Humangenetik 30, 197--205 (1975) © by Springer-Verlag 1975

Reviews

Recent Developments in Foetal Haemoglobin Research Hezekiah K a m u z o r a 1 M.t~.C. Abnormal Haemoglobin Unit, University Department of Clinical Biochemistry, Addenbrooke's Hospital, Hills goad, Cambridge Received June 2, 1975 / July 21, !975 Summary. There have been numerous new contributions to the knowledge of foetal haemoglobin over the last few years. It is, therefore, timely to review them together. They throw light on the arrangement on the chromosome of non-~ chain genes, and on the condition generally known as Hereditary Persistence of Foetal Haemoglobin (ttPFtI) and have contributed to other aspects of human ontogeny and physiology.

Foetal haemoglobin is a tetramer composed of two ~ and two 7 chains (~272). The 7 chains are very similar in p r i m a r y structure to the fi chains, each having 146 amino acid residues which differ only at 39 positions (Sehroeder et al., 1963). Most of the differences (22 residues) are found on the external surface with no significant effect on the properties of the molecule. The ~1 f12 and ~1 72 contacts, being invariant in all m a m m a l i a n globins (Dayhoff, 1972), are identical so t h a t both H b A and H b F would be expected to dissociate equally into c~fi or ~7 dimers. However, i m p o r t a n t changes occur in the internal p a r t of the molecule where m a n y residues are substituted, n o t a b l y the tyrosine fi130 which is substituted by t r y p t o p h a n in the 7 chain giving the H b F its characteristic ultraviolet spectrum ; and 3 other i m p o r t a n t residues at the ~1 fil contacts, fi51 Proline, which is substituted b y Alanine in 7, fill2 Cysteine, replaced b y Threonine in 7 and fi116 Histidine, replaced b y Isoleueine in 7. These few but i m p o r t a n t differences m a y account for the reduced t e n d e n c y of H b F to dissociate into monomers, and for its increased resistance to alkali denaturation. Recently Perutz (1974) has provided evidence for the involvement of fill2 Cysteine in alkali denaturation. The buried eysteine which lies at the al fil contact, being weakly aeidie, is ionised at raised pH. The ionised forms become easily h y d r a t e d thereby preventing association of subunits. I t is probable t h a t such other weakly ionising groups in some other part of the molecule m a y similarly account for the observed increased resistance to acid denaturation. Such differences could form the basis of the technique developed b y Betke and Kleihauer (1958) for qualitative estimation of H b F concentration within individual erythroeytes. The technique is based on differential resistance of H b F and I t b A to elution u n d e ~ m i l d acid conditions (pit 3.3). After acid elution, cells containing only I-IbA lose haemoglobin and 1 On study leave from Dares Salaam University as a Commonwealth Scholar, Churchill College, Cambridge. This review is part of a Ph.D. Thesis submitted to Cambridge University. Present address: Department of Biochemistry, University of Dares Salaam, Tanzania.

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appear as "ghosts" on haematoxylin-eosin staining, while cells with H b F retain haemoglobin and are stained pink. This technique has been used in classifying the distribution of I t b F among red cells in various disorders as either homogeneous or heterogeneous (unequal). In the normal adult, traces of H b F present are heterogeneously distributed among the red cells (Shepard et al., 1962). In some forms of Hereditary Persistence of Foetal Haemoglobin (HPFH) the foetal haemoglobin is homogeneously distributed among the erythrocytes, an indication t h a t both I-IbF and H b A can be present within the same red cells. Other important differences include replacement of Val NA 1 (N-terminal) in ~, by Gly in y, (part of which is acetylated) and His fi143 by Set in Y. The His fi143 residue is involved in binding 2,3 DPG to deoxyhaemoglobin. The