An alpha-thalassemic hemoglobinopathy: homozygosity for the HB Agrinio alpha 2-globin chain variant.

This report describes the first case of homozygosity for the Hb Agrinio [alpha 29(B10)Leu-->Pro] alpha 2-globin gene variant (codon 29, CTG-->CCG) in a Greek patient. At 12 months of age, the proband presented with a marked hypochromic, microcytic anemia, a very low level of Hb H (< 2.5%), rare Hb H inclusions, and a balanced alpha/non-alpha biosynthesis ratio. The mother had hematological findings and globin biosynthesis consistent with heterozygous beta-thalassemia, but paradoxically, red cell morphology demonstrated very rare Hb H inclusions. The father had mild microcytosis and hypochromia. Analysis of alpha- and beta-globin genotypes demonstrated that the patient was homozygous for the highly unstable Hb Agrinio variant, caused by a T-->C mutation in codon 29 of the alpha 2-globin gene. At the age of 13 years, the proband had a clinical phenotype compatible with mild thalassemia intermedia with moderate anemia (Hb 7-8 g/dL), normal growth and development, slight splenomegaly, and minimal bone changes, while Hb H and inclusion bodies were not detected.

The triplicated alpha-globin gene locus in beta-thalassaemia heterozygotes: clinical, haematological, biosynthetic and molecular studies.

Excess alpha-globin chains play a major role in the pathophysiology of homozygous beta-thalassaemia. In beta-thalassaemia carriers a minor effect of alpha-globin chain excess is reflected in a minimal or mild anaemia without clinical symptoms. Factors that increase alpha-chain excess in heterozygotes are expected to accentuate the severity of the clinical and haematological phenotype. We report the clinical, haematological, biosynthetic and molecular data in three beta-thalassaemia heterozygotes with the rare interaction of homozygosity for alpha-globin gene triplication, and in 17 heterozygotes with a single additional alpha-globin gene. The three patients homozygous for the alpha-globin gene locus (anti 3.7 kb arrangement) had beta(0)-thalassaemia mutations and a diagnosis of thalassaemia intermedia, preserving haemoglobin levels around 7-8 g/dl. Of the 17 beta-thalassaemia heterozygotes (six children and 11 adults), 16 had severe beta-thalassaemia mutations interacting with an additional alpha-globin gene (13 with alpha alpha alpha anti-3.7 and four with alpha alpha alpha anti-4.2). Compared to simple beta-thalassaemia heterozygotes, they had lower haemoglobin levels and red cell indices, but higher alpha/beta biosynthesis, HbF levels and reticulocytes. Our results suggest that homozygous alpha-gene triplication interacts with a severe beta-thalassaemia mutation to cause an alpha-chain excess equivalent to that observed in homozygous beta-thalassaemia intermedia. In heterozygotes for severe beta-thalassaemia mutations with one additional alpha-globin gene, the alpha-chain excess causes a more pronounced degree of anaemia than is usually seen in simple beta-thalassaemia heterozygotes.

The interaction of alpha zero thalassaemia with Hb Icaria: three unusual cases of haemoglobinopathy H.

The clinical, haematological, biosynthetic and molecular data of three Greek haemoglobin H (HbH) disease patients with a distinctive clinical phenotype are described. During infancy all three patients had unusually severe clinical manifestations for HbH disease, with anaemia necessitating blood transfusions, signs of bone changes, growth impairment, and splenomegaly. Molecular analysis identified a rare alpha-thalassaemia genotype (--Med/ alpha Ic alpha). Splenectomy resulted in marked amelioration of the clinical signs; post splenectomy all three patients preserve adequate haemoglobin levels (9-10 g/dl) with growth restored to normal. Despite the initial severe clinical phenotype in these patients, our experience indicates that splenectomy modifies the clinical course to that of mild thalassaemia intermedia. This observation should be considered carefully when giving genetic counselling to families carrying the rare Hb Icaria mutation and an alpha zero thalassaemia mutation.

Molecular characterization of homozygous (high HbA2) beta-thalassemia intermedia in Greece.

