Haemoglobin structure andfunction 154

Haemoglobin genes and chains 154

The spectrum of globin genemutations 155

Sickle-cell disease 156

�-Thalassaemia 159

�-Thalassaemia 161

��-Thalassaemia and hereditarypersistence of fetal haemoglobin 162

Interaction of different haemoglobinmutations 163

Summary 164

Multiple choice questions 165

Chapter contents Inherited disorders of the structure and function ofhaemoglobin, known collectively as the haemoglo-binopathies, hold a distinguished and prominent role inthe annals of human and medical genetics. Historically,haemoglobin was one of the first human proteins forwhich the underlying amino acid structure and molec-ular basis were elucidated. Sickle-cell disease, one ofthe commonest forms of haemoglobinopathy, is theprime example of an inherited disorder for which het-erozygote advantage and its underlying mechanismhave been demonstrated. Knowledge of the sickle-cellmutation was exploited by the research group whichdeveloped the polymerase chain reaction (p. 16), andsickle-cell disease was the first inherited condition forwhich prenatal diagnosis was undertaken using linkageanalysis (p. 91).

However, the importance of the haemoglobinopathiesin medical genetics lies not in their historical significancebut in their enormous impact on human morbidity andmortality. In 1994 it was estimated that approximately 5%of the word’s total population, now 6 billion, carries ahaemoglobin mutation and that over 350 000 childrenare born each year with a serious disorder of haemoglo-bin structure (e.g. sickle-cell disease) or synthesis (e.g. tha-lassaemia). Such is the impact of these disorders, both at an individual level and on a global scale, that theWorld Health Organization has actively promoted severalnational population screening programmes, with thedual goals of informing reproductive choice and, thereby,reducing the number of severely affected children. Theseprogrammes have met with variable success, as discussedin Chapter 7 (p. 149).

CHAPTER 8

Genes and haemoglobin

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Haemoglobin structure and functionThe haemoglobin molecule is made up of four poly-peptide chains, normally two � and two non-�, each ofwhich has a single haem moiety consisting of an ironatom located at the centre of a porphyrin ring. Thistetramer is spherical in structure, with the globinchains folded so that the four haem groups lie in sur-face clefts equidistant from each other (Fig. 8.1). Thetetramer is held together by bonds between the � andnon-� chains, and the quarternary structure changes as oxygen is taken up by oxygenation of each haemgroup.

Normally the oxyhaemoglobin dissociation curve hasa sigmoid shape, which means that large amounts ofoxygen are taken up or released by a small increase ordecrease in oxygen tension. This is facilitated by inter-action of the haem groups, so that when one haemgroup in a tetramer becomes oxygenated, conforma-tional changes result in increased oxygen affinity of the closely adjacent non-oxygenated haem groups.Abnormal forms of haemoglobin which lack � chainsshow abnormal subunit interaction, with a differentpattern of oxyhaemoglobin dissociation curve that pre-vents oxygen release at normal physiological oxygentensions. Fetal haemoglobin, as discussed below, hasgreater oxygen affinity than that of adults because ofreduced binding to the tetramer of 2,3-diphospho-glycerate, which normally stabilizes the deoxygen-ated form of haemoglobin, thus lowering its oxygen affinity.

Haemoglobin genes and chainsThe human haemoglobin genes show considerablehomology and almost certainly arose from a singlecommon ancestral gene. They are located in two clus-ters, an � or �-like complex on chromosome 16 and a � or �-like complex on chromosome 11 (Fig. 8.2). Each of these clusters contains at least one pseudogene.Pseudogenes closely resemble functional adjacentgenes but contain mutations which have renderedthem inactive. Both they and the almost identical func-tional genes are thought to have originated from acommon ancestral gene.

The �-like cluster is located close to the end of chro-mosome 16 at 16p13.3 and consists of three pseudo-genes (��, ��2 and ��1), three functional genes (�1, �2,and �) and one gene (�) of unknown function. The � gene encodes an �-like � (zeta) chain in early embry-onic life; �1 and �2, which arose from a duplicationevent approximately 60 million years ago, encode �-globin chains from late embryonic life onwards. Thuseach normal individual has four active �-globin genesencoding �-globin chains.

The �-like gene cluster is located on the short arm ofchromosome 11 at 11p15.5 and is spread over a regionof approximately 60 kb. It consists of a single pseudo-gene (��) and five functional genes (�, G�, A�, , and �),which encode the �, �, , and � chains respectively. The� chains are encoded mainly in fetal life, by G� and A�,when they combine with � chains to form fetal haemo-globin (Hb F). �-globin chain synthesis begins soon afterbirth. Normal adult haemoglobin (Hb A) consists of two�-globin and two �-globin chains. The �-globin and �-globin chains consist of 141 and 146 amino acidsrespectively. Adults also have a small quantity (2–3%) ofHb A2, which consists of two �-globin chains and two -globin chains (Table 8.1).

8 genes and haemoglobin154

Fig. 8.1. Three-dimensional structure of the haemoglobin molecule.Glu 6 marks the site of the amino acid substitution which causessickle-cell disease. X marks the site where 2,3-diphosphoglyceratebinds in the deoxygenated state. Reproduced with permission fromElliott WH, Elliott DC (2001) Biochemistry and molecular biology,2nd edn. Oxford University Press, Oxford.

Fig. 8.2. Structure of the � and � globin gene clusters onchromosomes 16 and 11. Reproduced with permission from LewinB (2000) Genes VII. Oxford University Press, New York.

