Molecular analysis of β‐thalassaemia patients in a high incidence area of southern Italy

The prevalence of eight mutations in 84 patients with β‐thalassaemia major and in 16 subjects with thalassaemia intermedia was investigated. All of the patients were Italian, originating from Eastern Sicily (Messina area) and some Calabrian regions. Genomic DNA was amplified by polymerase chain reaction (PCR). DNA molecular investigations were performed by allele‐specific oligonucleotide (ASO) hybridization, to identify the following β‐thalassaemia mutations: CD39 (C‐T), IVS1‐110 (G‐A), IVS1‐6 (T‐C), IVS1‐1 (G‐A), IVS2‐745 (C‐G), IVS2‐1 (G‐A), ?87 (C‐G), CD6 A (?A). Our data underline that in thalassemia intermedia two mutations were statistically prevalent: IVS1‐6 T→C (P < 0.001) and CD 6‐A (P < 0.05). CD 39 was statistically prevalent in β‐thalassaemia major patients (P < 0.01). The difference between the two groups was not statistically significant for all the other mutations. Five different genotypes were recorded among thalassaemia intermedia and 15 among β‐thalassaemia major patients. Twenty‐five percent of the intermedia patients and 4.5% of the major patients had homozygosity for mild mutations (group I); 62.5% of the intermedia patients and 26.2% of the major patients had combinations of mild/severe mutations (group II). In addition, homozygosity or double heterozygosity for severe mutations (group III) was found in 12.5% of the intermedia patients and 69% of the major patients. Some genotypes were restricted to thalassaemia intermedia, including heterozygosity ?87/IVS1‐6 and IVS1‐6/CD 6‐A. It is essential to understand the distribution and frequency of the relevant mutations in each population where β‐thalassaemias exist. This is of particular importance for genotype–phenotype correlation and for carrier detection, genetic counselling and prenatal diagnosis.     

When to consider transfusion therapy for patients with non‐transfusion‐dependent thalassaemia

Non‐transfusion‐dependent thalassaemia (NTDT) refers to all thalassaemia disease phenotypes that do not require regular blood transfusions for survival. Thalassaemia disorders were traditionally concentrated along the tropical belt stretching from sub‐Saharan Africa through the Mediterranean region and the Middle East to South and South‐East Asia, but global migration has led to increased incidence in North America and Northern Europe. Transfusionists may be familiar with β‐thalassaemia major because of the lifelong transfusions needed by these patients. Although patients with NTDT do not require regular transfusions for survival, they may require transfusions in some instances such as pregnancy, infection or growth failure. The complications associated with NTDT can be severe if not properly managed, and many are directly related to chronic anaemia. Awareness of NTDT is important, and this review will outline the factors that should be taken into consideration when deciding whether to initiate and properly plan for transfusion therapy in these patients in terms of transfusion interval and duration of treatment.     

Subclassification of HbS syndrome: is it necessary?

Sickle β‐thalassaemia (S β‐thalassaemia) is a condition, which results from coinheritance of a sickle cell gene and a β‐thalassaemia gene. The clinical phenotype depends on the type of β‐thalassaemia gene (β⁺ or β⁰). There are several similarities in clinical and haematological features, which sometimes pose a difficulty in correct diagnosis. A definitive diagnosis is required in order to initiate early supportive treatment in patients with homozygous sickle cell disease (SS disease) and to define the later clinical course. Forty‐seven cases of haemoglobin sickle syndrome (HbS syndrome) were studied. The clinico‐haematological features and high‐performance liquid chromatography (HPLC) results from 17 patients with S β‐thalassaemia were compared with those of SS disease (10 patients). Splenomegaly was more commonly found in patients with S β‐thalassaemia. Among the haematological features, red blood cell counts and Hb_A2 levels were significantly higher in patients with S β‐thalassaemia, while red cell indices, such as MCV, MCH were significantly lower than those seen SS disease. MCHC, PCV total haemoglobin (Hb), HbS, A and HbF were similar in the two groups.     

Common origin of a rare β‐globin initiation codon mutation (ATG→AGG) in Asians

In this report, we describe two Thai siblings presenting with mild hypochromic microcytic anaemia and splenomegaly since 2½ years of age. However, both patients were otherwise well with normal weight and height development and did not require transfusion during the 6‐year follow‐up period. Haematological and haemoglobin analyses were consistent with the clinical diagnosis of Hb E/β‐thalassaemia disease. To provide proper genetic counselling for this family, a definitive diagnosis of β‐thalassaemia was achieved using molecular analysis. We identified a rare initiation codon mutation (ATG→AGG) of the β‐globin gene in combination with the Hb E mutation (codon 26: GAG→AAG). The initiation codon mutation has previously been reported in several East Asian populations but has never been found in Southeast Asia and in combination with Hb E before. The haplotype analysis revealed a common origin of this mutation in the Asian population (5′: ? + ? + + ? +: 3′, type IV with framework 3 according to Orkin S, et al.). Although this rare mutation abolished the β‐globin expression and was considered as β⁰‐thalassaemia, the relatively mild phenotype in our patients may be attributed to a strong association between this mutation and the ?158 ^Gγ (C→T) polymorphism, an XmnI cleavage site (+), resulting in a high propensity of postnatal γ‐globin expression and ameliorating the clinical phenotypes.     

