Stepwise diagnostics of hemoglobinopathies

Sickle-cell disease

This term covers both homozygous sickle-cell disease (HbSS) and a series of mixed heterozygous hemoglobinopathies, such as HbS/β-thalassemia, HbSC or HbSD disease. Sickle-cell disease is based on a defect in the β-globin chain, which causes the formation of an abnormal hemoglobin called HbS. The disease originates with the mutation p.Glu7Val (historically Glu6Val) in the β-globin gene (HBB). The affected β-globin chain in its deoxygenated state is unstable and has an increased tendency to aggregate. Heterozygous HbS carriers (HbAS) show a normal blood count and are generally symptom-free. The percentage of HbS varies between 35% and 45%. The HbA₂ value may be increased. If the amount of HbS is below 30%, an existing iron deficiency or coexisting α-thalassemia should be considered [2, 4, 6, 10].

In the homozygous state, the mutation results in the so-called homozygous sickle-cell disease, which is characterized by chronic hemolytic anemia, acute crises due to vascular occlusion and the resulting tissue and organ damages. The characteristic sickle-shaped deformation of the red blood cells is due to the modified properties of hemoglobin S when deoxygenated. The blood count shows a markedly reduced hemoglobin level of 6–9 g/dL. The hemoglobin pattern shows >90% HbS, a variable percentage of HbF <10%, and a HbA₂ amount of <3.5%. HbA is undetectable (Table 2) [4, 6].

The HbS anomaly can also occur in combination with other abnormal hemoglobins or thalassemias, from which rarer forms of sickle-cell disease arise. For compound heterozygosities, the combination with β-thalassemia, HbC or HbD plays a particularly important role. The combination of HbS with β⁰-thalassemia is characterized by severity similar to the homozygous variant (HbSS). In addition to hypochromic microcytosis, these patients show a percentage of HbS>80%, HbF<20% and HbA₂>3.5%. The hemoglobin concentration is lowered to an extent similar to that in patients with homozygous sickle-cell disease [2, 4].

The hemoglobin pattern of a patient with HbS in combination with β⁺-thalassemia shows a percentage of HbS>55%, HbF>20% and HbA₂>3.5%. Unlike homozygous sickle-cell disease or the combination HbS/β⁰-thalassemia, the percentage of HbA is approx. 20% in this case [4].

HbC disease

HbC disease is based on the mutation p.Glu7Lys (historically Glu6Lys) in the HBB gene. The affected β-globin chain in its deoxygenated state is unstable and has an increased tendency to aggregate. Heterozygous HbC carriers (HbAC) are clinically inconspicuous. Only the level of MCHC is increased. The HbC amount of the total hemoglobin is approximately 50% while the rest is made up of HbA [1]. Homozygous HbC mutation carriers (HbCC) and patients with a combination of HbC and β-thalassemia or other abnormal hemoglobins, are refered to as suffering from HbC disease. An exception here is the combination with HbS (HbSC disease). Depending on the molecular background, patients with HbC disease exhibit mild to moderate hemolytic anemia. Table 3 summarizes the most important diagnostic parameters, in cases of homozygosity, for HbC and HbC-β-thalassemia [4].

HbE disease

HbE disease is based on the mutation p.Glu27Lys (historically Glu26Lys) in the HBB gene. On the one hand, this mutation causes an amino acid substitution in the β-globin chain, and on the other hand, a cryptic splice site is activated, whereby the expression of the affected β-globin chain is reduced in the sense of β⁺-thalassemia.

Heterozygous carriers of HbE generally show no clinical symptoms, but usually exhibit in their blood count a light, variable hypochromia (MCH≈25 pg) and microcytosis (MCV approx. 73 fL). The HbE percentage of the total hemoglobin can vary greatly. Generally it is 30%–45%. If the concentration of HbE is low, the possibility of a co-existing iron deficiency or a combination with α-thalassemia should be considered [4].

In the case of homozygous disease, affected patients exhibit hypochromic, microcytic anemia, clearly recognizable by the MCH value (≈20 pg) and MCV (≈62–67 fL), as well as a hemoglobin concentration between 10 and 14 g/dL. The hemoglobin pattern consists of more than 95% HbE, with the balance being HbF and HbA₂. Table 2 summarizes the most important diagnostic parameters [2, 4, 6].

β-Thalassemia

Beta thalassemia is based on a quantitative defect of β-globin chain synthesis, which in large part is caused by point mutations in the β-globin gene (HBB). These mutations trigger a reduced expression of structurally normal β-globin. Increased erythropoiesis produces an imbalance in the synthesis of globin chains and thus a surplus of free α-globin chains that denature rapidly due to their instability. Depending on the molecular phenotype of a partial or complete loss of β-chain synthesis, one can distinguish between β⁺-or β⁰-thalassemia. Relative to the severity of the disease, β-thalassemia can be divided into the clinical forms thalassemia minor, thalassemia intermedia and thalassemia major, which are each based on a different genetic constellation [1]. A conspicuous blood count (hypochromia, microcytosis) in combination with a conspicuous hemoglobin differentiation (elevated HbA₂) represents the typical laboratory diagnosis of heterozygous β-thalassemia.

Hypochromic, microcytic anemia is diagnosed in most of these cases. A MCH<25 pg (usually between 19 and 23 pg), combined with elevated HbA₂ of 3.5% and higher (generally, between 4.0 and 6.0 percent), gives rise to the suspicion of an underlying heterozygous β-thalassemia (thalassemia minor). However, the HbA₂ fraction may also be between 6.5% and 8.0% in individual cases. The HbF fraction varies. HbF is elevated up to 3% in approx. 30% of patients. In rare cases, the HbF level may be as high as 3%–15%. Cave: the presence of iron deficiency may lead to reduced HbA₂ and thereby could mask an existing heterozygous β-thalassemia because of the missing typical increase in HbA₂ (>4%) [4].

