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Angastiniotis M, Eleftheriou A, Galanello Ret al., authors; Old J, editor. Prevention of Thalassaemias and Other Haemoglobin Disorders: Volume 1: Principles [Internet]. 2nd edition. Nicosia (Cyprus): Thalassaemia International Federation; 2013.
The haemoglobinopathies include quantitative and/or qualitative genetic disorders caused by mutations affecting the genes responsible for haemoglobin synthesis. Based on the gene(s) involved and the type of defect, the haemoglobinopathies can be broadly classified into thalassaemias (α, β, δβ) and abnormal structural variants. However, there are also structural variants such as Hb Lepore and HbE that result in a thalassaemic phenotype. The term Hereditary Persistence of Foetal Haemoglobin (HPFH) is used to define a group of conditions characterised by increased levels of HbF in adults, due to a persistent synthesis of γ-globin chains after birth without any significant clinical or haematological manifestations.
In general terms, the haemoglobinopathies are autosomal recessive disorders and the homozygous or genetic compound states result in clinically significant phenotypes of variable severity (i.e. thalassaemia major, thalassaemia intermedia, sickle cell syndromes, HbE syndromes). Heterozygotes are symptom-free but present haematological characteristics, often useful for their identification. The heterozygous states for the most common haemoglobinopathies are summarised in Table 4.1.
Classification of carriers for most common haemoglobinopathies.
The aim of screening (or carrier testing) is to identify carriers of haemoglobin disorders in order to assess the risk of a couple having a severely affected child and to provide information on the options available to avoid such an eventuality. Ideally, screening is performed before pregnancy (1). There are several possible strategies for screening, depending on factors such as the frequency of the disease, heterogeneity of the genetic defects, resources available, and social, cultural and religious factors.
Knowing the frequency and heterogeneity of the haemoglobinopathies in a target population is a critical prerequisite in planning an adequate strategy of carrier identification and in selecting the most suitable laboratory methods (see below). In addition, however, the technical facilities, infrastructure and financial resources available affect both the strategy and the choice of methods for carrier identification.
There are two types of screening: mass screening, provided to the general population before and at childbearing age, and target screening, which is restricted to a particular population group, such as couples preparing to marry, before conception or in early pregnancy.
Mass screening is more organisationally demanding than target screening, requiring careful planning and adequate technical and financial resources. This approach is most appropriate where there is a high frequency of thalassaemia, placing particular emphasis on pregnant women when they first present for antenatal care. The laboratory methods for carrier identification are relatively expensive (electronically determined RBC indices, HPLC analysis of haemoglobin) and the flowchart is laborious and may include complex methods such as globin chain synthesis and DNA analysis. For countries with more limited resources, mass screening can be conducted using cheaper methods (such as single tube osmotic fragility tests, or chromatography for HbA2 determination) and a less complex flowchart. The laboratory methods for carrier identification are described in detail in the second volume of this book (Volume 2: Laboratory Protocols).
Screening may be “retrospective” - that is, when couples already have an affected child, or “prospective” – i.e. when carriers are identified before having an affected child. Retrospective screening is often performed in populations with a low frequency of thalassaemia, or at the initiation of a prevention program in a high frequency population. The method is relatively cheap and simple because it is restricted to a portion of the population. But the effect on the number of affected births is limited, since sick children may be born to undetected at-risk couples. For this reason, prospective carrier identification is more appropriate for populations with a high frequency of thalassaemia.
Screening can be targeted at different age groups (Table 4.2), with genetic counselling adjusted according to the age of the individual or target group being screened. Currently, newborn screening (discussed in detail chapter 10) is only recommended for sickle cell disease, since early recognition of the disease can prevent mortality and morbidity caused by bacterial sepsis or sequestration crisis in the first months of life. Newborn screening for β-thalassaemia is less frequent, as it requires expensive DNA analysis. Advances in molecular biology and biotechnology may in the future provide faster and cheaper methods (e.g. microchips) for newborn screening of the haemoglobinopathies and other genetic diseases and traits (i.e. cystic fibrosis, G6PD, Wilson disease, etc). However, even if neonatal carrier identification were to become feasible, the problem of providing genetic counselling and relevant information throughout adult life remains.
