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PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for beta-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant.

Hb Crete, an electrophoretically neutral, unstable, high oxygen affinity variant, was characterized by protein and DNA analyses in the homozygous state in a 32-year-old woman from Crete, with erythrocytosis and microcytosis. The proband and members of her family over 3 generations, including 5 carriers of Hb Crete, were subject to clinical, hematological and biochemical investigations, and DNA, RNA and protein studies were carried out. The proband demonstrated features associated with disturbed hemoglobin (Hb) structure and function, including erythrocytosis and additionally a state of functional anemia, the latter reflected by increased erythropoetin levels and cardiac output. In addition, all the carriers surprisingly had hematological and biosynthetic findings more usually associated with thalassemia trait. The structural change in Hb Crete only partly explains all the pathological manifestations of this variant, and other mechanisms are discussed.

Background: Phenylketonuria (PKU), an inborn error of metabolism, is treated with a low phenylalanine (Phe) lifelong diet, which can be characterized as vegetarian. 8-Hydroxy-2-deoxyguanosine (8-OHdG) is highly implicated in degenerative diseases., Objective: To evaluate the effect of plasma total antioxidant status (TAS) and Phe on the serum marker of DNA damage, 8-OHdG, in PKU., Methods: Twenty-four PKU patients on a strict diet (group A), 25 PKU patients on a "loose diet" (group B), and 24 healthy children (controls) participated in this study. Plasma TAS was evaluated spectrophotometrically. 8-OHdG and Phe were measured in blood with immunoassays., Results: TAS levels were significantly higher (P < 0.001) in group A (1458 +/- 140 micromol/L) and controls (1452 +/- 235 micromol/L) than those in group B (907 +/- 150 micromol/L). In contrast, 8-OHdG serum levels were 2-fold higher in group B (0.22 +/- 0.03 ng/mL) as compared with those in group A (0.11 +/- 0.02 ng/mL) and 3-fold higher than those in controls (0.08 +/- 0.02 ng/mL) (P < 0.001). As expected, Phe levels were also significantly higher in group B than those in the other study groups. Positive correlation coefficients were found between Phe and 8-OHdG levels, whereas negative correlations were evaluated between TAS and 8-OHdG in all groups., Conclusions: The high Phe and the low TAS plasma levels in PKU patients on a "loose diet" may induce DNA oxidation, as evidenced by the measured high 8-OHdG level in their sera. 8-OHdG evaluation may be a useful marker of increased risk for a neurodegenerative process.

Unlabelled: A 32-day-old girl with massive hypertriglyceridaemia and clinical signs of chylomicronaemia syndrome is described. Genetic study of the patient revealed compound heterozygosity for a common lipoprotein lipase gene mutation (G188E) and a novel missense mutation (M301R), consistent with reduced post-heparin plasma lipoprotein lipase immunoreactive mass observed., Conclusion: to the best of our knowledge, this is the first description of a patient with a M301R mutation in the lipoprotein lipase gene. In addition, dietary therapy with medium-chain triglycerides was successful supporting the effectiveness of this therapeutic approach in familial chylomicronaemia syndrome., (Copyright 2004 Springer-Verlag)

The most frequent defects resulting in alpha-thalassemia (thal) include large deletions that remove one or both of the duplicated alpha-globin genes on chromosome 16. Less commonly, alpha-thal mutations involve single nucleotide substitutions or micro-deletions, leading either directly to decreased alpha-globin chain synthesis by the affected allele, or indirectly through production of hyperunstable variant alpha-globin chains. Here we describe the characterization of a 33 bp in-frame deletion within the alpha1-globin gene, in a woman with hematological findings consistent with an alpha-thal trait. The amino acids predicted to be missing as a result of the 33 bp deletion are at the end of the E helix and the EF corner of the alpha-globin protein chain, and are not normally involved in the heme contact, although it is presumed that alpha-globin chain folding and hemoglobin (Hb) formation will be disrupted. The observation of inclusion and Heinz bodies indicates the synthesis of some abnormal Hb (or globin chains). An identical mutation has been previously observed in a single case, a Canadian individual of Greek descent, indicating that it is a rare mutation, and probably of the same origin. Possible mechanisms underlying the mutation at the DNA level are discussed.

