BrifirhJoumal of Haematology, 1976, 32, 5s.

The Kenya Form of Hereditary Persistence of Fetal Haemoglobin: Structural Studies and Evidence for Homogeneous Distribution of Haemoglobin F using Fluorescent Anti-haemoglobin F Antibodies P. E. NUTE,W. G. WOOD, G. STAMATOYANNOPOULOS, C. OLWENY AND P. J. FAILKOW Departnzents of Medicine (Division of Medical Genetics), Genetics and Anthropology, Regional Prinzate Research Center, University of Washington, Seattle, Washington, a d Uganda Cancer Institute, Kamyda, Uganda (Received I July 1975; accepted for publication

10July

1975)

SUMMARY. Several members of a Ugandan family wcre heterozygous for the yp fusion gene of Haemoglobin Kenya. Levels of Hb Keiiya were significantly highcr than those in subjects of previous reports, ranging from 20.68 to 23.35% of tlie total haemoglobin. Tlic individuals had also 5 4 % Hb F, consistiiig solely of a and Gy chains. Investigation of tlie distribution of Hb F among the red cells of Hb Kenya heterozygotes, using monospecific antibodies absorbed against pure Hb Kenya and rendered fluorescent by coiijugatioii with fluorescein isothiocyanate, showed the 'presence of fetal haemoglobiii in all red cells. The data suggest that the phenotype of the Hb Kenya trait resembles that of the Gy form of hereditary persistence of fetal haemoglobin rather than that of thalassaemia. Fusion haemoglobin chains are of unusual genetic significance because they provide insight into tlie linkage relationships between haemoglobiii genes and serve as tools for the iiivestiga: tion of gene transcriptioii aiid regulation of translation. Thus, the uncqual, nonhomologous crossovers that have led to the formation of the b p chains of the Lepore (Baglioni, 1962) aiid PS chains of the anti-Lepore (Ohta et al, 1971) haemoglobiiis have been instrumeiital to our understanding of the linkage rclationsliips between the p- and 8-chain loci and the patliogcnesis of the b p thalassaemias. A haemoglobiii of special genetic significance, Hb Kenya, has been described by Huisinaii et al (1972), Keiidall et af (1973) and Smith et a1 (1973). This variant contains non-a chaiiis that are 7-likc in sequence at residucs 1-80 and p-like at residues 87-146. Conscqueiitly, the production of this mutant is explicable in terms of a nonhomologous crossing-over between y- aiid p-chain genes. The discovery of Hb Kenya thus provided the first unequivocal evidencc that p aiid y loci are closely linked in humans. Furthermore, the fact that tlie synthesis of fetal liaenioglobiii containing only a and Gy chains continues through adulthood in heterozygotes for Hb Keiiya supports the suggestion that P- and b-gene Correspondence: Dr George Stamatoyannopoulos, Professor of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington 98195, U.S.A.

55

56

P. E. Nute et a1

deletions may bc associated with coiitiiiuatioii of 7-gcne transcription ; dcletioii of these two genes may thus be the underlying defect in some forms of hereditary persistence of fetal haemoglobiii (HPFH) (Huisman et a[, 1969) aiid in F thalassaemia (Stamatoyaiinopoulos et a!, 1971). hi this report, we describe findings from a family with Hb Kenya that was discovered during a population study in Uganda. Affccted members of this family possessed levels of the y! cliaiii that were coiisiderably higher than those in subjects of earlier rcports. Like other heterozygotes for Hb Kenya, the Ugandan carriers of this variant all had raised levcls of Hb F whose y chains were of the Gy type. Investigation of the distribution of Hb F ainoiig erythrocytcs, using moiiospccific aiiti-Hb F antibodies rendered fluorcscciit by conjugation with fluorescein isothiocyanate, indicated that Hb F was present in all red cells from the Ugandan heterozygotes. In this respect, the phenotype of the Hb Kenya trait is more like that of HPFH than that of F thalassaemia. MATERIALS AND METHODS