change makes 2,3 DPG less firmly bound in HbF, and this partly explains why I t b F has a higher oxygen affinity than ItbA. The elevated oxygen affinity is not a disadvantage to the foetus since the transfer of oxygen between the foetus and placerrga depend, among other factors, on their respective 02 dissociation curves. A higher oxygen affinity enhances the uptake of oxygen by the foetal blood from the placenta. The net uptake of oxygen is increased further by the high haematocrit of foetal blood. Conditions in the foetus are such t h a t the partial pressure of oxygen (p02) in foetal tissue can be as low as 16 m m Hg. N o v y (1972) compared the oxygen saturation curve of foetal blood to that of adult blood, and showed t h a t as much as half of the oxygen carried by the foetal erythrocytes is actually delivered to the tissues, at such a low pO 2. This p02, however, is not low enough to facilitate oxygen delivery from Haemoglobin Barts, and this explains the fate of Barts' hydropie infants. At the adult venous pO2 foetal haemoglobin can only release less than 20% of its oxygen compared to the 30% oxygen release by adult haemoglobin. Foetal haemoglobin is therefore less efficient as a respiratory pigment. There are pathological conditions where elevated levels of I t b F are observed in adults. In Hereditary Persistence of Foetal Haemoglobin ( I I P F H ) or some forms of ~-thalassaemia, H b F levels up to 100% can be found. Surprisingly, patients with such levels of haemoglobin F are neither hypoxie nor grossly polyeythaemie (Charaehe et al., 1968; Ringelhann et al., 19701. /

Dulolication. Unlike the d and/~ chains, whose synthesis is directed by single genes per chromosome, there is now evidence t h a t man has two or more genetic loci specifying y chains (Schroeder et al., 1968). When y chains are treated with cyanogen bromide or trypsin the 7CB-3 peptide (7134--146) or 7Tp )2IV (7133-144) reveal t h a t residue 136 is occupied b y either glycine or alanine (Schroeder et al., 1968; Weatherall and Clegg, 1972; Kamuzora et al., 1974, 1975). These chains are designated as G7 and a7 respectively. Variants of Hb~" have only one type of 7 chain containing either glycine or alanine at position 136, providing evidence t h a t 2 or more 7 chain loci exist in man. Examination of a number of cord bloods reveals t h a t normal HIbF contains 7 chains with a G7 : A7 ratio of 3 : 1, an indication t h a t 3 genes code for the G7 chains and 1 for the A7 chain making a total of 4 genes. I f all these genes are expressed equally, then, from a purely theoretical point of view the variants of 7 chains should account for no more than 25 % of total HHHbFin heterozygotes in individuals carrying a single variant chain gene.

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While the y chain variants observed to date have not exceeded the 25~o barrier, a lot of inconsistencies have been observed in final levels : a V variants, t t b F Malta 1 (25%) (Cauchi et al., 1969). I.ibF Malaysia (18.8%) (Lie-Injo et al., 1974). H b F Sardinia (25%) (Griffoni et al., 1975). Hbi~ Port Royal (14°/0) (BrimhM1 et al., 1974). Ay variants, l i b F Jamaica (13%) (Ahem et al., 1970). I I b F Hull (14%) (Sacker et al., 1967). H b F Texas 1 (12%) (Jenkins et al., 1967). t I b F Malta I I (5%) (Huisman et al., 1972a). One factor becomes immediately apparent. There must be 2 groups of ey variants comprising 18--25~o on one hand and about 14% on the other; and two groups of Ay variants comprising 12--14% on one hand and 5% on the other. Another important observation is that I-IbF Port Royal, which is a ey variant, occurs in the same proportion as the majority of the Ay variants. I t is through such observations that Schroeder and Huisman postulated that there exist four ?