A more pronounced microcytic, hypochromic blood count emerges in connection with β-thalassemia major. Usually, MCH is at 14–20 pg, and MCV at 50–60 fL. In addition, those patients typically suffer from anemia requiring transfusions, which is reflected in a hemoglobin concentration of less than 7 g/dL. The resulting hemoglobin pattern after chromatographic separation shows a significant increase in HbF of approximately 70%–90% and variable HbA₂ [2, 5].

A diagnosis of β-thalassemia intermedia and/or its differentiation from β-thalassemia major is difficult due to the hematologic phenotype and hemoglobin pattern that may potentially be similar [5]. The anemia in β-thalassemia intermedia (Hb of 6–10 g/dL) is generally not as pronounced as in the case of thalassemia major, but MCH (15–23 pg) and MCV (55–70 fL) are similarly reduced. The hemoglobin differentiation shows elevated HbF (up to 100%) with a variable HbA₂ fraction [2, 4].

A pathogenic mutation can be identified by molecular genetic analysis. The result of the genetic test, however, does not allow for an assessment of the clinical progression.

Molecular genetic diagnostics of β-thalassemia

The molecular genetic diagnostics regarding the β-globin-gene complex is carried out in stages – first, a sequence analysis is performed to locate a mutation in the HBB gene. If no mutation is detected deletion diagnostics of the β-globin-gene complex is performed. The β-globin-gene-complex can also be tested for gross deletions first when preliminary findings hint to a deletional genotype (for example, when δβ-thalassemia is suspected). The molecular genetic testing is used to verify hematological findings and to assess the risk to offspring to develop a severe form of hemoglobinopathy.

α-Thalassemia

α-Thalassemia is based on a quantitative defect of α-globin chain synthesis. The deficit of α-globin chains leads to the formation of excess hemoglobins that play a significant role in the clinical picture of α-thalassemia. The most common molecular genetic cause of α-thalassemia are large deletions in the α-globin gene cluster, which result in the loss of one or more α-globin genes. Since the α-globin gene cluster in a normal human cell consists of four α-globin genes (one HBA1 and one HBA2 gene on each chromosome 16), the severity of the disease depends on the number of deleted α-globin genes. The most common deletions are -α^3.7, -α^4.2, --^SEA, --^FIL, --^MED, -(α)^20.5 and --^THAI. In rare cases, point mutations, small deletions or insertions in α-globin-genes can also be responsible for α-thalassemia. Triplication of the α-globin-gene leads to the phenotype of thalassemia intermedia or thalassemia major only in combination with concurrent heterozygous β-thalassemia.

Symptoms can be assigned four levels of severity in clinical terms [14]:

• α-thalassemia minima (asymptomatic form): only one α-globin gene is deleted (-α/αα); this form is usually discovered only by chance, because all hematological parameters are normal.

• α-thalassemia minor: deletion of two α-globin-genes (--/αα, -α/-α), minor hematologic changes (hypochromia, microcytosis)

• HbH disease: only one functional α-globin-gene is left (--/-α), An essential feature is the varying degrees of hypochromic, microcytic anemia and the detection of hemoglobin H (HbH), a tetramer of four β-globin chains (β₄).

• Hb Bart’s/hydrops-fetalis-syndrome: complete loss of α-globin-genes (--/--), generally incompatible with life. Affected children usually die in utero or shortly after birth.

The forms minima and minor in the blood count. The hemoglobin level is in the normal range. It may, however, be slightly reduced in the case of heterozygous α⁰-thalassemia and homozygous α⁺-thalassemia. MCV and MCH vary depending on the form. In the case of thalassemia minima, MCH is within the normal range, but usually below 27 pg. With thalassemia minor, the value is significantly below the normal range (see Tables 4 and 5). Patients with hemoglobin H disease have Hb levels of 8–10 g/dL and an MCV of less than 22 pg [1, 2, 6, 15]. In the blood count of carriers of thalassemia minima, the key parameters (hemoglobin, MCV, MCH, MCHC) are barely decreased or are within the normal range (Table 4).

MCV and MCH are significantly reduced in patients with α-thalassemia minor (Table 5) [1, 4].

The hemoglobin differentiation by means of HPLC is normal in all three patients. This largely rules out the presence of β-thalassemia, and there is a suspicion of α-thalassemia or iron deficiency anemia. For the differentiation of thalassemia and iron deficiency, one can determine the iron status as well as use the Huber-Herklotz formula (only applies to MCH<27 pg): HH=(MCH×RDW)/(10×Ec)+RDW Readings below 21 indicate α-thalassemia, while those over 23 point to iron deficiency. Molecular genetic testing is necessary to confirm a suspected diagnosis of α-thalassemia [16].

Molecular genetic diagnostics of α-thalassemia

The molecular genetic testing of the α-globin-gene complex is carried out in stages. The first step is performed to detect frequent deletions by means of Gap-PCR or MLPA. If necessary, this can be followed up with a sequence analysis to detect mutations in the α-globin-genes. Diagnostic testing plays an important role for carriers wishing to have children in order to assess the risk of severe hemoglobinopathies in their offspring. Only the heterozygous alpha-zero deletion has clinical significance and may cause the hydrops fetalis syndrome if both parents are affected.