Thalassaemia

THALASSAEMIA was first recognized as a separate disease entity by Cooley (Cooley and Lee, 1925; Cooley, 1927). Since that time a number of related disorders have 'been describedland -these have been classified under the same general title. Because the main feature of these diseases was anaemia, associated with a deficiency of haemoglobin in the red cells, the primary abnormality has long been Ithought to be impaired haemoglobin synthesis. At first, the disease was classified in three groups: thalassaemia major, thalassaemia minor and Ithalassaemia initermedia according to the severity of the disease. Although it was soon found that there was a genetic relationship between the various ttypes, nothing was known of the underlying abnormality. Recent advances in 'the understanding of the structure, synthesis and genetic control of haemoglobins have led to a greater knowledge of 'the underlying abnormality in thalassaemia. Although the primary cause is still subject to speculation, the present concepts on the causation of the disease are leading to hypotheses which are directly open to experimental test. Furthermore, the classification of these diseases is of help in determining the prognosis in individual cases, as well as enabling one to give assistance by genetic counselling. Recently, several reviews on ithe subject of thalassaemia have appeared (Fessas, 1964; Motulsky, 1964a). There have also been a conference (Fink, 1964), and two monographs (Bannerman, 1961; Weatherall, 1965) devoted entirely to this disease.

THALASSAEMIA was first recognized as a separate disease entity by Cooley (Cooley and Lee, 1925;Cooley, 1927). Since that time a number of related disorders have 'been describedland -these have been classified under the same general title. Because the main feature of these diseases was anaemia, associated with a deficiency of haemoglobin in the red cells, the primary abnormality has long been Ithought to be impaired haemoglobin synthesis. At first, the disease was classified in three groups: thalassaemia major, thalassaemia minor and Ithalassaemia initermedia according to the severity of the disease. Although it was soon found that there was a genetic relationship between the various ttypes, nothing was known of the underlying abnormality.
Recent advances in 'the understanding of the structure, synthesis and genetic control of haemoglobins have led to a greater knowledge of 'the underlying abnormality in thalassaemia. Although the primary cause is still subject to speculation, the present concepts on the causation of the disease are leading to hypotheses which are directly open to experimental test. Furthermore, the classification of these diseases is of help in determining the prognosis in individual cases, as well as enabling one to give assistance by genetic counselling.

Normal Human Haemoglobins
In order to understand the present concepts of 'the aetiology of the disease, it is necessary to have a clear picture of the genetic control of human haemoglobin synthesis. This area has been reviewed in detail elsewhere (for references see . Adult Haemoglobins During adult life, the main pigment found in ,the red cells is called adult haemoglobin, or Hb-A. Each molecule of Hb-A consists of four globin (polypeptide)-chains arranged in two pairs, two so-called a-chains and two /-ohains. To each of these chains is attached a haem group. The structure of Hb-A can thus be written Hb-a2,82 (Schroeder, 1963). A second adult haemoglobin, called haemoglobin A2 (Hb-A2) is also found in the red cell. In the normal person, this amounts to 1.5-3% of Itotal haemoglobin (Kunkel, Ceppellini, Muller-Eberhard and Wolf, 1957;Silvestroni, Bianco, Muzzolini, Modiano, Vallesneri, 1957). This minor haemoglobin has a very similar structure to that of Hb-A, being composed of two a-chains and two 8-chains, and can thus be written Hb-a282 (Ingram and Stretton, 1962a and

b).
A trace of foetal 'haemoglobin (Hib-F) (less than 0.4%) (see below) is also found in the red cells of adults (Chernoff, 1953;Beaven, Ellis and White, 1960). Because it is very difficult to measure such small amounts accurately, the upper level of normal is 'taken as 1% when measured by the method of Betke, Marti and Schlicht (1959) or 2% when measured by the method of Singer, Chernoff and Singer (1951). Using starch gel electrophoresis with a itris-EDTA-borate buffer, pH 8.5, increases of between 1 and 2% of Hb-F can be detected Chernoff, Pettit and Northrop, 1965). Foetal Haemoglobin During foetal life, the main pigment present in tihe red cells is foetal haemoglobin, or Rb-F. This consists of two a-chains joined to two y-chains,   .
It should be noted 'that the three normal haemoglobins all contain the same a-chains and their own type of non-a-chains: 5-chains in Hb-A, 8 starch gel eleotrophoresis. This method also shows the presence of small amounts of Hb-74 (Fessas and ,Mastrokalos, 1959;Karaklis and Fessas, 1963;Weatherall, 1963;Huehns, Dance, Beaven, Heoht and Motulsky, 1964a). Hb-y4 has previously been called "fast foetal" haemoglobin (Fessas and Papaspyrou, 1957) or "Bart's" haemoglobin . That ithis haemoglobin consists of four y-chains i(the non-a-chain of foetal haemoglobin) was established Iby Hunt and Lehmann (1959) and Kekwick and Lehmann (1960). In normal cord blood samples the amount of Hlb-y4 does not exceed 0.5% I (Huehns, Beaven and Stevens, 1964).
During embryonic life, two further normal haemoglobins occur: embryonic haemoglobin (Gower) 1 and embryonic haemoglobin (Gower) 2 (Huehns, Flynn, Butler and Beaven, 1961;Huehns and others, 1964a). These haemoglobins also contain their own specific non-achain, the e-chain. The developmental changes in haemoglobin found in the red cells are given in Figure 1. The relative electrophoretic mobilities of 'the normal human haemoglobins are shown in Figure 2a. The Genetic Control of Haemoglobin Synthesis From ;the study of ithe abnormal haemoglobins it has 'been shown that the structure of each of the haemoglobin chains is controlled by a separate genetic locus and ,that each gene at each locus finds expression in its own messenger RNA "template", which, in turn, determines the synthesis of a particular polypeptide chain. The various polypeptide chains synthesized Ithen associate with each other and -their haem groups ,to form the haemoglobins found in vivo (Figure 2). It has been suggested ,that there is only one structural locus for the a-chains of Hb-A, Hb-F and Hb-A2. That this is so is confirmed by 'the finding 'that any abnormality of the a-chain affeots Ib-A2 and Hb-F as well as Hb-A (see  references).