~ chain loci, t I b ~ , , Ub~y, libmAy and I.ib~y producing the ), chains in the ratio of 4 : 2 : 2 : 1, respectively. They studied postnatal changes in the chemical heterogenity during the seven month period between the 32nd week of gestation and the fifth month after birth and found that the percentage of ay, ay, d and /3 chains among the non-~-ehains changed roughly from 70, 26, O, 3 to 2, 3, 1.5, 95.5°/0 (Sehroeder et al., 1971). This means that as a child grows to haematological maturity in the first 6 or 7 months of life the proportion of y136 glyeine drops from about 0.7 residues to about, 0.4 residues (i.e. the Gy: A~, ratio changes from 7 : 3 to 4 : 6 (or 2 : 3). Using the 4 : 2 : 2 : 1 gene activity as their model, they assumed that all the genes are fully expressed at birth so that theoretically the total G7 chain should account for 4 + 24+2 × 1O0 67%, the remaining 33% +'2÷1 should account for the AV chains. Indeed the ratio of 7:3 found in the newborn justifies this assumption. As the child grew to haematological maturity the ~y gene is inactivated so that the activities now become 0:2 : 2 : I. The glyeine value now becomes 0+2 × 100 or 40% which is the ratio normally found in adults (or 2:3). But the complexity arises when elevated levels of glycine are found in several other cases much in excess of 0.7 residues of glycine, as observed in acquired haematologieal disorders (I~osa et al., 1971). Such values could mean that Ay genes are either selectively suppressed, or the e?~ genes are activated. There are several pathological conditions in which the level of H b F is raised. The ItPt~I-I condition is probably the best documented, and has thrown some light on the arrangement and linkage of the non ~ genes on the chromosome. The H P F t t heterozygotes have normally operating genes for/3 h and )~ chains on one chromosome, and a total suppression of the function of the d and /3 loci on the homologous chromosome (Huisman et c~l., 1969). Knowledge regarding the arrangement of various non ~-chain genes on the chromosome has been obtained through studies of Haemoglobins Lepore and Kenya. Duma et al. (1968) found that patients homozygous for t{b Lepore lack both HbA and HbA t A chromosome carrying a gene specifying a Lepore chain must therefore lack both normal fi and d genes. The d/3 chain of lib Lepore is produced as a result of chromosomal mispairing during meiosis such that the d gene on one chromosome pairs with the/3 gene on the other, resulting in a d/3 gene. ttb Miyada (/3 d fusion) (Yanase et al., 1968) has an anti-Lepore chain whose NI-I~-terminal region is /3-like and

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whose COOI-I terminal end is identical to the 6 chain. The anti-Lepore chromosome contains both fi and c3 genes in addition to the fi-c~ gene, and individuals heterozygous for I-Ib Miyada are not thalassaemic, and have normal H b A 2 levels. This provides evidence t h a t the 6 and ~ chains are adjacent on the chromosome. Recently ttuisman et al. (1972c) have reported the discovery of I-Ib K e n y a which is a y fi cross over product. Individuals with this variant are associated with I-IPFH (Clegg et al., 1973) and their foetal haemoglobin contains only Gy chains. I t follows therefore t h a t in these individuals the C-terminal part of the Ay gene, the entire d gene and the N-terminal part of the fi gene have been deleted. Furthermore studies on individuals heterozygous for H b K e n y a and HbS showed they had no I-IbA (Kendall et al., 1973). The chromosome with I t b K e n y a gene must therefore lack the fl gene. Since H b Lepore homozygotes make large amounts of y chains (Fessas et al., 1962) the y loci are not deleted in these individuals; and since H b K e n y a is associated with production of Gy rather than Ay, the order of the genes on the chromosome must be Gy ay d ~. Recently Schroeder and Huisman (1974) have proposed the precise arrangement as I-IbmG7Hb~7 HbmAyHbAy I-Ib~ I-Ib/3, and have attempted to explain the genetic status of various conditions of I-IPFI-I. Complete suppression of Hb6 I-Ibfi genes would result in a H P F H condition where the y