Review Article J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from

Journal of Medical Genetics (1973). 10, 50.

Fetal and Embryonic Haemoglobins P. A. LORKIN MRC Abnormal Haemoglobin Unit, University Department of Biochemistry, Cambridge

Haemoglobin has been the subject of intensive form a nearly spherical molecule with extensive research for many years and is one of the most areas of contact between unlike chains; the two thoroughly understood of all protein molecules. main types of contact are denoted alp, and alg2 The amino-acid sequences of haemoglobins from The tetramer exhibits cooperative behaviour or many species of animals have been determined haem-haem interaction. As each haem combines (tabulated by Dayhoff, 1969) and the molecular with oxygen the affinity of successive haems in- structures of horse and human haemoglobins have creases. The oxygen affinity curve of the tetramer been determined in great detail by x-ray crystallo- is sigmoidal and may be represented approximately graphy (Perutz et al, 1968a and b; Perutz 1969). A by the Hill equation:* mechanism of action of haemoglobin has been pro- = kpo2n posed (Perutz, 1970a and b and 1972). The y haemoglobins of higher organisms share a common +kpo2n tetrameric structure built up of two pairs of unlike Oxygen affinity data are usually presented in copyright. chains; the a chains containing 141 amino-acid terms of P102, the partial pressure of oxygen re- residues and the non-a chains containing generally quired to attain half saturation with oxygen, and of 145 or 146 amino acids. In man, five types of n, the exponent of the Hill equation. These para- non-a chains have been recognized, /3, y, 8, E. and 4, meters respectively give measures of the oxygen corresponding to different stages of ontogenesis. affinity and of the degree of haem-haem interaction. The globin chains ofhaemoglobin and of myoglobin For normal haemoglobin n is approximately 3. have a similar overall tertiary structure composed of Myoglobin, isolated chains, and tetramers composed http://jmg.bmj.com/ segments of a helix interspersed with non-helical of one type of chain such as Hb H (4) show no regions forming a compact structure of great com- haem-haem interaction, their oxygen dissociation plexity. Amino-acid residues whose side chains curves are hyperbolic and the n value is 1. are of a polar nature, ie, those which bear an electric The haemoglobin tetramer has a lower oxygen charge or a strong dipole movement are located at affinity, more appropriate of its physiological func- the outer surface of the molecule in contact with the tion, than single chains. The cooperative behaviour whereas the of the tetramer renders it more efficient in releasing aqueous surroundings, non-polar on September 23, 2021 by guest. Protected hydrocarbon side chains are, in general, located oxygen in response to relatively small changes in within the interior of the molecule. The haem oxygen tension. The tetramer exhibits other groups are accommodated within deep cavities or physiologically advantageous properties; the oxygen pockets lined with non-polar side chains and are affinity varies with pH (Bohr effect) in such a manner anchored in position by links between the haem iron as to facilitate the release of oxygen in the tissues. and the imidazole side chains of histidines. The Haemoglobin binds CO2 at the N terminus of the non-aqueous environment of the haem promotes / chains (Kilmartin and Rossi-Bernardi, 1969) and reversible binding of oxygen and protects the iron assists its transport from the tissues to the lungs. from oxidation to the ferric form characteristic of Furthermore its oxygen affinity is reduced in methaemoglobin which is incapable of reversible the presence of organic phosphates particularly oxygen binding. 2,3 diphosphoglycerate (2,3 DPG) and inositol The four chains of the tetramer fit together to * Hill equation: y =fraction of haemoglobin in oxy form, P02 Received 27 November 1972. partial pressure of oxygen, k and n are constants. 50 Fetal and Embryonic Haemoglobins 51 J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from hexaphosphate (IHP) (Benesch and Benesch, 1969). postulated that a gradual divergence of the structure This provides an important method of regulating of the polypeptide chains led to their ability to the oxygen affinity in vivo. The properties can be associate with each other to form dimers and tetra- accounted for in terms of the molecular structure mers and the appearance of haem-haem interaction (Perutz, 1970a and b). and the Bohr effect. It seems likely that the Many species of animals contain multiple haemo- 'ancestral molecule' was probably longer than globins which can be separated by electrophoresis. modern globin chains because of the need to postu- The components are often present in unequal pro- late deletions and the fact that the globin chains of portions eg, in man Hb A and Hb A2 are present in more primitive animals eg, lamprey are longer than the ratio of about 40: 1. Some of the components those of higher animals. It is a matter of contro- may be chemically modified forms of other com- versy whether the differences in amino-acid se- ponents; for example the minor component Hb A,, quences of haemoglobin chains of different animals of human blood consists of Hb A modified by may be attributed to the action of Darwinian natural attachment of a carbohydrate residue at the N selection or to the action of genetic drift and the terminus of the chain (Bookchin and Gallop, fixation in different species of amino-acid substitu- 1968). Other multiple components represent the tions which cause no significant change in the pro- products of separate gene loci and contain globin perties of the haemoglobin and are selectively chains which differ by one or more amino-acid neutral (review by Harris, 1971). Some parts of substitutions, thus the : and 8 chains ofhuman Hb A the amino-acid sequence of the globin chain show and Hb A2 differ by 10 amino-acid substitutions. much less interspecies variability than others and Fingerprinting has revealed differences between some residues are invariant in all species. In the haemoglobins of different species (Muller, 1961) general, the amino acids whose side chains form the and in many cases the exact amino-acid sequences lining of the haem pocket and the surfaces of the have been determined (Dayhoff, 1969). In general ail92 interface show very little variability and any each species has one or more haemoglobin compon- substitutions are of a conservative nature ie, one ents unique to itself. The more closely related two amino acid is replaced by another of a similar charac- species are, the more similar are their haemoglobins. ter. In contrast, amino acids located at the outer In man and chimpanzee the major adult haemo- surface of the molecule show wide variability. copyright. globin Hb A is identical and the minor component In studying the haemoglobins of animals it is Hb A2 differs by only one amino-acid substitution practicable to examine the specimens from only a (de Jong, 1971). In various species of the genus small number of animals. The situation is different Equus haemoglobin components differ by one with the human population where blood samples amino-acid substitution (Kitchen and Easley, 1969). from many people have been examined by electro- When the amino-acid sequences of the globin chains phoresis during the course of routine haematological http://jmg.bmj.com/ of haemoglobins and myoglobins from many species examinations and surveys. are compared they show a great degree of homology These studies have given a measure of the degree if gaps are introduced at the appropriate positions ofintraspecic variation ofhaemoglobin in the human (Braunitzer et al, 1964). population and have led to the discovery of over 160 Such comparisons of globins and other proteins, different mutant forms ofhaemoglobin (reviewed by especially cytochrome c (Dayhoff, 1969), have pro- Huehns and Shooter, 1965; Lehmann and Carrell, vided a novel basis for the construction of phylo- 1969; Lehmann, 1972). The study of human genetic trees based on data quite independent of haemoglobin mutants has afforded much valuable on September 23, 2021 by guest. Protected conventional zoological characteristics. Further- information. Studies of the inheritance of abnor- more these comparisons have lent support to the mal haemoglobins showed that the a and f chains speculation that the haemoglobin and myoglobin were specified by separate gene loci, and this is also chains may themselves have diverged and evolved true for y and 8 chains and presumably also for the from a more primitive 'ancestral molecule'. The E and 4 chains. Some recent evidence (discussed general manner in which this may have come about below) suggests that, at least in some individuals, has been outlined by Ingram (1961), who postulated the a-chain locus is duplicated. The y-chain locus that an initial single gene locus specifying a globin is also duplicated and the polypeptide chains speci- chain may have undergone duplication followed by a fied by different loci differ by at least one amino- gradual divergence of the two genes by independent acid substitution (Schroeder et al, 1968). mutations and deletions. Successive duplications The study of rare pathological haemoglobins in may have given rise to the genes for the families of which the physiological functions are impaired has polypeptide chains observed in each species. It is played an important role in the understanding of 52 P. A. Lorkin J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from the normal functioning of haemoglobin (Perutz and at 12 months (summarized by Huehns and Beaven, Lehmann, 1968). Many of these haemoglobins 1971). It has been suggested that these different have mutations at sites which are normally in- haemoglobins represent adaptions to different modes variant. of obtaining oxygen (Huehns and Beaven, 1971). Despite great advances in our understanding of In the early embryo, oxygen is obtained from the the structure and function of haemoglobins many maternal interstitial fluid, in the developing fetus it problems remain to be solved. The factors which is obtained via the placenta, and after birth via the regulate the rate of production of a and non-a lungs. It is, however, possible for a fetus to survive chains and ensure that they are produced in equiva- with adult blood after an exchange transfusion lent amounts are not understood. In the thalas- (Dawes, 1967), and for certain rare adult individuals saemias there is an imbalance of chain production to survive although their red cells contain only the which results in the formation of one or other type of fetal type of haemoglobin. chain in excess with consequent disturbance of function (Weatherall and Clegg, 1972). The exact Embryonic Haemoglobins. The first definite nature of the lesion in the thalassaemic is not yet evidence for the existence of distinct embryonic known (Weatherall, 1968). It is not known why haemoglobins in man was reported by Huehns et al multiple haemoglobin components which are the (1961) who found two new haemoglobins in a small products of separate genetic loci are not necessarily embryo of 3-5 cm crown-rump length (CRL) from a produced in equal amounts (eg, Hb A and Hb A2) pregnancy of 9 weeks duration. No haemoglobins nor why there are changes in the proportions of different from Hb A and Hb F were found in 25 different components during development or under embryos from pregnancies of 10 weeks or more. conditions of stress. The discovery that haemo- The two haemoglobins, which were called Hb globin messenger RNA is longer than is required to Gower 1 and Hb Gower 2, migrated much more code for the structure protein and contains addi- slowly than Hb A on starch gel electrophoresis at tional sequences relatively rich in adenylic acid pH 8-6; Hb Gower 1 was the faster, migrating just (Lim and Canellakis, 1970) may have some bearing behind Hb A2. In a further study of haemoglobins on these questions. Possibly the untranslated from 12 embryos Huehns et al (1964b and c) found regions may be involved in the regulation of protein Hbs Gower 1 and 2 to be present in embryos of CRL copyright. synthesis. up to 6 cm; only traces of Hb Gower 1 were present Another fascinating problem is that ofthe changes in embryos up to 8-5 cm CRL and none was detec- of haemoglobin type which occur during fetal and ted in larger embryos. In the smallest embryo embryonic development and the changes which examined, CRL 2-5 cm, there was 24% Hb Gower 1 occur in some adult animals, in particular sheep and and 13o% Hb Gower 2. Small amounts of Hb goats, on exposure to the stress of anaemia. Al- Gower 2 were found in one out of 300 cord blood though the haemoglobin types involved have been samples. Other workers have reported similar http://jmg.bmj.com/ partly characterized in some cases, very little is findings, eg, Horton et al (1962) and Schneider et al known of the factors which control these develop- (1970) who found one case ofHb Gower 2 in a survey mental changes. An understanding of these factors of over 11,000 cord blood samples. Hb Gower 2 would be of both fundamental, theoretical, and also has also been found in small amounts (0-1-0-7%) of practical interest as a possible basis of therapy in red cells of newborns with the D1 trisomy abnor- for some haemoglobinopathies. mality. Examination of Hb Gower 2 by finger-