As has already Ibeen noted, the synthesis of embryonic haemoglobins is followed by the synthesis of foetal haemoglobin, which, in turn, (is replaced by the adult haemoglobins, Hb-A and IHb-A2. It has been suggested that 'these changes are brought about by a number of regulatory genes which control the rates of synthesis of the various polypeptide chains (Neel, 1961;Motulsky, 1962;Zuckerkandl, 1964). The Haem Groups The ihaem groups of the different human haemoglobins are all identical, ferrous protoporphyrin IX. These are synthesized separately from the globin chains by a complex series of enzymic reactions (Riimington, 1959). The genetic control of these processes is separate from that of Ithe protein part of the molecule. 720 POSTGRADUATE MEDICAL JOURNAL  <}i) (

THALASSAEMIA
The characteristic abnormality in thalassaemia is 'the reduction in the amount of haemoglobin in the red cells due to diminished haemoglobin synthesis. As haemoglobin consists of ithe haem group joined to 'the protein moiety, globin, impairment of haemoglobin synthesis could be due to diminished synthesis of either haem or globin. The finding !that there was impairment in the incorporation of l4C-glycine into haem and some haem precursors in in vitro bone marrow cultures prepared from patients with thalassaemia (Bannerman, Grinstein and Moore, 1959;Steiner, Baldini and Damashek, 1964) appears to suppont ithe idea 'that the abnormality in ,this disease is one of haem synthesis. Further support for this comes from the observation .that in 'thalassaemia rthere is a great increase in the excretion of early labelled bile pigment after the injection of 15N or 14C-lglycine ' (Grinstein, Bannerman, Vavra and Moore, 19160). On the other hand, the welldocumented clinical observation .that infants with thalassaemia major only develop the disease during the first year of life is opposed to the idea 'that tthe primary abnormality is a defeot in haem synthesis. This is because the haem groups of foetal haemoglobin and adult haemoglobins are identical, namely, ferrous protoporphyrin IX, and hence impairment of its synthesis should lead to anaemia during foetal as well as adult 'life. On the other hand, the protein parts of these molecules differ, containing y-chains in footal haemoglobin (Hb-a2y2) and l-chains in adult haemoglobin (Hlb-a4,2). Recent work has shown that haem exerts a negative feedback on the synthesis of 8-amino-laevulinic acid (ALA) in red cell precursors (Lascelles, 1960;Burnham and Lascelles, 1964;London, Bruns and Karibian, 1964;Steiner and others, 1964). The reduced rate of ihaem synthesis in ,thalassaemia can thus be explained by "feedback" inhibition of the synthesis of 8-ALA by the un-utilized haem formed in the Ithalassaemic red cell. The abnormality in bile-pigment labelling mentioned above is probably due ito the large amount of "'ineffective erythropoiesis" '(see later) found in this disease, rather than to an abnormality of haem synthesis. It would itherefore appear more likely !that ;thalassaemia is due to an abnormality of globin synthesis. This has recently been confirmed by the work of Marks and Burka (1964) who have shown ithat the synthesis of fl-chains is impaired in the "cell-free system", using ribosomes obtained from Pf-thalassaemic red cell precursors.
As has already been pointed out, the various globin chains (a-, f/-, -8, y-and e-chains) are separately synthesized, and any abnormality of globin synthesis would ithus be expected -to affect primarily only one type of chain. This implies that just as there are two main groups of abnormal haemoglobins, those affecting the a-chains and !those affecting the f-chains, there would also be two groups of thalassaemias, one affecting Ithe synthesis of a-chains and one affecting the synthesis of fP-chains, and these have been called a-thalassaemia and /-thalassaemia respectively. The finding of rare individuals without any ,Hb-A2 Warrington, Bell and Thompson, 1965) suggests that there might also be a mutation specifically inhibiting 8-chain synthesis, 8-thalassaemia. However, the absence of Hb-A2 on eleotrophoresis could also be explained if these individuals were the carriers of a 8-chain mutation 'which resulted in a Hb-A2 variant migrating with Hb-A (Baglioni, 1964). The existence of y-thalassaemia has also been claimed (Hamilton, Sheets and Brosseau, 1962). However, the only two established groups are a-and /f-thalassaemia.
The genes causing 'thalassaemia can, of course, occur in !the heterozygous or homozygous state. There would thus be a-and .3-thalassaemia trait and a-and f/-homozygous .thalassaemia. The action of the abnormal genes results in a reduction in the rate of synthesis of the particular polypeptide chain involved. The heterogeneity of clinical severity of the disease suggests 'that here are a number of different mutations causing thalassaemia. lit is 'thought that these differ in the degree of impairment of polypeptide chain synthesis caused. Some mutations would completely, or almost completely, inhibit the synthesis of a particular polypeptide chain, while others would only partially inhibit it. The former may be called "severe" thalassaemia genes and the latter "mild" thalassaemia genes.
In the simple thalassaemia heterozygote, it is difficult to measure the degree of inhibition caused by any panticular thalassaemia gene. However, an approximate measure of this is obtained from the proportion of Hb-A found in subjects who also carry an abnormal haemo-722 POSTGRADUATE MEDICAL JOURNAL

Clinical Features
The clinical features of the thalassaemias have been described by a number of authors (Astaldi, Tolentino, Sacchetti, 1951;Dacie, 1960;Wintrobe, 1963); only the main features will be outlined here and are summarized in Table 1. As ithe diseases caused by deficient a-or 8-chain synthesis are very similar, it is convenient to give a general description first. The specific features of each ,type will be discussed later.
Thalassaemia Major This is a severe anaemia with a haemoglobin of 3-6 g./100 ml. There is gross enlargement of the spleen and liver, partly due to infiltra-'tion with erythropoietic tissue. Besides extramyeloid erythropoiesis, ithere is gross expansion of the red marrow. This leads to typical X-ray appearances of 'the skeleton (Cooley, Witwe and 'Lee, 1927;Caffey, 1957;Mosely, 1962;Baker, 1964). In the most severe cases, even the small bones of the hands and feet are affected (Fig. 4a). Pathological fractures are not uncommon. The skull shows a distinctive appearance (Fig. 4b.). There is atrophy of ,the outer table, iwith a radiating growth of subperiostial bone, giving 'the so called "hair on end" appearance. The facial bones are also expanded, giving the typical mongoloid facies.
The mean -red cell survival is only moderately shortened to approximately 10-80 days, but there is also a small proportion of cells which are destroyed very rapidly (Kaplan and Zuelzer, 1950;Sturgeon and Finch, 1957;Erlandson, Schulman, Stern and Smith, 1958;Bailey and Prankerd, 1958). There is clearly a discrepancy between red cell productive capacity and the rate of red cell production. Sturgeon and Finch (1957) have shown that the rate of 59Fe uptake by the marrow greatly exceeds that appearing in the red cells. They argue that a large proportion of the red cells produced in the marrow are not released into 'the circulation but destroyed in situ, a process called "ineffective erythropoiesis".
Haematology. The total haemoglobin is extremely low: 3-6 g./100 ml. The red cells show gross variation of size and shape with marked hypochromia and 'target cells (Fig. 5 lysis. Occasionally, some cells are not even lysed by water. The plasma contains a raised bilirubin and also some metlhaemalbumen. As in other haemolytic diseases, the iron-binding capacity of the plasma is fully saturated. The bone marrow shows extreme hyperplasia of the erythropoietic cells, usually of the normoblastic type. Occasional cases with megaloblastic changes are seen (Chanarin, Dacie and Mollin, 1959).

Thalassaemia Minor
This is a mild disease, there being no, or only borderline, anaemia. The red cells show moderate variation in size and shape with some hypochromia. Target cells are often found.