chains will contain both ~y and Ay in the ratio of the newborn, while suppression of HbAy ttbd I-Ibfi would result in the same kind of situation but with a raised Gy:a7 ratio. Additional suppression of HbmAy would result in the H P F H of the G7 type (Negro type) while suppression of I-Ib~y and t t b ~ y genes would result in the Greek type of I-IPFI-I with only Ay chains. Theoretically therefore in an H P F H condition associated with both oy and Ay in the ratio of the newborn, the y chains should be synthesised about twice as much as in the H P F t I condition containing only Ay chains, and indeed the Greek heterozygous H P F H types have 10--15%, while the Negro H P F H with both ey and ay types have 2 5 - - 3 0 % HbF. The British type of H P F H which has just been described (Weatherall et al., 1975) can not be explained on the basis of the above model. I f we assume t h a t it arises from the deletion of the HbGmygene, then the remaining Hb~y gene should theoretically produce a condition where the Gy : Ay ratio is t h a t of normal adults in homozygotes, i.e. there should be 40% glycine at y136. A 10% glycine value that has been reported demands alternative explanation. Weatherall et al. (1975) propose t h a t in this condition there might be a mutation in some region of DNA in the y6fi gene cluster which would result in inefficient binding of suppressor I~NA leaving a "leaky" Ay locus. This is indeed most probable. An alternative explanation which I would like to propose for this British type of H P F I t involves a slight modification of the 4 : 2 : 2 : 1 gene model of Huisman and Schroeder. I f an extra gene coding for glycine (which I call ~ y ) i s introduced in the system, making a total of 5 genes coding for y chains; i.e. mY iY 10:~:: i :8 : 3 and if the activitiesof these genes are i0 :4 :i :5 :3 respectively, then the British type of H P F H can be explained by a deletion of mYG and ~y genes which would leave the ~y gene to account for the observed i0~o glycine. This model would

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similarly explain other forms of H P F H . The heterogeneity in distribution of I t b F in the British type of H P F H is difficult to explain but this m a y indicate t h a t this condition has a different etiological basis. Clinically H P F H is a benign condition. I t affects the synthesis of both d and fi chains, but complete compensation is achieved through production of appropriate amounts of y chains so as to balance the a/non ~ globin chain synthesis (Natta et al., 1974). The red cell indices are normal. Even the 4 homozytes for I I P F H which have been studied so far (Baglioni, 1963; Charache et al., 1968; l~ingelhann et al., 1970; Siegel et al., 1970), have been found to be normal, except in the latter case (Siegel et al., 1970) where the condition was associated with microcytosis, anisocytosis, poikilocytosis, numerous target cells and erythrocytosis. When these H P F H conditions are associated with fi thalassaemia the levels of H b F in simple heterozygotes is increased. The nature of y chains in such conditions has been recently summarised by Huisman et al. (1974). They found t h a t all heterozygotes m a y be placed into one of the two groups in which the Sy:Ay ratio is either about 2:3 (as in the I t b F of normal adults) or about 3:1 as in the H b F of newborn. Some structural variants, particularly those of /3 chains, are known to be associated with high levels of HbF. Sickle cell patients of Arab populations for example, have high t I b F levels, about 30~o, and their condition is known to be mild as a result (Ali, 1970; Perrine et al., 1972). This I I b F is heterogenously distributed among the red cells (Huisman et al., 1972b), with a Gy : A), ratio of 3 : 1. I n contrast, studies carried out b y Serjeant et al. (1968), showed t h a t in Jamaicans with sickle cell disease the H b F levels are usually below 10~o with a Gy : Ay ratio of 3:2 (Perrine et