printing (Huehns et al, 1964c) indicated that it is on September 23, 2021 by guest. Protected Fetal and Embryonic Haemoglobins composed of a chains and another type of chain, Ontogeny ofHaemoglobin. In several species denoted E, which differs from both the : and y of animals it has been observed that there is a change chains. Hb Gower 2 probably has the structure in the type of haemoglobin during embryological a2E2. Hybridization studies indicated that the Hb and fetal development. In man, early embryos Gower 1 probably consists entirely of E chains ie, contain two embryonic haemoglobins; these are re- HbE4. The E chains must be considerably more placed successively by fetal haemoglobin Hb Fa2 basic than the fl or y chains as Hb Gower 2 differs y2 and finally by the adult haemoglobins Hb A and from Hb A by about 6 units of positive charge. Hb A2 beginning before birth (Huehns and Shooter, Other mammals show a similar development of 1965). The amount of Hb F present at birth is specific embryonic and fetal haemoglobins. Klei- generally about 70-90% of the total haemoglobin. hauer, Brauchle, and Brandt (1966) examined This declines during the first 12 months after birth; haemoglobins from embryos of Simmentaler cattle less than 8 % is present at 6 months and less than 2 % of 0-5-6-0 cm CRL. The haemoglobins were Fetal and Embryonic Haemoglobins 53 J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from separated by starch gel electrophoresis. Embryos from Hb A, it shows a fine structure band at 289-8 of 3 0-7 0 CRL had fetal haemoglobin and two nm which is absent from Hb A (Beaven, Hoch, and embryonic haemoglobins which migrated slowly Hobday, 1951); the difference is due to the higher towards the anode on electrophoresis at pH 8-6. content of tryptophan in Hb F. Both glycine and Smaller embryos (1-01-5 cm) sometimes had three valine are present as N terminal amino-acid residues which were denoted 1,2, and 3 in order of decreasing in Hb F; only valine is found at the N terminus of anodal mobility; fraction 1 had the same mobility as Hb A; isoleucine is present in Hb F and absent human Hb A2. No fetal haemoglobin was de- from Hb A. tected in the smallest embryo (0-5 cm) which con- tained only fractions 2 (900,%) and 3 (10%). In all Structure of Hb F. Haemoglobin F is a cases fraction 2 was present in greater amounts than tetrameric molecule composed of a pair of a chains fractions 1 and 3. Embryos of 3 0 cm and longer identical to those in Hb A and Hb A2 (Schroeder had only fractions 1 and 2. In embryos of 6-0-7-0 et al, 1963b) and a pair of y chains. The y chains cm the embryonic haemoglobins amounted to only contain 146 amino acids with the sequence shown 2% of the total haemoglobin and only traces were in Fig. 1; this differs from that of the :l chains in 39 detected in larger embryos. Kleihauer and Stoffler positions as indicated (Schroeder et al, 1963a). (1968) detected two haemoglobin fractions analogous Although the three-dimensional structure of to Hbs Gower 1 and 2 in early embryos of cattle, Hb F has not been determined in detail it is likely sheep, and pig; these fractions were gradually re- that it is similar to that of adult haemoglobin. placed by fetal haemoglobins. Embryonic haemo- Approximately two thirds of the amino-acid sub- globins have been found in chickens (Manwell et al, stitutions in the y chain are on the external surface 1963) and ducks (Borgese and Bertles, 1965). and are likely to have relatively little effect on the Changes in haemoglobins have also been observed physiological properties of the molecule. There during the development of the adult frog R. are nine changes of internal residues including the catesbiana from the tadpole (Moss and Ingram, exchange of tyrosine 130 for tryptophan which is 1968a and b). responsible for the different ultra-violet spectrum of It is not certain whether Hb A is present in early Hb F. These substitutions are mainly of a con- human embryos. A protein which migrated in the servative nature in which one amino acid is replaced copyright. position of Hb A at pH 8-6 was found to have dif- by another of a similar type. Four substitutions ferent mobility from Hb A at pH 6-2 (Kaltsoya, occur at the a,/3, interface. These may account for Fessas, and Stavropoulos, 1966). This compo- the reduced tendency of Hb F to dissociate into nent may be identical with Hb Portland 1 (Capp, monomers which is the probable cause of its re- Rigas, and Jones, 1967) which is thought to be a sistance to alkaline denaturation (Baglioni, 1963;

