype(s), and analysis of the gamma gene promoters revealed three known polymorphisms (in linkage disequilibrium), with minimal influence on gamma-globin levels. However, the overall distribution of beta-thalassemia mutations in the 56 simple beta-thalassemia heterozygotes was significantly different (P<0.0002) compared to that in 986 simple beta-thalassemia heterozygotes with <2.5% Hb F, implicating an association between beta-thalassemia mutations and moderately increased Hb F levels, most notably codon 39 (C-->T), IVS-II-1 (G-->A), codon 6 (-A), and codon 8 (-AA), which accounted for 41/56 (73%) cases with >2.5% Hb F. In the remaining 15/56 (27%) cases, no common underlying globin genotypes could explain the raised Hb F levels. Overall, this study indicates that the control of Hb F levels in beta-thalassemia heterozygotes is heterogeneous and multi-factorial.

Molecular, haematological and clinical studies of the -101 C --> T substitution of the beta-globin gene promoter in 25 beta-thalassaemia intermedia patients and 45 heterozygotes.

We report the clinical, haematological, biosynthetic and molecular data of 25 double heterozygote beta-thalassaemia intermedia patients and 45 beta-thalassaemia heterozygotes with the C --> T substitution at nucleotide position -101 from the Cap site, in the distal CACCC box of the beta-globin gene promoter. This mutation is considered the most common amongst the silent beta-thalassaemia mutations in Mediterranean populations. Of the 25 compound heterozygotes for the beta -101 C --> T and common severe beta-thalassaemia mutations, all but one had mild thalassaemia intermedia preserving haemoglobin levels around 9.5 g/dl and haemoglobin F levels < 25%. The only transfused patient was characterized to have an additional alpha-globin gene. Strict assessment of haematological and biosynthetic findings in the heterozygotes for the beta -101 C --> T mutation (excluding six cases with an alpha-globin gene defect) demonstrated that less than half of them had completely normal (silent) haematology; the remainder had either high haemoglobin A2 values (in the range of 3.7-5.1%) and/or low red cells indices and/or raised haemoglobin F values. The alpha/non-alpha-globin chain synthesis ratios were generally raised, with mean 1.44 (1.07-2.10). Amongst the parents of the compound heterozygotes, who were not selected for molecular analysis following haematological screening, half of the cases were completely silent. Interaction with severe beta-thalassaemia mutations always resulted in the clinical phenotype of mild non-transfusion-dependent thalassaemia intermedia.

Interaction of an alpha(+)-thalassemia deletion with either a highly unstable alpha-globin variant (alpha2, codon 59, GGC-->GAC) or a nondeletional alpha-thalassemia mutation (AATAAA-->AATAAG): comparison of phenotypes illustrating "dominant" alpha-thalassemia.

Thalassemia syndromes and unstable hemoglobins traditionally represent two phenotypically separate disorders of hemoglobin synthesis. Highly unstable hemoglobin variants, however, often have phenotypic characteristics associated with both ineffective erythropoiesis (thalassemias) and peripheral hemolysis (unstable hemoglobins). Many highly unstable beta chain variants cause a dominant thalassemia-like phenotype, in which simple heterozygotes for such mutations have a clinical expression similar to thalassemia intermedia. The phenotypic expression of highly unstable alpha-globin variants is usually less severe, due mainly to a gene dosage effect, and they are often only characterized on interaction with other alpha-thalassemia mutations, whence they are classified as nondeletional alpha-thalassemia determinants. This study reports the clinical and hematological findings in five cases with rare alpha-thalassemia genotypes: a single patient with the thalassemic alpha2-globin gene codon 59 Gly-->Asp hemoglobin variant in trans to an alpha(+)-thalassemia deletion, and four compound heterozygotes for the nondeletional alpha-thalassemia polyadenylation mutation (alpha2 gene AATAAA-->AATAAG or alpha(T-Saudi)alpha/-alpha) and an alpha(+)-thalassemia deletion. Evaluation of the clinical and hematological features in these two analogous genotypes clearly demonstrates the more severe clinical expression associated with the alpha-thalassemic unstable hemoglobin variant. In addition, the case in this study with the codon 59 alpha chain variant provides a further example illustrating the spectrum of phenotypes associated with the alpha-thalassemic hemoglobinopathies.

Molecular, haematological and clinical studies of a silent beta-gene C-->G mutation at 6 bp 3' to the termination codon (+1480 C-->G) in twelve Greek families.

We report the clinical, haematological, biosynthetic and molecular data of 12 beta-thalassaemia intermedia patients and their heterozygous parents, all of whom carried a rare C-->G mutation at nucleotide position 6 3' to the termination codon (term. cd +6 C-->G) in the 3' untranslated region (3' UTR) of the beta-globin gene (+1480 C-->G). This mutation has been reported previously in a single beta-thalassaemia intermedia patient of Greek origin. The 12 patients of the present study had the clinical phenotype of mild non-transfusion-dependent thalassaemia intermedia, preserving haemoglobin levels around 9 g/dl and haemoglobin F levels <25%. All were compound heterozygotes for the +1480 C-->G mutation and common severe beta-thalassaemia mutations. The haematological parameters of heterozygotes with this mutation were within the normal range with the exception of a slightly raised alpha/non-alpha-globin chain synthesis (1.2-1.9). mRNA analysis demonstrated a 20-34% reduction in mRNA levels associated with the +1480 C-->G mutation compared to normal beta-globin alleles. These findings confirm that the C-->G mutation at position 6 3' to the termination codon is a mild beta-thalassaemia mutation causing slight reduction in beta-globin mRNA levels and beta-globin chain synthesis. It becomes clinically relevant when co-inherited with a severe beta-thalassaemia mutation in trans.

Correlation of clinical phenotype to genotype in haemoglobin H disease.

Clinical assessment, haematological studies, and globin gene mapping were performed in 21 Greek subjects with haemoglobin H disease. Clinical phenotypes ranged from mild, and virtually asymptomatic, to severe cases requiring transfusion. The severe clinical phenotype was exclusively associated with non-deletion genotypes, whereas the mild and intermediate phenotypes occurred with deletion genotypes. Patients with non-deletion genotypes had higher levels of Hb H. For deletion genotypes of haemoglobin H disease, the value of antenatal diagnosis is questionable. In non-deletion genotypes, antenatal diagnosis should be considered, because of the more severe clinical course observed in these patients.