There is increased resistance of the red cells to osmotic lysis. The most characteristic finding is a low MCH and a reduction in -the MCV. Occasionally the spleen may be palpable. There are no X-ray changes in 'the bones. The mean red cell survival is normal (Kaplan and Zuelzer, 1950;Malamos, Belcher, Gyftaki and Binopoulov, 1961). The clinical picture is often difficult to distinguish from that of iron deficiency anaemia.

Thalassaemia Intermedia
In this syndrome, the haemoglobin varies from ca 6 to 10 g./100 ml. The spleen is usually palpable and may be very large. In many patients there is also liver enlargement. The expansion of 'the bone marrow varies from patient to patient, depending on the severity of the disease. Thus, in the more severely affected gross X-ray changes in the bones are found, whilst in milder cases small or no changes may be detectable. The blood picture is typical of thalassaemia.
Classification of Thalassaemia (0 fl-thalassaemia In l-;thalassaemia, the rate of synthesis of the fl-chains is suppressed whilst that of the other polypeptide chains is not primarily affected. It can be seen from Figure 1 that at birth the y-ohains of foetal haemoglobin are dominant, and no disease would be expected at lthis stage of development but would commence at ca three months of age. Any deficiency of fl-chains would lead to a relative increase in yor 8-chains if these are produced at a normal rate. The y-chains might also compensate for the /l-chain deficiency if they are produced in quantity after the neonatal period (see Fig. 3). The specific features of f-thalassaemia are, therefore, commencenient of the disease after birth, together with a relative increase in Hb-A2 and/or Hb-F in the red cells.
There is another group of patients with the blood picture of thalassaemia minor and a raised HEb-F, but with a normal Hb-A2 (Zuelzer, Robinson and Booker, 1961;Fessas, 1962b;Gabudza, Nathan and Gardner, 1964;Weatherall, 1964a). These patients may have Hb-F levels higher than in classical 8f-thalassaemia trait. This form of f-,thalassaemia -trait also interacts with ithe classical 'type of P-thalassaemia ,trait with a high Hb-A2 Ito give the picture of fl-ithalassaemia major or intermedia. The explanation for the failure to find an increase of Hb-A2 in these cases is not clear. Some patients may be suffering from concomitant iron deficiency, which is known to reduce the level of Hb-A2 (Josephson and others, 1958;Chernoff, 1964). However, this does not explain all cases. Some authors have suggested that the mutation not only affects the synthesis of the 8-chains but also that of the 8-chains (Bernini, Colucci, De Michele, Piomelli, and Siniscalco, 1962;Fessas, 1964;Motulsky, 1964a), and the latter authors have coined the term ",88-thalassaemia". In other patients, the failure to find a raised Hb-A2 might be caused by the inheritance of a f/-thalassaemia gene which only partially suppresses p-chain synthesis. Occasionally, the clinical picture of 'thalassaemia minor is due to Ithe presence of an abnormal haemoglobin with a low rate of synthesis, such as IHb-'Lepore (see later) or Hb-Caserta (Ventruto, Baglioni, Rosa, Bianchi, Colombo and Quattrin, 1965).
From the above description it will be seen that the phenotypic expression of f-rthalas-  degree of anaemia and morphological changes in the red cells, which are subject to many other genetic and environmental factors, but also the more specific features such as the level of Hb-A2 and of Hb-F found (see, for example, Beaven, Stevens, Ellis, White, Bernstock, Masters and Stapleton, 1964;Lee, Haut, Cartwright and Wintrobe, 1964).
(b) Homozygous f-thalassaemia. Studies of parents of children with thalassaemia major show ,that usually both have thalassaemia trait with a raised iHb-A2 (Kunkel and others, 1957;Gerald and Diamond, 1958a), ,and Ithis disease is therefore !the homozygous form of ft-thalassaemia. Because f-chain synthesis is (almost) completely inhibited, a severe disease results which begins at the time in development when y-chain synthesis normally ceases and f-chain synthesis commences. The severe anaemia produces a stress situation in the erythroid marrow, which, by a mechanism not yet understood, attempts to compensate for the failure of f-chain synthesis by the continued production of y-chains. However, the rate at which these are produced is far below that necessary for complete compensation.* It appears that the Hb-F produced by such a situation is unevenly distributed among the red cell population. This has been shown in sickle cell disease (Singer and Fisher, 1952) and B/-thalassaemia minor (Shepard and others, 1962). As Hb-F is the only pigment produced in quantity in 3-thalassaemia major, this variation in the amount of Hb-F synthesized leads to cells containing varying amounts of haemoglobin. Those cells which contain very little haemoglobin are destroyed in the marrow, the so called "ineffective erythropoiesis" already referred to. The remaining, relatively few, cells reach the circulation. Some of these cells are rapidly destroyed, whilst the remainder have a moderately shortened survival. (For references see earlier). Gabudza, Nathan and Gardner (1963) found that the short-lived cells contain relatively less Hb-F and, on the above hypothesis, would contain less total haemoglobin than the remainder. The red cells in the circulation contain mostly Hb-F. This usually forms 60%/ or more of the total haemoglobin, but levels from as low as 10% up to 100% have been reported (see Weatherall, 1965). Hb-A2 is usually within normal limits (Kunkel and others, 1957;Silvestroni and others, 1957). The remainder is Hb-A. A trace of Hb-a is also present (Fessas and Loukopoulos, 1964). *The situation in which complete compensation occurs is called hereditary persistence of foetal haemoglobin, referred to later, and does not lead to anaemia.
One of the features of a-thalassaemia (see later) is the occurrence of the non-a-chain haemoglobins, Hb-,f4, Hb-y4 and Hb-8 in significant amounts in the red cells (see next section), and it might be expected that in p-,thalassaemia free a-chain haemoglobin, Hb-a, might be found in quantity in the red cells of severely affected individuals, whereas only trace amounts have been detected. The reasons for this are not yet clear, but it has been suggested that the rate of a-chain synthesis is closely linked to that of the P-, y-, and 8-chains (Huehns and Shooter, 1962). Although this has not yet been proved, there is some evidence supporting this hypothesis (Baglioni and Colombo, 1964). Alternatively it has been suggested that the excess a-chains formed precipitate in the red cells (Fessas, 1963).
One of the puzzling features of /-,thalassaemia is that though ,the proportion of Hb-A2 is raised in PB-thalassaemia trait, it is usually normal or low in Ithe homozygous state. If 8-chain synthesis were proceeding at a normal rate, it might be expected that Hb-A, would form a much higher proportion of total haemoglobin in the homozygous condition than in the trait form. A possible explanation is that the continued synthesis of y-chains suppresses that of the 8-chains by some kind of "feedback" mechanism.
(c) /-thalassaemia intermedia. There are many cases which do not fit the clinical picture of ,f-thalassemia major or minor. These are called fl-thalassaemia intermedia. Hlb-F forms from 10-80% of ,total haemoglobin in the red cells, whilst Hb-A, is usually raised above normal and in some cases is above 6% (Kunkel and others, 1957;Josephson and others, 1958;Gabudza and others, 1963;Pearson, 1964).