Epistasis and the sensitivity of phenotypic screens for beta thalassaemia

Genetic disorders of haemoglobin, particularly the sickle cell diseases and the alpha and beta thalassaemias, are the commonest inherited disorders worldwide. The majority of affected births occur in low‐income and lower‐middle income countries. Screening programmes are a vital tool to counter these haemoglobinopathies by: (i) identifying individual carriers and allowing them to make informed reproductive choices, and (ii) generating population level gene‐frequency estimates, to help ensure the optimal allocation of public health resources. For both of these functions it is vital that the screen performed is suitably sensitive. One popular first‐stage screening option to detect carriers of beta thalassaemia in low‐income countries is the One Tube Osmotic Fragility Test (OTOFT). Here we introduce a population genetic framework within which to quantify the likely sensitivity and specificity of the OTOFT in different epidemiological contexts. We demonstrate that interactions between the carrier states for beta thalassaemia and alpha thalassaemia, glucose‐6‐phosphate dehydrogenase deficiency and Southeast Asian Ovalocytosis have the potential to reduce the sensitivity of OTOFTs for beta thalassaemia heterozygosity to below 70%. Our results therefore caution against the widespread application of OTOFTs in regions where these erythrocyte variants co‐occur.     

Laboratory diagnosis of a compound heterozygosity for Hb Hekinan [α27(B8) Glu‐Asp] and a deletional α‐thalassaemia 2 in Thailand

We report the haematological and molecular characterization of a previously undescribed condition of compound heterozygosity for haemoglobin (Hb) Hekinan [α27(B8) Glu‐Asp] and a deletional α‐thalassaemia 2 detected in a Thai individual. Hb analysis demonstrated that although this Hb variant co‐migrates with Hb A on cellulose acetate electrophoresis and cation‐exchange high‐performance liquid chromatography (HPLC), the HPLC procedure using a weak cation‐exchange material with polyaspartic acid could clearly differentiate the two Hb. The variant could then be confirmed using the polymerase chain reaction‐restriction fragment‐length polymorphism (PCR‐RFLP) analysis of the amplified α1‐globin gene.     

α‐thalassaemia masked by β gene defects and a new polyadenylation site mutation on the α2‐globin gene

We report three examples of chronic anaemia involving complex combinations of α‐ and β‐globin gene defects. The first case had a potential Hb H disease caused by the classic SEA/RW deletions masked by Hb E [β26(B8)Glu→Lys] in the homozygous state. The second had an unusual Hb H disease caused by compound heterozygosity for two different α2 polyadenylation site mutations masked by a β‐thalassaemia heterozygosity. The third had an intermediate α‐thalassaemia with considerable anaemia caused by an as yet unknown polyadenylation site (AATAAA>AATAAC) mutation in combination with a common RW deletion masked by a common Hb C [β6(A3)Glu→Lys] heterozygosity. Diagnostic methods, genotype/phenotype correlations and the chance of overlooking these combinations during risk assessment in a multiethnic society are discussed.     

The use of real‐time PCR technique in the detection of novel protein 4.2 gene mutations that coexist with thalassaemia alpha in a single patient

α‐Thalassaemia is a very rare disease in Northern Europe in contrast to hereditary spherocytosis that is associated with red blood cell membrane defects. We report here α‐thalassaemia case who was also found to bear the erythrocyte membrane protein 4.2 gene mutations. mRNA relative quantification of red cell membrane protein genes in a Polish patient with α‐thalassaemia trait indicated EPB42 as the gene that could also be involved in anaemia pathogenesis. Sequencing revealed the presence of two novel mutations in the protein 4.2 gene: a G1701A genetic change that predicts an alanine to threonine at position 567 of the protein (A567T) and a T→A substitution that is located at position +6 of the donor splice site of intron 2 (IVS2nt+6T>A). This is the sixth variant of the erythrocyte membrane protein 4.2 gene mutations identified outside the Japanese population.     