Timing for haemoglobinopathy screening.
Experience with adolescent screening suggests that acceptance rates are high (usually above 80%) and the information is well understood and maintained (2-4). The advantages of screening in schools include the ability to reach a majority of the population and a sense of providing increased options to those identified as carriers (i.e. not to marry another carrier). However, the approach requires an intense and well-organised educational programme, relying on the support of motivated teachers and professional genetic counsellors.
Premarital testing is carried out in several Mediterranean countries (Greece, Italy and Cyprus) but it is not suitable in countries where the identification of a genetic risk prior to marriage can result in stigmatisation, particularly for the woman.
Preconception screening is directed at couples planning a pregnancy, while antenatal screening focuses on pregnant women. Screening during pregnancy may be disadvantageous in at-risk cases, since the only option is prenatal diagnosis. Furthermore, carrier identification may be too late to allow prenatal diagnosis, resulting in marked emotional stress.
Numerous carrier screening programmes are conducted around the world (for a review, see Cousens et. al., 2010, (5). They can be divided into mandatory or voluntary programmes. Despite the WHO recommendation that no compulsory genetic testing should be carried out, some countries, including Iran, Saudi Arabia and Palestinian territories have laws in place making haemoglobinopathy screening mandatory for all couples before having the approval to get married. In Cyprus, couples waiting to get married are required by the church to be screened and counselled. In other countries, including Sardinia, Greece, Guandong province of China and in England, haemoglobinopathy screening programmes are offered on a voluntary basis.
Inductive screening (also known as cascade screening or extended family testing) involves the testing of relatives of identified carriers and/or patients, and is a powerful means of improving the efficiency of carrier identification. In Sardinia for instance, such a policy has led to the detection of 90% of expected at-risk couples through tests on only 15% of the adult population (5).
The β-thalassaemias are very heterogeneous at the molecular level, with more than 200 point mutations and deletions of different severity described (Annex 2) (6, http://globin.cse.psu.edu/). The degree of severity generally corresponds to the magnitude of the residual output of the defective β-globin gene, and accordingly, the β-thalassaemia mutations are classified into severe, mild and silent types. The different types of β-thalassaemia mutation produce clinical and haematological phenotypes of variable severity, both in homozygotes (or genetic compounds) and in carriers. The clinically significant forms of β-thalassaemia (major and intermedia) are described in Chapter 7.
β-Thalassaemia carriers of either the βo or severe β+ type are characterised by modified red blood cell indices, haemoglobin pattern and globin chain synthesis ratio. The red blood cell count (RBC) is relatively high, while mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) are markedly reduced (MCV 60-70fl, MCH 19-23pg). Mean haemoglobin levels may be up to 2g/dl lower than normal, but vary widely. Red cell morphology is modified and typically includes microcytosis, hypochromia and variation in the size and shape of red cells (anisopoikilocytosis), target cells and basophilic stippling. The most characteristic haematological finding related to the β-thalassaemia trait is an elevated level of HbA2, typically between 4–6%. Occasionally, β-thalassaemia carriers may have unusually elevated levels of HbA2 (>6.5%). Such carriers generally have point mutations, or more rarely, deletions involving the 5’ promoter region of the β-gene. HbF may be slightly elevated (1–3%) in about 30% of carriers, and globin chain synthesis analysis shows an imbalanced α/β-globin ratio, with values ranging from 1.5-2.5. Finally, the decrease in osmotic fragility is another characteristic haematological finding in β-thalassaemia carriers (see Table 4.3).
Atypical β-thalassaemia carriers.