tetramer composed of y chains and a fifth type of Huehns et al, 1964a). The residues at the a1/32 http://jmg.bmj.com/ chain called the 4 chain ie, Y2A2 (Capp, Rigas, and interface are unchanged. Jones, 1970). The 4 chain has been found in cord An important difference between Hb F and Hb A bloods from several normal and abnormal infants arises from the substitution of histidine / 143 by (Todd et al, 1970). serine in the y chain. Perutz (1970a and b) has suggested that 2,3, DPG binds to deoxy haemo- Fetal Haemoglobin. In 1866, Korber dis- globin in a cavity between the f/ chains lined with covered that blood obtained from the human pla- positively charged groups, the a amino groups of the centa was more resistant to denaturation by acids N terminal residues, the E amino group of lysine 82, on September 23, 2021 by guest. Protected and alkalis than normal blood. This discovery was and the imidazole group of histidine 143; this has the first indication that there was a difference be- recently been confirmed by x-ray crystallography tween the haemoglobins of adults and of fetuses and (Arnone, 1972). DPG stabilizes the deoxy form of newborns. The early history of this work has been haemoglobin which results in a reduced oxygen reviewed by Bertles (1969). Human fetal haemo- affinity. In Hb F the replacement of histidine by globin Hb F differs from adult haemoglobin Hb A serine removes two positively charged residues from in several physical properties. It is much more the cavity resulting in DPG being less strongly resistant to denaturation in alkaline solution and this bound. Consequently the oxygen affinity of Hb F property provides the simplest way of detecting and is not reduced by DPG so much as that of Hb A. assaying it (Singer, Chernoff, and Singer, 1951). Hb F and Hb A can be distinguished immuno- Haemoglobin F Variants. Extensive surveys logically (Darrow, Nowakovsky, and Austin, 1940). of thousands of cord blood samples have been car- Hb F has a slightly different ultra-violet spectrum ried out and a number of fetal haemoglobin variants J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from 54 P. A. Lorkin have been found. Both a- and y- chain variants have (Na-Nakorn et al, 1969). In the homozygous con- been detected. When the mutation is in the a chain dition there is complete suppression of a-chain syn- an abnormal haemoglobin is also present during thesis and the fetal cells contain only Hb Barts. adult life, but when the mutation is in the y chain an Death in utero results from the failure of the haemo- abnormal haemoglobin is present only during fetal globin to release its oxygen (Lie Injo, 1962). life and for a few months after birth. The known y- chain variants are listed in Table I; they all appear Fetal Haemoglobins in Other Animals. Many to be harmless. In some cases the amino acid species of mammals and other animals have been present at position y 136 has also been identified. examined for the presence of fetal haemoglobins. It has been found that resistance to alkaline denatura- TABLE I tion is not a universal property of fetal haemoglobin. FETAL HAEMOGLOBIN MUTANTS Beaven and Gratzer (1959) detected a fetal haemo- globin in the rhesus monkey, Macaca mulatta, by Name Substitution 136 Reference agar gel electrophoresis. The fetal haemoglobin F Texas I 5 A2 Glu Lys Ala Jenkins et al (1967), migrated more slowly than the adult haemoglobin Ahern et al (1972) in this system, the order of migration being the F Texas II 6 A3 Glu Lys ? Larkin et al (1968) opposite to that in humans. M. mulatta fetal F Alexandra 12 A9 Thr Lys ? Loukopoulos et al (1969) haemoglobin showed a tryptophan fine structure F Jamaica 61 E5 Lys Glu Ala Ahern et al (1970) F Malta, 117 G19 His Arg Gly Cauchi et al (1969) band at 289-7 nm and was resistant to alkaline de- F Malta2 ? Huisman et al naturation. Sen, Das, and Aikat (1960) measured (1972) F Hull 121 GH4 Glu Lys (Ala)* Sacker et at (1967) the rate of disappearance of alkali resistant haemo- F Houston ? Glu Ala ? Schneider et al (1966) globin in infant M. mulatta. The amount of alkali F Roma ? Silvestroni and resistant haemoglobin averaged 48-90% in four new Bianco (1963) F Warren ? ? Huisman et al born monkeys and fell to the adult level (average (1965) F Dickenson 97 FG4 His Arg ? Schneider et al 2-9% in 60-90 days; only one component was de- (1970) tected in both adult and fetal haemoglobin. Other F Kuala 22 B4 Asp Gly Ala Lie Injo et al Lumpur (1973) species of Macaca show interesting differences. Kitchen, Eaton, and Stenger (1968) examined copyright. * Ahern et al (1973). haemoglobins from fetal and adult Macaca speciosa (stump-tailed macaque) by starch gel electro- Haemoglobin Barts Y4. Haemoglobin Barts phoresis and peptide mapping. Two adult and (Ager and Lehmann, 1968) is a tetramer composed two fetal haemoglobin components were detected. of four normal y chains and is analogous to Hb H In adults the electrophoretically faster component the and the slower com- (f4). On electrophoresis at pH 8-6 it migrates more represented 60-65 % of total http://jmg.bmj.com/ rapidly towards the anode than Hb A and for this ponent 35-40% although this ratio was reversed in reason was formerly called 'fast fetal haemoglobin' some animals. The two components differed in (Fessas and Papaspyrou, 1957). It may be iso- their a chains, a difference in peptide a Tp 3 was lated by chromatography on IRC-50 ion exchange detected by peptide mapping. No embryonic resin. Hb Barts shows a more well resolved trypto- haemoglobins were detected, but embryos younger phan fine structure band at 289-6 nm than Hb F; it than 47 days gestation were not examined. is more resistant to alkaline denaturation than Hb A In contrast to the situation in M. speciosa, Nute but less so than Hb F (Dance, Huehns, and and Stamatoyannopoulos (1971) found evidence for on September 23, 2021 by guest. Protected Beaven, 1963). Haemoglobin Barts has a very high two types of chain in Macaca nemestrina (pig-tailed oxygen affinity and shows no haem-haem interaction macaque). They examined haemolysates from or Bohr effect (Horton et al, 1962); it is physiologi- fetal and infant macaques ranging from 60 days cally useless haemoglobin as it is unable to release gestation to 86 days after birth (gestation period 170 its oxygen to tissues. days). Haemolysates from fetuses contained either Small amounts of Hb Barts have been detected in two (in the younger ones) or three components. normal cord bloods by starch gel electrophoresis The third component had the same electro- and staining for haem protein with benzidine phoretic mobility as the single band found in adult (Fessas and Mastrokalos, 1959); it is also found in animals. The two fetal haemoglobins, F slow and early fetal and embryonic life. Increased amounts F fast, were present in the ratio of approximately of Hb Barts are found in the cord bloods of indi- 2: 1. The proportions in seven fetuses between viduals suffering from thalassaemia. In the al 132 and 167 days gestation estimated by DEAE thalassaemia trait there is about 5-10% Hb Barts Sephadex chromatography were F slow 63-68%, J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from Fetal and Embryonic Haemoglobins 55 F fast 30-35%', and A 0-5°O. Fingerprinting of lation is composed of nucleated cells probably the isolated a and y chains indicated that the a derived from the yolk sac blood islands; the second chains were probably identical but that there were population is composed of smaller non-nucleated several differences between the y chains. The y cells from the fetal liver and developing bone chain of the fast component appeared to have a marrow (Craig and Russell, 1964). The first lower content of tryptophan. population contained at least three haem proteins It is possible that M. speciosa may contain two and the second contained a single haemoglobin types of y chain which are electrophoretically indis- different from those in the first. The haemoglobins tinguishable as is the case in man and also in the (EI, EII, and EIII) from the yolk sac cells were chimpanzee (de Jong, 1971). In view of the dis- separated into their constituent chains which were covery that the ratio of the two types of y chains in of five types denoted x, y, z, a, and / (Fantoni, Bank, humans changes after birth, it would be interesting and Marks, 1967) Hb EI contained x and y chains; to know whether this also occurs in M. tnemestrina; EII contained a and y chains, and Hb EIII contained technically this would be easier because it is possible x and z chains. The haemoglobin from the liver- to separate the two components by electrophoresis. derived cells was indistinguishable from the adult Alkali resistance is not a property specific to fetal haemoglobin composed of a and : chains. The haemoglobins. Buettner-Janusch and Twichell Hbs EII and EBII differed only in the nature of their (1961) examined several species of prosimian pri- non-a chains. It was pointed out that one of the mates for alkali resistant haemoglobins. High embryonic chains might differ from another only levels of alkali resistant haemoglobins (40-80°O) by the presence of an N terminal acetyl group, as the were found in adult individuals from several species case in human Hb F (Schroeder et al, 1962) and in of Lemuriformes and Lorisiformes (10-600o). bullfrog haemoglobin (de Witt and Ingram, 1967). Adults from several species of anthropoid apes Possibly the two erythroid cell lines may develop showed, in general, low levels of alkali resistant from different precursor cell lines in the respective haemoglobins. High levels of alkali resistant tissues or alternatively the erythroid cells of the yolk haemoglobin were found in infant and fetal Macaca sac blood islands may seed the developing liver and mulatta and in infant Cebus apella. change the types of haemoglobin produced in re- Fetal haemoglobins have been detected in some sponse to the new environment. copyright. common domestic mammals and other animals. Kleihauer et al (1966) and Kleihauer and St6ffler (1968) demonstrated that cattle, sheep, and pig Detection and Isolation of Fetal Haemo- embryos all go through a type of development simi- globin. The most common method of detecting lar to human embryos first producing two embryo- and estimating Hb F is based on its resistance to