Haemutologic, electrophoretic arid chvoriiatographic tcchniqr ies. Blood samplcs wcre collectcd in ACD and flown uiidcr rcfrigeratioii to Scattlc. Rcd-cell haemolysates wcre screcned by starcli-gel electrophorcsis in Tris-EDTA-borate, pH 8.6 (Weatherall & Clegg, 1972), and whole bloods were subjectcd to routiiic haematological analyses by standard techniques. Electrophoresis of haemolysates on ccllulose-acetate strips, elution of Hbs A, A, and Kenya, and spectrophotonictric dctcrmiiiatioii of tlic haemoglobin coiiceiitratioiis of the cluates provided estimatcs of the relative coiiceiitratioiis of these tlirce haemoglobiiis in the red cells of all available family members. Levels of Hb F wcre ineasurcd by alkali denaturation (Betke et a/, 1959). All measureinents were carricd out in duplicate, and were confirmed by calculation of tlie proportions of Hbs A, A, F aiid Kenya in the red cells of the propositus following the elution of these haemoglobins from a coluiiiii of DEAE-Sephadex (A-so) (Huisman & Dozy, 1965). The chromatographic bed measured 2.0 x 45 cin, aiid resolution of the various compoiients in approximately 300 mg of liaemoglobin was achievcd by application of a pH gradient produced by mixing I litrc of 0.05 M Tris-HC1, pH 8.2 (starting buffer) with I litre of 0.05 M Tris-HC1, pH 7.3 (limiting buffer). All buffers contained IOO mg KCN/l. Stnrctiira2 analysis of Hb Kenya. The electrophoretically abnormal haemoglobiii in the propositus was isolated by chromatography 011 DEAE-Sepliadex as describcd above. Following conversion to globin by precipitation in cold, acidified acetone, the 01 and non-01 chains were separatcd by chromatography 011 CM-cellulose (Whatmaii CM-52) in 8 M urea buffers made 0.05 M in 2-mercaptocthanol (Clegg et d, 1968). The abnormal chain was aminoetliylated (Jones, 1964) and digested with trypsiii-TPCK (Worthingtoii Biochemical Corp.). Peptides wcre separated by paper electrophoresis at pH 6.4 (Ingram, 1958), followed by descciidiiig chromatography using a solvent of pyridine, isoamyl alochol, aiid water (Baglioiii, 1965). After staining with 0.03% ninhydrin in acctoiie, peptides were eluted from the maps with 6 N HCI (Saiiger & Tuppy, 1951)~hydrolysed in sealcd capillary tubes at 108°Cfor 24 h, and subjected to analysis on a Bcckmaii IZOBamino acid aiialyser. Striictirral unalysis of Hb F. Following clucidatioii of the structure of the abnormal liaemoglobin chain, Hb F from tlie propositus was aiialyscd to determine the presence of glycyl

Hacirioglobin Kenya

57

and/or alaiiyl residues in position I 36 of its y chains. After acid-acetone precipitation, the ether-dried globin was cleaved with cyanogen bromide; tlic C-terminal 7-chain fragiiieiits (yCB-3) wcrc isolated by gel filtration in a 1.8 x 292 ciii column of Scpliadex G-SO(fine), equilibrated and developed with 10g/l. formic acid (Nute ct nl, 1973). The YCR-3 fragments were recovered by lyophilizatioii aiid hydrolysed in duplicate at 108°C in 6 N HCl for 24 11. The glycine/alaninc ratios were determined followiiig amino acid analysis. Prcyaratioii m i d ynrificntioii ofJtioucscc'izt anti-Hh F antibodies. Details of the preparation and purification of antibodies against human Hb F are presented elsewhere (Wood et al, 1973). Briefly, aiittbodies against Hb F were raised in rabbits, and iioiispccific activity was removed from die antiserum by absorption against Hb A (plus the minor derivatives A,, AI,, etc.) aiid Hb A,, all of wliicli were linked to agarose beads (Poratli ct al, 1967). The aiiti-Hb F aiitibodies were removed from the absorbed antiserum by passage through a column of CNBractivated agarose to wliicli Hb F was bound. After tliorougli washing of tlie agarosc-Hb Faiiti-Hb F complex, tlic antibodies were dissociated from the agarose-Hb F by dcvelopniciit of tlie columii with CO,-saturated water (pH 4.0) at 4°C. As some H b F was also eluted during this step, further purification was carried out by loading tlie aiitibody-liaemoglobin mixture onto a column of CM-cellulose equiiibrated with C0,-water. Antibodies wcre eluted with 0.05 M NaHCO,, the haemoglobiii remaining bound to tlic column bed (Tozcr ef a!, 1962). After dialysis atid recovery by lyophilizatioii, tlie specificity of tlie anti-Hb F antibodies was tested separately against purified Hb F and Hb Kenya in Oucliterloiiy double-diffusion plates; tlie antibodies reacted with both liacmoglobiiis. An aliquot of tlie antibody solution was subsequently absorbed against purified, agarosc-bound Hb Kenya, the use of agarosebound liaciiiogiobin ensuring reinoval of both precipitating and iionprecipitating antibodies from tlic solution. All traces of reactivity with Hb Kenya, as measured in Oucliterloiiy plates, were rciiiovcd by this procedure, while the anti-Hb F activity was reduced. Samples of absorbed (against Hb Kenya) and unabsorbed antibodies were then coupled with fluorescein isotliiocyanatc (Nairn, 1969) and used to label erythrocytes in peripheral blood smears as described by Dan & Hagiwara (1967).