al., 1972). Recently we have found a mild form of sickle cell disease in which the H b F is high (25%), the H b F was evenly distributed with a Gy : Ay ratio of 2 : 3 forming an entirely different class of sickle cell disease (Kamuzora and Lehmann, unpublished). The ratios suggest that increased synthesis of ey chains m a y account for the increased H b F in Arabs, whereas the increased synthesis of Ay chains m a y account for increased H b F in Jamaicans and the patient we studied. Several other pathological conditions, particularly those associated with anaemia, cause elevation of H b F levels. They include aplastic anaemia, leukemias, pernicious anaemia,/~-thalassaemia and certain instances of endocrinopathy. In aplastic anaemia, whether acquired or congenital, H b F levels range from 3 - - 1 5 % (Shahidi et al., 1962; Aksoy and Secer, 1964). I t has even been proposed t h a t in such conditions H b F m a y be of prognostic value, significant patient survival being directly correlated to elevated levels of H b F (Bloom and Diamond, 1968). Recent workers, however, have indicated that this correlation is a weak one (Li et al., 1972). In acute leukemias of childhood, significant elevated levels of H b F have been observed (Bartolozzi and Marianelli, 1966). Hardisty etal. (1964) noted t h a t in one of the chronic myelocytic leukemias of childhood associated with the Philadelphia (Ph 1) chromosome no elevation of H b F was found, whereas in those lacking the Ph ~ chromosome, high H b F levels were found exceeding 40o/0 . Weatherall et al. (1968) in studying one patient with juvenile myelocytie leukemia over a 12-month period noted t h a t apart from raised t t b F levels, erythrocyte carbonic anhydrase and erythrocytc I antigen titres were decreased to foetal

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levels, suggesting that the condition may represent regression towards the foetal state. Such phenomenon could probably apply to other conditions with high I-IbF levels like paroxysmal nocturnal haemoglobinuria (Weatherall and Walker, 1965), refractory normoblastie anaemia (Weatherall and Clegg, 1972), Bronchogenie Carcinoma (Nyman et al., 1970) and untreated Addisonian pernicious anaemia (Beaven st al., 1960). It has also been observed that blood of women in the second trimester of pregnancy (Rueknagel and Chernoff, 1965; Pembrey et al., 1973), and of patients with thyrotoxicosis (Lie-Injo, 1967) have high t t b P levels, presumably indicative of hormonal influence on H b F synthesis. On the whole it appears that increased H b F synthesis is distinctly advantageous in red cell survival, and hence to patient survival in many of the haematologieal abnormalities. In I-IbS/S disease or f thalassaemia, this is distinctly so; and so any efforts aimed at increasing H b F synthesis is clinically worthwhile. Direct stimulation of H b F synthesis in humans has been reported, HIM1 and Motulsky (1968) carried out probably the most exciting experiments in this line. They cultivated human bone marrow cells for up to 10 days in vitro at 27°C in the dark and noted a striking increase in I-IbF synthesis. Actinomycin was observed to cause more depression of H b F than HbA synthesis suggesting an different transcription of mRNA for ?/chain in the bone marrow; thereby providing a tool for the study of mechanisms controlling HbF synthesis. Unfortunately, subsequent workers (Wood, 1974) have not yet succeeded in reproducing these results. Other workers have studied the effect of erythropoietin on I-IbF and HbA synthesis. Busch (1972) noted that'in human foetal liver the ratio HbA/HbF did not change in spite of 40% overall increase in Itb production, while Gabuzda et al. (1970), using cell cultures of neonatal calf marrow had noted significant response to erythropoietin, with marked stimulation of adult haemoglobins and had suggested that erythropoietin might play a significant role, directly or indirectly, in the gradual switch over