nic haemoglobins analogous to human Hbs Gower 1 alkaline denaturation. In the method devised by http://jmg.bmj.com/ and 2 which are later replaced by a fetal fraction and Singer et al (1951) a dilute haemolysate is exposed finally by the adult form. The amino-acid sequence to NaOH solution at pH 12-7 for 1 minute, and the of the y chain of bovine fetal haemoglobin has been solution is then neutralized. Oxyhaemoglobin A is determined (Babin et al, 1963). It contains 145 denatured by this treatment and is precipitated with amino acids, one fewer than in human y chain. It ammonium sulphate; Hb F remains in solution and has N terminal methionine and differs from the is estimated spectrophotometrically. The method human 3 chain in 32 positions and from the human has the advantage of simplicity but tends to give y chain in 40 positions. A nearly complete seq- imprecise results at low levels of Hb and results on September 23, 2021 by guest. Protected quence for the y chain of sheep Hb F was obtained that are too low with high levels of Hb F (Jonxis and by Wilson et al (1966). Sheep of genotypes AA, Huisman, 1956). More precise estimates may be BB, and AB, which share a common ax chain, have obtained by measuring the actual rate of denatura- the same type of fetal haemoglobin. In goats, two tion of the sample under standard conditions (re- types of fetal haemoglobins are found according to viewed by Huehns and Beaven, 1971). The the genotype of the goat. These haemoglobins have method has been adapted to a microscale for study- a common y chain and differ in their a chains. This ing small samples from embryos. variation reflects a similar variation in the adult A modification of the Singer method using cyan- haemoglobin types. The rather complex pattern methaemoglobin for measuring samples containing of haemoglobins in the goat has been summarized less than 10°o Hb F has been described by Betke, by Huisman et al (1969a). Marti, and Schlict (1959). In fetal mice of strain C57/BL 6J there are two Intracellular Hb F can be detected by the Klei- distinct populations of erythrocytes. One popu- hauer test (Kleihauer, Brown, and Betke, 1957) 56 P. A. Lorkin J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from which is based on the observation that Hb F is less blood would enable it to take up oxygen from the readily eluted than Hb A from cells in citrate buffer, adult blood in the maternal circulation. Purified pH 3-2. After elution of the haemoglobin the cells human Hb F has been reported to show the same are stained with haematoxylin and eosin. The oxygen affinity (Allen, Wyman, and Smith, 1953) or degree of staining is proportional to the amount of a lower affinity than purified Hb A (McCarthy, Hb F in the cells. Cells which contained only Hb 1943; Tiyuma and Shimizu, 1969). The explana- A are visible as 'ghosts' while cells containing tion why the human fetal cells nevertheless show a large amounts of Hb F, eg, cord cells are heavily higher oxygen affinity lies on the different responses stained. of Hb A and Hb F to 2,3 DPG. Hb A binds 2,3 The cellular distribution of Hb F may also be DPG strongly and its oxygen affinity is consider- determined by using a fluorescent-labelled antibody ably reduced; Hb F binds 2,3 DPG less strongly. against Hb F (Tomoda, 1964; Hosoi, 1965; Dan and In the presence of equivalent amounts of 2,3 DPG Hagiwara, 1967). Both these methods illustrate the oxygen affinity of Hb A is reduced much more that individual erythrocytes are capable ofsynthesiz- than that of Hb F (Tiyuma and Shimizu, 1969). ing both Hb A and Hb F. Fetal and adult cells have approximately the same concentration of 2,3 DPG (Guest and Rapoport, Isolation of Hb F. Human Hb F is not readily 1941) and consequently fetal cells have a higher separated from Hb A by paper electrophoresis. oxygen affinity than adult cells. A further factor Satisfactory separations for diagnostic purposes may assisting oxygen transfer across the placenta is the be achieved by electrophoresis in agar gel or starch change in pH occurring in the fetal and maternal gel (Huehns, 1968; Huehns and Beaven, 1971). blood. Fetal blood reaching the placenta undergoes Haemoglobin F may be separated best by ion a rise in pH from 7-3 to 7-4 increasing its oxygen exchange chromatography. Prins and Huisman affinity (Bohr effect) and assisting oxygen uptake. (1955) separated Hbs A, S, C, and F on IRC-50, a Maternal blood undergoes a fall in pH assisting its cation exchange resin using sodium citrate buffers. release (Walker, 1959). Allen, Schroeder, and Balog (1958), using a phos- phate buffer containing cyanide, obtained a good Conditions in which there is a Retention separation of Hb F from Hb A, and observed that or Reappearance of Hb F in Adult Life copyright. Hb F from cord blood splits into two fractions F, Normally Hb F is replaced virtually completely (20%) and F,, (80%). These two components are by Hb A within about one year after birth. How- equally resistant to alkaline denaturation (Matsuda ever in a number of abnormal or pathological con- et al, 1969), and differ only in that the N terminal ditions the synthesis of Hb F may persist into adult residue of the y chain in Hb F is acetylated. life or may re-appear. N terminal residues have been found Acetylated http://jmg.bmj.com/ in haemoglobins of other animals, and an enzyme Hereditary Persistence of Fetal Haemo- has been found which acetylates the N terminus of globin (HPFH). A benign hereditary condition in chicken haemoglobin (Marchis-Mouren and which the synthesis of Hb F persists into adult life Lipmann, 1965). was first described by Edington and Lehmann Haemoglobin F may also be separated by chro- (1955a and b) in Ghana. It was subsequently found matography on DEAE Sephadex (Huisman and in Uganda, Jamaica, USA, Greece, and Italy Dozy, 1965; Dozy, Kleihauer, and Huisman, 1968; (summarized by Ringelhann et al, 1970) and in