RESULTS Haeriiatologicul findings and rcsrilts of haemglobin uiialyscs. The patterns produced by starchgel electrophoresis of lysates from the propositus, his parents, aiid liis sibliiigs appear in Fig I. Tlie father's liacmoglobins were elcctroplioretically normal, whereas tlie remaining individuals all had high lcvcls of Hb F that migrated aliead of the abnormal compoiieiit subsequently identified as Hb Kenya. Hacmatological fiiidiiigs appear in Table I. Red cell morphology was iiormal in the propositus and his parents, but liis brother aiid sister showed mild microcytosis, liypocliromia and poikilocytosis. Proportioiis of Hb Kenya, measured spectrophotometrically followiiig elution of tlie various fractions from cellulose-acetate strips, raiigcd from 20.68 to 23.3 5% (iiicaii = 21.87%). Levels of Hb A, in tlie four affected persons ranged from 1.96 to 2.34% (mean = z.Is%), while Hb F (measured by alkali denaturation) constituted from 4.72 to 7.93% (mean = 7.06%) of the total liaemoglobin. Proportiotis of the various haemoglobins

P. E. Nirte et a1

58

of the propositus, determined by chromatography 011DEAE-Sephadex, were: A, = 1.20% ; F (plus A3) = 11.91%; Kenya = 31.43%; A = 65.46%. With the exception of the lower value for Hb A2, the chromatographic data correspond closely to those derived from cellulose-acetate electrophoresis. Structiire of the abitormal hacmoglobiti. Tryptic peptide maps prepared from the hydrolysed 11011-a chains of thc abnormal compoiieiit showed ninliydrin-positive spots in positions

0y-chain peptides b-chain peptides

t

:'. . ...._

I

; '

.;

...,..: .

-

Electrophoresis

2

I+

FIG2. Tracing of a map produced by the tryptic peptides of the aminoethylated, non-a chains from the abnormal haemoglobin of the propositus. Note the presence of ninhydrin-positive spots in the positions typical of those found on either normal y- or echain maps.

TABLE I. Haematological data and results of haemoglobin analysis

Source

Father Mother Propositus Sister Brother

Age (yr) 35 30 8

6 4

Hb (g/d)

RBC ( x IO"/~.)

PCV

MCV

(fl)

13.9 11.3 10.1 11.9 9.9

4.93 4.03 3.76 4.92 4.03

0.42 0.33 0.29 0.35 0.30

85.2 81.9 77.1 71.1 74.4

MCH (pg)

MCHC

H6Az

H6 F

H6 Kenya

(gi4

(73

(94

(%)

28.2 28.0

33.1 34.2 34.9 34.0 33.1

2.78 2.14 2.17 1.96 2.34

4.72 7.93 7.82 7.76

26.9 24.2 24.6

1.11

0

23.35 21.99 21.44 20.68

typical of either P- or 7-chain patterns (Fig 2). Judging from their positions on the maps, peptides numbered I-Ioa (using the numbering system employed by Smith ct al, 1973) were identical to peptides 1-10 (consisting of residucs 1-83) of normal y chains. Compositional analyses of peptides 3 , 9 and Ioa (Table 11) confirmed this judgment. The positions of peptides Iob-15, and amino acid analyses of Iob, 12b and 14(Table 11) indicated that residues 83-141

Haemoglobin Kenya

+ enya 2

FIGI. Patterns produced by starch-gel clectrophoresis of haeinolysatcs from the propositus (P) and his father (F), mother (M), sister (S) and brother (B).