Sickle-cell and beta-thalassemia syndromes are priority genetic diseases for prevention programs involving population screening with the option of prenatal diagnosis for carrier couples. Preimplantation genetic diagnosis (PGD) represents a specialized alternative to prenatal diagnosis and is most appropriately used for couples with an unsuccessful reproductive history and/or undergoing assisted reproduction. However, clinical application of PGD has been hindered by difficulties in reliably transferring molecular diagnostic protocols to the single-cell level. We standardized and validated a protocol involving first-round multiplex PCR, amplifying the region of the beta-globin gene containing most of the common disease mutations world-wide and two unlinked microsatellite markers (GABRB3 and D13S314), followed by: 1) analysis of beta-globin genotypes with real-time PCR and 2) microsatellite sizing to exclude chance contamination. The protocol was standardized on 100 single lymphocytes from a beta-thalassemia heterozygote, including 15 artificially contaminated samples, the latter demonstrated through microsatellite analysis. PCR failure and allele drop-out (ADO) were observed in one (uncontaminated) sample each (1.2%). A pilot study in six clinical PGD cycles with five different beta-globin genotype interactions achieved results (in 5-6 hr) in 46 out of 50 single blastomeres (92%), all concordant with results from an established PGD method applied simultaneously; microsatellite analysis detected only parental alleles, excluding contamination. Beta-globin genotypes were also confirmed in two blastomeres through prenatal diagnosis (twin pregnancy), and in 11 out of 12 spare embryos, revealing one incident of ADO. Overall, the protocol proved to be sensitive, accurate, reliable, rapid, and applicable for many genotype interactions, with internal monitoring of contamination, thus fulfilling all requirements for clinical PGD application., (Copyright 2004 Wiley-Liss, Inc.)

The lipoprotein lipase coding gene sequence was analysed on a 10-year-old girl with new-onset Type 1 diabetes mellitus (DM), ketoacidosis and severe hypertriglyceridaemia (TG > 112.9 mmol/l), revealing that the patient was a compound heterozygote for two mutations, D9N in exon 2 and S447X in exon 9. Although these two mutations usually do not considerably impair lipolytic enzyme activity, the combination of both in this patient may play a role in the development of severe hypertriglyceridaemia.

Beta-thalassemia is uncommon (0.5%) in the Romanian population, but it must be considered in the differential diagnosis of hypochromic anemia. The molecular characterization of beta-thalassemia is absolutely necessary for molecular diagnosis, as well as any genetic epidemiological study in this region. Molecular analyses consist of mutation detection by molecular scanning of beta-globin gene. This gene has 3 exons and 2 introns, involved in beta-thalassemic pathogenesis. Clinical application of DNA analysis on beta-thalassemic chromosomes allowed characterization of 29 persons with different beta-thalassemia mutations among 58 patients with anemia. The experimental strategy was based on sequential PCR amplification of most of the beta-globin gene and running on denaturing gradient gel electrophoresis of amplification products. Definitive characterization of mutations in samples identified with shifted DGGE patterns was performed ARMS-PCR and/or PCR-restriction enzyme analysis methods. Eight different beta-thalassemia alleles were identified, the most common being IVS I-110 (G-A) and cd 39 (C-T). Comparison of overall frequency of mutations in the neighboring countries, shows that these results are in the frame of overall distribution of these mutations in Mediterranean area, especially in Greece and in Bulgaria. Molecular diagnosis is useful for differentiating mild from severe alleles, for genetic counseling, as well as for mutation definition in carriers, identified by hematological analysis necessary for prenatal testing and genetic counseling.

The coinheritance of beta-thalassemia major with the genotype of Hb H disease is extremely rare, with few reported cases. We investigated the hematological, biochemical, biosynthetic, molecular and pathophysiological parameters to evaluate a rare male patient with this compound syndrome. The patient was studied at first diagnosis during hospitalization at 50 years of age and subsequently followed up for more than a year. Examinations included full hematological, biochemical, biosynthetic, molecular, pathophysiological and clinical parameters. Besides standard parameters, we additionally measured reticulocyte hemoglobin content (CHr), erythropoietin (Epo), soluble transferrin receptors (sTfR), oxygen pressure at 50% hemoglobin saturation (P50), 2,3-bisphosphoglycerate (2,3-BPG), total glutathione (GSHt), oxidized glutathione (GSSG), malonyldialdehyde (MDA), nontransferrin-bound iron (NTBI), vitamins A and E. The male patient was first hospitalized for a 2-day period at 50 years of age, following the finding of marked anemia (hematocrit 20%) during a blood test to investigate the cause of fatigue in the absence of weight-loss or other notable symptomatology. He had never been transfused, maintaining Hb 85-95 g/l. Definitive diagnosis was achieved through DNA studies, which showed coexistence of beta-thalassemia major (IVSI-6 T > C/IVSI-I G > A) with Hb H disease (-alpha(3.7)/-(Med)). Alpha/non-alpha globin chain biosynthesis was completely balanced. Parameters demonstrated a well-compensated anemia with ineffective erythropoiesis and oxidative stress, which was ameliorated following splenectomy. In conclusion, this case is a remarkable example that the coinheritance of severe forms of beta-thalassemia and alpha-thalassemia interact in a "synergistic" manner to almost complete balance the symptoms of classic thalassemia syndromes.