Huehns, 1968) though this method does not resolve Thailand (Wasi, Pootrakul, and Na-Nakorn, 1968). on September 23, 2021 by guest. Protected it from one of the minor components of Hb A. The condition occurs with a frequency of about Separation can also be achieved on CM Sephadex 0-1% in the Negro population in Baltimore and (Zade-Oppen, 1963; Honig, 1967). Boston (USA) and of about 0 25% in Greece. Negro heterozygotes have about 25% Hb F whereas Function of Fetal Haemoglobin. The oxy- the Greek heterozygotes have about 14% Hb F gen affinity curves of human and most mammalian (Fessas and Stamatoyannopoulos, 1964). The fetal and avian embryonic bloods lie to the left of the haemoglobin is uniformly distributed. The trait is corresponding curves for adult blood (Darling not associated with any other haemological abnor- et al, 1941; Bartels, Hiller, and Reinhardt, 1966). mality. Another type of HPFH with only 2-3% This is also true for suspensions of cells in buffered Hb F was found in about 1% of about 3000 recruits saline, ie, the fetal cells have a higher oxygen affinity from southern Switzerland (Marti and Butler, than adult cells. This would be expected on 1961). Homozygotes for HPFH have been found general grounds since the higher affinity of fetal (Wheeler and Krevans, 1961; Polosa et al, 1964; Fetal and Embryonic Haemoglobins 57 J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from

Ringelhann et al, 1970); the cases described by 1963; Hardisty, Speed, and Till, 1964). Two Wheeler and Krevans and Polosa were quite young forms of childhood myelocytic leukaemia are recog- children (three to four years old at the time of in- nized (Hardisty et al, 1964). A juvenile type is vestigation) but the person described by Ringelhann associated with a high level of Hb F, absence of the and his co-workers was a 45-year-old man. Philadelphia chromosome and a poor response to therapy. An adult type is associated with the pre- Persistence ofFetal Haemoglobin in Haemo- sence of the Philadelphia chromosome, low levels globinopathies. Persistence of synthesis of small ofHb F, and a betterresponse to therapy. Weatherall, amounts of Hb F has been observed in haemo- Edwards, and Donohoe (1968) observed a gradual globinopathies affecting the synthesis of chains reversion of haemoglobin and carbonic arhydrase (Beaven, Ellis, and White, 1960b). In homozygous to the fetal type in a study of one patient, a 2-year- sickle-cell disease Hb F is found in adults (Singer old girl, with juvenile myelocytic leukaemia. The I et al, 1951) in amounts ranging from about 2% up to antigen titre (see below) also fell to values approach- 20-30% (Beaven, Ellis, and White, 1961). The ing that characteristic of cord blood. Hb F is not distributed uniformly but varies from Slightly increased amounts of Hb F (1-3%) cell to cell (Bertles and Milner, 1968). Hb F levels have been found in children with lymphoblastic are reported to decrease with age in men but not in leukaemia and monocytic leukaemia (Weatherall et women (Bickers, 1966). G. R. Serjeant (personal al, 1968). communication) also observed that female SS Small amounts (1-2%) of Hb F were reported in homozygotes tend to have somewhat higher levels approximately 50% of patients with untreated of Hb F than male. This, and other findings dis- Addisonian pernicious anaemia (Beaven et al, 1960b). cussed below, suggest that Hb F synthesis may be In some cases there was an initial rise in Hb F level subject to some degree of hormonal control. followed by a fall to normal levels in response to In heterozygous thalassaemia, which causes a vitamin B12 therapy. variable degree of suppression of /-chain synthesis, there is a moderately elevated level of Hb F not Maternal Synthesis of Hb F in Pregnancy. usually exceeding about 6%h which is unevenly dis- Pembrey and Weatherall (1971) in a short report tributed through the erythrocyte population. In observed that of 45 women followed throughout copyright. heterozygous P8 thalassaemia (F thalassaemia) pregnancy and six months after, 20-25% had a which causes suppression of both ,B- and 8-chain raised level of Hb F in the second trimester, which synthesis there is a more marked increase of Hb F fell to normal at 36 weeks. The rate of increase and up to about 35 % (reviewed by Motulsky, 1964). In non-uniform distribution suggested that the Hb F homozygous thalassaemia where there is more severe was of maternal origin. This was confirmed by