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Each globin gene has a similar structure consisting of a 5′ promoter region, a cap site, a 5′ untranslatedregion, an initiation codon, three exons, two introns, atermination codon, a 3′ untranslated region, and apoly(A) signal tail. The �- and �-like gene clusters shareseveral features in common. They both have upstreamlocus control regions (LCR� and LCR�), which regulatetranscription of the actively expressed genes within therelevant cluster. The genes in each cluster share temporaland spatial expression patterns, which are regulated bythe relevant locus control regions and more closely adja-cent enhancer sequences and promoter regions. In bothclusters the genes are arranged sequentially in order oftheir temporal developmental expression, a characteris-tic which they share with the HOX gene family clusters(p. 169). Spatially, the globin genes are expressed initiallyin the yolk sac and then by the liver in embryonic life,and the spleen and bone marrow in fetal life.

The different temporal expression patterns of the �- and �-globin chains explain the different ages of onset of the various forms of haemoglobinopathy. Severe �-thalassaemia presents in utero or at birth, whereas �-thalassaemia and sickle-cell disease do not manifestuntil the synthesis of �-globin chains begins in earlyinfancy.

The spectrum of globin genemutationsGlobin biosynthesis is a complex process involvingtranscription to produce a messenger RNA precursor(pre-mRNA); post-transcriptional processing with 5 ′ -endcapping and methylation; translation, which proceedsthrough three separate phases of initiation, elongation,and termination; and finally interaction of the haemo-globin chains to form mature haemoglobin. Given thisdegree of complexity, it is not surprising that theprocess is error-prone. Generally disorders of haemo-globin are divided into two main groups consisting ofthose due to structural abnormalities and those thatarise because of reduced globin synthesis.

Abnormalities of haemoglobin structureOver 700 abnormal haemoglobins have been described,although not all of these are pathogenic. They can beclassified on the basis of the type of underlying mu-tation or according to their clinical consequences (Table 8.2). Originally haemoglobin variants were iden-tified by protein electrophoresis, but this has largelybeen superseded by direct DNA sequencing. Most of therecognized variants are the result of point mutationsleading to single amino acid substitutions. Some ofthese are clinically silent, but others result in clinicalproblems through a variety of effects on the haemo-globin molecule, which include:

◆ instability of the tetramer

◆ formation of an abnormal three-dimensional struc-ture

◆ prevention of ferric iron reduction

◆ prevention of normal haem binding.

Several other mutational mechanisms have also beenshown to result in structural haemoglobin variants(Table 8.2). These include frame-shift and in-framedeletions/mutations, chain termination mutations, andmispairing of homologous sequences leading tounequal crossing-over and the formation of fusion �

genes. This latter mechanism accounts for a rarehaemoglobin variant, Hb Lepore. Several differentforms of Hb Lepore have been identified, together withcomplementary forms of Hb anti-Lepore. They have allarisen through mispairing of the closely homologous -and �-chains with subsequent unequal crossing-over.The resulting � fusion chain contains 5′ polypeptidesand 3′ � polypeptides. The complementary anti-Leporefusion chain consists of 5 ′ � polypeptides and 3 ′

the spectrum of globin gene mutations 155

Key Point

Fetal haemoglobin (Hb F) consists of two �- and two�-chains. Adult haemoglobin (Hb A) contains two �- and two �-globin chains. Adults also have a smallamount of Hb A2, which is made up of two �- andtwo -globin chains.

Human haemoglobin

Haemoglobin Chain Expression % in structure pattern normal

adults

Gower I �2�2 Embryonic 0

Gower II �2�2 Embryonic 0

Portland �2�2 Embryonic 0

Fetal (Hb F) �2�2 Fetal 1

Adult (Hb A)a �2�2 Infancy → 97

Adult (Hb A2) �22 Infancy → 2–3

a A small amount of Hb A is glycosylated with a glucose moiety at the N-terminus. This is known as Hb A1c. Hb A1c is raised in individuals withdiabetes mellitus, in whom it gives an indication of the quality of diabeticcontrol over preceding months (p. 183).

TABLE 8.1

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polypeptides, and the corresponding haemoglobin isknown as Hb anti-Lepore or Hb Miyada.

Note that when letters of the alphabet had beenexhausted variant haemoglobins were generally namedafter the hometown of the first reported patient—e.g.Hb Leiden, Hb Louisville, Hb Madrid, etc.). Sometimesthe name of the family in which the variant was firstidentified was used, and occasionally the hospital atwhich the variant was first recognized (e.g. Hb Barts).

Abnormalities of haemoglobin synthesisInherited disorders associated with reduced or absentsynthesis of one or more of the normal globin chainsare known collectively as the thalassaemias. (Thalassais the Greek for sea, and the disorders were so namedbecause of their concentration around the Mediter-ranean.) The thalassaemias are classified on the basis of the globin genes involved (e.g. �-, �-, and �-thalassaemia) and whether gene expression is com-pletely (e.g. �º) or only partially (e.g. �+) suppressed. As a group, the thalassaemias constitute the mostcommon autosomal recessive disorders in the world.

The mutational basis of the common �- and �- formsof thalassaemia is complex and heterogeneous. �-tha-lassaemia is usually caused by large deletions, whereas�-thalassaemia shows marked mutational heterogene-ity with point mutations and small deletions or inser-tions. These can influence the synthesis of �-globinchains at various stages from transcription throughRNA processing, cleavage, and polyadenylation to trans-lation. The most common �-thalassaemia mutationfound in individuals originating from the Mediterran-ean region results in the creation of a new acceptor AGsplice site in the first intron of the �-globin gene. In

contrast, the most common �-thalassaemia mutationfound in the Indian subcontinent is a small 619-bpdeletion involving the 3′ end of the �-globin gene.

Sickle-cell diseaseSickle-cell disease (MIM 603903) is one of the most com-mon inherited disorders in the world, with an estimat-ed incidence in African-Americans of approximately 1 in 625. The disorder was first described in 1910 andwas shown to be due to an abnormality in haemoglobinin 1949 (Box 8.1). The molecular basis and underlyingpathophysiology are well understood but, despite ex-tensive research, treatment remains largely supportiverather than curative. Sickle-cell disease is still a majorcause of ill-health in the black population throughoutthe world.