(Facing p 58)

P. E. Nute et a1

FIG 3. Pcripheral blood snicar froni thc propositus stained with fluorescein-labelled anti-Hb F antibodies not absorbcd against Hb Kcnya. Note the uniformly bright fluorescence ovcr all red cells. FIG 4. Peripheral blood sinear from the propositus stained with fluorescein-conjugated anti-Hb F antibodies after absorption of the antibodies against purified Hb Kenya. Note fluorescence over all red cells, although its intensity is appreciably less than that depicted in Fig 3 , and the variation in fluorescencc from cell to ccll. FIG 5 . Mixture of red cells from a normal individual and an Hb Kenya heterozygote, smeared and labelled with fluorescein-conjugated antibodies absorbed previously against Hb Kenya. The proportion of labelled cells closely approximates the proportion of cells from the Hb Kenya heterozygote (about 10%) plus the proportion of Hb F-containing cells in the blood of the normal subject (2.6%). See text for details.

Ham oglobin Ket 1ya

59

of the abnormal chain were identical to those of the normal p chain. The residues occupyiiig positioiis 80 and 87 arc of particular iiiiportancc, as they differ in fl and y chains and delimit the area of transition from 7- to 0-like sequciices in thc fusion chains of Hb Kenya. The elcctrophoretic mobility of peptide Ioa was idcntical to that of peptide 10from normal y chains, indicating that aspartic acid was prcsciit in position 80, rather than thc asparaginyl residue found at this position in normal 0 chains. Whereas residues 81-86 are identical in fl and y chains, thrcoiiinc and glutamine reside at b87 and y 8 7 rcspcctively. Thc composition of peptide Iob (Fig 2, Table 11) indicated that thrconinc occupied position 87 of the abnormal chain. Hencc, thc crossover between y- and p-chain genes took place in the regions specifying residues 81-86, producing the fusion gciie of Hb Kenya (Huisinan ct d, 1972; Kendall et ul, 1973 ; Sniitli ct al, 1973).

Asp Thr

2.00 0.90

Ser

Glu GlY Ala Val Ile Leu Phe LYS AE-Cys His

2.16 2.93 0.93

A%

0.91

2.02

0.92

I .07

1.02

1.04 1.89 0.97 1.09 0.99

I .oo

1.02

0.91 0.90 1.08

1.08 3.96 2.78

2.00

1.00

1.09

0.98 0.97 0.86 0.84

1.10

2.06

0.94 0.94

0.96 0.91 1.98 0.91

2.00

0.96 0.87

1.00

1.00

1.00

1.93

1.06

Striicfrirc ofHb F. Duplicatc analyses of the y CB-3 fragments from the Hb F of the propositus yielded glyciiie/alanllie ratios of I .05/2.03 and 1.08/2.00, illustrating that position was occupicd solcly by glycinc while positions 13 8 and 140 contained the two alanyl residues norm3lly found in all human y CB-3 fragments. These observations are in complete accord with those made on the Hbs F of otlicr hetcrozygotes for Hb Kenya (Huisman c t d,1972; Kendall ct a/, 1973 ; Smith et d , 1973) and suggest that the Ay gene was iiivolved in the crossover responsible for the anomalous chain of Hb Kenya. Distribiifion of Hh F’ anzorg red cells. Antibodies against Hb F, absorbed against normal adult haemoglobins, were cross-reactive with Hb Kciiya in Ouchtcrloiiy plates. When thesc antibodies were conjugated with fluoresccin isothiocyanate and applied to blood sinears from heterozygotcs for Hb Kenya, uiiiforinly bright fluorescence was observed over all erythrocytes (Fig 3). Given the extent to which the noii-cl chains of Hb Kciiya are identical to those of Hb F, the likelihood that this result reflected the detcction of both Hbs F and Kenya in red cells seemed quite strong. Hence, a second aliquot of the antibody solution was

