Alpha-thalassaemia

Abstract

Alpha-thalassaemia is inherited as an autosomal recessive disorder characterised by a microcytic hypochromic anaemia, and a clinical phenotype varying from almost asymptomatic to a lethal haemolytic anaemia.It is probably the most common monogenic gene disorder in the world and is especially frequent in Mediterranean countries, South-East Asia, Africa, the Middle East and in the Indian subcontinent. During the last few decades the incidence of alpha thalassaemia in North-European countries and Northern America has increased because of demographic changes. Compound heterozygotes and some homozygotes have a moderate to severe form of alpha thalassaemia called HbH disease. Hb Bart's hydrops foetalis is a lethal form in which no alpha-globin is synthesized. Alpha thalassaemia most frequently results from deletion of one or both alpha genes from the chromosome and can be classified according to its genotype/phenotype correlation. The normal complement of four functional alpha-globin genes may be decreased by 1, 2, 3 or all 4 copies of the genes, explaining the clinical variation and increasing severity of the disease. All affected individuals have a variable degree of anaemia (low Hb), reduced mean corpuscular haemoglobin (MCH/pg), reduced mean corpuscular volume (MCV/fl) and a normal/slightly reduced level of HbA2. Molecular analysis is usually required to confirm the haematological observations (especially in silent alpha-thalassaemia and alpha-thalassaemia trait). The predominant features in HbH disease are anaemia with variable amounts of HbH (0.8-40%). The type of mutation influences the clinical severity of HbH disease. The distinguishing features of the haemoglobin Bart's hydrops foetalis syndrome are the presence of Hb Bart's and the total absence of HbF. The mode of transmission of alpha thalassaemia is autosomal recessive. Genetic counselling is offered to couples at risk for HbH disease or haemoglobin Bart's Hydrops Foetalis Syndrome. Carriers of alpha+- or alpha0-thalassaemia alleles generally do not need treatment. HbH patients may require intermittent transfusion therapy especially during intercurrent illness. Most pregnancies in which the foetus is known to have the haemoglobin Bart's hydrops foetalis syndrome are terminated due to the increased risk of both maternal and foetal morbidity.

Figure 1

The world distribution of haemoglobinopathies overlaps the geographic distribution of malaria. The prevalence has increased in previously non-endemic areas as a consequence of historical and recent immigration flows, slave-trade, trading activities and colonization. In all these regions there is a high prevalence of a thalassaemia. It is believed that carriers of α thalassaemia are protected against malaria and that natural selection is responsible for elevating and maintaining their gene frequencies.

Figure 2

The Haemoglobin Bart's hydrops syndrome. a. peripheral blood film with immature red-cell precursors and hypochromic, microcytic, red cells showing anisocytosis and poikilocytosis; b. stillborn hydropic infant [5].

Figure 3

The structure of the α-globin gene cluster on chromosome 16. The telomere is shown as an oval, genes in the region are shown as boxes. The α-globin regulatory region (MCS-R 1 to 4) is indicated as vertical bars. The scale is in kilobases as indicated above. The alpha-gene cluster is enlarged showing the traditional gene names above and the HGVS gene names below. The table below shows the classification of gene defects and phenotypic expression.

Figure 4

Deletions that cause α⁺-thalassaemia. The homologous duplication units X, Y and Z in which the α-genes are embedded are indicated as colored boxes. A cross-over between the mis-paired Z boxes during meiosis gives rise to the -α^3.7 and ααα^{anti 3.7} chromosomes. Cross-over between misaligned X-boxes give rise to -α^4.2 and ααα^{anti 4.2}.

Figure 5

Deletions of one α-gene giving rise to α⁺-thalassaemia. The extent of the deletion is shown as bars, thin lines indicate regions of uncertainty of the breakpoints.

Figure 6

Deletions of two α-genes giving rise to α⁰-thalassaemia.

Figure 7

(continuation of figure 6) a. Large deletions involving both α-genes and b. deletions of the α-globin regulatory region leaving the α-genes intact.

Figure 8

Red blood cell indices in patients with various genotypes associated with α-thalassaemia. The bar shows the mean and standard deviation. a. Haemoglobin level (Hb in g/dl), b. Red Cell Count (RBC indicated as × 10¹²/l), these are sex-dependent (blue for male distribution, pink female distribution). (adapted from Higgs 1993, Wilkie 1991) [44,101].

Figure 9

(continuation of figure 8) a. Mean Cellular Volume (MCV in fl) and b. Mean Cell Haemoglobin (MCH in pg) (adapted from Higgs 1993, Wilkie 1991) [44,101].

Figure 10

HPLC and Capillary Hb electrophoresis patterns of an adult with HbH disease. The HbH (β4 tetramers) peak elutes from the column as a compressed fraction, and as a fast moving fraction in electrophoresis.

Figure 11

HPLC and Capillary Hb electrophoresis patterns of a neonate with α thalassaemia trait (--/αα) and a significant amount of Hb Bart's (γ4 tetramers). Hb Bart's in newborns with α thalassaemia disappears rapidly after birth. In newborns with Hb H disease, Hb Bart's will be substituted by HbH after birth. In Hb Bart's hydrops foetalis syndrome due to homozygosity of α⁰-thalassaemia only Hb Bart's is seen.

Figure 12

Mean and standard deviation of HbA₂ in different α-thalassaemia genotypes.

Figure 13

An inclusion body positive cell seen in Brilliant Cresyl Blue stained red cells of a α⁰-thalassaemia carrier. Inclusion Bodies are β4-tetramers precipitating on the red cell membrane, which damages the membrane and induces haemolysis. HbH is unstable and inclusion body positive cells are more difficult to find in older blood samples. The number of inclusion body cells seen after staining is much lower in α⁰-thalassaemia carriers than in patients with HbH disease (1 in 5-10 fields versus several per field at 1000× microscopic magnification).

Figure 14

The principle of Multiplex Ligation dependent Probe Amplification (MLPA). a. Probe pairs at different locations along the region of interest are hybridised specifically head-to-tail to the target sequence and subsequently ligated. The ligated probes are amplified by quantitative PCR using fluorescent labelled primers complementary to the tag-sequences and separated by capillary electrophoresis on an automated fragment analyzer. b. peak heights represent the amount of amplified product of each separate probe pair. c. By dividing the peak heights of the patient sample and a normal control for each fragment, the ratio's of 0.5 shown in the graph mark the deletion of certain probes located along the genome, indicating the presence of a deletion of one allele.