Genetics and epidemiologySickle-cell disease shows autosomal recessive inheri-tance. Carriers, who are described as having the sickle-cell trait, are generally entirely healthy, although theycan develop clinical problems, such as vaso-occlusiveepisodes (see below), in conditions of very low oxygensaturation such as may be encountered in deep-seadiving, flying in unpressurized aircraft, or duringgeneral anaesthesia.

The frequency of the sickle-cell trait is high in allpopulations originating from equatorial Africa, where

8 genes and haemoglobin156

Key Point

Mutations in the haemoglobin genes can be sub-divided on the basis of whether they affect haemo-globin structure or haemoglobin synthesis.

Examples of variant structural haemoglobins and their clinical effects

Abnormal haemoglobin Mutation Outcome and clinical effects

Hb S Single nucleotide substitution (�Glu6Val) Hb polymerizes causing sickling and haemolytic anaemia

Hb C Single nucleotide substitution (�Glu6Lys) Hb cystallizes causing mild haemolytic anaemia

Hb M (Boston) Single nucleotide substitution (�His58Tyr) Hb has low O2 affinity causing asymptomatic cyanosis associated with methaemoglobinaemia (hence Hb M)

Hb E Single nucleotide substitution (�Glu26Lys) → Reduced synthesis of � chains causing very mild activation of cryptic donor splice site anaemia

Hb Tak Frameshift insertion → elongated � chain Hb has high O2 affinity causing polycythaemia

Hb Gun Hill Deletion (� - codons 91–95) Unstable Hb tetramer leading to haemolytic anaemia

Hb Constant Spring Substitution in stop codon → elongated Unstable � chain causing mild thalassaemia� chain

Hb Lepore Unequal crossing over between and � chains Reduced synthesis of the � fusion chain

TABLE 8.2

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carriers have a relative resistance to falciparum malar-ia. Carrier frequencies as high as 20% have been foundin countries such as Kenya and Uganda. Lower carrierfrequencies of 5–10% are seen in the Middle East and incountries around the Mediterranean where malaria isendemic. The carrier frequency in African-Americans isapproximately 8%, consistent with the incidence ofhomozygotes of 1 in 625 (i.e. 1/12.5 × 1/12.5 × 1/4 =1/625—see p. 136)

Molecular pathogenesisThe sickle-cell mutation involves a base change of A toT in the second nucleotide of the sixth codon in the �-globin gene, resulting in a substitution of valine for glu-tamic acid (i.e. GAG to GTG) (Fig. 8.3). This leads to theformation of an �2�2 tetramer which is unstable in thedeoxygenated form. When the oxygen saturation fallsbelow 85%, the �2�2 tetramers polymerize to form par-

allel rod-like structures, causing the red blood cells tobecome sickle-shaped.

Sickling of the red blood cells has two main conse-quences:

sickle-cell disease 157

BOX 8.1 LANDMARK PUBLICATION: THE BASIC DEFECT IN SICKLE-CELL DISEASE

The technological advances of the last 50 years havebeen such that it is now difficult to contemplate howlittle used to be understood about the basic cause ofmost inherited disorders. In many ways sickle-cell dis-ease, because of the ready accessibility of blood fromaffected individuals, served as the prototype for howinherited disorders might be investigated. This can be illustrated by a review of two major landmarkpublications.

The first, by Linus Pauling and colleagues at theCalifornia Institute of Technology in Pasadena, waspublished in 1949. Having observed that red bloodcells underwent sickling if the haemoglobin wasdeoxygenated, they compared haemoglobin fromaffected and unaffected individuals by electrophore-sis. The results clearly indicated that these haemoglo-bins behaved differently. They also showed thathaemoglobin from people with sickle-cell trait wasmade up of two types, one identical to that found innormal individuals and the other identical to thatfound in patients with sickle-cell disease. Finally,Pauling and his colleagues compared the haem moi-eties from normal and sickle-cell haemoglobin andfound them to be identical. Taken together, theseobservations strongly suggested that the basic defectsin sickle-cell disease resided in the globin componentof sickle-cell haemoglobin.

The next major step in the understanding of sickle-cell disease was reported by Ingram, from Cambridge,in 1957. He separated the polypeptides in normal and

sickle-cell haemoglobins by digestion with trypsin andthen subjected them to a combination of electrophore-sis and partition chromatography. One peptide showeda consistently different pattern and on subsequentanalysis was found to differ in just one amino acid, i.e.glutamic acid in normal Hb A and valine in Hb S. Basedon these results Ingram proposed that replacement of asingle base pair in the DNA of the globin gene couldaccount for the observed amino acid change and theensuing clinical problems seen in sickle-cell disease.This was the first time that a single-gene mutation hadbeen shown to cause an amino acid change in an inher-ited Mendelian disorder.

Against the backdrop of modern science theseachievements seem tame by comparison. However, intheir day they were groundbreaking, paving the wayfor the extraordinary revolution in molecular biologythat followed. Linus Pauling in particular is widelyregarded as one of the founding fathers of molecularbiology. Amongst his many distinctions he is the onlyindividual ever to receive two unshared Nobel prizes,for Chemistry in 1954 and for Peace in 1962.

ReferencesIngram VM (1957) Gene differences in human haemoglobin:

the chemical difference between normal and sickle-cellhaemoglobin. Nature, 180, 326–328.

Pauling L, Itano HA, Singer SJ, Wells IC (1949) Sickle-cellanemia, a molecular disease. Science, 110, 543–548.

Codon

5 6 7

Hb A C C T G A G G A G(Pro) (Glu) (Glu)

Hb S C C T G T G G A G(Pro) (Val) (Glu)

Hb C C C T A A G G A G(Pro) (Lys) (Glu)

Fig. 8.3. The A to T mutation in the �-globin gene which causessickle-cell disease. The G to A mutation which causes HbC is alsoshown.