Heterozygotes for mild β-thalassaemia mutations generally have higher MCV and MCH values compared to those in β-thalassaemia heterozygotes with severe βo and β+ mutations. Their HbA2 levels usually range from borderline-normal to slightly increased values (3.4-4%), as in carriers of the IVSI-6 (T
C) mutation, up to manifestly increased levels (4.5-6%), as in carriers of mild β-gene promoter mutations
In carriers of very mild or silent β-thalassaemia alleles, the minimal deficit of β-globin production is not associated with any consistent or significant phenotype. In most cases, MCV, MCH, HbA2, total haemoglobin and even α/β-globin synthesis ratios are within the normal range, although sometimes borderline-raised HbA2 and/or slightly reduced red cell indices are observed, indicating the presence of a thalassaemic allele, and thus the need for further investigation.
The δβ-thalassaemias may be divided into (δβ)+ and (δβ)o, based on the residual output of δ- and β-chains from the affected chromosome (7). (δβ)+-Thalassaemia includes Hb Lepore determinants and more complex disorders resulting from the presence of two different mutations within the same β-like gene cluster (Corfu and Chinese δβ-thalassaemia determinants). (δβ)o-Thalassaemias are due to large deletions involving the εγδβ-gene cluster, removing the δ- and β-genes, but leaving one or both γ-genes intact.
Carriers of (δβ)o-thalassaemia deletions are characterised by milder haematological changes than those observed in the β-thalassaemia trait. The Hb level may be normal or slightly reduced and the red cell changes (i.e. microcytosis, hypochromia, anisopoikilocytosis) are mild (MCV 70 fl and MCH 24 pg). HbA2 is normal or slightly reduced but HbF is characteristically increased (5-20%) with a heterogeneous distribution amongst the red cells. Globin chain imbalance is mild (α/non-α ratio around 1.5).
Hb Lepore is a haemoglobin variant that results from non-homologous crossing-over between the δ- and β-globin genes, the product of which is a hybrid δ- and β-globin chain. Four types of Hb Lepore have been identified to date (Hb Lepore Boston, Hollandia, Leiden and Baltimore), differing at the exact point at which the crossover has occurred. The four types have similar electrophoretic and chromatographic properties, whereby at alkaline pH the electrophoretic mobility is slightly anodal to HbS. In carriers of Hb Lepore, the abnormal fraction constitutes between 5-15% of total haemoglobin, with reduced HbA2 levels (approximately 2%) and mildly increased HbF (2-5%). The haematological picture is characterised by mild anaemia (Hb 11-13g/dl), microcytosis (MCV: 70-75fl) and hypochromia (MCH: 20-24pg). The mean α/non-α ratio is approximately 1.5.
Corfu δβ-thalassaemia results from the presence of two different mutations in the same chromosome (in cis): partial deletion of the δ-gene and an IVSI-5 (GA) β-thalassaemia mutation. Carriers of this form of (δβ)+-thalassaemia have haematological findings comparable to the β-thalassaemia trait, but with normal or slightly reduced HbA2.
The Sardinian δβ-thalassaemia is a non-deletion allele, characterised by a (CT) substitution at position –196 of the Aγ-globin gene in cis to the common βo Cd 39 (CT) nonsense mutation. Sardinian δβ-thalassaemia heterozygotes show typical but mild thalassaemic blood changes, normal HbA2 levels (2-3%) and increased HbF levels (10-20%). The α/non-α globin chain synthesis ratio is only mildly imbalanced (approximately 1.5).
The classical phenotype of heterozygous β-thalassaemia, essentially characterised by reduced MCV and MCH and increased HbA2, may be modified by several genetic determinants, with resulting potential problems in carrier identification. The most common forms of atypical carriers, with the corresponding genotype, are summarised in Table 4.3. The category of atypical β-thalassaemia carriers includes β-thalassaemia heterozygotes with an unusually severe haematological and clinical phenotype.