Abnormal globin chain biosynthesis may result in deficient quantity (thalassemia) or structural variation (abnormal hemoglobins) and traditionally, they represent two phenotypically distinct groups of disorders. However, the phenotypic expression of unstable hemoglobin variants often combine features of thalassemia together with variable peripheral hemolysis. To achieve definitive diagnosis in a child presenting with hemolytic anemia along with features associated with thalassemia intermedia, we evaluated clinical, hematological, biochemical, globin biosynthetic and molecular data. Definitive diagnosis was achieved by DNA analysis which characterized the proband to be a compound heterozygote for a common alpha-thalassemia-2 deletion (3.7 kb) and Hb Questembert (alpha131[H14] Ser>Pro) caused by a C>T mutation in codon 131 of the alpha1 globin gene in trans. The phenotype of thalassemia intermedia with marked dyserythropoiesis, found in patients inheriting alpha-thalassemia mutations along with unstable alpha-globin variants (i.e., alpha-thalassemic hemoglobinopathies), represents a distinct type of thalassemic syndrome. The proband in this study additionally had variable peripheral hemolysis, presumably related to characteristics of the unstable Hb Questembert. There is minimal experience for the management of such atypical cases and this case illustrates that it is probably insufficient to monitor clinical status in patients with such hemoglobinopathies based only on the levels of hemoglobin.

Background and Objectives: Ferrokinetic studies and erythroid cell ultrastructural studies have indicated some degree of ineffective erythropoiesis in heterozygous beta-thalassemia, although a wide case-to-case variation was observed. In this study we applied rapid biochemical and hematologic measurements to assess erythroid marrow activity (sTfR) and reticulocyte hemoglobin content (CHr) in iron-sufficient individuals with heterozygous beta-thalassemia and investigated the correlation with the degree of globin polypeptide chain imbalance by comparing parameters between beta-thalassemia heterozygotes with genotypes of variable severity., Design and Methods: We studied 57 iron-sufficient adults with heterozygous beta-thalassemia, divided into groups according to genotype: group A: beta(silent)-thalassemia heterozygotes, group B: beta(+)-thalassemia heterozygotes and group C: beta(0)-thalassemia heterozygotes. Twenty-one hematologically normal individuals served as controls (group D). We measured hematologic parameters including CHr with a Bayer-Advia 120 hematology analyzer. Hemoglobins were analyzed by high performance liquid chromatography, while biochemical parameters of iron status (iron, ferritin, transferrin and sTfR) were measured with chemical, luminometric and nephelometric methods., Results: We found significant positive correlations between sTfR values for all beta-thalassemia heterozygote groups when plotted against Hb A(2) and Hb F levels (r=0.566, p<0.0001 and r=0.283, p<0.03, respectively) and significantly negative correlation between CHr and Hb A(2) values (r=-0.790, p<0.00001). These data reflect the fine association of globin polypeptide chain imbalance with erythron expansion and the greater degree of ineffective erythropoiesis in beta-thalassemia heterozygotes with more severe genotypes., Interpretation and Conclusions: This study is the first demonstration that sTfR and CHr are useful parameters for evaluating the relative severity of different genotypes in heterozygous beta-thalassemia.