The exaot genetic causation of these syndromes is not clear, but they may represent the interaction of two 8--thalassaemia genes, one of Which is of 'the severe type, causing (almost) complete suppression of P/-chain synthesis, whilst the other causes only partial suppression. Studies of the parents of these patients have shown that in some families both have thalassaemia trait with a raised Hb-A, (Pearson, 1964;Weatherall, 1965), whilst in others only one parent shows this type (Zuelzer and others, 1961;Wolf and Ignatov, 1963;Gabudza, Nathan and Gardner, 1964). interaction is the reduction in the number of A A-chains synthesized, leading to an alteration in the ratio of Hb-A to abnormal haemoglobin found when compared to that present in the simple heterozygous haemoglobinopathy. If a 3-'thalassaemia gene which completely suppresses /A-chain synthesis is inherited, no Hb-A will be found. With other genes, various proportions of Hb-A are found. The ratio of Hb-A ,to abnormal haemoglobin appears constant in any one family (Fessas, 1964).
The most common of these syndromes is sickle cell-thalassaemia disease. This disease has been recognized for a long time and is described in detail by Singer, Singer and Goldberg (1955 Weatherall, 1965)].
Hlb-A2 levels appear to distinguish between the two conditions, although the available data are very scanty. In homozygous sickle cell disease the Hb-A2 level is usually normal, whilst in sickle cell-thalassaemia it is usually raised, but some overlap does occur. In homozygous sickle cell disease the range for Hb-A2 reported is 1.7-4.6%, with 21 cases within the normal range of 1.5-3% and 2 above (Huisman and Dozy, 1962;Aksoy, 1963); in sickle cell-/B-thalassaemia 16 cases examined, range 2.4-7.0% (Henderson, Potts and Burgess, 1962;Aksoy, 1963;Monti and others, 1964). Other syndromes of this type are Hb-C-thalassaemia disease (Singer, Kraus, Singer, Rubenstein and Goldberg, 1954;Zuelzer and Kaplan, 1954), Hb-D-thalassaemia disease (Hynes and Lehmann, 1956), -Hb-E-lthalassaemia disease (Na-Nakorn and MinniCh, 1957; Kochhar and Kathpalia, 1963), and Hb-J-thalassaemia disease (Sydenstrycker, Horton, Payne and Huisman, 1961). Clinically, these are variants of /f-ithalassaemia of intermediate to mild severity. The diagnosis is made from the clinical picture and the electrophoretic analysis of the haemolysate. non-homologous crossing-over leading to the formation of the IHb-Lepore genes (from Baglioni, C. (1962), Proc. Nat. Acad. Sci. (Wash.), 48, 1880, with permission of the National Academy of Sciences, Washington). very similar to the true thalassaemias. Furthermore, this haemoglobin is found not infrequently in areas of the world where thalassaemia is commonly found. Haemoglobin Lepore was first described by Gerald and Diamond (1958) in an Italian family and has also been reported from Indonesia (Neeb, Beiboer, Jonxis, Kaars Sijpesteijn and Muller, 1961). Several other reports of a similar haemoglobin have appeared in the 'literature: "Hb-Pylos" was found in Greece (Fessas, Stamartoyannopoulos and Klaraklis, 1962) and "Hb-G"* in Ittaly (Silvestroni and Bianco, 1958), and these are probably other examples of this haemoglobin. Lepore haemoglobin migrates with Hb-S on starch block or gel electrophoresis.
Structural studies of these haemoglobins show that they are composed of itwo normal a-chains joined to 'two non-a-chains; ithese non-a-chains consist of the same number of amino acids as the Pf-chains, but ithe amino acid sequence is like that of the 8-chain at the N-terminal end and the B-chain at ithe C-terminal end (Baglioni, 1962); they have tlherefore been called /B-chains. In .haemoglobin Lepore Boston (B;agl,ioni, 1965) ithe 8$f-chain consists of 8-chain sequence from the N-terminal amino acid to ca amino acid no. 87 and of P-chain sequence from ca no. 116 to the C-terminal end. The amino acids ,in the region 88-115 could belong to either the 8or Pf-chain. In Hb-Lepore Hollandia amino acids 1-22 have the 8-chain sequence, 50-146 the f8-chain sequence and 23-49 could have arisen from either chain (Barnabas and Muller, 1962). Baglioni (1962) has discussed the genetic event that could have led Ito Ithe formation of a 8fi-polypeptide chain. Two possibilities are mentioned, both of which require the Hbp and Hba loci to be closely linked (see later). The first possibility is a deletion of part of -the  same length as the Por the 8-chain. The second, and apparently more likely, situation, which accounts for ithe joining of the 8 to the f, gene in such an exactly complementary way, is a non-homologous crossing over between corresponding points of the 8f and the 8 genes, resulting in the formation of unequal products, one of which is Hb-Lepore , (Figure 6).

Clinical Features
Hb-Lepore trait (Gerald and Diamond, 1958b;Neeb and others, 1961;Fessas and others, 1962). Individuals carrying this trait (have a blood picture similar to that found in thalassaemia trait. Starch gel or starch block electrophoresis reveals the abnormal haemoglobin, which amounts to 10-15% of total haemoglobin. 'Hb-A2 is within normal limits, and Hb-F may be normal or slightly raised. Homozygous Lepore disease. (Neeb and others, 1961;Fessas and others, 1962). Clinically, ithis condition is a severe type of thalassaemia intermedia. No Hb-A or Hb-A2 is found; IHb-Lepore amounts to 12% of total haemoglobin, the remainder being Hb-F.

Most of ,the haemoglobin in the red cells is
Hb-F; 'Hb-A2 is low or normal in amount and Hb-Lepore forms 5-10% of total haemoglobin. No HbIA is found.
(i7) The interaction of a-with fP-thalassaemia . Clinically, this disease is a form of thalassaemia minor. The diagnosis can only be established by family studies.
(iii) a-thalassaemia As the a-chains of Hb-A, Hb-F and Hb-A2 are identical, a-thalassaemia affects haemoglobin production both before birth as well as later in life, and it appears that Ithe effect is clinically more severe during foetal life than later.
(a) a-thalassaemia trait. Clinically, this condition is even milder than B-thalassaemia trait.