The co-inheritance of heterozygous β-thalassaemia with homozygous α+-thalassaemia (-α/-α) or heterozygous αo-thalassaemia (--/αα) has a significant effect on the red cell indices, particularly the MCV and MCH, which may be normalised (8). The effect of interacting heterozygous α+-thalassaemia (-α/αα) is usually less evident (Figure 4.1). Of note is that the HbA2 levels in these double heterozygotes remain elevated within the range for β-thalassaemia carriers, the practical consequence of this being that if HbA2 determination is always performed in screening programmes, double heterozygotes for β- and α-thalassaemia will not escape diagnosis. On the other hand, a primary screen carried out using red cell indices followed by HbA2 determination only in those individuals with reduced MCV and MCH may result in failure to identify these double heterozygotes.
Effect of co-inheritance of different α-thalassemia alleles an β-thlassemia carriers.
Some β-thalassaemia carriers have normal or borderline-raised HbA2 levels but the MCV and MCH values are within the typical carrier range. Such carriers include heterozygotes for some mild mutations, such as IVSI-6 (TC), double heterozygotes for δ- and β-thalassaemia (in cis or in trans), carriers of the Corfu δβ-thalassaemia allele and, rarely, carriers of εγδβ-thalassaemia, which involve large deletions of the β-like gene cluster but which spare the β-genes (Spanish, English, Dutch types). To differentiate these atypical β-thalassaemia carriers from α-thalassaemia heterozygotes, it is necessary to perform family studies and/or globin chain synthesis and/or globin gene analysis.
A third group of atypical carriers are those with normal MCV, MCH and normal/borderline HbA2 values (silent β-thalassaemia carriers). Subjects with this phenotype may be carriers of very mild or silent β-gene mutations (associated with high residual β-globin chain output), or carriers of the triple α-globin gene arrangement (ααα/αα), in whom the excess α-globin chain synthesis is equivalent to that in carriers of very mild β-thalassaemia alleles. Recently mutations at KLF1 gene have been associated with borderline HbA levels and normal MCV and MCH (9). Identification of silent carriers is usually retrospective in parent(s) of patients with mild thalassaemia intermedia. However, if a silent carrier is suspected on the basis of borderline red cell indices and/or borderline HbA2 levels, a definitive diagnosis may be obtained using globin chain synthesis analysis (sometimes slightly imbalanced), or more often in most laboratories, through characterisation of the mutation by DNA analysis. Because of their silent phenotype these carriers may escape identification in general population-screening programmes. However, this will not have critical consequences, since homozygosity for very mild and silent mutations, or compound heterozygosity for mild with even severe β-thalassaemia mutations, usually results in attenuated forms of thalassaemia intermedia (11).
An extreme, although rare, instance of a complex thalassaemia gene combination is the co-inheritance of α-, δ- and β-thalassaemia alleles, which may lead to a silent phenotype and pitfalls in carrier diagnosis (10).
In rare instances, β-thalassaemia carriers may have a significant clinical phenotype. Co-inheritance of heterozygous β-thalassaemia with the triple or quadruple α-globin gene arrangement generally results in mild thalassaemia intermedia (12-15). On the other hand, the presence of an HbH disease genotype (--/-α or --/αNDα) in interaction with heterozygous β-thalassaemia, results in a moderate to severe anaemia (Hb 8-10g/dl) with marked microcytosis (MCV < 60fl) and hypochromia (MCH < 19pg). It should be pointed out that HbA2 is usually in the β-thalassaemia carrier range and that HbH inclusion bodies are absent (16, 17).
Some rare molecular lesions of the β-gene, most commonly in exon 3, produce highly unstable β-globin chain haemoglobin variants, which precipitate in erythroid bone marrow precursors because they fail to assemble in functional haemoglobin tetramers. This results in ineffective erythropoiesis and a clinical phenotype of thalassaemia intermedia with an increased HbA2 and an imbalanced α/β ratio. Because of the precipitation in early red cell precursors, the β-chain variant is usually undetectable in peripheral blood. Since the inheritance of a single β-thalassaemia allele results in a clinically evident phenotype, these forms are also known as dominant β-thalassaemias (14).