Background: Hemoglobinopathies are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples., Methods: As a model, we designed a protocol based on the LightCycler technology to screen for a spectrum of beta-globin gene mutations in the Greek population. Design was facilitated by dual fluorochrome detection and close proximity of many mutations. Three probe sets were capable of screening 95% of beta-globin gene mutations in the Greek population, including IVSII-745C-->G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1G-->A, IVSI-5G-->A, IVSI-6T-->C, IVSI-110G-->A, and Cd39 C-->T., Results: The protocol, standardized by analysis of 100 beta-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek beta-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations HbS/Cd6 and IVSI-5/IVSI-1, indicating a need of alternative methods for definitive diagnosis. One sample demonstrating a unique melting curve was characterized by sequencing as Cd8/9+G. Three samples carried mutations outside the gene region covered by the probes. The protocol was 100% accurate in 25 prenatal diagnosis samples, with 14 different genotype combinations diagnosed. The protocol was also flexible, detecting five beta-globin gene mutations from other population groups (IVSI-1G-->T, IVSI-5G-->C, IVSI-116T-->G, Cd37 TGG-->TGA, and Cd41/42 -TCTT)., Conclusions: The described LightCycler system protocol can rapidly screen for many beta-globin gene mutations. It is appropriate for use in many populations for directing definitive mutation diagnosis and is suited for rapid prenatal diagnosis with low cost per assay.

Cystic fibrosis (CF) mutation analysis on 437 CF patients, characterized 80 different mutations (20 so far specific to our population) accounting for 91% of CF genes and generating 103 different genotypes. Eight mutations were common [F508del (53.4%), 621+1G>T (5.7%), G542X (3.9%), N1303K (2.6%), 2789+5G>A (1.7%), 2183AA>G (1.4%), E822X (1.4%), R1158X (1%)], 12 showed frequencies between 0.5% and 1%, while the remaining (60) were very rare (1 to 3 alleles). Denaturing gradient gel electrophoresis (DGGE) screening of 12 exons (3, 4, 7, 10, 11, 13, 14b, 16, 17b 20 and 21) detected 85.5% of CF alleles. Haplotypes for eight diallelic and three microsatellite markers have been characterized for the common, a few rare and novel Greek mutations. Results of 165 prenatal diagnoses (including 49 due to bowel hyperechogenicity), testing a total of 41 different parental genotypes, are reported. One hundred and sixteen prenatal tests resulted in 22 affected, 59 heterozygous, 34 normal fetuses and one incomplete diagnosis. Of the 49 echogenic bowel fetuses, 3 were heterozygotes. Carrier screening was initiated, with emphasis on individuals and couples in high-risk groups - with a family history of CF, one partner with CF, and couples with male infertility seeking in vitro fertilization (IVF). Mutation analysis on 672 individuals (120 couples, 91 unaffected CF siblings, 283 CF family relatives and 58 general population subjects), identified a total of 176 heterozygotes and 7 couples where both partners were CF heterozygotes. Prenatal diagnosis was performed in 4 cases and 3 were counseled on the availability of a prenatal test.

A single patient with a rare Haemoglobin H (HbH) disease genotype (- -Med/alphaTSaudialpha) was observed to have exceptionally high levels of HbH (> 60%) and paradoxically high total haemoglobin levels. Studies of haematological parameters, blood biochemistry and oxygen transport properties revealed a severe functional anaemia, associated with marked erythropoietic stimulation and a markedly raised cardiac output. This rare case illustrates the complexity of interactions that may be associated with the clinical course of HbH disease, highlighting that haematological parameters alone may lead to spurious evaluation of clinical status. Issues related to the therapeutic management of unusual cases are raised.

Cystic fibrosis (CF) is one of the most important genetic diseases requiring prevention programmes. Preimplantation genetic diagnosis (PGD) represents an alternative to prenatal diagnosis, and is especially appropriate for couples with an unsuccessful reproductive history. For clinical application, protocols must be optimized to minimize PCR failure, allelic drop-out (ADO) and contamination, while simultaneously detecting a wide spectrum of CF genotypes. We have developed a flexible multiplex PCR protocol allowing analysis of sequence variations in any combination amongst seven CFTR gene exons (4, 10, 11, 13 in two parts, 14b, 17b and 21) by nested PCR and denaturing gradient gel electrophoresis analysis, along with analysis of a fluorescently labelled intragenic microsatellite (IVS8CA). The experiments were carried out on 390 single lymphocytes from three CF patients, one heterozygote and one non-CF individual. PCR efficiency of the exons ranged from 90 to 100%, and ADO from 0 to 3.8%. IVS8CA was co-amplified with a PCR efficiency of 92.4 and 10.8% ADO. The present method overcomes the need for separate assays for each CFTR gene mutation. Additionally, it facilitates analysis of any informative linked polymorphic sequence variation (within the seven exons) along with analysis of a microsatellite, which is useful (when informative) for minimizing misdiagnosis and/or indirect diagnosis. This method proved robust and flexible for diagnosing diverse CF genotype combinations in single cells.