Presumably, ;this is because the remaining normal Hba gene is able to give almost full compensation. The clinical features of the trait have 4been investigated by Malamos and others (1962) and Weatherall 1(1964b). At (birth, these individuals carry 5-10% Hb-y4, and their red cells already show !the typical morphology of thalassaemia minor. During ithe neonatal period, this disappears, together with THb-F . Later in life, Ithe patient has slight anaemia with a blood film suggesting thalassaemia. The Hb-A2 level is normal. It is, therefore, clearly difficult to distinguish these cases from /8I-thalassaemia mentioned earlier or from mild iron deficiency. (The latter can be excluded by estimating the serum iron. Occasionally, iron deficiency and ,thalassaemia minor occur in the same patient. The blood picture in these is more severe than in the simple trait). The finding of Hb-?4 or Hb-y4 in any relatives helps in Ithe diagnosis. Malamos, Fessas and Stamatoyannopoulos (1962) have described the presence of occasional red cells with inclusion bodies after incubation with brilliant cresyl blue, presumably due to the presence of Hb-P/4. In a few patients, a trace of Hb-y4 is found in the haemolysate. The above description of a-thalassaemia trait presumably applies to a gene which causes complete suppression of a-chain synthesis, since in individuals in whom there is interaction of an a-'thalassaemia gene of this type with an a-chain mutation of haemoglobin no normal aA-chains are formed (Dormandy, Lock and Lehmann, 1961; Lie-Injo Luan Eng and Hart, 1963).
The situation in a-thalassaemia trait which only partially suppresses the synthesis of achains is not clear. In adults, this is probably undetectable. At 'birth, 'these may have a slight increase in Hb-y4, in the region of 1-2% of total haemoglobin. Such individuals are rare (<1%) in England but are more frequent (ca 12%) in individuals originating overseas (author's unpublished ,observations). One case has been studied in some detail. This infant was an offspring of a patient with Ib-H disease (Case 2, Dance, IHuehns and . Total haemoglobin in the cord blood was 17.1 g./100 ml., and the red cells showed considerable anisocytosis but no hypochromia; the serum bilirubin was 2.2 mg./100 ml. (b) Homozygous a-thalassaemia. This is due to the inheritance of two a-thalassaemia genes causing severe a-chain suppression (see above). No a-chains are synthesized, and death in utero apparently occurs at approximately 32 weeks' gestation. The foetus shows severe hydrops foetalis, anaemia, and erythroblastosis (Lie-Injo Luan Eng, 1962).
(c) a-thalassaemia intermedia or haemoglobin H disease. Haemoglobin H (Hb-,4) was first described in a Chinese family by Rigas, Osgood (1955, 1956) and in a Greek family by Gouttas, Fessas, Tsevrenis and Xefteri (1955). It was shown to consist of four p-chains by Jones, Schroeder, Balog and Vinograd (1959)  Hb-H disease suffer from a mild form of thalassaemia intermedia, with haemoglobin levels of 8-12 g./100 ml. There may be a rough correlation between the severity of the disease and the amount of Hb-/84 found in the red cells. In our own cases, the most affected (Case 2, Bingle, Huehns and Prankerd, 1958) had a haemoglobin of 10 g./100 ml. with 15% Hb-/4, whilst a recent patient (unpublished) had only 2% Hb-/4 with 12.5 g. haemoglobin/ 100 ml. The clue to the diagnosis of Hb-H disease is the finding of the characteristic inclusion bodies in the red cells on incubation with brilliant cresyl blue (Fig. 5b). Hb-H is best demonstrated using starch gel electrophoresis in phosphate buffer at pH 7.0 (Huehns, Flynn, Butler and Shooter, 1960) (Fig. 2b).

Recent work on Hb-H disease leaves little
doubt that it is caused solely by a deficiency in a-chain synthesis, and is therefore a form of a-thalassaemia*. The finding that Hb-H consists of four /A-chains (Jones and others, 1959;Jones and Schroeder, 1963) and that /A-chains obtained from Hb-A will also form Hb-H (Huehns, 1962;Huehns and others, 1965) indicates that in vivo Hb-/,8 is not due to a structural abnormality of its /3-chain or the presence of an abnormal enzyme causing the ./-chains to form Hb-/34. As the a-chains for the formation of the three normal haemoglobins are derived from a common metabolic pool (see , a-chain deficiency should lead not only to the formation of Hb-,8 but to the formation of Hb-y4 and Hb-8 in the same individual. This has been demonstrated in four patients with Hb-H disease (Dance and others, 1963). The finding that Hb-/34 occurs together with Hb-y4 has previously been reported in a number of patients (Ramot, Sheba, Fisher, Ager and Lehmann, 1959;Fessas, 1960;Huehns and others, 1960). The possibility that other patients with Hb-H disease may also carry Hb-8 is suggested by the low levels of Hb-A2 commonly found in the disease (Gerald and Diamond, 1958a;Ramot and others, 1959;Dittman, Haut, Wintrobe and Cartwright, 1960;Koler and Rigas, 1961;Dance and others, 1963), some 8-chains being used to form Hb-8 rather than Hb-A2.
Two other points need explanation. The first is the finding that Hb-y4 can occur without any Hb-/4, although plenty of /-chains are being *Hb-H also occurs in occasional patients with erythroleukaemia (Bergren and Sturgeon, 1960 (Choremis, Zannos-Mariolea, Ager and Lehmann, 1959). It appears that a-chains combine preferentially with ,8-chains to form Hb-A rather than with y-chains to form Hb-F (Huehns and others, 1960;Huehns, Beaven and Stevens, 1964). On this hypothesis, the relative amounts of Hb-y4 and 'Hb-/84 formed at a given level of a-chain deficiency depend solely on the number of y-chains available. Thus, when y-chains are available in quantity and 8-chains relatively scarce, as, for example, in foetal life and early infancy or in the case reported by Choremis and co-workers (1959), deficiency in a-chain formation could result in all the 8-chains being used up in the formation of Hb-A, while those y-chains left over after the formation of Hb-F would polymerize to produce Hb-y4. Secondly, if it is true that the formation of Hb-/84 and Hb-y4 are both due to diminished a-chain production, it would be expected that all carriers of Hb-y4 at birth would later develop Hb-/4. Follow-up studies have shown that this is not so Malamos and others, 1962;Weatherall, 1964b). The explanation for this is not at all clear but could be based on a falling off in the absolute rate of non-a-chain synthesis in the postnatal period (Huehns and others, 1960).
When the published pedigrees of families with Hb-H disease are studied, it appears likely that the disease is caused by the interaction of two genes (Huehns and others, 1960;Koler and Rigas, 1961). Huehns and others (1960) have suggested that these are two a-thalassaemia genes. The occurrence of the "severe" type of a-thalassaemia trait in some relatives is well documented. However, the work of Lie-Injo Luan Eng (1962) indicates that the inheritance of two such genes leads to intrauterine death.