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◆ the sickled red cells are fragile, with a shortened sur-vival time, resulting in a chronic haemolytic anaemia

◆ they clump together to form aggregates, which canlead to occlusion of the peripheral circulation.

This tendency to increased viscosity and peripheralvaso-occlusion accounts for most of the more seriouscomplications of sickle-cell disease.

Laboratory diagnosisSickled cells may be present in a peripheral bloodsmear (Fig. 8.4). A definitive diagnosis is made byhaemoglobin electrophoresis, which shows a high levelof Hb S, (>80%), or by direct mutation analysis for theHb S mutation using a PCR-based technique. Carrierscan be detected by a simple ‘sickling’ test which

8 genes and haemoglobin158

Fig. 8.4 Blood film showing red-cell sickling. Courtesy of Dr ClaireChapman, Leicester Royal Infirmary, Leicester.

BOX 8.2 CASE HISTORY: SICKLE-CELL DISEASE

The following case history, taken from the first docu-mented report of a patient with sickle-cell disease,was published in 1910.

A 20-year-old student from the island of Grenada inthe Caribbean sought medical advice because of a 5-week history of cough and fever. During childhoodhe had suffered from yaws, an infectious illnesscaused by a spirochaete. This is common in the trop-ics and causes pustular granulomatous lesions whichheal slowly, leaving ulcers and scars. On leaving schoolat the age of 17 years he had gone to study in Chicago,where he began to experience palpitations and short-ness of breath with a general lack of energy. He alsonoted that on occasions his sclerae had a yellowishtinge.

On examination it was confirmed that he didindeed have yellow sclerae, an indication of jaundice,which in his case was almost certainly caused byhaemolysis. His mucous membranes were describedas pale, this being a typical feature of anaemia. Healso showed clear signs of chest infection, togetherwith evidence of cardiac enlargement.

Investigations included a full blood count, whichrevealed a level of haemoglobin of 40% (i.e. <6 g/100 ml)with microcytes and nucleated red cells. The bloodfilm showed a large number of ‘thick elongated, sickle-shaped and crescent-shaped’ forms. A trace of bile wasobserved in the patient’s urine. No infectious agent that would account for the clinical findings could beidentified.

The patient was treated with rest and ‘nourishingfood’ for a period of 4 weeks, following which he was discharged with a haemoglobin level of 58%(8.4 g/100 ml). Over the next year or two he was seenon several occasions with recurrent bronchitis, per-sistent anaemia, a painful swollen knee, and finallywith what was almost certainly a haemolytic crisiswhen he experienced pain in his back and limbs inassociation with pallor, jaundice, shortness of breath,and pyrexia. Following this the patient was lost to fol-low-up. Subsequent research has revealed that hereturned to Grenada, where he practised as a dentistuntil his death at the age of 32 years. He is buried inthe Catholic cemetery in Sauteurs, close to the cliffedge where the indigenous native Carib Indians com-mitted mass suicide rather than submit to subjuga-tion by invading Europeans.

With the benefit of hindsight, we can see that thisman had relatively typical features of sickle-cell dis-ease with chronic anaemia, haemolysis, and recurrentinfection. It says much for his stoical courage that hemanaged to qualify as a dentist and survive to the ageof 32 years without the benefits of either a diagnosisor any effective treatment.

ReferenceHerrick JB (1910) Peculiar elongated and sickle-shaped red

blood cells in a case of severe anaemia. Archives of InternalMedicine, 6, 517–521.

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involves inspection for sickle-cells in a peripheral bloodfilm exposed to low oxygen saturation, or more reliablyby either haemoglobin electrophoresis or direct muta-tion analysis.

Clinical featuresOnset is usually in infancy as �-globin gene expressionovertakes �-globin gene expression. Affected childrenpresent with anaemia and jaundice, due to haemolysis,which persists throughout life. The disease course ismarked by recurrent painful sickle-cell crises caused byanaemia, haemolysis, and vaso-occlusive ischaemia,particularly involving the abdominal viscera and thelong bones (Box 8.2). Other organs including the brainand lungs can also be affected. Splenic infarctionresults in increased susceptibility to infection with bac-teria such as Streptococcus pneumonia and Haemophilusinfluenza. The long-term outlook is unpredictable. Someaffected individuals succumb to infection in early life,particularly in areas of socio-economic deprivation,whereas others survive well into middle age. Con-comitant high levels of Hb F tend to be associated witha milder disease course.

Treatment and preventionSeveral approaches have proved effective in reducingmorbidity and mortality. These include immunizationagainst pneumococcal infection, regular prophylactictreatment with penicillin to prevent infection, and folicacid supplementation to prevent folate deficiency whichcan exacerbate the anaemia. Crises are treated withfluid replacement, oxygen, and analgesics. Drugs suchas hydroxyurea and butyrate lead to an increase in fetalhaemoglobin production, with a reduction in theincidence of painful crises. Hydroxyurea has beenapproved specifically for the treatment of sickle-celldisease. Individuals with the sickle-cell trait, i.e. carri-ers, do not require any treatment other than adviceabout avoiding activities, such as deep sea diving andflying in unpressurized aircraft, which could exposethem to low levels of oxygen saturation.

Bone marrow transplantation has been carried out ina relatively small number of patients with sickle-celldisease, with success rates of around 90%. Pre-existingorgan damage due to vaso-occlusion is associated with apoorer outcome. One of the major problems in sickle-cell disease is that it can be very difficult to predict theoutcome in a young child, making it equally difficult todetermine whether bone marrow transplantation isindicated. Once it has become clear that the disease isfollowing a severe course, the success rate for bone

marrow transplantation becomes suboptimal. Thusideally a method is needed for predicting future diseaseseverity so that criteria for treatment can be estab-lished.