or complete suppression of /-chain synthesis Hb F in-vitro labelling studies of reticulocytes of women http://jmg.bmj.com/ may constitute 80-90% of the total haemoglobin. in the first trimester of pregnancy. The results suggested that a factor might be synthesized in early Hb F in Chromosomal Abnormalities. There pregnancy which is capable of inducing fetal haemo- is a delayed postnatal disappearance of Hb F in in- globin synthesis. fants with D1 (13) trisomy and the appearance of Rucknagel and Chernoff (1955) also reported Hb A2 is slightly delayed (Huehns et al, 1964d; slightly raised levels (2.8-3.5%) of Hb F in about Powars, Rohde, and Graves, 1964). Trace amounts 10% of women during the second trimester of pre-

of Hb Gower 2 were also found. These findings nancy. Bromberg, Salzburger, and Abrahamov on September 23, 2021 by guest. Protected have suggested that some structural or control genes (1957) reported that Hb F was raised in four affecting haemoglobin synthesis might be located on patients with molar pregnancy. These patients the chromosome triplicated in D1 (13) trisomy excreted large amounts of chorionic gonadotrophin. (Huehns et al, 1964d). Since no fetus was present in these cases, the source of the Hb F must have been the mother. Reappearance ofHb F. Reappearance of Hb F In an attempt to investigate possible hormonal (Beaven, Ellis, and White, 1960a) has been observed influences on fetal haemoglobin, Davies and Bull in acquired aplastic anaemia, leukaemia, perni- (1971) reported that the administration of prolactin cious anaemia, and in certain cases of excessive to hamsters induced the production of a haem pro- endocrine activity eg, thyrotoxicosis. tein with the same electrophoretic mobility as ham- High levels of Hb F, up to 50%, have been ob- ster fetal haemoglobin. The authors tested a served in acute granulocytic leukaemia of children number of tranquillizing drugs which have been below the age of five years (Beaven and White, reported to induce lactation or breast enlargement 58 P. A. Lorkin J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from and which might presumably induce endogenous levels of true cholinesterase, glyoxylase, carbonic prolactin production. Most of the drugs were in- anhydrase, and catalase from cord blood cells com- active in inducing the production of the haem pro- pared to the levels in cells from adults. Adult teins, and two, fluphenazine and imipramine, blood cells have an antigen (I antigen) which is induced very small amounts. absent from cord cells (McGinniss, Schmidt, and Lie Injo, Hollander, and Fudenberg (1967) Carbone, 1964). Cord blood cells have a different found three patients with increased Hb F (2-9, 5-1, antigen, the i antigen, which is maximally developed and 19-5%0 ) in 13 patients with thyrotoxicosis. at birth and decreases during the first year of life Nine patients had a deficiency of carbonic anhy- (Marsh, 1961). The increase of i antigen is associ- drase activity, another characteristic of fetal erythro- ated with a reciprocal increase in I antigen which is cytes. The patient with the high level (19-5%) of poorly developed at birth. A high i antigen titre Hb F was treated for thyrotoxicosis and showed a and the fetal type II hexokinase isoenzyme have decrease of Hb F to 4-4% and a rise in carbonic been found in an individual homozygous for HPFH anhydrase activity to normal levels. (Charache, Schrueffer, and Bias, 1968). Increased In all cases in which reappearance of Hb F is ob- anti-i activity without any corresponding decrease served, there is a heterogenous distribution of Hb F in I antigen has been reported in patients with / among the erythrocyte population, this also applies thalassaemia major (Giblett and Crookston, 1964). in thalassaemia and in haemoglobinopathies Increased anti-i activity has also been reported in (Shepard, Weatherall, and Conley, 1962). How- patients with hypoplastic anaemia and acute leu- ever in hereditary persistence of fetal haemoglobin kaemia. There was no correlation between the the Hb F is always uniformly distributed. amount of Hb F and i antigen. Other Changes Occurring During the Switch Changes in the Ratio of Gy and Ay Chains. from Fetal to Adult Haemoglobin. The change One of the most interesting recent developments from the production of fetal to adult haemoglobin is has been the discovery of multiple structural genes accompanied by changes in some of the enzymes for the y chain of fetal haemoglobin (Schroeder et al, present in the cell and in other properties of the cell. 1968). When globin from Hb F is treated with Cord blood cells have a MCV about 20% larger cyanogen (CNBr) the methionyl bands are cleared. copyright. than that of cells from adults (Neerhout, 1968) and One of the resulting peptide fragments yCB3 con- contain more K+ and water (Zipursky, Larue, and tains residues 134-146 of the y chain with the Israels, 1960). Cells from newborns have a higher amino-acid sequence shown in Figure 1. Amino- lipid content although the proportions of the lipid acid analysis of this peptide gave integral values for components are similar to that in adult cells with all the amino acids except glycine and alanine. in the of various Analysis of yCB3 from many cord bloods gave only minor variations proportions http://jmg.bmj.com/ phospholipid fractions and of fatty acids (Neerhout, average values close to Gly0.75 and Ala2.25. This 1968). There is a lower level of carbonic anhy- indicated that there might be two types of y chain; drase and a different hexokinase isoenzyme in cord one with glycine and one with alanine at position blood cells (Holmes et al, 1967). 136. The yCB3 obtained from normal cord blood Hexokinase activity in human erythrocytes can be evidently contained a mixture of the two types. If separated into three distinct bands by starch gel the composition of yCB3 were Glyo Ala3 then the electrophoresis at pH 8-6, the bands are designated chains would all be of the Ay type; if it were Gly,

I, II, and III in order of increasing electrophoretic Ala2 they would all be of Gy type. Intermediate on September 23, 2021 by guest. Protected mobility. Normal adult erythrocytes contain hexo- values of glycine indicate a mixture of the two types. linase types I and III. Type II is present in the Evidence that the two types of chain were the pro- erythrocytes of newborn infants and is absent from ducts of distinct genes rather than, for example, the those of normal adults. It is, however, present in result of ambiguous translation, is provided by ana- the erythrocytes of adults with hereditary persis- lysis of mutants of Hb F where the abnormal y tence of Hb F. Type II hexokinase and Hb F chain must be the product of a single gene. In the appeared to be associated. Neonatal erythrocytes cases where this has been examined it has been are also reported to be more susceptible to haemo- found that there is either all alanine or all glycine at lysis in dilute H202 than adult erythrocytes position 136 and not a mixture. The ratio of Gy to (Gordon, Nitowsky, and Cornblath, 1955), this is Ay has been studied in Hb F from cases of HPFH in presumably due to the lower levels of catalase and Negroes (Huisman et al, 1969b) and Greeks glutathione perioxidase (Gross et al, 1967). Jones (Huisman et al, 1970); in the Hb F from / thalas- and McCance (1949) found significantly reduced saemia (Schroeder et al, 1970; Schroeder and J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from Fetal and Embryonic Haemoglobins 59