The genetic basis of asymptomatic codon 8 frame‐shift (HBB:c25_26delAA) β0‐thalassaemia homozygotes

Two 21‐year old dizygotic twin men of Iraqi descent were homozygous for HBB codon 8, deletion of two nucleotides (–AA) frame‐shift β⁰‐thalassaemia mutation (FSC8; HBB:c25_26delAA). Both were clinically well, had splenomegaly, and were never transfused. They had mild microcytic anaemia (Hb 120‐130 g/l) and 98% of their haemoglobin was fetal haemoglobin (HbF). Both were carriers of Hph α‐thalassaemia mutation. On the three major HbF quantitative trait loci (QTL), the twins were homozygous for G>A HBG2 Xmn1 site at single nucleotide polymorphism (SNP) rs7482144, homozygous for 3‐bp deletion HBS1L‐MYB intergenic polymorphism (HMIP) at rs66650371, and heterozygous for the A>C BCL11A intron 2 polymorphism at rs766432. These findings were compared with those found in 22 other FSC8 homozygote patients: four presented with thalassaemia intermedia phenotype, and 18 were transfusion dependent. The inheritance of homozygosity for HMIP 3‐bp deletion at rs66650371 and heterozygosity for Hph α‐thalassaemia mutation was found in the twins and not found in any of the other 22 patients. Further studies are needed to uncover likely additional genetic variants that could contribute to the exceptionally high HbF levels and mild phenotype in these twins.     

Glutathione reductase activity and its relationship to pyridoxine phosphate activity in G6PD deficiency

Per cent stimulation of GR activity by FAD in vitro and PNP oxidase activity were measured in G6PD deficiency, heterozygous β-thalassaemia and controls. It is confirmed that, in contrast to the high stimulation of GR by FAD commonly found in thalassaemia indicating red-cell deficiency of FAD, and shown here to be greater in the Italian subjects, GR is usually saturated with FAD in G6PD deficiency, leading to high in vitro activity. Unexpectedly, on the other hand, low FMN-dependent PNP oxidase activity due to red-cell deficiency of FMN, confirmed by response to oral riboflavin, was found in the majority of subjects with G6PD deficiency, similar to that found in heterozygous β-thalassaemia. Whereas this is explained in thalassaemia by an inherited slow red-cell metabolism of riboflavin to FMN, it is suggested that in G6PD deficiency an increased rate of red-cell metabolism of FMN to FAD leads to the low FMN and high FAD. When G6PD deficiency occurs with heterozygous β-thalassaemia, GR is usually saturated with FAD as in G6PD deficiency alone, unless there is an inherited, very slow red-cell metabolism of riboflavin to FMN. The part played by GR in haemolytic crises in G6PD deficiency is discussed.     

No evidence for myocardial iron overload and free iron species in multitransfused patients with sickle/β0‐thalassaemia

Iron overload (IO) in the heart is a life‐threatening complication in transfusion‐dependent patients with thalassaemia major (TM) and to a lesser extent in sickle cell disease (SCD), while no data are available in patients with sickle/β⁰‐thalassaemia. Iron deposition in the heart, liver and pancreas was assessed using T2* MRI sequences, as well as free iron species assays – non‐transferrin bound iron (NTBI) and labile plasma iron (LPI), in addition to serum ferritin, percentage transferrin saturation and serum hepcidin, in 10 multitransfused patients (>30 yr) with sickle/β⁰‐thalassaemia. None of the patients had iron deposition in the heart. Three patients had mild, one had moderate, and two had severe liver IO. Two patients had mild iron deposition in the pancreas. In all the patients, serum hepcidin levels were normal – NTBI and LPI were not detected. Possible explanations of these findings are discussed.     

Beta-thalassaemia intermedia: is it possible consistently to predict phenotype from genotype?

Eighty-seven patients with β thalassaemia of intermediate severity were investigated in our Unit to determine whether it is possible to consistently predict phenotypic severity from genotypic factors. The subjects were from the following ethnic backgrounds: Asian Indian (35.1%), Middle Eastern (24.3%), Mediterranean (21.6%), Northern European (14.9%) and South-East Asian/Chinese (4.1%). There was a wide spectrum of phenotypic severity; 49 had mild disease, 22 moderate and 16 severe disease. 22/87 patients had inherited only a single copy of a β-thalassaemia allele, of whom 11 had also co-inherited triplicated α genes (ααα/αα or ααα/ααα) and seven had dominantly inherited β thalassaemia. In four of the heterozygotes no explanation was found for the thalassaemia-intermedia phenotype. 65/87 patients were homozygous or compound heterozygous for 26 mutations (40 genotypes) which ranged from very mild β⁺ to β° thalassaemia alleles. All patients with two mild or very mild β⁺ thalassaemia alleles had mild to moderate disease. Although concurrent inheritance of extra α genes with heterozygous β thalassaemia results in thalassaemia intermedia, the disease is mild. Co-inheritance of α thalassaemia as a modulating factor was not evident in this cohort of patients. Presence of the in-cis Xmn I-^Gγ site was a modulating factor but insufficient to explain the high fetal haemoglobin levels encountered. In conclusion, apart from the two categories of triplicated α genes with heterozygous β thalassaemia and inheritance of mild β⁺ thalassaemia alleles, it was not possible to consistently predict phenotype from α and β genotypes alone, due to the influence of modulating factors, some implicated (such as inheritance of HPFH determinants) and others as yet unidentified.     