In theory, the births of many affected children couldbe prevented through population carrier screening pro-grammes. However, as discussed in Chapter 7 (p. 150),these have often been received with hostility and indif-ference because of poor planning and suggestions ofracist overtones. Prenatal diagnosis, based on directmutation detection, can be offered when both parentsare known to be carriers.

�-Thalassaemia�-Thalassaemia (MIM 141800) is a major cause of illhealth in South-east Asia, in parts of which the carrierfrequency is as high as 1 in 5. The condition is also com-mon in those parts of Africa and in Mediterraneanregions where malaria is endemic. The high carrier fre-quencies are thought to reflect heterozygote advantagemediated by relative resistance to severe malaria, par-ticularly the cerebral form, but the underlying mecha-nism for this remains unclear.

Genetics and molecular pathogenesis�-Thalassaemia is caused by a deficiency of �-globinchain synthesis, with the most common underlyingmutational mechanism being deletion of one or both ofthe contiguous �-globin genes. These deletions arecaused by unequal crossing-over between homologoussequences in the �-globin gene cluster (Fig. 8.5).

Normally each individual should have four function-al �-globin genes. Loss of one gene (��/�–) constitutes asilent carrier state of no clinical importance (Table 8.3).Loss of two genes (�–/�– or ��/– –) results in mildanaemia and is referred to as �-thalassaemia trait. Loss of three genes (�–/– –) results in the formation of�4 tetramers, known as Hb H, and accordingly isreferred to as Hb H disease. Finally, loss of all four �-globin genes (– –/– –) causes a lethal condition knownas hydrops fetalis in which the fetus cannot make any

�-thalassaemia 159

Key Point

The sickle-cell mutation results in a substitution ofvaline for glutamic acid in the sixth codon of the �-globin gene. Homozygotes have sickle-cell diseasein which red blood cells sickle when exposed to low levels of oxygen saturation. This causes haemo-lytic anaemia and painful vaso-occlusive sickle-cellcrises.

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fetal haemoglobin, but instead makes haemoglobin con-sisting of �4 tetramers known as Hb Barts. The ��/– –genotype is found mainly amongst South-east Asians, sothat homozygosity for the – – form of thalassaemia traitis seen almost exclusively in this population.

Clinical featuresIndividuals with �-thalassaemia trait (��/– – or �–/�–)are usually asymptomatic, with only a mild anaemiaand haemoglobin levels of 10–12 g/100 ml (normal

range 12–14 g/100 ml). In contrast, individuals withHb H disease (�–/– –) have a more severe anaemia withhaemoglobin levels of 7–10 g/100 ml, associated withinfection, haemolysis, and splenomegaly. Loss of allfour �-globin genes presents in mid-pregnancy withsevere anaemia and fluid overload, giving the clinicalpicture of hydrops fetalis. This presentation almostalways results in death in utero.

The diagnosis of the various forms of �-thalassaemiais suspected on the basis of abnormal haematological

8 genes and haemoglobin160

A �2 �1

�2 �1

�2 �1

B

C D

A B

Normal (��)

alignment (��)

Misalignment

Crossover

C D�2 �1

A D�2 �2 �1

C B �1

Duplication (���)

Deletion (�-)

Fig. 8.5 How misalignmentwith unequal crossing over inmeiosis can generate an �-globin gene deletion.

Classification of �-thalassaemia

Condition Number of functional Main type of haemoglobin Clinical outcome�-globin genes

Normal 4 (��/��) HbA (�2�2) Normal

Silent carrier or 3 (��/�–) HbA (�2�2) Normal�+-thalassaemia trait

�º-thalassaemia trait 2 (�–/�– or ��/– –) HbA (�2�2) Mild anaemia

Hb H disease 1 (�–/– –) HbA (�2�2) and Hb H (B4) Moderate haemolytic anaemia

Hydrops fetalis 0 (– –/– –) Hb Barts (�4) and Hb Portland (�22) Death before birth

TABLE 8.3

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indices (Table 8.3) and subsequently confirmed byhaemoglobin electrophoresis and PCR-based molecularanalysis.

�-ThalassaemiaThe first description of severe thalassaemia is creditedto Cooley et al. in the 1920s who reported a group ofchildren seen at the Children’s Hospital of Michigan. Tothis day the condition continues to be referred to asCooley’s anaemia. The various forms of �-thalassaemia(MIM 141900) now constitute a major challenge for health services in many parts of the world, not only because of their high frequency but also becauseof the severity of the anaemia which, in manyinstances, results in lifelong transfusion dependency. �-Thalassaemia is also important as it serves as a modelfor how a radical therapy, bone marrow transplanta-tion, can, in some instances, be curative.

Note that the nomenclature used for different gradesof severity of �-thalassaemia is potentially confus-ing. Severe transfusion-dependent �º-thalassaemia isdescribed as thalassaemia major. Milder �+-thalassaemia,which is not transfusion dependent, is sometimes clas-sified as thalassaemia intermedia. Heterozygous carriers,who are usually asymptomatic, are described as havingthalassaemia minor or thalassaemia trait.

Genetics and epidemiology�-Thalassaemia shows autosomal recessive inheritancewith high carrier frequencies occurring in a broad beltrunning from the Mediterranean through North Africaand the Middle East to the Indian subcontinent andSouth-east Asia. As with sickle-cell disease and �-thalas-saemia, the high carrier frequencies are attributed torelative resistance to falciparum malaria. Frequenciesas high as 20–25% have been observed in some Mediter-ranean islands, such as Cyprus and Rhodes, with lowerfrequencies of around 3–10% in the Indian sub-continent and South-east Asia. Many other forms ofabnormal haemoglobin, notably Hb S and Hb E, arealso extremely common in many of these regions. Thismeans that the overall incidence of severely affected

homzygotes and compound heterozygotes is higherthan would be predicted if only the carrier frequency of�-thalassaemia is taken into account.