10 GL Y HIS PHE THR GL U GLU ASP LYS ALA THR ILE THR SER LEU TRP VAL LEU PRO GLU SER ALA VAL ALA 20 30 GLY LYS VAL ASN VAL GLU ASP ALA GLY GLY GLU THR LEU GLY ARG ASP GLU VAL ALA 40 LEU LEU VAL VAL TYR PRO TRP THR GLN ARG PHE PHE ASP SER PHE GLU 50 60 GLY ASN LEU SER SER ALA SER ALA ILE MET GLY ASN PRO LYS VAL ASP THR PRO ASP VAL 70 LYS ALA HIS GLY LYS LYS VAL LEU THR SER LEU GL Y ASP ALA ILE ALA PHE SER ASP GLY LEU 80 90 LYS HIS LEU ASP ASP LEU LYS GLY THR PHE ALA GLN LEU SER GLU ALA ASN THR 100 LEU HIS CYS ASP LYS LEU HIS VAL ASP PRO GLU ASN PHE L YS LEU ARG 110 120 LEU GLY ASN VAL LEU VAL THR VAL LEU ALA ILE HIS PHE GLY LYS CYS HIS -yCB3 - 130 GLU PHE THR PRO GLU VAL GLN ALA SER TRP GLN LYS MET VAL THR PRO TYR VAL ALA 140 C,LY VAL ALA SER ALA LEU SER SER ARG TYR HIS or ASN ALA HIS LYS ALA

YCB3 -

FIG. 1. Amino-acid sequence of the y chain of human Hb F*

Huisman, 1970) and combinations of HPFH and f not only the ratio of the Gy and Ay chains but also copyright. thalassaemia (Huisman et al, 1971), from F thalas- that of the : and 8 chains. saemia (Stamatoyannopoulos et al, 1971), in Some interesting differences have been observed acquired haemological disorders (Rosa, Beuzard, and in Gy/Ay ratio in Hb F from heterozygotes and homo- Toulogaot, 1971) and the postnatal changes in the zygotes for HPFH and double heterozygotes for G7/Ay ratio have been studied by Schroeder et al HPFH and : thalassaemia. Negro heterozygotes (1971). The results of some of this work have been for HPFH produced Hb F containing only the G y summarized by Huisman and Schroeder (1971). chain. There were two subgroups, one producing http://jmg.bmj.com/ Analysis of 94 cord blood samples from various on average about 50' Hb F and another producing parts of the world give mean values of 0-72 and 2-32 10-200o Hb F (average 13-5%); one person who for the numbers of glycine and alanine residues in was a double heterozygote for Hb S and HPFH yCB3. The phenomenon appears to be universal produced 30% Hb F containing only the G-y chain. for the samples were taken from babies of a wide On the other hand, Greek HPFH heterozygotes variety of races. The ratio Gy to Ay changes during produced Hb F containing only Ay chains; again the period after birth and eventually attains a ratio of there were two subclasses-one producing on on September 23, 2021 by guest. Protected approximately 2:3. This ratio is also characteristic average about 5% Hb F the other about 17°o Hb F. of Hb F isolated from normal adults. At present it The majority of HPFH heterozygotes produced is difficult to account for how this change in ratio Hb F containing both types of chains. These might come about. Huisman and Schroeder (1971) were subdivided into three groups. In the major speculate that perhaps similar mechanisms control group there was 20-30%' Hb F containing an average of 0 4 residues of glycine; a second, smaller group of individuals producing the same amount of * The residues which differ in the and y chains are given in Hb F but with a higher average value of 0-6 residues italics. The corresponding residue in the chain is shown beneath. Position 136 is occupied by either glycine or alanine. of glycine. A third subgroup produced less Hb F, Amino-acid differences between the ,3 and y chains. (The about 15%', containing only about 0-15 residues of remaining differences are at external sites.) glycine. It was observed that when a s gene was 1 Internally sited residues positions 11, 27, 54, 74, 75, 130, 133 Haem contacts positions 70,71 in trans to the HPFH determinant in the major 2 aPl6 contact positions 51,112,116,125 group the amount of Hb F produced was increased 3 Central cavity positions 104, 139 4 DPG-binding site positions 1, 143 but the Gy/Ay ratio was unchanged. In a family in J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from 60 P. A. Lorkin which there was a combination of HPFH and /3 is possible that there might be a heterogeneity with- thalassaemia the Gy/Ay ratio in double heterozygotes in the population, some individuals having a single for HPFH and / thalassaemia was intermediate be- a-chain locus and others two loci. tween the Gy/Ay ratio in single heterozygotes for HPFH and for /3 thalassaemia. In the various Haemoglobin Changes Occurring in Sheep acquired haematological disorders exhibiting raised and Goats in Response to Anaemia Hb F studied by Rosa et al (1971) the amount of A somewhat different change in haemoglobin glycine in yCB3 varied between about 0-5 and 0 94 type occurs in sheep and goats in response to anae- residues. There was no obvious correlation be- mia. Sheep may be classified into three main tween the nature of the disease, the amount of Hb F genotypes with respect to their adult haemoglobins; synthesized, the state of the bone marrow, or the AA producing only Hb A, AB producing Hb A and nature of the treatment. Two embryos of 16 and Hb B, and BB producing only Hb B. Haemo- 20 weeks were also examined, they showed the globins A and B have a common a chain and differ same Gy/Ay ratio as newborns. in their / chains, Hb B has a higher oxygen affinity The observations of Hb F in the HPFH condition than Hb A. An additional rare allele of the a chain have been explained by assuming deletions of (aD) occurs in some sheep; the aD chain differs from various gene loci (Huisman et al, 1969b). However, the normal a chain by a Gly->Asp substitution at these schemes do not explain some important ob- position 15 (Huisman et al, 1968). When sheep of servations particularly the change in Gy/Ay ratio genotypes AA and AB are made anaemic by re- after birth and the heterogeneity of the condition. peated bleeding a new type of haemoglobin, Hb C, Clearly the situation is complex, but it now seems appears. This change can also be induced in non- well established that there are at least two genetic anaemic sheep by injection of plasma from anaemic loci for the y chain; possibly there may be as many sheep (Boyer, 1967; Gabuzda et al, 1968). Sheep as four (Huisman et al, 1972). The different loci Hb C contains a new type of /3 chain the /c chain are capable of being expressed to different extents which contains only 141 amino-acid residues and during development and in some abnormal con- differs from the /A chain in at least 16 positions. fB not ditions. These important observations still await a Animals containing only the gene do produce copyright. full explanation which will undoubtedly greatly in- Hb C. Hb C has also been found in young lambs in crease our understanding of the factors regulating the first few months after birth (Huisman et al, haemoglobin synthesis. 1969a). At birth the haemoglobin was 95-98%' There is some evidence indicating that the a- Hb F, this decreased after birth and none was de- chain locus is duplicated (Lehmann and Carrell, tectable at 40 days. At birth no Hb C was detec- 1968). It is frequently observed that abnormal table, but the level rose during the 2 weeks following human haemoglobins with mutations in the a chain birth and reached 5-6% at 14-20 days and after- http://jmg.bmj.com/ make up about a quarter of the total haemoglobin wards fell, reaching about 1-2% at 76 days. Other whereas /-chain mutants make up nearer to half the haematological changes were observed during this total in heterozygotes. The / chain is known to be period. There was an initial fall in PCV and hae- the product of a single gene locus: if there were two moglobin concentration and an increase in the num- a-chain gene loci then the abnormal a chain would ber of reticulocytes during the first 20 days. The be expected to amount to about a quarter of the production of Hb C was stimulated by injected total if all genes were expressed equally. The most erythroprotein. convincing evidence for there being at least two a- The same phenomenon was observed to a much on September 23, 2021 by guest. Protected chain loci comes from the observation by Hollan more marked degree in young goats. Adult goats et al (1972) of an individual whose blood contained of genotypes AA, AB, and BB all have a common/ two different a-chain abnormal haemoglobins in chain and differ in their a chains. A variant of /3 addition to normal Hb A. As three different a chain (/D) is found in some goats, the /D chain differs chains cannot arise from a single locus, there must be from the normal / chain by an Asp->His substitu- at least two a-chain loci in this individual. How- tion at position 21. When goats are made severely ever, individuals have been found in New Guinea anaemic, a new type of chain, the /3C chain is pro- (Beaven et al, 1972) who appear to be homozygous duced in all phenotypes including those with the for the a-chain abnormal haemoglobin Ja Tongariki. /D chains. The /c of the goat is very similar to that They did not appear to be suffering from a thalas- of the sheep and possibly differs only by a Thr ->Ser saemia and since it is highly improbable that they substitution at position 50 (Huisman et al, 1967). could have inherited four abnormal a-chain genes The /c chain is present in small amounts in non- they may have only a single a-chain gene locus. It anaemic goats. In newborn goats Hb F constituted J Med Genet: first published as 10.1136/jmg.10.1.50 on 1 March 1973. Downloaded from Fetal and Embryonic Haemoglobins 61