Homozygous beta-thalassemia is usually characterized by severe anemia requiring regular blood transfusion for survival. For homozygous patients with milder clinical manifestations and no dependence on transfusion therapy, the term thalassemia intermedia is usually applied. Genetic mechanisms that may ameliorate the clinical expression of homozygous beta-thalassemia include coinheritance of alpha-thalassemia, inheritance of mild beta-globin gene mutations, and increased gamma-globin chain production, which may partially compensate for the lack of beta-globin chain synthesis. To identify which of these factors may contribute to the modification of childhood homozygous, high-hemoglobin A2 (HbA2) beta-thalassemia in Greece, the interaction of alpha-thalassemia, types of beta-thalassemia mutations, and the presence of a polymorphic site 5' to the G gamma-globin gene, which has been described as associated with increased gamma-globin chain production in some cases, was assessed. The results were analyzed in light of similar studies in 150 randomly selected, homozygous, high-HbA2 beta-thalassemia patients with the aim of assessing whether thalassemia genotypes can provide information useful for prognosis and/or more appropriate management of homozygous beta-thalassemia patients. The results indicate that, in general, the major factor modifying the clinical expression of homozygous, high-HbA2 beta-thalassemia in Greece is the inheritance of mild beta-thalassemia mutations. Although there is not always a complete correlation of genotype with clinical phenotype, the inheritance of two mild beta-thalassemia alleles results in almost all cases (11 of 12 cases in this study) in thalassemia intermedia phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Hematologic phenotype of the mutations IVS1-n6 (T-->C), IVS1-n110 (G-->A), and CD39 (C-->T) in carriers of beta-thalassemia in Greece.

The hematologic phenotype was characterized in heterozygotes for three of the most common beta-thalassemia mutations in the Greek population. The study included 17 carriers of beta++ IVS1-n6 (T-->C), 21 carriers of beta+ IVS1-n110 (G-->A), and 17 carriers of beta 0 CD39 (C-->T). The 55 beta-thalassemia heterozygotes were selected from among parents of patients on regular transfusion regimens, and the beta-thalassemia mutation was identified by means of the polymerase chain reaction to amplify the appropriate region of the beta-globin gene and then by allele-specific oligonucleotide hybridization. The assessment of hematologic phenotype included complete blood count and quantitation of hemoglobin HbA2 and HbF and of the globin chain biosynthesis ratio. Comparison and statistical analysis of the hematologic parameters for the three mutations demonstrated no consistent correlation among the three mutations relative to Hb levels, hematocrit, and red cell indices, although heterozygotes for the IVS1-n6 mutation produce red blood cells with slightly higher mean corpuscular volume; significantly lower values of HbA2 (mean, 3.81% +/- 0.62% with four values less than 3.60%) in IVS1-n6 heterozygotes compared with IVS1-n110 heterozygotes (mean, 4.69% +/- 0.48%) and CD39 heterozygotes (mean, 4.75% +/- 0.50%, P < 0.001); and significantly higher HbF levels in CD39 heterozygotes (mean, 2.31% +/- 1.52%) compared with IVS1-n6 heterozygotes (mean, 0.79% +/- 0.45%, P < 0.01) and IVS1-n110 heterozygotes (mean, 1.17% +/- 0.75%, P < 0.01). With respect to the HbA2 levels, the findings are in agreement with previous studies in Mediterranean populations; the slightly higher levels of HbF in CD39 heterozygotes appear to be reported for the first time.

The molecular basis of normal HbA2 (type 2) beta-thalassemia in Greece.

Heterozygotes for beta-thalassemia usually have raised levels of HbA2, but in Greece about 5% of beta-thalassemia carriers are observed to have normal or borderline levels. It is postulated that such cases have mild beta+ thalassemia mutations or coinheritance of delta-thalassemia. We selected 18 heterozygotes with the hematological phenotype of normal HbA2 (type 2) beta thalassemia who were negative for the delta beta Corfu mutation, and screened them for previously defined Mediterranean beta-thalassemia and delta-thalassemia mutations. The coinheritance of beta and delta-thalassemia was demonstrated in four cases with the following genotypes: in cis beta+ IVSII -n745/delta+ 27, beta 0NS39/delta 059(-A), beta+ IVSI-n110/delta 059(-A) and in trans beta+ IVSI-n6 and delta+ 27. A further nine heterozygotes had mild beta(+)-thalassemia mutations (eight with the beta+ IVSI-n6 mutation, one with the beta+ polyA (A-->G) mutation). In four heterozygotes with severe beta-thalassemia chromosomes (2 beta+ IVSI-n110, 1 beta 0 FSC-6, 1 beta 0 IVSI-n1) no known delta-thalassemia mutations were observed. One case had a delta beta deletion chromosome. These results indicate that the hematological phenotype of normal HbA2 (type 2) beta-thalassemia in Greece is genetically heterogeneous; it is mainly associated with the delta beta Corfu mutation or coinheritance of beta and delta thalassemia mutations or with very mild beta(+)-thalassemia mutations, mainly beta+ IVSI-n6. In the rare cases with severe beta-thalassemia mutations, the normal levels of HbA2 may be due to coinheritance of as yet undefined delta thalassemia mutations.