Preimplantation genetic diagnosis (PGD) represents an alternative to prenatal diagnosis and allows selection of unaffected IVF embryos for establishing pregnancies in couples at risk for transmitting a genetic disorder.

Preimplantation genetic diagnosis (PGD) requires the combined efforts of geneticists and workers in the field of reproductive medicine. This was studied on the basis of a questionnaire, sent to 35 members of the PGD Consortium of the European Society of Human Reproduction and Embryology (ESHRE). A reply was obtained from 20 centres. They represent the majority of activities in the field of PGD in the world. It is obvious that many of the activities (in vitro fertilisation, embryo culture and biopsy) take place in IVF units while others (counselling and diagnosis) are the responsibility of genetic diagnostic centres. The distances between both units vary considerably. In all but one centre sex determination is offered. Aneuploidy screening is offered in 13 out of 20 centres. PGD of translocations and other structural chromosome abnormalities is offered in all but one centre. The number of monogenic diseases offered varies considerably. In comparison to prenatal diagnosis PGD is more expensive. The majority of these costs are due to the IVF or ICSI procedure. The charges for PGD vary between about 600 euro and 4000 euro. In 16 out of 20 centres the parents to be must sign an informed consent form., (Copyright 2001 John Wiley & Sons, Ltd.)

Background and Objectives: The degree of globin chain imbalance and tissue hypoxia are important determinants of clinical severity in thalassemia syndromes. Thus phenotypic expression may be modified by interaction of alpha- and beta-thalassemia defects, level and type of hemoglobin synthesized and oxygen release to the tissues. We evaluated hematology, erythroid marrow activity and functional anemia in patients with the rare interaction of a single a-globin gene and heterozygous beta-thalassemia (HbH/beta-thal trait)., Design and Methods: In 7 patients characterized by DNA analysis to have HbH disease genotypes with beta-thalassemia trait, we assessed hematologic findings, serum transferrin receptor (sTfR), serum erythropoietin (Epo), red cell 2,3-disphosphoglycerate (2,3-DPG) and whole blood oxygen releasing capability., Results: Patients with HbH/beta-thal trait had moderate anemia, marked hypochromasia and microcytosis, normal or raised HbA2, and no electrophoretically/chromatographically detectable HbH. Epo and sTfR levels were significantly higher than in beta-thalassemia heterozygotes, but lower than in patients with HbH disease; 2,3-DPG levels were highest in HbH/beta-thal trait. Oxygen binding studies and simulations showed reduced oxygen affinity (P50) in HbH/beta-thal trait, resulting in increased oxygen release (O2R)., Interpretation and Conclusions: Hematologic findings and bone marrow activity in patients with HbH/b-thal trait were consistent with the modified globin chain imbalance and hemoglobin synthesis expected from interaction of HbH disease with heterozygous b-thalassemia, although this rare complex genotype may elude diagnosis based on hematology alone. Significantly higher red cell 2,3-DPG levels were an unexpected finding, and the consequent increase in oxygen release capability resulted in a compensated functional anemia relative to hemoglobin levels.

Hb Mont Saint-Aignan [beta128(H6)Ala-->Pro] is a mildly unstable variant, associated with hemolytic anemia, marked microcytosis and increased alpha/beta biosynthetic ratio (1.55 versus 1.1 +/- 0.1 in the control). The abnormal chain was isolated by selective precipitation with isopropanol and the structural modification determined by protein chemistry methods (reversed phase high performance liquid chromatography and mass spectrometry). Possible mechanisms underlying the beta(+)-thalassemia-like expression of this variant are discussed.

Hb Sitia [beta128(H6)Ala-->Val] was found in a Greek female with slightly reduced red blood cell indices. The abnormal hemoglobin was indistinguishable from Hb A by electrophoresis but eluted after Hb A on cation exchange high performance liquid chromatography. DNA sequence analysis revealed a GCT-->GTT mutation at codon 128, which is predicted to encode an Ala-->Val substitution. This was confirmed by mass spectrometry analyses of the beta-globin chain. Since alanine at beta128(H6) interacts with several amino acids of the alpha1beta1 contact, its replacement by a larger residue results in a mild instability of the molecule and slight modifications of the oxygen binding properties.