60

P. E. Nute et a1

absorbed against purified Hb Kenya to remove all antibodies that cross-reacted with tlie abnormal haemoglobin. The resulting antibody preparation showed no traces of anti-Hb Kenya activity in Ouchtcrloiiy plates; moreover, much of the anti-Hb F activity was also eliminated. Although quantitative studies were not performed, these results suggest that the most strongly antigenic sites presented by chains, when combined with a chains to forni the intact Hb F tetramer, involve residues occupying the first 80 positions in the 7-chain sequence. However, tlie presence of antibodies reactive against areas of the C-terminal portion of the y chain is illustrated by the fact that all cells from Hb Kenya heterozygotes still fluoresced upon application of fluorescent antibodies from which all traces of anti-Hb Kenya activity had *beenremoved, although the intensity of fluorescence was less and varied more from cell to cell than it did prior to absorption against Hb Kenya (Fig 4). Further tests conducted on mixtures of cells from normal subjects and heterozygotes for Hb Kenya illustrated the specificity of the reaction betwecii cells and fluorescent antibodies. Cells from an Hb Kenya heterozygote were mixed with those from a normal individual (2.6% ofwhose erythrocytes bound fluorescent anti-Hb F antibodies) in an approximate ratio of I :10; smears of the mixture were exposed to fluorescein-conjugated antibodies previously absorbed against Hb Kenya. In three separate smears, fluorescent cells constituted 11.7, 13.2 and 14.4% of the 2000 cells counted per sinear (Fig 5 ) , further indicating that all cells from Hb heterozygotes contained Hb F.

DISCUSSION Haenioglobiii Kenya has been found in a total of 18 individuals from western Kenya (Huisman et al, 1972; Keiidall et al, 1973 ; Smith et al, 1973). Of these, four were also heterozygous for the pS allele and showed no signs of Hb A synthesis, an observation suggesting that no pchain gene lies in cis to the yp fusion gene. Analyses of the Hbs F of six affected individuals showed that their chains had only glyciiie in position 136 (Schroeder et al, 1968; Huismaii et al, 1972; Keiidall et al, 1973 ; Smith rt al, 1973), as did the y chains of the propositus described in the present report. Structural analyses of the non-a chains of Hb Kenya and the y chains of tlie Hbs F of affccted individuals support the contentions that the p- and y-chain genes are linked, and that the Hb Kenya variant originated by nonhomologous crossing over between the A? and /? loci, producing a deletion chromosome bearing only the Gy and AyP genes (Huisman et al, 1972; Keiidall et al, 1973; Smith et al, 1973). The discovery of Hb Kenya has shed light on the molecular mechanisms underlying the phenotype of hereditary persistence of fetal haemoglobin (Kendall et al, 1973 ; Smith et al, 1973). Tn the majority of heterozygotes for the African variety of HPFH, the Hb F contains both Ay and Gy chains; however, some individuals produce only Gy chains (Huisman et al, 1969). Given closely linked Gy, Ay, 6 and p loci, one might attribute HPFH to deletions of various loci, deletion o f p and 6 loci producing the Ay Gy form of HPFH and deletion o f p , 6 and A? loci producing the Gy form of HPFH. The findings in cases of Hb Kenya lend support to this hypothesis. In this view, deletion of portions of the Ay and p (and possibly all of the 6) genes would lead to continuation of transcription of the Gy gene in cis to the fusion gene, producing a phenotype characteristic of the Gy form of HPFH (Huisman et al, 1969; Keiidall ef a!, 1973). This explanation for tlie presence of Hb F in heterozygotes is plausible, but there