Molecular pathogenesisThe many mutations associated with �-thalassaemiaeither reduce �-globin gene expression (�+-type) orcompletely suppress it (�º-type). The net effect is areduction in, or a complete absence of, the synthesis of�-globin chains resulting in an � : � chain imbalancewith excess �-globin chains. These are unstable andprecipitate in the red cell precursors to form inclusionbodies. Unfortunately these interfere with red cell mat-uration, so erythropoiesis is impaired. Those red cellswhich do enter the circulation are destroyed prema-turely by the spleen.

This combination of reduced erythropoiesis andincreased red cell destruction results in severe hae-molytic anaemia, with increased intramedullary ery-thropoiesis, which leads in turn to expansion of thebone marrow cavities with bone deformity and a risk ofpathological fracture.

Laboratory diagnosisIn severe cases the level of haemoglobin ranges from 3 to 8 g/100 ml with hypochromic, microcytic red cells(Fig. 8.6). The bone marrow shows marked erythroidhyperplasia. In �-thalassaemia there is no Hb A, soelectrophoresis shows only Hb A2 and Hb F. Carriers of�-thalassaemia show mild anaemia, with haemoglobinlevels of 9–11 g/100 ml, and slightly elevated levels ofHb F (1–3%) and Hb A2 (4–6%).

�-thalassaemia 161

Key Point

�-Thalassaemia is usually caused by deletion of �-globin genes. Loss of three �-globin genes causesHb H (�4) disease. Loss of all four �-globin genescauses death in utero due to hydrops fetalis with HbBarts (�4).

Fig. 8.6 Peripheral blood film in severe �-thalassaemia showinghypochromia, microcytosis, and nucleated red cells. Courtesy of Dr Claire Chapman, Leicester Royal Infirmary, Leicester.

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Clinical features and treatmentChildren with �º-thalassaemia (thalassaemia major)usually present at around the age of 6 months when �-globin chains should normally be replaced by �-globin chains. Symptoms include poor feeding, fail-ure to thrive, and recurrent infection. Without regularreplacement transfusion the disease runs a downhillcourse with death in childhood due to general malaiseand infection. Regular transfusion restores good gener-al health, but leads to excess iron deposition in theheart, pancreas, and liver. This will prove fatal in earlyadult life unless prevented by adherence to a strictregime of iron chelation therapy with a drug such asdesferrioxamine With a combination of regular trans-fusion and appropriate chelation therapy, affectedindividuals can survive well into adult life. In poorcountries where such therapeutic regimes are notavailable, survival into adulthood is rare.

Bone marrow transplantation using HLA-compatibleunaffected related donors is potentially curative and hasbeen carried out in over 1000 children with success ratesof over 90%. This possibility of successful therapy hasprompted some parents to request in vitro fertilizationwith pre-implantation genetic diagnosis to enable anunaffected HLA-compatible ‘saviour’ sibling to be deliv-ered as a potential donor. Understandably this has raisedserious ethical concerns about the possible use of a‘designer baby’ as an uninformed and non-consentingdonor for the affected older sibling (pp. 144, 237).

PreventionCarrier detection programmes for �-thalassaemia havebeen introduced in several Mediterranean regions andhave met with considerable success in countries suchas Cyprus where participation is strongly encouraged(p. 149). Ethical concerns about issues such as termina-tion of pregnancy and forced participation have to bebalanced against the suffering experience by affectedchildren in countries where limited financial resourcesmean that there is little hope of effective treatment.

��-Thalassaemia and hereditarypersistence of fetal haemoglobin�-Thalassaemia and hereditary persistence of fetalhaemoglobin (HPFH—MIM 142470) are rare conditions oflimited clinical importance, particularly when comparedto the devastating impact made by the severe forms of �-and �-thalassaemia. However, their inclusion in a discus-sion of the haemoglobinopathies is justified by theinsight that they provide into possible approaches totherapy based on modification of gene expression.

Both conditions are characterized by reduced orabsent expression of the - and �-globin genes. In �-thalassaemia this is caused by a deletion involvingthe contiguous (closely adjacent) - and �-globin genes(Fig. 8.7). Heterozygotes are asymptomatic, and homo-zygotes show only a mild haemolytic anaemia. This isin marked contrast to the severe transfusion-dependentanaemia seen in homozygous �º-thalassaemia. For rea-sons which are not fully understood, expression of the�-globin genes is not suppressed in �-thalassaemia, soheterozygotes and homozygotes show levels of Hb F of

8 genes and haemoglobin162

Key Point

�-Thalassaemia shows marked mutational hetero-geneity. Homozygotes for severe �º-thalassaemiarequire life-long treatment with blood transfusionand iron chelation therapy. Alternatively, bone mar-row transplantation from a histocompatible donoris potentially curative.

Fig. 8.7 The effects of different deletions in the �-globin genecluster. Reproduced with permission from Lewin B (2000) Genes VII.Oxford University Press, New York.

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4–18% and 100% respectively. This increased expressionof the �-globin genes continues throughout life andprotects the individual concerned from the harmfuleffects of the � : �-like globin chain imbalance whichimpairs erythropoiesis in �-thalassaemia.

Similarly, in HPFH, which can be caused either bydeletions involving the - and �-globin loci or by variouspoint mutations in the �-globin gene cluster, increasedexpression of the �-globin genes compensates forreduced or absent expression of the - and �-globin genes.Heterozygotes and homozygotes are asymptomatic, withHb F levels of around 20% and 100% respectively. It is notknown why the expression of the �-globin genes is main-tained in these conditions. Possible explanations includethe prevention of normal suppression of postnatal �-glo-bin gene expression by point mutations in, or deletionsof, �-globin gene regulatory regions.

Whatever the correct explanation, which may wellinvolve several different regulatory mechanisms, these‘accidents of nature’ serve to emphasize the potentialvalue of therapeutic approaches aimed at increasing

�-globin gene expression in sickle-cell disease (p. 159).This is further illustrated by the outcomes seen inindividuals who inherit two different forms of haemo-globinopathy, as described in the next section.