of haemoglobin types during development. Erythropoietin has, at least, been observed to have a direct effect on f chain switching in anaemic sheep; when a sheep homozygous for ttbA becomes anaemic, a switch in f chain production occurs from fA to the structurally unique fie chain (Van Vliet and I-Iuisman, 1964; Nienhuis and Anderson, 1972). Other areas of hormonal research have included thyroid hormones. Experimental acceleration of ontogenie switch from larval haemoglobin to adult haemoglobin synthesis has been achieved by adding thyroid hormone to aqueous environment of tadpoles (Moss and Ingram, 1968). But the applicability of this achievement to humans is still far fetched because in frogs exclusively different cell lines for adult and larval haemoglobins occur whereas in humans the foetal and adult haemoglobins seem to coexist in the same cells. Whatever methods are applied to solving haemoglobin disorders, a realistic hope, at least in the forseeable future, lies in selective stimulation of I-IbF synthesis. References Ahern, E. J., Jones, R. T., Brimhall, B,, Gray, R. H. : Haemoglobin F Jamaica ((~2~261LyS--> Glu, 136 Ala). Brit. J. Haemat. 18, 369--375 (1970) Aksoy, M., Secer, F. : Fetal hemoglobin in acquired aplastic anemia. Acta haemat. (Basel) 112, 188--192 (1964)

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Ali, S. A. : Milder variant of sickle cell disease in Arabs in Kuwait, associated with elevated levels of foetal haemoglobin. Brit. J. liaemat. 19, 613--620 (1970) ]3aglioni, C. : A child homozygous for persistence of tbetal haemoglobin. Nature (Lond.) 198, 1177--1179 (1963) Bartolozzi, G., ~{arianelli, L. : Estimation of fetal hemoglobin in leukemia. Aeta haem~t. (Basel) 35, 214--220 (1966) ]3asch, R. S. : Hemoglobin synthesis in short term cultures of fetal hemoglobin tissues. Blood I~9, 530--535 (1972) Beaven, G. H., Ellis, M. J., White, J. C. : Studies on human foetal haemoglobin. II. Foetal haemoglobin levels in healthy children and adults in certain haematological disorders. Brit. J. I-Iaemat. 6, 201 222 (1960) Betke, K., Kleihauer, E. : Fetaler und bleibender ]31utfarbstoff in Erythrozyten und Erythroblasten yon mensehlichen Feten und Neugeborenen. ]31ut 4, 241 (1958) Bloom, G. E., Diamond, L. K. : Prognostic value of fetal hemoglobin levels in acquired aplastie anemia. New Engl. J. Med. 278, 304--307 (1968) Brimhall, ]3., Vedwiek, T. S., Jones, R. T., Ahem, E., Palomino, E., Ahem, V. : Haemoglobin F Port Royal (~2Gy2 125 Glu --> Ala). Brit. J. liaemat. 27, 313--318 (1974) Caughi, M. N., Clegg, J. B., Weatherall, D. J. : Haemoglobin F Malta: a new foetal haemoglobin variant with a high incidence in Maltese infants. Nature (Lond.) 223, 190--191 (1969) Charaehe, S., Schruefer, J. J., Bias, W. B. : Hereditary persistence of fetal red cells. J. clin. Invest. 47, 17A (1968) Clegg, J, B., Weatherall, D. J., Gilles, H. M. : Hereditary persistence of foetal haemoglobin associated with a yfi fusion variant, haemoglobin Kenya. Nature (Lond.) New Biol. 246, 184--186 (1973) Conley, C. L., Weatherall, D. J., l~iehardson, S. N., Shepard, M. K., Charaehe, S. : Hereditary persistence of fetal hemoglobin: A study of 79 affected persons in 15 negro families in Baltimore. Blood 21, 261--281 (1963) Dayhoff, M. O. : Atlas of protein sequence and structure, Vol. 5. Washington: National Biomedical Research Foundation 1972 Duma, H., Efremov, G., Sadikario, A., Teodosijev, B., Mladenovski, D., Vlaski, g., Andreeva, M. : Study of nine families with haemoglobin Lepore. Brit. J. }Iaemat. 15, 161--172 (1968) Fessas, P., Stamatoyannopoulos, G., Karakhis, A.: Hemoglobin "Pylos". Study of a hemoglobinopathy resembling thalassemia in the heterozygous and double heterozygous state. Blood 19, 1~-22 (1962) Gabuzda, T. G., Silver, R. K., Chui, L. C. : The formation of fetal and adult hemoglobin in cell cultures of neonatal calf marrow. Brit. J. Haemat. 