Abnormal haemoglobins

Historical background
In 1948, Horlein and Weber showed that the hemoglobin molecule could be the cause of a disease. This was done by demonstrating that, in some congenital methaemoglobinaemias, the disease was not linked to a heme defect but to a haemoglobin molecule abnormality inherited according to the autosomal mode.

In 1949, Pauling demonstrated by liquid vein electrophoresis that patients suffering from sickle cell disease carried a haemoglobin, which differed in charge from that of normal subjects. This hemoglobin was named Hb S (from sickle). Ten years later, Ingram et al. found that Hb S had a Glu>Val substitution at position β6. Further studies of hemoglobin in various populations, revealed the existence of several other Hb variants. Those having a high prevalence, such as Hb C, Hb D-Punjab, Hb E were among the first chemically characterized. In a first step, the hemoglobin mutants were named by a letter according to their electrophoretical mobility or to some other specific properties. A variant having a mobility similar to that of Hb S at alkaline pH, but with normal solubility was named Hb D. When the variant moved slightly more towards the anode, between the positions of Hb S and Hb F, it was named Hb G. Hb C, Hb E and Hb O migrate almost at the position of Hb A2. Hb J and Hb I are fast moving variants. Rapidly, it was observed that many variants sharing an identical mobility in zone electrophoresis at alkaline pH differed in their amino acid substitution. To avoid confusion, at the exception of the first variants that have been found, the new described hemoglobins were no more named by a single letter but by their geographical origin and by the corresponding amino acid substitution (ex.: Hb D-Punjab β121 Glu>Gln, Hb D-Iran β22 Glu>Gln).

Today about 1160 hemoglobin variants, affecting the various globin genes, have been reported. An interactive database (HbVar), describing the main properties of each of those, is freely available on the web (http://globin.cse.psu.edu). Since HbVar is connected to DNA resources, two variants carrying the same structural abnormality at the protein level, but resulting from a different mutation at the nucleotide level, or affecting the same nucleotide but in paralogous gene (HBA1 or HBA2, HbG1 or HBG2), are considered as distinct variants. Up to now, 812 variants have been identified in the β globin gene and 702 in the two α globin genes. About 100 δ globin gene variants are known, they may be a cause of pitfalls in the measurement of Hb A2 level during diagnosis of β thalassaemia carriers. Most of the abnormal haemoglobins result from a single point mutation, but more complex mechanisms may be observed. Post-translational modifications following a mutation, two mutations in the same codon, insertion or deletion of one or more codons or unequal crossing-over between two genes are examples of these rare events.

General considerations on haemoglobin variants
A first group of variants is made by the very common ones. Each of those affects several million individuals. Very likely they have been selected because of some protective effects against malaria in heterozygous carriers. This is the case for Hb S and Hb C in populations from African origin. The large incidence of these variants is similar to that observed for the thalaassemias in other populations. Nevertheless, in the case of thalassaemias, the selective advantage against malaria is brought not by the success of a single point mutation but by a series of several hundred different mutations. Each of these abnormalities leads to a decreased or abolished synthesis of a globin chain and results in microcytosis and hypochromia, a phenotype resulting in a protective effect against malaria.

A second group of abnormal haemoglobins involves variants limited to some populations in which they may reach, in some cases, a polymorphism level of around 1% and be considered as genetic markers. These variants are usually clinically silent but some of those may interact with another RBC abnormality present in the same geographical region resulting then in severe phenotypes. Their identification is, therefore, required for diagnosis and for genetic counseling. This is essentially the case for mutations, such as Hb D-Punjab or Hb O-Arab that may be found associated with Hb S.

A last group is made up of rare haemoglobin variants. They result from mutations only observed in a few families or individuals. Most of these variants are clinically silent and have been found during systematic population screening or because of their interference with another biological test, such as cation exchange HPLC done for measuring glycated hemoglobin.

Finally, a few rare variants are found because they lead to haematological disorders. Examples are unstable haemoglobins responsible for chronic haemolytic anaemia, high oxygen affinity variants leading to polycythaemia, low oxygen affinity haemoglobins  resulting in a well tolerated cyanotic anaemia or Hb M causing a hereditary methaemoglobinaemia.

Frequent variants
Hb S is the most common severe haemoglobin (Hb) variant. The mutation β6(A3) Glu>Val (HBB: c.20A>T) creates in the deoxygenated state of the Hb molecule a hydrophobic interaction site between two β chains leading first to the association of Hb tetramers into fibers and further to sickling of the RBC. Worldwide about 2.3 millions of patients display severe sickle cell anemia syndromes because being homozygous for this mutant or compound heterozygous for Hb S and a few other Hb abnormalities. Hb S is easily detected by all the classical analytical systems, but in each method, other variants, may behave as Hb S, leading to false positive diagnosis. Thus in the absence of a priority given to one of the few tests that recognize specifically the Hb S mutation (solubility test, electrophoresis on agar gel at acidic pH, identification of the mutation at the protein or DNA level…), a single test would never be sufficient to attest certainly for the presence of the Hb S mutation.

Hb C [β6(A3) Glu>Lys (HBB: c.19G>A)] is the second most frequent variant in the African population. It is harmless in the heterozygous state. But in the homozygous state, due to a dehydration of the RBC resulting from the charge difference of the Hb altering the intra-erythrocytic pH, it leads to a moderate haemolytic anemia, microcytosis with dense cells, splenomegaly, and infrequently, hyperbilirubinemia. In compound heterozygous state with Hb S, this increase of intra-erythrocytic haemoglobin concentration enhances Hb S polymerization. Hb C patients and their families may benefit from genetic counseling with special mentioning of the risk of having children with sickle cell disease if the partner is a carrier of the sickle trait.