Interaction of different haemoglobinmutationsAs a result of the high gene frequencies of many differ-ent forms of haemoglobinopathy in areas where malar-ia is endemic, individuals are often encountered whohave inherited two or more mutations in the �- and/or�-globin gene clusters. In practice it can be difficult topredict the outcome, but some consistent observationshave been made.

interaction of different haemoglobin mutations 163

Key Point

Individuals with �-thalassaemia and HPFH are onlymildly affected because of the protective effects ofsustained �-globin gene expression.

BOX 8.3 CASE CÉLÈBRE: TIONNE ‘T-BOZ’ WATKINS

Tionne ‘T-Boz’ Watkins is the ‘T’ in the all-female rapgroup TLC. As well as receiving numerous musicalaccolades, including two Grammies and two AmericanMusic Awards, she has also achieved success as a song-writer, producer, actor, and poet. All of this has beenachieved against a background of chronic ill-healthbrought on by her underlying diagnosis of sickle-celldisease, caused by compound heterozygosity for thesickle-cell trait and �-thalassaemia.

In her semi-autobiographical book, Thoughts, sheprovides a graphic description of what it is like toexperience a sickle-cell crisis:

I start aching, sometimes in really excruciating pain. Whenit’s really bad, it’s as if someone is stabbing me with a butcherknife over and over where it hurts. The only parts of my bodythat have never hurt are my feet and my fingers. I’ve had paineverywhere else at one time or another. It can affect my wholebody, or sometimes it can be just a leg. If it’s my legs, I can’twalk and I have to learn how to walk again. If it’s my arms, Ican’t hold anything, so people have to help feed me. I feelpainful and weak. I’ve been in so much pain that I get deliri-ous, not knowing where I’m at. Or my face might swell up to apoint where you wouldn’t recognize me.

This is followed by an account of the treatment:In the hospital, I have to drink lots of fluids and get hooked

up to an IV to flush the blood. I also get hooked up to oxygenuntil the blood recovers. Meanwhile, my body fights against

itself because the doctors prescribe drugs that constipate, thenthey give me other drugs that act as laxatives. They’re alsodrugging me up to cover the pain. And these are some power-ful painkillers. You go into the hospital with one problem andleave with another. Now you feel like a crackhead or a heroinaddict. Once I get off the drugs I go through withdrawal, withhot and cold flashes, shaking and jumping with crazy dreams,and it’s hard to breathe. In the meantime, my body is sweat-ing out the drugs so I smell like all these chemicals.

Despite this depressing account of life as a sickle-cell patient, the underlying theme of this youngwoman’s life, and her book, is very positive. She con-cludes the chapter entitled ‘Monster in my veins’, inwhich she describes her illness, with an appeal to themedical community that ‘we need more research andto get more aggressive about helping the minoritiesout there who suffer from this disease’. The final sen-tence aptly sums up her attitude to life and to her ill-ness. ‘As for me, I don’t intend to let it keep me from doingthe things I love to do, that make me who I am. You can callthat stubborn, but I just call it living’.

ReferenceWatkins, Tionne ‘T-Boz’ (1999) Thoughts. HarperCollins, New

York.

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�-Thalassaemia/�-thalassaemiaAn individual who is a carrier of both �-thalassaemiaand �-thalassaemia is referred to as a double hetero-zygote (p. 74). Such individuals are usually entirelyhealthy. Curiously, the presence of an �-thalassaemiamutation tends to reduce the severity of the anaemiaseen in homozygotes for �-thalassaemia. This is almostcertainly because the imbalance of � : � globin chains,which leads to impaired erythropoiesis, is reduced bythe presence of an �-thalassaemia mutation.

�-Thalassaemia/Hb SIndividuals who are compound heterozygotes for �º-thalassaemia and Hb S have a severe disorder similarto sickle-cell disease (Box 8.3). This is predictable, giventhat the �º-thalassaemia gene is silenced so that the only�-globin chains manufactured are Hb S. Compound het-erozygotes for HbS and milder �+-thalassaemia mutationsusually have only mild anaemia or are asymptomatic.

Haemoglobin S/C diseaseThis results from compound heterozygosity for muta-tions in the sixth codon of the �-globin gene (Fig. 8.3), ifan individual inherits Hb S from one parent and Hb Cfrom the other. Clinically this results in a relativelymild form of sickle-cell disease, which presents in latechildhood or adult life, but which is associated with a high incidence of vaso-occlusive complications, par-ticularly involving the lungs and the retinal vessels.

Other �-globin variants which react with Hb S tocause sickle-cell disease include Hb D, Hb E andHb O Arab. Hb D exists in several different forms, ofwhich the most common, Hb D Punjab, shows a partic-ularly high frequency in India and China. Hb E is thecommonest haemoglobin variant in the world, and inthe homozygous state usually presents as a mild formof �-thalassaemia. Some compound heterozygotes for�º-thalassaemia and Hb E can be so severely affectedthat they are transfusion dependent.

SummaryThe inherited disorders of haemoglobin structure and synthesis, known collectively as the haemoglo-binopathies, together constitute the most commonautosomal recessive disorders in the world. The haemo-globin molecule contains four globin chains, �2�2 infetal haemoglobin and �2�2 in adult haemoglobin.These chains are encoded by genes in two globin geneclusters, an � or �-like cluster on chromosome 16 and a� or �-like cluster on chromosome 11.

Sickle-cell disease is the most common disorder ofhaemoglobin structure and affects approximately 1 in625 African-Americans. It is caused by an A to T pointmutation in the sixth codon of the �-globin gene,resulting in a substitution of valine for glutamic acid.The resulting globin tetramer is unstable in the de-oxygenated form, causing the red blood cells to becomesickle-shaped. This results in chronic haemolytic anaemiaand peripheral vaso-occlusion leading to extremelypainful sickle-cell crises.