Differential gene expression analysis in early and late erythroid progenitor cells in β‐thalassaemia

β‐ thalassaemia is a disorder of globin gene synthesis resulting in reduced or absent production of the β‐globin chain in red blood cells. In this study, haematopoietic stem cells were isolated from the peripheral blood of six transfusion dependent β‐thalassaemia patients and six healthy controls. Following 7 and 14 d in culture, early‐ and late‐ erythroblasts were isolated and purified. No morphological difference in maturation was observed following 7 d in culture, while a delayed maturation was observed in the patient group after 14 d. Following RNA isolation and linear amplification, gene expression analyses were performed using microarray technology. The generated data were analysed by two methods: the BRB‐ArrayTools platform and the Bioconductor platform using bead level data. Following 7 d culture, there was no difference in gene expression between the control and patient groups. Following 14 d culture, 384 differentially expressed genes were identified by either analysis. A subset of 90 genes was selected and the results were confirmed by Quantitative‐Real‐Time‐polymerase chain reaction. Pathways shown to be significantly altered in the patient group include apoptosis, MAPKinase and the nuclear factor‐κB pathway.     

Haemoglobin disorders in Australia: where are we now and where will we be in the future?

Inherited disorders of haemoglobin (Hb), such as thalassaemia and sickle cell disease (SCD) are common and responsible for significant morbidity and mortality on a global scale. As Australia becomes increasingly ethnically diverse, their prevalence will increase. However, we lack important demographic and epidemiological data to manage these disorders and their consequences and to support affected individuals and communities. Thalassaemia and SCD are lifelong conditions. Affected individuals have reduced life expectancies, poorer quality of life and complex healthcare needs. Treatment strategies currently focus on prenatal diagnosis, red blood cell transfusion, iron chelation, management of iron‐related complications, haemopoietic stem cell transplantation (HSCT) and hydroxyurea. Currently, the only curative therapy is HSCT; however, gene therapy offers the possibility of cure and trials are currently underway. These therapies are associated with significant complications and substantial costs; there is also evidence of variation in approaches to diagnosis and care. Optimal strategies for many aspects of management are not yet defined and more research is necessary to inform clinical care and health service delivery.     

Association of G6PD202A,376G with lower haemoglobin concentration but not increased haemolysis in patients with sickle cell anaemia

The genetic bases of the highly variable degrees of anaemia and haemolysis in persons with Hb SS are not fully known, but several studies have indicated that G6PD deficiency is not a factor. The G6PD^202A and G6PD^376G alleles and α‐thalassaemia were determined by molecular genetic testing in 261 children and adolescents with Hb SS in a multicentre study. G6PD^202A,376G (G6PD A?) was defined as hemizygosity for both alleles in males and homozygosity in females. Among the participants 41% were receiving hydroxycarbamide. The prevalence of G6PD^202A,376G was 13·6% in males and 3·3% in females with an overall prevalence of 8·7%. G6PD^202A,376G was associated with a 10 g/l decrease in haemoglobin concentration (P = 0·008) but not with increased haemolysis as measured by lactate dehydrogenase, bilirubin, aspartate‐aminotransferase, reticulocyte count or a haemolytic component derived from these markers (P > 0·09). Similar results were found within a sub‐group of children who were not receiving hydroxycarbamide. By comparison, single and double α‐globin deletions were associated with progressively higher haemoglobin concentrations (P = 0·005 for trend), progressively lower values for haemolytic component (P = 0·007), and increased severe pain episodes (P < 0·001). In conclusion, G6PD^202A,376G may be associated with lower haemoglobin concentration in sickle cell anaemia by a mechanism other than increased haemolysis.     

Antenatal screening for haemoglobinopathies: current status,barriers and ethics

Sickle cell disease (SCD) and thalassaemia are genetic disorders that are caused by errors in the genes for haemoglobin and are some of the most common significant genetic disorders in the world, resulting in significant morbidity and mortality. Great disparities exist in the outcome of these conditions between resource- rich and resource-poor nations. Antenatal screening for these disorders aims to provide couples with information about their reproductive risk and enable them to make informed reproductive choices; ultimately reducing the likelihood of children being born with these conditions. This review provides an overview of the current status of antenatal, pre-marital and population screening of SCD and thalassaemia in countries with both high-and low prevalence of these conditions, methods of screening in use, and discusses some of the pitfalls, ethical issues and controversies surrounding antenatal screening. It also discusses outcomes of some screening programmes and recognises the need for the establishment of antenatal screening in areas where their prevalence is highest; namely sub-Saharan Africa and India.