86-98 % of the haemoglobin in 12 animals exa- Allen, D. W., Wyman, J., and Smith, C. A. (1953). The oxygen equilibrium of fetal and adult hemoglobin. Journal of Biological mined by Huisman et al (1969a). Haemoglobins Chemistry, 203, 81-87. containing /c chains were virtually absent at birth Arnone, A. (1972). X-ray diffraction study of binding of 2, 3 dipho- spoglycerate to human deoxy haemoglobin. Nature, 273, 146-149. but rapidly increased in amount after birth and by Babin, D. R., Schroeder, W. A., Shelton, J. R., Shelton, J. B., and 40-50 days constituted more than 80% of the total Robberson, B. (1963). The amino acid sequence of the y chain of haemoglobin. Haemoglobins containing chains bovine fetal hemoglobin. Biochemistry, 5, 1297-1309. flA Baglioni, C. (1963). Correlations between genetics and chemistry of showed a slight rise in the first 4-10 days followed human hemoglobins. In Molecular Genetics, ed. by J. M. by a decline to very low levels. After 60 days the Taylor. Part I, pp. 405-475. Academic Press, New York. Bartels, M., Hiller, G., and Reinhardt, W. (1966). Oxygen affinity /3C-contaiaing haemoglobins were gradually re- of chicken blood before and after hatching. Respiration Physiology, placed by flA_containing haemoglobins. During 1, 345-356. this period Beaven, G. H., Ellis, M. J., and White, J. C. (1960a). Studies on initial of growth of the animals other human foetal haemoglobin. I. Detection and estimation. haematological changes occurred. There was a British Journal of Haematology, 6, 1-22. large increase in red blood cell count from about Beaven, G. H., Ellis, M. J., and White, J. C. (1960b). Studies on human foetal haemoglobin II. Foetal haemoglobin levels in 9 x 106 per mm3 to more than 20 x 106 per mm3, a healthy children and in certain haematological disorders. British decrease in MCV and a rise in the number of Journal of Haematology, 6, 201-222. Beaven, G. H., Ellis, M. J., and White, J. C. (1961). Studies on reticulocytes. Injection of extracts containing human foetal haemoglobin III. The hereditary haemoglobino- erythroid-stimulating factors stimulated the pro- pathies and thalassaemias. British Journal of Haematology, 7, duction of the /c chain and led to almost complete 169-186. Beaven, G. H. and Gratzer, W. B. (1959). Foetal haemoglobin in formation of the g3c chain. In sheep similar experi- the monkey. Nature, 184, 1730. ments produced only about 10% of haemoglobin Beaven, G. H., Hoch, H., and Hobday, E. R. (1951). The haemo- containing the chain. globins of the human foetus and infant. Electrophoretic and flc spectroscopic differentiation of adult and foetal types. Bio- These findings are of great interest in that they chemicalJournal, 49, 374-381. indicate that the production of different haemoglo- Beaven, G. H., Hornabrook, R. W., Fox, R. H., and Huehns, E. R. (1972). Occurrence of heterozygotes and homozygotes for the a bin types may be subject to hormonal control. chain haemoglobin variant Hb J Tongariki in New Guinea. Nature, 235, 46-47. Beaven, G. H. and White, J. C. (1963). The occurrence of Hb F or Hb H in the leukaemic state. (Abstract.) In Proceedings of the 9th Conclusion Congress of the European Society of Hematology, Lisbon, p. 543. Karger, Basel. copyright. Further studies of the developmental changes in Benesch, R. and Benesch, R. E. (1969). Intracellular organic phos- haemoglobin and of the accompanying changes in phates as regulators of oxygen release by haemoglobin. Nature, the red cell should continue to be a fruitful field of 221, 618-622. Bertles, J. F. (1969). The occurrence and significance of fetal hemo- research. The means by which these changes are globins. In Regulation of Hematopoiesis, ed. by A. S. Condon. brought about and the causes of the reversion to Appleton-Century-Crofts, New York. Bertles, J. F. and Milner, P. A. (1968). Irreversibly sickled erythro- fetal haemoglobin production in certain disorders cytes: a consequence of the heterogeneous distribution of haemo-

are not understood. Some evidence suggests that globin types in sickle cell anemia. Journal of Clinical Investiga- http://jmg.bmj.com/ may to tion, 47, 1731-1741. the change be subject hormonal control and Betke, K., Marti, H. R., and Schlict, I. (1959). Estimation of small the evidence is stronger in the case of the related percentages of foetal haemoglobin. Nature, 184, 1877-1878. changes occurring in sheep and goats. A fuller Bickers, J. N. (1966). Alkali resistant haemoglobin in sickle-cell disease. Annals of Internal Medicine, 64, 1028-1033. understanding of these processes might provide a Bookchin, R. M. and Gallop, P. M. (1968). Structures of haemo- basis for the treatment of some serious haematologi- globin Alc; nature of the N-terminal P chain blocking group. cal disorders such as sickle-cell and :3 thalassaemia. Biochemical and Biophysical Research Communications, 32, 86-93. Borgese, T. A. and Bertles, J. F. (1965). 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