The Corfu delta beta thalassaemia mutation in Greece: haematological phenotype and prevalence.

The Corfu delta beta thalassaemia mutation, a 7.2 kb deletion partially removing the delta-globin gene and a single nucleotide mutation (G----A) at intervening sequence I (IVSI-n5) in the beta-globin gene in cis, was first described in a family from Corfu; the carriers for this mutation had the unusual haematological phenotype of heterozygous beta-thalassaemia with normal levels of HbA2. To investigate the frequency and haematological characteristics of Corfu delta beta thalassaemia in Greece we analysed 25 unrelated normal HbA2 type 2 beta-thalassaemia heterozygotes and their 23 clinically affected offspring. Gene mapping demonstrated that nine (36%) of the 25 normal HbA2 beta-thalassaemia heterozygotes were in fact Corfu delta beta thalassaemia heterozygotes and of the 23 patients, two were Corfu delta beta thalassaemia homozygotes and five compound heterozygotes for Corfu delta beta thalassaemia and another beta-thalassaemia defect. Detailed haematological analysis demonstrated that: the Corfu delta beta thalassaemia mutation does not completely abolish the expression of the beta-globin gene; the HbA2 levels are slightly lower (P less than 0.01) and the HbF levels slightly higher (P less than 0.01) in Corfu delta beta thalassaemia heterozygotes compared to beta-thalassaemia heterozygotes with the normal HbA2-type 2 phenotype who do not have the Corfu delta beta chromosome.

Clinical, haematological, and genetic studies of type 2 normal Hb A2 beta thalassaemia.

The clinical and haematological phenotype as well as chain synthesis data were studied in 35 doubly heterozygous patients with either normal Hb A2 and Hb F, type 2 beta thalassaemia and beta (high A2) thalassaemia (26 patients), or type 2 and other rare beta or delta beta variants (nine patients). Patients doubly heterozygous for type 2 and beta zero or delta beta zero thalassaemia variants had no detectable Hb A, indicating that the type 2 normal A2 beta thalassaemia is primarily the result of a beta zero gene. The clinical phenotype varied from severe thalassaemia major to mild thalassaemia intermedia, and was mainly related to the thalassaemia variant with which the type 2 normal A2 beta thalassaemia was combined, and the proportion of Hb A produced in beta + thalassaemia patients. Haematological and chain synthesis data were similar in heterozygotes with type 2 and beta zero or beta + (high A2) thalassaemia. Hb A2 levels were within the normal range (2.3 to 3.6%) though absolute values (Hb A2 per RBC) ranged from low normal (0.5 pg/RBC) to increased levels (1.0 pg/RBC.) The variation of Hb A2, as well as the presence of Hb A2 in a type 2/delta beta high F patient and the complete absence of HbA2 in a homozygous type 2 patient, indicate that there are at least two genotypes of type 2, one beta zero and the other delta beta zero. This has been recently proven by gene mapping studies. For clinicians, routine haematological and family studies are sufficient for the proper treatment and prevention of doubly heterozygous type 2 patients.

Frequency of alpha-thalassemia in Greece.

Using hematological and gene mapping techniques, a cord blood survey was carried out to estimate the frequency of alpha-thalassemia in the Greek population. Out of 227 newborns studied, 16 (7.05%) were found by gene mapping to be alpha-thalassemia 2 heterozygotes (-alpha/alpha alpha), and of these only two had increased levels of hemoglobin Bart's in the cord blood (1.2 and 2.0%). Similarly, one heterozygotes for the common Mediterranean alpha-thalassemia 1 haplotype (-/alpha alpha) and one for the 20.5-kb deletion type (-(alpha)20.5/alpha alpha) were found, showing increased levels of Bart's of 4.8 and 6.6%, respectively. Four (1.76%) heterozygotes for the triple alpha gene arrangement (alpha alpha alpha/alpha alpha) were found. One individual with a level of Bart's in the cord blood of 8% was found to be a double heterozygote for alpha-thalassemia 2 and a dysfunctional alpha gene arrangement (-alpha/-(alpha)?). These results give an overall incidence for alpha-thalassemia in the Greek population of 8.4%.

The molecular basis of HbH disease in Greece.