It has therefore been suggested that Hb-H disease is caused by the interaction of a "severe" a-thalassaemia gene (causing complete suppression of a-chain synthesis) with a "mild" a-thalassaemia gene (causing partial suppression of a-chain synthesis) or an a-chain haemoglobin mutation (Huehns, 1962). Analysis of the published family data of Hb-H disease are consistent with this hypothesis (Huehns and Motulsky, unpublished;Wasi, Na-Nakor and Svingdamrong, 1964). As the incidence of the "severe" a-thalassaemia trait in Greece is about 0.4% (Malamos and others, 1962), the occurrence of families in which Hb-H appears in several generations (Gouttas and others, 1955;Bingle, Huehns and Prankerd, 1958) indicates that the postulated "mild" a-thalassaemia gene must be relatively common. The finding that copyright. on 24 July 2018 by guest. Protected by http://pmj.bmj.com/  slight increases of Hb-y4 occur relatively commonly in non-Caucasian cord bloods examined (see above) lends some support to this hypothesis. Further support would be obtained if all newborns from parents with Hb-H disease carried increased Hb-y4. Six such infants have been reported; five of these carried Hb-y4 (Fessas, 1960; Benesh, Ranney, Benesh and Smith, 1961;Huehns, unpublished); one infant did not show any abnormal haemoglobin (Gouttas, Tsevrenis and Papaspyrou, cited by Fessas, 1960). It is, however, not known whether the latter case was examined by sufficiently sensitive methods to detect the small increases postulated to occur in some of these. Another such infant did not show any Hb-y4 by paper electrophoresis at the age of five weeks (Brain and ).
An alternative genetic explanation is that the Hb-H disease results from the presence of an a-thalassaemia gene with a second gene (replacing the "partial" a-thalassaemia gene postulated above) which segregates independently Weatherall, 1965). In favour of this hypothesis is the finding that 7 out of 29 offspring from parents with Hb-H disease also had the disease and that one newborn from these parents did not carry Hb-y4.
(d) The interaction of a-thalassaemia with an a-chain variant of haemoglobin A. Hb-Q-athalassaemia disease clinically resembles Hb-H disease, except that instead of Hb-A, Hb-Q is found (Vella, Wells, Ager and Lehmann, 1958;Dormandy and others, 1961;Lie-Injo Luan Eng and Hart, 1963). Because no Hb-A is present in these cases, the a-thalassaemia would be of the "severe" type. One patient with Hb-I-athalassaemia disease has been described (Atwater, Schwartz, Erslev, Montgomery and Tocantins, 1960). Clinically, the picture was that of thalassaemia. The patient's haemolysate showed approximately 80% Hb-I and 20%/ Hb-A; Hb-A2 and Hb-I2 were also present in trace amounts (Atwater, Huehns and Shooter, 1961). In this case, the a-thalassaemia allowed the formation of significant amounts of Hb-A and was therefore of the "mild" type.
(e) The occurrence of a-thalassaemia with /,-chain variants of haemoglobin A. The occurrence of a-thalassaemia with Hb-E (Tuchinda, Rucknagel, Minnich, Boonyaprakos, Balankura and Savatee, 1964), Hb-C and Hb-S (Zuelzer, Neel and Robinson, 1956;Weatherall, 1964b) has been described. In each case, the relative proportion of abnormal haemoglobin to Hb-A was reduced as compared to that found in the simple trait. It is difficult to explain this interaction at the level of the gene because the a-thalassaemia locus is not linked to the Uf-chain structural locus (see . It is therefore possible that this is due to the preferential formation of Hb-A rather than the abnormal haemoglobin (Weatherall, 1964b). If this is so, then perhaps some Hb-f,, Hb-,8c or Hb-i3s respectively might be found in these cases. Although Hb-,fP and Hb-3S have already been described (Huisman, 1960) they were not found in the cases investigated by Weatherall, neither was Hb-PE by found Tuchinda and others (1964). The Significance of Haemoglobin F after the Postnatal Period The finding of Hb-F after the postnatal period is not always a sign of thalassaemia. It appears that it can persist in a number of haematological conditions which commence during the neonatal period. The commonest of these are the haemoglobinopathies, but it has also been reported in other congenital haemolytic anaemias , trisomy 13-15 (Huehns and others, 1964a) and some cases of Down's syndrome (trisomy 21-22) (Weinstein and Rucknagel, 1964). High levels are often found in acute leukaemia in young children. In adult patients, small increases in Hb-F are occasionally found in leukaemia (Beaven and others, 1961), aplastic anaemia (Jonxis, 1963), untreated pernicious anaemia (Beaven and others, 1961), molar pregnancy (Bromberg, Salzberger and Abrahamov, 1957) or during the second trimester of pregnancy (Rucknagel and Chernoff, 1955). Iron deficiency, acquired haemolytic anaemias and congenital heart disease are not associated with a raised Hb-F.

Hereditary Persistence of Foetal Haemoglobin
In this inherited condition there is depression of 8f-and S-chain synthesis without haematological abnormality, since there is good compensation by the continued production of Hb-F. In contrast to Hb-F in thalassaemia or sickle cell anaemia, Hb-F is evenly distributed in the cells (Thompson, Mitchener and Huisman, 1961;Bradley, Brawner and Conley, 1961;Fessas and Stamatoyannopoulos, 1964).
Several different types have been described.
(i) African type of Persistent Foetal Haemoglobin (Thompson and others, 1961). This trait occurs in approximately i% of Negroes. In the heterozygous form, 20-30% of haemoglobin is Hb-F, the remainder being Hb-A and Hb-A2. In the homozygous form (Wheeler and Krevans, 1961) only Hb-F is found. In neither condition is there any significant pathology. The interaction of this trait with p-thalassaemia is called persistent haemoglobin F-thalassaemia disease. These patients have the clinical picture of thalassaemia intermedia (Kraus, Koch and Burckett, 1961;Wheeler and Krevans, 1961; Barkhan and Adinolfi, 1962), Hb-F amounting to 65-70%, with a normal level of Hb-A2. The diagnosis can only be made by family studies.
This trait occurs in about 0.25% of Greeks.
In the heterozygous form, 11-18% of Hb-F is found in the red cells, the remainder being Hb-A and Hb-A2. There are no other haematological abnormalities. This trait has also been described in association with 8-thalassaemia, and is known as the Greek type of persistent Hb-F-thalassaemia disease. Clinically, it is a form of /-thalassaemia intermedia with 20-40% Hb-F and a raised Hb-A2.
(iii) A third type of persistence of Hb-F occurs in 1% of the population of Southern Switzerland (Marti, 1963). In this trait, Hb-F is slightly elevated (1-3%); there is no haematological abnormality.

Treatment of Thalassaemia
At present, there is no specific remedy for thalassaemia and the principal forms of treatment available are blood transfusions and splenectomy. As both these have serious disadvantages they should only be used for specific indications. Patients with relatively mild thalassaemia who stabilize with a haemoglobin level above 7.0 g./100 ml. should not receive any treatment except, of course, to tide them over intercurrent illness (Erlandson, Brilliant and Smith, 1964).