19, 621--633 (1970) Griffoni, V., Kamuzora, H., Lehmann, H., Charlesworth, D. : A new Hb F variant: F Sardinia 775 (El9) isoleueine + threonine found in ~ family with I-Ib G Philadelphia, fl chain deficiency and a Lepore-like haemoglobin, indistinguishable from lib Ao. Acta haemat. (Basel). In press (1975) liall, J. G., Motulsky, A. G. : Production of foetal haemoglobin in marrow cultures of human adults. Nature (Lond.) 217, 569--571 (1968) I-Iardisty, R. M., Speed, D. E., Till, M. : Granuloeytie leukaemia in childhood. Brit. J. liaemat. 1O, 551 556 (1964) Huisman, T. li. J., Sehroeder, W. A., Bannister, W. H., Greeh, J. L. : Evidence for four nonallelic structural genes for the y chain of human fetal hemoglobin. Biochem. Genet. 7, 131--139 (1972a) Huisman, T. H. J., Schroeder, W. A., Bouver, N. G., Miller, A., Shelton, J. R., Shelton, J. B., Apell, G. : Chemical heterogeneity of foetal haemoglobin in subjects with sickle cell anaemia, homozygous Hb C disease, SC-disease and other combinations of haemoglobin variants. Clin. chim. Aeta 38, 5--16 (1972b) Huisman, T. H. J., Schroeder, W. A., Dozy, A. M., Shelton, J. R., Boyd, E. M., Apell, G.: Evidence for multiple structural genes for gamma chain of human fetal hemoglobin in hereditary persistence of fetal hemoglobin. Ann. N.Y. Aead. Sei. 165, 320--331 (1969)

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Huisman, T. H. J., Schrocder, W. A., Efremov, G. D., Duma, H., Mladenovski, B., Hyman, C. B., Rachmilewitz, A. E., Bouver, N., Miller, A. B., Brodie, A., Shelton, J. R., Shelton, J. B., Apell, G. : The present status of the heterogeneity of fetal hemoglobin in fl-thalassemia : an attempt to unify some observations in thalassemia and related conditions. Ann. N.Y. Acad. Sci. 282, 107--124 (1974) Huisman, T. H. J., Wrightstone, R. N., Wilson, J. B., Schroeder, W. A., Kendall, A. G. : Hemoglobin Kenya, the product of fusion of y and # polypeptide chains. Arch. Bioehem. Biophys. 158, 850--853 (1972c) Jenkins, G. C., Beale, D., Black, A. J., Huntsman, R. G., Lehmann, H. : Hemoglobin F Texas I (a2Y2 5 Glu-+ Lys) a variant of haemoglobin F. Brit. J. Haemat. 18, 252--255 (1967) Kamuzora, H., l~ingelhann, B., Konotey-Ahulu, F. I. D., Lehmann, H., Lorkin, P. A.: The y-chain in a Ghanaian adult, homozygous for hereditary persistence of foetal haemoglobin. Acta haemat. (Basel) 51, 179--184 (1974) Kamuzora, H., l~ingelhann, B., Konotey-Ahulu, F. I. D., Lehmann, H., Lorkin, P. A. : Further investigations on the y-chain of a Ghanaian adult, homozygous for hereditary persistence of foetal haemoglobin: Isolation of yCB-3 fragment and determination of Gy:Ay ratio in human haemoglobins. Acta haemat. (Basel) 53, 315--320 (1975) Kendall, A. G., Ojwang, P. J., Sehroeder, W. A. : Hemoglobin Kenya, the product of y--fl fusion: Studies in the family. Amer. J. hum. Genet. 25, 548 (1973) Li0 F. P., Alter, B. P., Nathan, D. G. : The mortality of acquired aplastic anemia in children. Blood 40, 153--162 (1972) Lie-Injo, L. E., Hollander, L,, Fudenberg, H. H. : Carbonic anhydrase and fetal hemoglobin in thyrotoxicosis. Blood 80, 4 4 2 4 4 8 (1967) Lie-Injo, L. E., Kamuzora, H., Lehmarm, I-I.: Haemoglobin F Malaysia a2y~ (NAI) Gly -~ Cys 136 Gly. J. reed. Genet. 1l, 25--30 (1974) Moss, B., Ingram, V. M. : Haemoglobin synthesis during amphibian metamorphosis II synthesis of adult haemoglobin following thyroxine administration. J. molee. Biol. 32, 493-502 (1968) Natta, C. L., Niazi, G. A., Ford, S., Bank, A. : Balanced globin chain synthesis in hereditary persistence of fetal hemoglobin. J. clin. Invest. 