Globin gene mapping in 16 Greek individuals with HbH disease and their parents has demonstrated the occurrence of several HbH genotypes brought about by the interaction of two alpha zero-thalassaemia and two alpha+-thalassaemia haplotypes. Eight of the 16 patients had the genotype - -Med/-alpha 3.7, four the genotype -(alpha)20.5/-alpha 3.7 and three the genotype - -Med/alpha alpha T. In one patient the restriction data are consistent with two possible genotypes alpha alpha T/alpha alpha T or - -/alpha alpha T. It is demonstrated that HbH disease in Greece is heterogeneous, with the deletion haplotypes - -Med and -alpha 3.7 being more prevalent than the -(alpha)20.5 and non-deletion (alpha alpha T) haplotypes.

The triplicated alpha gene locus and beta thalassaemia.

In five families, the coinheritance of beta thalassaemia and an additional alpha gene (alpha alpha alpha/alpha alpha) have been observed. Among the beta thalassaemia heterozygotes, no phenotypic effect of the triplicated alpha gene was detected clinically or at the haematological level. Unexpectedly, however, four out of five beta thalassaemia homozygotes with alpha alpha alpha/alpha alpha gene complement had the milder clinical condition of thalassaemia intermedia and in at least one case there was evidence to suggest that this might be due to the alpha alpha alpha gene arrangement actin as an alpha thalassaemia allele.

The clinical phenotype of beta and delta beta thalassemias in Greece.

Based on precise evaluation of hematological findings and clinical manifestations, the relationship between genotype and clinical phenotype was studied in 475 Greek patients with beta and delta beta thalassemias. Almost all known genotypes are included in this series, but the most frequent was homozygous beta th high A2 (71.6%), beta th/beta th silent (7.4%), beta th/delta beta oth high F (6.3%) and beta th/beta th Dutch (6.3%). In general, the phenotype was related to the genotype, though clinical heterogeneity was detected among patients with the same genotype. The severe type of thalassemia major was most commonly found in homozygous beta th patients mainly of beta o/beta o and beta o/beta + genotypes while homozygous beta + patients had milder clinical manifestation. Furthermore a small group of patients, characterized as homozygous beta ++ (HbF less than 30%) had mild thalassemia intermedia. In addition mild thalassemia intermedia was principally related with homozygous delta beta oth, and compound heterozygous beta th/beta th silent I, and less frequently with other genotypes such as compound heterozygous with beta th/beta th Dutch, beta th/beta th silent II, beta th/delta beta oth high F or Lepore. It was shown that precise genetic characterization and clinical evaluation is of primary importance in predicting the prognosis and formulating the proper treatment for the individual patient with thalassemia.

Developmental changes in hemoglobin F levels during the first two years of life in normal and heterozygous beta-thalassemia infants.

To study the developmental pattern of hemoglobin F (HbF) during the first two years of life, levels of HbF were estimated in two groups of infants: 117 normal infants and 98 heterozygotes for beta-thalassemia, all aged between 1 and 24 months. The results may be summarized as follows: (1) Levels of HbF in beta-thalassemia heterozygotes were significantly higher than those of normal infants of the same age (P less than .01). (2) A reference curve for the decline of HbF in infants with beta-thalassemia trait was established to facilitate the diagnosis of heterozygotes during this period of life. (3) Hemoglobin A2 (HbA2) was also higher in beta-thalassemia heterozygotes than in normal infants of the same age. HbA2 increases with increasing age, reaching normal adult values at age 5 to 6 months. It is postulated that the higher level of HbF in heterozygous infants during the first two years of life is associated with the presence of the beta-thalassemia gene, which influences the increased synthesis of HbF in red cell.

Globin gene mapping in normal Hb A2 types of beta-thalassaemia.

Globin-gene mapping of DNA from 13 families with normal Hb A2 beta-thalassaemia (both type 1 and type 2) failed to detect any difference from normal in their globin-gene arrangement. We conclude that deletions such as those responsible for gamma beta-thalassaemia or a 'silent' Hb Lepore are not responsible for this type of beta-thalassaemia in Greece.

Haemoglobin Bart's hydrops syndrome in Greece.

A case of haemoglobin Bart's hydrops syndrome was characterised in a Greek family with a history of three other fetuses with hydrops. Family studies showed that both the mother and father carried alpha-thalassaemia genes, and globin-chain synthesis analysis of the present fetus showed a total absence of alpha-chain production. The haemoglobin composition of the fetus was similar to that seen in cases in south-east Asia, and analysis of DNA from the Greek case confirmed the total deletion of the alpha-chain genes. The extent of the deletion, however, differed from that seen in south-east Asian cases and included the loss of one of the embryonic zeta-chain genes. Thus the severe form of alpha-thalassaemia occurs in Greece but has arisen independently from the similar condition in south-east Asia. The condition must be considered in any woman of this racial background who gives a history of unexplained stillbirths.