Background and Objectives: The occurrence of Hb CS is usually limited to the geographic area which includes Southern China and South East Asia. In 1968 Hb CS was also found to occur in the Mediterranean area where it was originally described as Hb Athens. We investigated the independent origin of these termination codon mutations of the alpha 2-globin gene by determining the alpha-cluster haplotype and comparing the hematologic data from Hb CS-Hb H patients and their family members., Design and Methods: We studied one Hb CS-Hb H patient of Greek origin and a Sicilian family in which one individual was affected by Hb CS-Hb H. The haplotype of the Hb CS allele was determined and compared to the haplotype of an Hb CS-Hb H individual of Chinese origin., Results: The haplotype found for the Greek and Sicilian Hb CS was the same but differed significantly from the Asiatic Hb CS mutation., Interpretation and Conclusions: The Hb CS mutation found in both Mediterranean patients arose independently in the Mediterranean area. The difference in clinical manifestation of the Hb CS-Hb H disease in both patients is less common but consistent with similar variation in the clinical expression of analogous Hb Icaria-Hb H disease patients.

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.

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.

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., (Copyright 2000 Academic Press.)

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.

Preimplantation genetic diagnosis (PGD) allows the selection of unaffected IVF embryos for transfer in couples that are at risk for transmitting genetic diseases. For monogenic diseases, polymerase chain reaction (PCR)-based diagnosis is usually performed on single blastomeres. In Greece, up to 10 per cent of the population are carriers for beta-thalassaemia and related haemoglobinopathies, and more than 20 pathological mutations in the beta-globin gene have been described. In this study we report a strategy which includes a first round of PCR, allowing subsequent nested PCR and DGGE analysis for at least 95 per cent of beta-thalassaemia major genotypes in the Greek population. The use of DGGE for beta-globin genotype analysis is advantageous: it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it detects the presence of normal alleles and monitors the occurrence of allelic drop-out (ADO) through the expectation that heterozygous samples have more than one electrophoretic band on DGGE analysis. The optimization, accuracy and reliability of the method was evaluated by genotyping 325 single blastomeres, 110 amniocytes and 55 lymphocytes. Results confirmed that PCR efficiency and occurrence of ADO are improved by higher denaturation temperatures in the first cycles of first-round PCR, influenced by the size of the fragment amplified in the first round of PCR and additionally by the quality and type of cells being genotyped. The proposed strategy was accurate and reliable, and thus for application to PGD should ensure the transfer of unaffected embryos. Furthermore it is widely applicable in most of the populations worldwide where beta-thalassaemia is common.

In this report we describe a case of Hb H disease due to the interaction of the --(MED 1) deletion with a new alpha(+)-thalassemia determinant. The molecular analysis of the proband's genomic DNA was carried out by polymerase chain reaction amplification and sequencing of both alpha genes of the alpha(+)-thalassemia chromosome and revealed a deletion of codon 62 of the alpha1 gene. This DNA triplet codes for a valine residue at the E11 alpha helix, which is located in the interior of the heme pocket. Substitutions of valine E11 with other amino acid residues in the alpha as well as beta polypeptide chains lead, in the heterozygous carrier, either to Hb M disease or to congenital non-spherocytic hemolytic anemia. We assume that the deletion of valine at alpha62(E11) disrupts the conformation of the alpha chain to such an extent that the mutated subunit is rapidly removed by proteolysis. The final result is an alpha-thalassemia phenotype rather than an unstable hemoglobin syndrome. This conclusion is supported by the apparent absence of an abnormal alpha chain in the peripheral blood of the patient.

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.

We report here the functional and structural characterization of Hb Acharnes [beta53(D4) Ala --> Thr], an unstable and electrophoretically silent variant, that was found associated in trans with a beta(0)-thalassemic mutation (IVSI-1 G --> A), in a patient with thalassemia intermedia syndrome. This case is discussed in comparison with other sporadic cases that we have previously investigated, resulting from the co-inheritance of a beta(0)-thalassemic mutation (CD39 C --> T) with two other types of unstable hemoglobins, Hb Köln [beta98(FG5) Val --> Met], and Hb Arta [beta45(CD4) Phe --> Cys]. It may be concluded that, in these associated forms, both the degree of instability of the variant and the altered oxygen binding properties (affecting the degree of tissue hypoxia) are major determinants of their clinical expression., (Copyright 1999 Wiley-Liss, Inc.)