α-Thalassaemia is usually caused by α-globin gene mutations that either reduce (α+) or completely abolish (αo) the production of α-globin chains by the affected allele. α-thalassaemia is most frequently caused by deletions that remove part or all of the α-globin gene cluster. Less commonly, the mutations are nucleotide changes within either of the duplicated α-globin genes (the so-called non-deletion determinants) and, very rarely, deletions that include the HS-40 region but leave α-genes intact (Annex 3).
α+-thalassaemia is an asymptomatic carrier state in which one α-globin gene is dysfunctional. Red cells are often not microcytic, and HbA2 and HbF levels are always normal. In the neonatal period, small amounts (1-3%) of Hb Bart’s (γ4) may be detected. Reliable diagnosis of the α+-thalassaemia trait can only be achieved by DNA analysis.
The αo-thalassaemia trait results when two α-globin genes are dysfunctional. It is usually associated with a slight reduction in haemoglobin concentration and red cell indices (MCV, MCH), hypochromia, microcytosis and anisopoikilocytosis, with decreased erythrocyte osmotic fragility and HbA2 levels in the low to low-normal range (1.5-2.5%). The α/β-globin biosynthetic ratios average 0.7. During the neonatal period, there are moderate amounts of Hb Bart’s (3-8%) and cord blood erythrocytes are microcytic. At the DNA level, the phenotypic presentation of α-thalassaemia-1 trait may be caused by deletions that remove both loci from the same chromosome (αo-deletions), or homozygosity for α+-thalassaemia deletions.
HbH disease occurs when α-globin synthesis is reduced to about one-quarter of normal levels. It is characterised by the presence of the abnormal haemoglobin component, HbH, a homotetramer of β-globin chains (β4). HbH is detected on electrophoresis of freshly prepared haemolysate at alkaline or neutral pH, and typically amounts to 3-30% of total haemoglobin. The clinical presentation of HbH disease varies widely, from a mild asymptomatic to a severe anaemia requiring intermittent red blood cell transfusions. In addition to anaemia, clinical features may include jaundice and hepatosplenomegaly.
At the DNA level, HbH disease most commonly results from co-inheritance of αo-thalassaemia with α+-thalassaemia deletions. Interactions of an αo-thalassaemia deletion with a non-deletion α-thalassaemia allele or co-inheritance of non-deletion α-thalassaemia mutations may also give rise to HbH disease. Studies that have correlated haematological, biochemical and clinical findings with genotypes indicate that patients with non-deletion HbH disease mutations have more severe clinical expression, and patients with the severest phenotypes usually have α-thalassaemic (hyperunstable) globin variants (18-20). Phenotypic severity is not simply related to the degree of α-globin deficiency; in addition, HbH is unable to deliver oxygen to the tissues and is unstable, tending to precipitate within the red cells. Thus high HbH levels may exacerbate anaemia by negatively influencing tissue oxygenation, and both HbH and α-thalassaemic hyperunstable haemoglobin variants appear to reduce red cell survival within the bone marrow and circulation (20).
The moderate haemolytic anaemia may be exacerbated in the febrile state (haemolytic crisis). However, compared to patients with β-thalassaemia major, they have relatively little ineffective erythropoiesis. The very rare forms of unusually severe HbH disease associated with hydrops foetalis, for which prenatal diagnosis can be indicated, are described in Chapter 7.
Hb Bart’s hydrops foetalis This is the most severe form of α-thalassaemia and is discussed in Chapter 7.
These should involve an evaluation of the incidence of αo-thalassaemia and α+-thalassaemia in the population group, reflected by the prevalence of symptomatic forms of α-thalassaemia (HbH and/ or Hb Bart’s).
HbH disease is not considered to be amongst those haemoglobinopathies targeted for prevention, while couples at risk for having a child with Hb Bart’s hydrops foetalis should be detected and prenatal diagnosis is always indicated in such cases to avoid the severe toxaemic complications that frequently occur in pregnancies with hydropic foetuses and which are potentially detrimental to the pregnant mother. The approaches for prenatal diagnosis of Hb Bart’s hydrops foetalis are described in Chapter 7.