Hb E [β26(B8) Glu>Lys (HBB: c.79G>A)] is the most frequent variant observed in populations from South East Asia. Since the mutation creates an alternative splice site near exon I, a fraction of the mRNA is defectively processed and results in a thalassaemic decreased expression. The major fraction of the mRNA is translated into a protein with an aminoacid change at position β26 which does not change the functional properties of this variant. In total, Hb E behaves as a mild β+ thalassemia: homozygous carriers display a mild disease while compound heterozygotes with a β0 thalassaemia defect will suffer from a thalassaemia intermaedia that may be severe.

Hemoglobin variants interacting with Hb S
Hb D-Punjab [β121(GH4) Glu>Gln (HBB: c. 364G>C)] is a rare variant often observed in populations from North of India but also found in many individuals from Balkan region, Turkey, North and West Africa. Using conventional electrophoretic methods, this variant was frequently confused with Hb S but is now clearly identified by cation exchange HPLC, capillary electrophoresis or a solubility test (8). Hb D-Punjab is harmless alone, even in homozygous state, but is the only Hb of the “D family” which leads to a severe form of sickle cell disease when associated with Hb S.

Hb O-Arab [β121(GH4) Glu>Lys (HBB:c. 364G>A)] is found mostly in individuals from East Africa but has also been observed in the Balkan region, in North Africa and in several other populations. As Hb C, it causes a RBC dehydration, and thus to a severe sickle cell disease when associated with Hb S.

Rare hemoglobin variants
Hundreds of those, which usually are clinically neutral, are found during systematic studies done for diagnosis of sickle cell or thalassaemia carriers or during measurement of glycated haemoglobin. Investigation of patients presenting with some haematological disorders is another completely different circumstance under which one may found some abnormal variant displaying unusual functional properties.



Unstable haemoglobins were described some 50 years ago when studying patients with non spherocytic hereditary congenital Heinz body haemolytic anaemia. In the RBC of these patients, inclusions bodies made from haemoglobin precipitates (Heinz bodies) were observed either spontaneously or after incubation with an oxidant dye. Electrophoretic or chromatographic studies of haemolysates reveal inconstantly an abnormality but when the lysate is incubated at 37°C in the presence of 17 % isopropanol during a length of time insufficient to precipitate Hb A, a haemoglobin component is observed which precipitates. Today about one hundred variants belonging to this group with various degree of instability are known,. The structural modifications is often localized near the haeme pocket as shown in Figure 1.

When the haemoglobin variant is highly unstable, it may be in too small amounts in the circulating blood to lead to a clear precipitate. The instability tests may be then erroneously considered as negative. The same situation may occur for unstable haemoglobins caused by an α globin chain abnormality. A special group made of hyper unstable haemoglobins behaves as dominantly inherited β thalassaemias, they result mainly from mutations localized in the third exon of the β globin gene, in regions coding for the α1β1 contact but not always,. Unstable variants affecting the α globin chain are frequently responsible for non-deletional α+ thalassemia phenotypes and when associated with another α chain abnormality they may lead to the presence of some Hb H.

Rare hemoglobin variants with altered oxygen affinity


In 1966, S. Charache et al., reported the first example of a patient who displayed an erythrocytosis resulting from the presence of an abnormal haemoglobin with increased oxygen affinity. About 100 variants with increased oxygen affinity are now known. Some of those display a very high oxygen affinity and are responsible for a marked secondary erythrocytosis. Nevertheless, the two thirds of the variants reported in the literature as having an increased oxygen affinity are not associated with secondary erythrocytosis. This is explained either because of a mild or moderate increase in oxygen affinity found only during in vitro studies, or because of a too low expression of the variant to significantly disturb the delivery of oxygen to the tissues. The most frequently observed structural modifications are localized in the α1β2 interface, or at the C-terminal. The most classical example concerns variants at position β99, for which all the possible changes have been described (Figure 2) resulting in an almost similar increase in oxygen affinity. They impair formation of a stable deoxygenated state. Other mutations modify directly or indirectly the surrounding of the heme and the site where oxygen binds. hemoglobins with increased oxygen affinity are important to be diagnosed because albeit usually well tolerated in a young patient they frequently lead to thrombotic complications in older ones or when they are associated with another cause that increases a thrombotic risk. Evidence for a high oxygen affinity haemoglobin can only be obtained through the study of the oxygen binding properties of a freshly drawn blood.

hemoglobins with decreased oxygen affinity are extremely rare. They result from a stabilization of the deoxygenated structure and lead therefore to a cyanotic anemia usually well tolerated when the variant is not unstable. In the case of unstable variants with low oxygen affinity (such as Hb Hammersmith), since the hemoglobin level is already low in the steady state, any hemolytic crisis may lead to a strong severe anemia.

Hemoglobins M
This group of rare variants results from a globin abnormality leading to a particular oxidation of the haeme. The structural change consists usually in the replacement for a tyrosine of one of the histines bound to the haeme. This change leads to a permanent binding of the haeme to the globin with formation of a stable phenolate and no possibility for oxygen binding. The diagnosis of Hb M is suggested by a typical chocolate brownish color of the blood and specific spectrophotometric properties. Haemoglobins M have no real population specificity, and result from neomutation in several observations. The same kind of abnormality was found affecting γ chains leading thus to a cyanosis at birth that disappeared with the switch from fetal to adult haemoglobin. In the heterozygous form Hb M are well tolerated and the greatest hazard for a carrier is a misdiagnosis with the risk of heavy cardiovascular investigations, which is specially the case for newborn babies,.

Conclusions
The presence of an abnormal haemoglobin is a frequent observation. It is important to recognize those which are, or may be responsible, for a pathological manifestation from the large majority of variants which are simple polymorphisms or display only mild haematological consequences.