Haernoglobirz Kenyn

61

are alternative possibilities. Synthesis of Hb F is characteristic of F tlialassaeiiiia (Stamatoyaiiiiopoulos ~f al, 1969) and is also found in scvcral (though iiot all) 6/3-(Lcpore)-thalassaemia hetcrozygotes. Moreover, continuation of the activity of the y-chain gene in cis to thc y/3 fusion gciie does not immediately suggest an HPFH type of defect, since a similar situation might also exist in a /3y thalassaeiiiia (Staniatoyaiiiiopoulos, 1971) ; in addition, synthesis of y chains directed by gciies iii cis to a fl-thalassaemia dctcrmiiiaiit has also been postulated (Huisiiiaii et al, 1971). The results of red-cell labelling by fluorescent antibodies provide evidence that tlie unequal crossover uiiderlyiiig tlic production of Hb Kenya yields a plieiiotype more like that of an HPFH tliaii a thalassacmia. Distributions of Hb F ainoiig crythrocytes, as well as erythrocytic morphology and tlic values of M C H aiid MCV, are iiistruineiital in distinguishing between HPFH and F thalassaciiiia (Weatherall & Clegg, 1972; Stamatoyaiiiiopoulos ct a / , 1969). Lcvcls of Hb F in lieterozygotes for F tlialassaemia can be as high as those in lietcrozygotes for the Grcck (Ar) typc of HPFH. However, with the acid elution technique, thc distribution of Hb F in Ftlialassaemia is heterogeneous whilc in HPFH, Hb F is dctcctcd in every rcd cell, although cells appear to vary in the amounts of Hb F they contain (Fessas & Stamatoyaiiiiopoulos, 1964). Thcsc diffcrciiccs in distribution of fetal haeiiioglobiii in red cells are also observed whcn samples from F-thalassaemia and HPFH lieterozygotcs arc studied with fluorescent antibody procedurcs (Wood et al, 1975 ; Staiiiatoyannopoulos ef al, unpublished observations). The prcsciice of Hb F in all rcd cells froin lieterozygotes with clcvatcd proportioiis of this haeiiioglobiii is thus coiisidered a characteristic of HPFH. Kciidall et al (1973) and Smith ct al (1973) have uscd tlic acid-elution tcchniquc to deiiioiistrate that Hb F is prcsciit in all red cells from licterozygotes froin Hb Kenya. Howcver, the ‘liomogciicous’ distribution of fetal liaeiiioglobiii observcd may reflect a tciidciicy of tlie a,(yP), haemoglobiii to remain, like Hb F, in the erythrocyte. Siiicc tlic acid-clutioii propcrtics of Hb Kenya arc uiikiiowii, estimation of proportioiis of Hb F-coiitaiiiiiig cells by the method of Klcihaucr et al(1957) may be unreliable in the preseiicc of this abnormal liacmoglobiii. For this rcasoii, we employed fluorescent antibodies to dcteriiiiiie if the 5-8% Hb F in tlie erytlirocytcs of hcterozygotes for Hb Kciiya were licterogciicously or liomogeiicously distributcd. Our experiments uiiainbiguously deinoiistratc that Hb F is present in all rcd cells from Hb Kenya heterozygotcs, a finding coiisistciit with thc interpretation that Hb Kenya trait more closcly resembles an HPFH than a SP thalassacmia. The low levels of y/3 chains found in all Hb Kenya lieterozygotcs dcscribed previously leads one to suspect that the phenotype of Hb Kenya trait might be that of a /3y tlialassacinia. Haeinoglobiii Kenya coiistituted 5.7% of the total liaemoglobiii in the hcterozygote studied by Kclidall ct al(1973) ; this corresponds to only I .4 pg of H b Kenya or 0.7 pg of y/3 chain pcr cell. The average proportion o f H b Kenya in the hctcrozygotcs aiialysed by Smith et a[ (1973) was 9.18%, a level corrcspoiidiiig to 2.3 pg of the abnormal liaeinoglobiii or 1.14 pg of tlie fusion chain per cell. Such low lcvcls of Hb Kenya are comparable to the amounts of Sp chain in tlie cells of lietcrozygotcs for Hb Lepore (Fessas et al, 1962). Lcvels of Hb Kenya in affectcd members of the Ugandan family described herein arc in striking contrast to those yrcseiited above, raiigiiig from 20.68 to 23.35%, with 5.67 pg of Hb Kenya or 2.83 pg of the y P chain pcr ccll. These levels are comparable to thosc of the PS chains in heterozygotes for anti-Lepore liaemoglobiiis (Olita et al, 1971 ; Badr ct al, 1973). W e can offcr no explaiiatioii

62

P. E. Nute et a!

for the different levels of Hb Kenya described in the subjccts of this arid previous reports (Keiidall et al, 1973; Smith et a!, 1973). Our demonstration of the homogeneous distribution of Hb F among red cells from Hb Kenya heterozygotes and of levels of Hb Kenya approximating those of the anti-Lepore haemoglobiiis support the suggestion by Kendall et a! (1973) and Smith ct al(1973) that the Hb Kenya phenotype is etiologically related to the Gy form of HPFH, the sole difference being in the extciit of the region deleted from the series of linked 11011-a chain genes. ACKNOWLEDGMENTS