54, 433--438 (1974) Nienhuis, A. W., Anderson, W. F.: Hemoglobin switching in sheep and goats: Change in functional globin mRNA in reticulocytes and bone marrow cells. Proc. nat. Acad. Sci. (Wash.) 69, 2184--2188 (1972) Novy, M, J. : Alterations in blood oxygen affinity during fetal and neonatal life. In: Oxygen affinity of hemoglobin and red cell base status (eds. M. Rorth, P. Astrup), pp. 696--712. Alfred Benzon Symposium IV Copenhagen. New York: Academic Press 1972 Nyman, M., Skolling, P~., Steiner, H. : Acquired macrocytic anemia and hemoglobinopathy - a paraneoplastic manifestation. Amer. J. Med. 48, 792--797 (1970) 1)embrey, M. E., Weatherall, D. J., Clegg, J. R. : Maternal synthesis of hemoglobin F in pregnancy. Lancet 1973 I, 1350--1355 Perrine, t~. P., Brown, M. J., Clegg, J. B., Weatherall, D. J., May, A.: Benign sickle cell anaemia. Lancet 1978 II, 1163--1167 Perutz, M. F. : Mechanism of denaturation of haemoglobin by alkali. Nature (Lond.) 247, 341--344 (1974) Ringelhann, B., Konotey-Ahuly, F. I. D., Lehmann, H., Lorkin, P. A.: A Ghanaian adult homozygous for hereditary persistence of foetal haemoglobin and heterozygous for elliptocytosis. Acta haemat. (Basel) 43, 100--110 (1970) Rosa, J., Beuzard, Y., Bran, B., Toulgoat, N.: Evidence for various types of synthesis of human y chains of haemoglobin in acquired haemolytic disorders. Nature (Loud.) New Biol. 288, 111--113 (1971) Rucknagek, D. L., Chernoff, A. I. : Immunological studies of hemoglobins, 111. Fetal hemoglobin changes in circulation of pregnant women. Blood 10, 1092--1099 (1965) Sacker, L. S., Beale, D., Black, A. J., Huntsman, R. G., Lehmann, H., Lorkin, P. A. : Haemoglobin F Hull @121 glu --> lys) homologous with haemoglobin 0 Arab and 0 Indonesia. Brit. med. J. 1967 llI, 531--533 Schroeder, W. A., Huisman, T. H. J. : Multiple cistrons for fetal hemoglobin in man. Ann. N.Y. Acad. Sci. 241, 70--79 (1974)

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Schroeder, W. A., Huisman, T. H. J., Shelton, J. R., Shelton, J. B., Kleihauer, E. F., Dozy, A. M., Robertson, B. : Evidence for multiple structural genes for the ~ chain of human fetal hemoglobin. Proc. nat. Acad. Sei. (Wash.) 60, 537--544 (1968) Sehroeder, W. A., Shelton, J. B., Cormiek, J., 3ones, R. T.: The amino acid sequence of 7 chain of human foetal haemoglobin. :Biochem. (Wash.) 2, 992--1008 (1963) Serjeant, G. R., Riehards, R., Barbar, P. R. N. : Relatively benign sickle cell anaemia in 60 patients aged over 30 in West Indies. Brit. reed. J. 1968 III, 86 91 Shahidi, N. J., Gerard, P. S., Diamond, L. K. : Alkali-resistant hemoglobin in aplastie anemia of both acquired and congenital types. New Engl. J. Med. 266, 117--120 (1962) Shepard, 5I. K., Weatherall, D.J., Conley, G.L.: Semi-quantitative estimation of the distribution of fetal hemoglobin in red cell populations. Bull. Johns Hopk. Hosp. 110, 293--310 (1962) Stegel, W., Cox, t~., Sehroeder, W. A., I-Iuisman, T. It. J., Penner, D., Rowley, P. T.: An adult homozygous for hereditary persistence of fetal hemoglobin. Ann. intern, Med. 72, 533--542 (1970) Van Vliet, G., Huisman, T. H. J. : Changes in haemoglobin types of sheep as a response to anaemia. Bioehem. J. 98, 401--409 (1964) Weatherall, D. J., Cartner, R., Clegg, J. R., Wood, W. G., Maerae, I. A., Mackenzie, A.: A form of hereditary persistence of foetal haemoglobin eharacterised by uneven cellular distribution of haemoglobin F and production of haemoglobins A and A2 in homozygotes. Brit. J. tIaemat. 29, 205--220 (1975) Weatherall, D. J., Clegg, J. B. : The thalassaemia syndromes, 2nd ed. Oxford: Blackwell 1972 Weatherall, D. J., Edwards, J. A., Donohoe, W. T. A. : Haemoglobin and red cell enzyme changes in juvenile myeloid leukaemia. Brit. reed. J. 1968 I, 679--681 Weatherall, D. J., Walker, S. : Changes in the chromosome and haemoglobin patterns in a patient with erythroleukaemia. J. reed. Genet, 2, 157--220 (1965) Wood, W. R. : Haemoglobin synthesis in suspension cultures of bone marrow. Brit. J. ttaemat. ~6, 451--458 (1974) ¥anase, T., ]C[anada, M., Seita, M., Ohya, I., Ohta, ¥., Imamura, T., Fijimura, T., Kawasaki, K., ¥amaoka, K. : Molecular basis of morbidity - - from a series of studies of haemoglobinopathies in Western Japan. Jap. J. hum. Genet. 13, 40--53 (1968) Dr. It. Kamuzora, }I. R. C. Abnormal Haemoglobin Unit University Department of Clinical Biochemistry Addenbrooke's Hospital, Hills Road Cambridge CB2 2QR, England