The thalassaemias are disorders of haemoglobin syn-thesis. �-Thalassaemia results from reduced synthesis of�-globin chains, usually because of deletion of one ormore of the two �-globin genes on each number 16 chro-mosome. Loss of three �-globin genes results in Hb H (�4)disease characterized by moderately severe lifelonganaemia. Loss of all four �-globin genes results in severeanaemia before birth leading to hydrops fetalis and deathin utero. The chief haemoglobin in these infants consistsof four �-chains (�4), and is known as Hb Barts.

�-Thalassaemia results from reduced synthesis of �-globin chains with marked underlying mutationalheterogeneity in the �-globin genes on chromosome11. The resulting � : � chain imbalance results in pre-cipitation of �-chains and impaired erythropoiesis withsevere chronic haemolytic anaemia. Severely affectedchildren are dependent on regular blood transfusion,which conveys a risk of chronic life-threatening ironoverload. Bone marrow transplantation is potentiallycurative.

Both �- and �-thalassaemia are common in areaswhere malaria is endogenous, with carrier frequenciesof up to 1 in 5 in South-east Asia (�-thalassaemia) andMediterranean regions (�-thalassaemia). Together withsickle-cell disease, these conditions are major causes ofchronic morbidity and premature mortality, particular-ly in some of the world’s poorest countries where facil-ities and resources for health care are limited. Themanagement of these disorders and the suffering thatthey cause remains a major challenge for health careagencies throughout the world.

Further readingOld J (2002) Hemoglobinopathies and thalassemias. In:

Rimoin DL, Comra JM, Pyeritz RE, Korf BR (eds)Principles and practice of medical genetics, 4th edn.Churchill Livingstone, Edinburgh, pp. 1861–1898.

Rodgers GP (1998) Sickle cell disease and thalassaemia.Baillière’s Clinical Haematology, 11(1). Baillière Trindall,London.

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Sergeant GR (1992) Sickle cell disease, 2nd edn. OxfordUniversity Press, Oxford.

Weatherall DJ, Clegg JB (2001) The thalassaemia syn-dromes. Blackwell Science, Oxford.

multiple choice questions 165

1 The following statements about haemoglobin arecorrect:

(a) fetal haemoglobin contains two �-globin and two-globin chains

(b) adult haemoglobin contains two �- and two �-glo-bin chains

(c) the �- and �-globin gene clusters are arranged intandem on chromosome 11

(d) normal humans have two �- and four �-globingenes

(e) expression of the globin genes involves chro-matin remodelling

2 The following statements about abnormal forms ofhaemoglobin are correct:

(a) Hb H contains four � chains

(b) Hb Barts contains four � chains

(c) Hb S is caused by a single point mutation

(d) Hb S and Hb C are caused by different pointmutations in the same codon

(e) Hb Lepore is caused by formation of a fusion gene

3 Healthy, unaffected parents have a 6-month-oldinfant who has just been diagnosed with sickle-celldisease. It would be correct to tell them that

(a) the chance that their next baby will be affected is1 in 4

(b) babies with sickle-cell disease are anaemic frombirth

(c) treatment involves regular folic acid supplemen-tation

(d) early treatment of infection is important

(e) it is important to try to avoid dehydration

4 Healthy, unaffected parents have a 6-month-oldinfant who has just been diagnosed with �-thalas-saemia. It would be correct to tell them that

(a) the chance that their next baby will be affected is1 in 4

(b) their next baby is likely to have hydrops fetalis

(c) most affected babies present at birth with severeanaemia

(d) treatment involves regular iron supplementation

(e) bone marrow transplantation is potentially cura-tive

5 The following statements about thalassaemia are correct

(a) �-thalassaemia is usually caused by a frame-shiftinsertion

(b) �-thalassaemia is usually caused by a completedeletion of both �-globin genes

(c) loss of three �-globin genes causes Hb H disease

(d) homozygotes for �-thalassaemia are usuallyonly mildly affected

(e) someone who inherits an �-thalassaemia mutationfrom one parent and a �-thalassaemia mutationfrom their other parent will have severe anaemia

Multiple choice questions

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Answers1 (a) false—fetal haemoglobin contains two �- and two

�-globin chains

(b) true

(c) false—the �-globin gene cluster is on chromo-some 16

(d) false—normal humans have four �- and two �-globin genes

(e) true—this is initiated by the relevant locuscontrol regions

2 (a) false—Hb H contains four �-globin chains

(b) false—Hb Barts contains four �-globin chains

(c) true—a point mutation in the sixth codon of the�-globin gene

(d) true—glutamine to valine in Hb S and glutamineto lysine in Hb C

(e) true—it is the result of a small fusion gene gener-ated by unequal crossing-over

3 (a) true—inheritance is autosomal recessive

(b) false—presentation is from 3 months onwards as�-globin chains start to replace �-globin chains

(c) true—this is to prevent coincidental folic aciddeficiency anaemia

(d) true—children with sickle-cell disease are verysusceptible to bacterial infection because of auto-splenectomy

(e) true—this can predispose to vaso-occlusive crises

4 (a) true—inheritance is autosomal recessive

(b) false—this is a presentation of �-thalassaemia

(c) false—as with sickle-cell disease presentation isfrom 3 months onwards as �-globin chains startto replace �-globin chains

(d) false—iron overload is a major hazard in �-thalassaemia

(e) true—bone marrow transplant from a histocom-patible donor is very successful

5 (a) false—the most common mutational mechanismis a deletion of the entire gene

(b) false—there is marked mutational heterogeneitywhich can include small intragenic deletions

(c) true—the excess �-globin chains form Hb H

(d) true—there is a compensatory increase in Hb Fdue to persistent �-globin chain synthesis

(e) false—such a person is a double carrier and isusually not anaemic

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