The Bio-Rad Variant Hemoglobin testing system is an automated high performance liquid chromatography analyzer marketed with a beta-thalassemia short program to quantify Hbs F and A2, and assist in detecting Hbs A, S, D, C, and E. Although the two hemoglobins present in Hb H disease, Hb Bart's and Hb H, are separated by the system, they are not quantitated. In this study we modified the beta-thalassemia short program in order to facilitate quantitation of Hb Bart's and Hb H. Blood samples from 60 patients with Hb H disease, with various underlying genotypes, were studied. Analyses were performed on the day of blood collection or on hemolysates stored at -80 degrees C in cyanide or carbomonoxy forms. The mean sum of Hb Bart's and Hb H levels in all patients was found to be 12% (range 1.8-35%). Patients with nondeletional mutations (or association of alpha(0) deletion and nondeletional mutations) had notably higher Hb Bart's and Hb H levels when compared to patients with deletional genotypes.

Purpose: To determine serum immunoreactive erythropoietin (Epo) and soluble transferrin receptors (sTfR) levels in patients with hemoglobin H (HbH) disease and the correlation with HbH levels and alpha-globin genotype., Patients and Methods: Twenty patients with HbH disease were studied. Methods applied included cation-exchange high pressure liquid chromatography for HbH levels, chemoluminescence for Epo concentration, immunoassay for sTfR concentration, and DNA analysis for alpha-globin genotype characterization., Results: Serum Epo and sTfR levels were significantly elevated (46.6+/-26.8 IU/l and 5.6+/-1.8 mg/l, respectively) in patients with HbH disease compared to controls (9.2+/-3.3 IU/l and 1.8+/-0.7 mg/l, respectively). Epo and sTfR levels correlated positively with HbH concentration (r = 0.93 and 0.80, respectively). The highest Epo and sTfR values were observed in three patients with the highest HbH levels who all had nondeletion alpha-thalassemia mutations., Conclusion: Epo and sTfR levels are increased in patients with HbH disease; this increase is directly related to the HbH concentration that usually reflects the degree of globin polypeptide imbalance. The correlation of Epo, sTfR, and reticulocyte production index in these patients indicates that anemia in HbH disease mainly is caused by ineffective erythropoiesis and a mild degree of peripheral hemolysis.

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.

Patients with the nondeletion genotype of hemoglobinopathy H (HbH or beta4) disease have higher proportions of HbH and more severe tissue hypoxia than patients with the deletion genotype. Because these patients' red blood cells (RBCs) contain mainly two Hb species, HbH and HbA, the high proportion of HbA can be exploited by lowering its oxygen affinity; this would probably increase oxygen delivery to the RBCs and improve the patients' clinical phenotype. Allosteric effectors that induce a low-affinity Hb may be useful in this regard. We investigated the effect of a bezafibrate derivative, RSR-4, on the oxygen affinity of RBCs and purified hemolysates containing HbA and HbH. This allosteric effector crosses RBC membranes and binds reversibly to the alpha-chains of deoxy-Hb, decreasing hemoglobin oxygen affinity. The blood used was obtained from a patient with HbH disease (alphaTSaudi homozygote) whose HbH level was 33.5% as measured by high-performance liquid chromatography. Oxygen binding studies were performed in RBCs and purified hemolysates. RBCs incubated in the presence of 500 microM RSR-4 (2-[[[(3,5-dichloroanilino)-carbonyl]methyl]phenoxy]-2-methylpropi onic acid) in standard conditions (pH 7.4, 0.14 M NaCl, 37 degrees C) displayed an increase in their P50 value from 14.5 to 35.2 mm Hg. Oxygen binding studies in purified stripped hemolysates (pH 7.2, 0.1 M NaCl, 25 degrees C) showed that addition of both 500 microM RSR-4 and 1 mM of 2,3 diphosphoglycerate (DPG) led to an 11-fold decrease in oxygen affinity, whereas the addition of the natural effector DPG or RSR-4 alone produced a 2.7- and 5.7-fold decrease, respectively. In both cases, the oxygen equilibrium curves (OECs) were biphasic due to the presence of the noncooperative, high-oxygen-affinity HbH (beta4) component. After addition of RSR-4, the lower part of the OEC (corresponding to HbH) was not shifted compared with the upper part (corresponding to HbA). These results were confirmed by kinetic studies of CO recombination. Both experiments demonstrated that RSR-4 does not affect beta4 Hb. Our findings provide an experimental model for lowering the oxygen affinity of HbA in HbH-containing cells and suggest that the oxygen delivery capability of the latter would be thereby improved.