In order to avoid unnecessary (expensive) investigations and the engendering of anxiety, it is recommended that DNA analysis for definitive characterisation of the αo-thalassaemia trait be carried out only when BOTH partners have an MCH < 25pg, after iron deficiency has been excluded.
It should be noted that the presence of the β-thalassaemia trait may mask the simultaneous presence of the αo-thalassaemia trait. Therefore, in certain ethnic groups (e.g. Chinese), a detailed investigation is indicated if one partner has the β-thalassaemia trait and the other is a probable carrier of the αo-thalassaemia trait.
The asymptomatic sickling disorders include sickle cell trait (HbAS) and the doubly heterozygous condition of HbS and Hereditary Persistence of Foetal Haemoglobin (HbS/HPFH) (21). The symptomatic sickling disorders can be divided into mild and severe conditions (see Table 4.4). The milder conditions include HbSC disease, HbS/δβ-thalassaemia, HbS/β+-thalassaemia and the homozygous condition HbSS (or sickle cell anaemia) associated with the Arab-Indian β-globin haplotype (22). The severe sickling disorders are HbSS, associated with the Cameroon, Benin, Senegal and Bantu β-globin haplotypes, HbS/βo-thalassaemia and, finally, the HbS trait combined with the β-globin chain variants Hb D-Punjab, Hb O-Arab and the rare doubly substituted HbS variants.
Average haematological findings of some sickling disorders.
HbS heterozygotes are normally asymptomatic, although sickle cell formation leading to vascular occlusion may occur under certain conditions of significant hypoxia, e.g. at high altitude or under anaesthetic. HbAS individuals without interacting α thalassaemia have 35-40% HbS. The interaction of α-thalassaemia reduces the percentage of HbS and the red cell indices (see Table 4.5). The HbA2 level is often slightly above normal (3.5-4%), but this never signifies the presence of a β-thalassaemia gene unless the HbS is greater than 50% of total haemoglobin.
Percentage HbS and MCV values in HbS individuals with and without α+-thalassaemia.
Patients doubly heterozygous for HbS and Hereditary Persistence of Foetal Haemoglobin (HPFH) are either asymptomatic or have an extremely mild form of sickle cell disease. There is usually no anaemia and patients have very few episodes suggesting sickle cell crises, although occasional mild bone pains have been reported. The condition has been reported in Africans with HbS trait and either the black HPFH1 or the Ghanaian HPFH2 deletion, and also in Indians with the Indian HPFH3 deletion. Patients have nearly normal red cells, each with 20-30% HbF in a pancellular distribution. However, this condition may be difficult to diagnose haematologically because many patients have reduced red cell indices due to co-existing α+-thalassaemia.
HbS in the homozygous state or in combination with either HbC, Hb O-Arab, Hb D-Punjab or β-thalassaemia causes sickle cell disease. HbS is less soluble than normal haemoglobin during deoxygenation, crystallising out into polymers in the form of long fibres that cause the classical sickle-shaped deformation of the red cell. The sickle-shaped cells are more rigid than normal red cells and tend to block small arteries, resulting in an inadequate oxygen supply to the tissues and organs. In addition, the sickle-shaped cells have a shorter lifespan, resulting in a lifelong haemolytic anaemia. The sickle cell disease genotype interactions are discussed in Chapter 7.
HbE was the fourth abnormal haemoglobin to be identified by haemoglobin electrophoresis, in 1954 (23), and in 1961 it was characterised as having the substitution of lysine for glutamic acid at position 26 of the β-globin chain (24). Many types of HbE syndromes are observed, due to various interactions with α- thalassaemia, β-thalassaemia or other haemoglobin variants. The symptomatic and asymptomatic forms are summarised in Tables 4.6 and 4.7. The asymptomatic are discussed below; forms for which prenatal diagnosis may be considered are discussed in Chapter 7.