This work was supported by grants GM-15253 and RR-00166 and coiitracts N O I - E S - ~ - ~ I ~ I and NOI-CM-71 343 from thc National Institutes of Health, U.S. Public Health Service. REFERENCES BADR,F.M., LORKIN,P.A. & LEHMANN, H. (1973) Haemoglobin P-Nilotic containing a 8-6 chain. Nature: N e w Biology, 242, 107. BAGLIONI, C. (1962) The fusion of two peptide chains in hemoglobin Lepore and its interpretation as a genetic deletion. Proceedings of the National Academy of Sciences ofthe United States ofAmerica, 48, 1880. BAGLIONI, C.(1965) Abtiormal human hemoglobins. X. A study of hemoglobin Lepore,,,,,,. Biochiririca et Biophysica Acta, 97, 37. I. (1959) EstimaBETKE,K., MARTI,H.R. & SCHLICHT, tion of small percentages of foetal hacnioglobin. Nature, 184, 1877. CLEGG, J.B., NAUGHTON, M.A. & WEATHERALL, D.J. (1968) Separation of the LY and /+chains of human haemoglobin. Nature, 219, 69. DAN, M. & HAGIWARA, A. (1967) Detection of two types of hemoglobin (Hb A and Hb F) in single erythrocytes by fluorescent antibody technique. Japanese Journal of Hnnian Genetics, 12, 5 5 . FESSAS,PH. & STAMATOYANNOPOULOS, G. (1964) Hereditary persistence of fetal hemoglobin in Greece. A study and a comparison. Blood, 24, 223. FESSAS, PH., STAMATOYANNOPOULOS, G. & KARAKLIS, A. (1962) Hemoglobin ‘Pylos’: Study of a hemoglobinopathy resembling thalassemia in the heterozygous, homozygous and double heterozygous state. Blood, 19, I. HUISMAN, T.H.J. & DOZY,A.M. (1965) Studies on the heterogeneity of hemoglobin. IX. The use of tris(hydroxymethy1)aminomethane-HC1 buffers in the anion-exchange chromatography of hemoglobins. Journal of Chromatography, 19, 160. S., HUISMAN, T.H.J., SCHROEDER, W.A., CHARACHE, BETHLENFALVAY, N.C., BOUVER, N., SHELTON, J.R., SHELTON, J.B. & APELL,G. (1971) Hereditary persistence of fetal hemoglobin. Heterogeneity of fetal hemoglobin in homozygotes and in conjunction

with b-thalassemia. N e w England Jorrrrzal ofMedicine,

285, 711. HUISMAN, T.H.J., SCHROEDER, W.A., DOZY,A.M., SHELTON, J.R., SHELTON, J.B., BOYD,E.M. & APELL, G. (1969) Evidence for multiple structural gcnes for thc gamma-chain of human fetal hemoglobin in hereditary persistence offetal hemoglobin. Annals of the N e w York Academy of Sciences, 165, 320. HUISMAN,T.H. J., WRIGHTSTONE, R.N., WILSON, J.B., SCHROEDER, W.A. & KENDALL, A.G. (1972) Hemoglobin Kenya, the product of fusion of y and B polypeptide chains. Archives of Biocheniistry and Biophysics, 153, 850. INGRAM,V.M. (1958) Abnormal human haemoglobins. I. The comparison of normal human and sickle-cell haemoglobins by ‘fingerprinting’. Biochiniica et Biophysica Acta, 28, 539. JONES,R.T.(1964) Structural studies ofaminoethylated hernoglobins by automatic pcptide chromatography. Cold Spring Harbor Syniposia on Quantitative Biology, 29, 297. KENDALL, A.G., OJWANG,P.J., SCHROEDER, W.A. & HUISMAN,T.H.J. (1973) Hemoglobin Kenya, the product of a y-p fusion gene: studies of the family. American Joitmal of Human Genetics, 25, 548. E., BRAUN,H. & BETKE,K. (1957) KLEIHAUER, Demonstration von fetalem Hamoglobin in den crythrocyten eines Blutausstrichs. Klinische Wochcnschriji, 35, 637. NAIRN,R.C. (1969) Appendix. Fluorescent Proteirz Tracing (ed. by R. C. Nairn), pp 303-309. Livingstone, Edinburgh. NUTE,P.E., PATARYAS, H.A. & STAMATOYANNOPOULOS,G. (1973) The Gy and Ay hemoglobin chains during human fetal development. American Jonrnal of Human Genetics, 25, 271. OHTA,Y., YAMAOKA, K., SUMIDA,I. & YANASE, T. (1971) Haemoglobin Miyada, a 8-6 fusion pcptide

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