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.

This study reports the characterization of 60% of low density lipoprotein receptor (LDLR) gene mutations in 150 unrelated Greek familial hypercholesterolaemia (FH) heterozygous children by the analysis of six LDLR gene mutations. The linkage disequilibrium of two polymorphic microsatellites (D19S394 and D19S221) flanking the LDLR gene on chromosome 19 to the four most common mutations strongly suggests that each mutation is identical-by-descent in the probands included in this study (this is also supported by the geographical distribution of FH families with these mutations throughout Greece) and permits an estimation of the number of generations from a common ancestor for each mutation. The characterization of 60% of LDLR mutations in a representative sample of Greek FH heterozygotes provides a basis for the diagnosis of FH through DNA analysis in Greece, by using single-strand conformation polymorphism analysis followed by allele-specific oligonucleotide hybridization (exon 6 mutations) or restriction endonuclease analysis (C152R, V408M). A rapid diagnostic assay positive for the mutation has been developed for the most common mutation, G528D. The application of simple DNA diagnostic assays for LDLR mutation analysis are appropriate for the early identification of FH heterozygotes in Greece and are useful for the primary prevention of coronary artery disease.

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 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.

To initiate the complete characterization of mutations in the CFTR gene in Greek cystic fibrosis (CF) patients, we screened 184 patients for six relatively common mutations (delta F 508, G542X, G551D, 621 + 1 G-->T, N1303K, W1282X) using allele-specific hybridization and, in addition, analyzed exons 4, 5, 7, 8, 10, 11, 17b, 19, 20 and 21 using the method of denaturing gradient gel electrophoresis (DGGE). Six mutations accounted for 65.9% of the CF alleles in Greek patients, of which the delta F 508 mutation had a frequency of 52.7%. A further 15 previously described mutations accounted for another 8.3% CF alleles and one previously undescribed mutation (3272-4A-->G) was found in one chromosome. The W1282X mutation was not detected at all. Thus, so far, we have identified 21 mutations in the CFTR gene in Greek CF patients, accounting for 74.5% of the CF alleles.

Two novel CFTR mutations were detected in Greek cystic fibrosis patients. One was a missense mutation, A46D, and the other a splice mutation, 296 + 1G-C. Neither was detected on normal chromosomes.

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)

The presence of nine mutations in the phenylalanine hydroxylase (PAH) gene, previously described in phenylketonuria (PKU) patients of other Mediterranean and European populations, was assessed in 47 Greek PKU and 3 hyperphenylalaninaemia (HPA) patients. Of the nine mutations investigated, only five were detected, characterizing 31% of the PKU alleles in our patients.

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.

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.

We have identified three individuals of Greek or Greek Cypriot origin with an atypical form of HbH disease characterized by a severe hypochromic microcytic anaemia associated with relatively small amounts of HbH in the peripheral blood. Molecular analysis has shown that each is a compound heterozygote for a previously described mutation affecting the poly A addition signal (AATAAA-->AATAAG) and a previously undescribed mutation involving a T-->C transition in codon 29 of the alpha 2 gene causing a leucine-->proline substitution. Although this mutation would be expected to produce an unstable haemoglobin and hence a haemolytic anaemia, simple heterozygotes for the alpha 29Leu-->Pro mutation have the phenotype of alpha-thalassaemia trait.

alpha-Thalassemia is usually due to deletions within the alpha-globin gene cluster, leading to loss of function of one (-alpha) or both [-(alpha) or --] alpha-globin genes. Nondeletion mutations (denoted alpha alpha T or alpha T alpha) are less frequent and in Greece are not well defined. We report the analysis of 16 nondeletion alpha-thalassemia chromosomes using a polymerase chain reaction method to amplify specifically the alpha 2-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 (alpha PolyA alpha), two the IVSI 5' pentanucleotide deletion (alpha Hph alpha), two the Hb Icaria mutation (alpha Ic alpha), and one the initiation codon mutation (alpha Nco alpha). In two, the defects are still undefined. These findings show that nondeletion alpha-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.

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.