Summary of the common HbE syndromes.
Haematologic data in various HbE syndromes.
HbE heterozygotes are clinically normal, with minimal changes in blood counts and erythrocyte indices. Red cell morphology is similar to that in thalassaemia minor with normocytic or slightly microcytic red cells (MCV 84±5fl). A few target cells may be present in the blood smear. Osmotic fragility curves may be within normal limits or moderately shifted to the right, indicating slightly decreased osmotic fragility. Haemoglobin electrophoresis reveals both HbA and HbE. HbE quantification is crucial for the diagnosis of HbE syndromes arising from the interaction of HbE with other inherited haemoglobin abnormalities or non-genetic factors (see Table 4.6). HbE constitutes 25-30% of the haemolysate in simple HbE trait or compound heterozygosity for HbE and α+-thalassaemia, and in general these two conditions cannot be differentiated by haematological screening. HbE levels are reduced by co-inherited αo-thalassaemia to 19-21% (25), and markedly reduced in individuals who co-inherit heterozygous HbE and HbH disease (HbAE Bart’s disease syndrome) to 13-15% (26). HbE levels above 39% suggest the interaction of β-thalassaemia with HbE. HbE heterozygotes deficient in iron also have reduced amounts of HbE and lower MCV and MCH, depending on the degree of iron deficiency.
HbE homozygotes usually have normal haemoglobin levels (although some may be mildly anaemic) and clinical symptoms, such as jaundice and hepatosplenomegaly, are rare. Bone changes are not present. Reticulocyte counts are consistently normal and nucleated red cells are absent from the circulation, but a characteristic finding is 20-80% target cells and osmotic fragility is markedly decreased (see Table 4.7). Haemoglobin analysis reveals about 85-95% HbE with remainder HbF. There is defective βE-globin chain synthesis in all HbE homozygotes with an average α/non-α ratio of 2, equivalent to the ratio found in β+-thalassaemia heterozygotes (23). Defective βE-chain synthesis is due to decreased βE mRNA production, a result of abnormal RNA splicing caused by HbE mutation (28,29).
Carrier identification strategies should ensure that no carrier eludes detection. There are two possible methodological approaches for β-thalassaemia carrier identification:
A primary screening approach is recommended in countries with low frequency and limited heterogeneity of thalassaemia, while complete screening is recommended in populations where both α- and β-thalassaemias are common, and where interaction of α- and β-thalassaemias could lead to missed diagnoses due to the normalisation of red cell indices.
A flow-chart illustrating the strategy used to identify carriers in high risk areas is shown in Figure 4.2, while the recommended methods with cut-off indices are summarised in Table 4.8. These cut-off indices are the most widely used, however appropriate reference values should be independently defined for each population as there may be slight differences according to the types of thalassaemia alleles present. In addition, there should be regular quality control programmes in order to monitor the accuracy of laboratory results. The techniques for carrier identification are extensively treated in Prevention Book 2.
Flow chart for thalassaemia screening.
Screening for β-thalassaemia carriers: recommended methods and cut-off values (see also Prevention Book 2).
Screening programmes for the haemoglobinopathies have become well-established in many countries over the last 20 years. But despite the large amount of accumulated knowledge, several problems in carrier identification remain. The most common problem is the presence of microcytosis with normal HbA2 and HbF, which may be due to iron deficiency, the α-thalassaemia trait or δβ+-thalassaemia (see Figure 4.2). Iron deficiency anaemia produces a wide range of red cell abnormalities (reduction of MCV, MCH, and Hb levels, and raised RBC), depending on the severity at the time of haematological analysis. For this reason, iron deficiency anaemia is easily mistaken for some forms of heterozygous thalassaemia. Besides iron deficiency, similar haematological findings are associated with α-thalassaemia and δβ+-thalassaemia. Iron deficiency may be distinguished from forms of α- or δβ+-thalassaemia by an increase in zinc erythrocyte protoporphirin (ZnPP) and a reduction in serum iron, transferrin saturation and serum ferritin. Alternatively, an imbalance in the α/β-globin chain synthesis ratio supports the presence of α-thalassaemia (α/β ratio < 0.9) or δβ+-thalassaemia (α/β ratio > 1.2). Finally, family studies may also be useful for distinguishing the diagnosis.