In the more severe cases, once it has been decided to transfuse a child regularly, a definite regime should be planned. In some clinics, the indication for transfusion is the appearance of symptoms in the child; in these cases, the haemoglobin may fall to a very low level and there appears to be general agreement that this is not the best criterion. Alternatively, transfusion is recommended whenever the haemoglobin falls to a selected level, and various workers have chosen different values between 7 and 10 g./100 ml. It can be shown theoretically that the amount of blood transfused per year is approximately the same regardless of the level of haemoglobin chosen. Therefore, there is no advantage to the patient, such as reducing the risk of transfusion siderosis, in choosing the lower level. There are, however, definite advantages to the patient in maintaining the higher level. The patient would feel much better and be able to exert greater physical and mental activity. It has already been shown that children treated in this way develop more rapidly than patients kept on lower haemoglobin levels (Erlandson and others, 1964;Schorr and Radel, 1964;Wolman, 1964). There is also evidence that the deposition of iron in the tissues is not as deleterious as once thought, provided the oxygen supply to the tissues is kept near normal (Wolman, 1964). With the maintenance of high haemoglobin levels, the absorption of iron from the gut might also be less than if anaemia is constantly present. Finally, if high haemoglobin levels are maintained by transfusion the great stimulus to marrow expansion present in disease will disappear, and it might be expected that the gross bone changes usually present will regress or not develop in the first place. In this connection it might be pointed out that fresh blood has a better survival than stored blood and by its use the transfusion requirement may be reduced by 10-20%.
Recently, chelating agents have been used in the treatment of iron overload in thalassaemia . Both desferrioxamine and diethylenetriaminepenta acetate (DTPA) were used. Smith suggests that the most effective way to remove iron from heavily iron-laden thalassaemic children is to combine large doses of DTPA at the time of the transfusions with daily injections of desferrioxamine between transfusions. However, the long term efficiency of these drugs in removing iron from the body has not yet been proved, and the possibility of toxic manifestations must be borne in mind (Fairbanks, Watson and Beutler, 1963). It must also be remembered that alimentary iron absorption continues in thalassaemia despite iron overload, and total iron uptake of the body would be reduced if this could be blocked.
Investigations on the effect of oral chelating agents on iron absorption suggest that these may only be marginally useful adjuncts to treatment (Bannerman, 1964). Splenectomy does not improve thalassaemia unless there is hypersplenism usually shown by an increased transfusion requirement or the appearance of thrombocytopenia (Smith, Erlandson, Stem and Schulman, 1960;Bouroncle and Doan, 1964). In these patients, it can be shown that there is considerable copyright. on 24 July 2018 by guest. Protected by http://pmj.bmj.com/ splenic destruction of donor blood with a consequent shortened red cell survival. Following splenectomy, the survival of donor cells often returns to normal. A second indication for splenectomy is gross splenic enlargement such as to cause mechanical interference in the patient's life, with constant discomfort.
In thalassaemia there is an increased demand for folic acid, and several patients with megaloblastic bone marrow changes have been reported. Usually, this is associated with pregnancy (Goldberg and Schwartz, 1954;Jandl and Greenberg, 1959) but may occur at other times (Chanarin, Dacie and Mollin, 1959).
There is also some evidence that folic acid deficiency may increase the anaemia of thalassaemia Luhby, Cooperman, Feldman, Ceraldo, Herrero and Marley, 1961). Such patients should clearly be treated with folic acid. The question arises whether there is subclinical folic acid deficiency in most cases of thalassaemia due to its high utilization by the increased cell division of the bone marrow. Some of the work on folic acid deficiency in sickle cell disease (Watson-Williams, 1965) may be relevant. This author reports four adult patients with normoblastic erythropoietic marrow, severe sexual immaturity and a very rapid plasma clearance of folic acid. All four showed startling improvement in their development on treatment with folic acid. In thalassaemia, the response to treatment has not been so dramatic, but several authors have reported a general improvement and a reduction in transfusion requirement (Luhby and others, 1961). It therefore seems reasonable to give supplements of folic acid to patients with thalassaemia, although startling response to this therapy will be rare. Iron therapy, particularly parenteral iron, should not be given in thalassaemia unless iron deficiency has been proved by estimation of the serum iron.
Treatment of Haemoglobin H Disease Since its discovery, Hb-H has been known to be less stable than normal haemoglobin. This leads to intraerythrocytic precipitation of haemoglobin, formation of red cell inclusion bodies (Fig. 5b) and the rapid destruction of the erythrocytes in the spleen at a red cell age of 40-45 days . The formation of these inclusion bodies and the consequent red cell destruction are considerably increased by oxidation to methaemoglobin. For this reason, the methaemoglobin-inducing drugs (for example, nitrites, sulphonamides and phenacetin) should be avoided in patients with Hb-H disease. Rigas and Koler (1961) have also shown that after splenectomy the red cell survival is significantly increased to near normal values, with concomitant decrease in anaemia.
This beneficial effect of splenectomy has also been reported by other workers (Gouttas and others, 1955;Lie-Injo Luan Eng and Hart, 1963;Woodrow, Noble and Martindale, 1964). Splenectomy should therefore be considered in the more severe cases of Hb-H disease with splenic enlargement, provided splenic destruction of red cells can be demonstrated.

The Distribution of Thalassaemia
The general distribution of thalassaemia in the world is shown in Fig. 7. Very few studies have differentiated between a-and 8thalassaemia. It appears, however, that both occur in the same regions, a-thalassaemia having been reported from Italy, Greece, Israel and other Mediterranean countries, South East Asia, and Africa (see Motulsky, 1964, for references). A general review of the distribution of thalassaemia is given by Allison (1961). (Further references are to be found in the work of the following authors: Lie-Injo Luan Eng, 1964;Flatz, Pik and Sringam, 1965;Malamos and others, 1962;Silvestroni and Bianco, 1963;Motulsky, 1964;Weatherall, 1965).
It is difficult to see why thalassaemia, which, in the homozygous form, is fatal in early life, should be common in such a large part of the population. Haldane (1949) suggested that death from the homozygous disease might be balanced by protection against death from malaria by the trait form (heterosis). A great deal of research to support this idea has been carried out. Comparison of the overall distribution of malaria with that of thalassaemia in the world shows quite good correspondence.
However, when the detailed distributions in one country are compared, there are some very serious discrepancies. The details of this fascinating aspect of the thalassaemia story have recently been discussed by Motulsky (1964b).
From the above description, it may appear that thalassaemia only occurs in the areas shown on the map and in individuals originating from them. Recent work has shown that -both a-thalassaemia and p-thalassaemia may rarely be found in British or other Northern European individuals (Callender, Mallet and Lehmann, 1961;Buchanan, Kinloch, Hutchison, Pinkerton and   The loci controlling the structure of the a-and 8-chains (Hba locus and Hbp locus) are not linked. In contrast to this, the Hba locus is closely linked to the Hbp locus, no crossovers occurring in at least 45 possibilities.
It is also thought that the Hby locus is linked to the Hbp + Hba complex because the mutation causing persistence of foetal haemoglobin is closely linked or allelic to it, no crossovers occurring in 44 possible instances (for references see . Motulsky (1964a) has recently collected the published evidence for linkage between the Hb, locus and the l-thalassaemia locus.
Analysis of the offspring of matings of heterozygotes for both fl-thalassaemia and a fl-chain structural mutant showed that among 80 offspring there were two possible, but not proved, crossovers. These findings indicate that the f-thalassaemia genes are closely linked (or allelic, if the possible crossovers are disproved) to the Hbp structural locus. The close linkage between Hb-and Hba loci mentioned above implies that fl-thalassaemia is also linked to the latter. There are only relatively few families which throw light on this linkage (Huisman, Punt and Schaad, 1961; Moore and Pearson, 1964), the latter authors reporting one definite crossover. The finding of one crossover (out of 19 possibilities) between the Hb locus and fl-thalassaemia while there are none (out of 45 possibilities) between the Hbp and Hbs loci suggests that the Hba locus is further from the ,8-thalassaemia locus than the Hbp locus, i.e. that the /-thalassaemia locus is separate from the Hbp structural locus. Clearly, more family studies are required to resolve this point. The a-thalassaemia locus is not linked to the Hbp locus or the P--thalassaemia locus (Cohen, Zuelzer, Neel and Robinson, 1959;Tuchinda and others, 1964), and this is consistent with the idea that the a-thalassaemia locus is linked to the Hbo locus. However, there are no studies as yet to resolve this point. The biochemical interactions between a-thalassaemia and a-chain mutants of haemoglobin are similar to those 'between S-thalassaemia and the various /-chain haemoglobin variants, and this suggests that the genetical situation might be analogous to that for 8/-thalassaemia.
One of the curious features of the thalassaemia genes is that they have not been found in coupling with a haemoglobin mutant. In those cases where the thalassaemia gene completely suppresses the synthesis of the haemoglobin chain concerned there is, of course, no way to determine the nature of the suppressed chain. However, it would be copyright. on 24 July 2018 by guest. Protected by http://pmj.bmj.com/ expected that in some cases the mutations for an abnormal haemoglobin and thalassaemia would occur on the same chromosome. In these, the proportion of the variant haemoglobin would be less than in the simple abnormal haemoglobin heterozygote, and both genes would be inherited together. The lack of these cases supports the close linkage between the haemoglobin structural loci and the thalassaemia genes.
(ii) The Genetic Abnormality in Thalassaemia At present, the specific abnormality in thalassaemia is unknown. However, it is useful to consider the different possibilities so that experiments may be devised to test them.
The theories on the causation of thalassaemia fall into several groups. The first of these is a mutation of the corresponding Hb structural locus. Ingram and Stretton (1959) suggested that one cause of thalassaemia might be a hidden amino acid substitution, that is, one that did not result in a difference in electrophoretic mobility of any "haemoglobin A" still being made. This hypothesis could be tested by examining the Hb-A found in cases of Hb-S-/,thalassaemia disease (or other thalassaemia/ haemoglobin mutant interactions). Guidotti (unpublished, quoted by Baglioni, 1963) has examined the amino acid composition of the isolated peptides from two such samples of "Hb-A" and found them to be identical with that of the corresponding peptides from Hb-A obtained from a normal adult. Although these findings are strong evidence against this hypothesis, they do not altogether exclude it. Recently, Itano (1965) has advanced another theory. This idea depends on the degeneracy of the genetic code, that is, the ability of more than one "triplet code word" to code for the same amino acid (see Stretton, 1965). In normal persons, the normal triplet code word is present, but in thalassaemia this is replaced by another which codes for the same amino acid. A hypothetical example would be the change from GGU to GGA, both coding for glycine. One of the factors that limits the rate of addition of any particular amino acid to the growing end of the polypeptide chain during synthesis is the availability of amino acid transfer-RNA fitting any particular coding triplet, and it is argued that there is plenty of transfer-RNA to fit the normal coding triplet but a deficiency of transfer-RNA for the abnormal triplet. This deficiency of availability of an amino acid leads to a block or hold-up at a particular point in the synthesis of the affected polypeptide chain and the decreased normal globin production seen in thalassaemia. Ingram (1964) has suggested that in thalassaemia the messenger RNA for the affected chain is abnormal and blocks some of the ribosomal sites of protein synthesis so that they are not available for haemoglobin synthesis. Another possibility is that the abnormal messenger RNA formed is less stable than normal mRNA. Each mRNA would then only be available for the synthesis of a smaller number of polypeptide chains than its normal counterpart.
Recent work on the "amber" and "ochre" mutants of certain phage types has shown that some mutations lead to the release of partially completed polypeptide chains (see Stretton, 1965). It is possible that in thalassaemia similar mutations occur.
The above hypotheses are mutations of either the Hba or Hb p structural loci, and proof that the corresponding thalassaemia loci are distinct from these would, of course, rule them out.
The discovery that haemoglobin Lepore (which closely resembles /-thalassaemia in its clinical haematological features) consists of the N-terminal part of the 8-chain joined to the C-terminal part of the P-chain and is probably due to an unequal crossover (Baglioni, 1962(Baglioni, , 1965 has led to the speculation that this type of mutation may account for other cases of thalassaemia (Nance, 1963;Smithies, 1964).
Although it seems unlikely that this hypothesis is of general application, one situation deserves further discussion. When the amino acid sequences of Hb-A and Hb-A2 are compared (see , it can be seen that if the crossover occurs in the first 24 nucleotide pairs (i.e. 8 amino acids), the resulting chain will have the same amino acid sequence as Hb-A. This would appear to fit very well to the finding of Guidotti mentioned above. However, it has to be remembered that during the crossover the Hbs loci are in effect deleted, and heterozygotes should have a low (or normal) Hb-A2, as is found in Hb-Lepore trait (Gerald and Diamond, 1958b). Thus, the crossover hypothesis would not fit the high A2 /-thalassaemia but only those cases with a normal Hb-A2 (the /38-thalassaemia of Fessas, 1964). If the crossover occurred a little further along the gene, the "Hb-A" formed would contain one or two "silent" amino acid substitutions (at positions 9 and 12), allowing this hypothesis to be tested. If the crossover included position 22 of the 8-chain, of course the electrophoretically separable Hb-Lepore would result. Finally, it has been suggested that thalassaemia is due to a mutation of a closely linked ratecontrolling locus. This hypothesis would, of course, be favoured if a definite crossover between the Hbp (or Hb,) locus-and / (or a-)thalassaemia were discovered. Such a ratecontrolling locus has been postulated in the control of bacterial protein synthesis, the operator locus of Jacob and Monod (1961). Although there is no evidence for such regulatory loci in mammals, a number of workers have discussed this concept in relation to thalassaemia (Neel, 1961;Motulsky, 1962;Sturgeon, Schroeder, Jones and Bergren, 1963;Zuckerkandl, 1964). However, these hypotheses do not explain all the findings in thalassaemia.
The multiplicity of hypotheses put forward to explain the findings in thalassaemia indicates that at the present time the data on which a sound theory can be based are not yet available. Finally, it has to be remembered that the thalassaemias are phenotypes which may be caused by different mutations in different individuals.