In rare cases when β-thalassaemia carriers have concomitant iron deficiency, HbA2 levels may be reduced, although they usually remain within the β-thalassaemia carrier range. In exceptional cases where β-thalassaemia carriers have very severe iron deficiency, HbA2 levels may fall to within the normal range. In practice, if an individual has very severe iron deficiency anaemia with normal HbA2, it is preferable to treat the patient with iron to correct the anaemia before repeating tests to determine HbA2 (30).
In the course of screening to identify β-thalassaemia carriers, it is not uncommon to find individuals with borderline HbA2 levels (3.2–3.5%) and normal or mildly reduced red cell indices (31, 32). The majority of cases with borderline HbA2 levels and normal red cell indices have normal β- and α-globin genes, and the borderline HbA2 levels may be explained by the extreme distribution of the normal range of HbA2. However, these cases are sometimes carriers of silent β-thalassaemia mutations (-101 C T, IVSII-844 C G, +1480 C G, +33 C G) or a triplicated α-gene locus. As previously mentioned some cases of borderline HbA2 with normal RBC indices are associated with mutations at KLF1 gene (8). Individuals with borderline HbA2 values (3.4-3.8%) and reduced MCV and MCH are usually carriers of a mild β-thalassaemia allele (e.g. IVSI-6 T C, CAP+1 A C, polyA T C), or compound heterozygotes for δ- and β-thalassaemia.
Diagnosing the atypical carriers involves family studies and/or globin chain synthesis and/or DNA analysis, and it is recommended that the presence of a β-thalassaemia allele be excluded in any subject with a borderline HbA2 level, especially if their spouse is a typical β-thalassaemia carrier (see Figure 4.3).
The most common forms of δβo-thalassaemia and HPFH are characterised by normal HbA2 and increased HbF levels. Red cell indices in HPFH are normal compared to δβo-thalassaemia carriers, in whom the MCV and MCH are usually slightly reduced. Differentiation between δβo-thalassaemia and HPFH is facilitated either by globin chain synthesis (normal or minimally imbalanced α/β synthesis ratio in HPFH, mild to moderate imbalance [α/β ratio 1.4–2] in δβo-thalassaemia), or by DNA analysis. HbF distribution in red cells is reported as homogeneous or pancellular in HPFH and heterocellular or uneven in δβo-thalassaemia, although the distinction is less clear if more sensitive immunological methods are used. It is also important to differentiate between HPFH and δβo-thalassaemia in the context of genetic counselling: genetic compounds with β-thalassaemia and HPFH result in a silent or very mild clinical phenotype, while δβo-thalassaemia carriers are at risk of producing thalassaemia major in combination with classical β-thalassaemia.
Carriers of structural haemoglobin variants are usually detected during screening programmes for the haemoglobinopathies. The identification of a variant is based on a series of specific tests, summarised in Figure 4.4. The simple sickling test allows correct diagnosis of HbS. For all the other variants, even if a presumptive diagnosis may be achieved by careful interpretation of several laboratory methods (such as relative electrophoretic mobility, chromatographic elution times, amount and analysis of globin chains), DNA analysis remains the only approach to provide a precise identification. Finally, due to the influence of co-existing α-thalassaemia on haematological parameters it is sometimes difficult to accurately diagnose the phenotypes of HbSS, HbS/βo-thalassaemia, HbS/δβ-thalassaemia and HbS/HPFH based on a haematological assessment of the patient, and family studies are therefore recommended.
Flow-chart for Hb variant identification.
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The publication contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the Thalassaemia International Federation.
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