Proc. Natl. Acad. Sci. USAVol. 77, No. 11, pp. 6874-6878, November 1980Medical Sciences

Proportion of hemoglobin G Philadelphia (a268 Asn-.LYs#2) inheterozygotes is determined by a-globin gene deletions

(restriction endonuclease mapping/a-thalassemia/American Blacks/hemoglobin variants)

GWENDOLYN B. SANCAR*, BASIL TATSISt, MARISOL M. CEDENO*, AND RONALD F. RIEDER*t*State University of New York-Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203; and tQueens Hospital Center Affiliation of LongIsland Jewish-Hillside Medical Center, Jamaica, New York 11432; and Health Sciences Center, State University of New York, Stony Brook, New York 11794

Communicated by Helen M. Ranney, August 20, 1980

ABSTRACT In humans the a-globin genes are duplicatedand closely linked. Whereas individuals heterozygous for mosta-chain mutations possess approximately 25% abnormal he-moglobin, heterozygotes for the a-chain variant Hb G Phila-delphia synthesize either 33% or 50% Hb G. Both variable genedosage and interaction with a-thalassemia have been proposedto explain this observation. To differentiate between thesemodels, we have performed restriction endonuclease mappingand hematological studies on individuals with Hb G from fourfamilies. In every case the aG locus was carried on an EcoRIor EcoRI + BamHI fragment approximately 4 kilobases shorterthan that bearing the two linked aA loci of hematologicallynormal individuals. Bgl II digestion revealed that the a geneis the only a locus on the affected chromosome. Erythrocyteindices and a/# synthesis ratios indicated that the aG chro-mosome confers a-thalassemia. In addition to the aG gene,subjects who synthesized 33% Hb G possessed two aA genes onthe homologous chromosome and exhibited the mild form ofa-thalassemia trait ("silent carrier"). Subjects who synthesized50% Hb G possessed a single aA gene trans to the acy locus anddisplayed the more pronounced form of a-thalassemia trait. Onesubject, who synthesized 100% aG chains and had Hb G-HbH disease, was found to have a single nonfunctional a gene transto the aC gene. Thus the proportion of Hb G synthesized byheterozygotes is determined by interaction with a-globin genedeletions cis and trans to the aG locus.

The number and arrangement of the human a-globin geneshas been established by utilizing the techniques of molecularhybridization and restriction endonuclease analysis. In mostpopulations the a loci are duplicated and closely linked on

chromosome 16 (1-4), each diploid cell containing foura-globin genes. Deletion of variable numbers of a genes occursin subjects from several racial groups (2, 5-7) and is associatedwith a decrease or absence of a-globin synthesis characteristicof the different a-thalassemia syndromes (8). In Chinese andThai populations, the reduction in a-chain synthesis is generallyproportional to the number of genes deleted; individuals witha-thalassemia 2 ("silent carrier"), a-thalassemia 1 (a-thalas-semia trait), Hb H disease, and the Hb Bart's hydrops fetalissyndrome usually have deletions of one, two, three, or foura-genes, respectively (2, 5, 7).Hemoglobin from individuals heterozygous for a stable

structural at variant (aix) generally contains approximately 75%Hb A and 25% Hb X (1, 9), as predicted by the four a genemodel. However, several a variants are present in unusuallyhigh proportions, composing 30-50% of the circulating he-moglobin in heterozygotes. Included among these "high ex-

pression" variants are Hb J Tongariki (10), J Mexico (11), GPhiladelphia, Anantharaj, J Nyanza, G Chinese, J Rovigo, JCape Town (12), Q Thai (13), and Norfolk (14). Three different

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6874

mechanisms have been proposed to explain these observations:(i) increased transcriptional activity at the variant a locus orincreased translation of variant mRNA (15); (ii) interaction witha-thalassemia determinants cis or trans to the variant locus (16);and (iii) heterogeneity in the number of a-globin genes thatdoes not produce an imbalance in globin chain synthesis-i.e.,variable gene dosage (9, 17, 18). Subjects synthesizing 31% or55% Hb J Mexico appear to fit the first model because theseindividuals possess four a genes (15) and do not display clinicalstigmata or al// synthesis ratios suggestive of a-thalassemia(19). In contrast, both heterozygotes and homozygotes for HbJ Tongariki possess a single a locus on each chromosome andexhibit microcytosis, hypochromia, and reduced a/l mRNAratios compatible with a-thalassemia trait (20). The third modelhas been proposed by Baine et al. (18) to explain the resultsobserved for Hb G Philadelphia heterozygotes.Hb G Philadelphia (a68 Asn-Lys) is the most common a-chain

variant and occurs in approximately 1 in 5000 American Blacks(21). Most studies indicate that Hb G heterozygotes synthesizeeither 33% or 50% of the abnormal chain (17, 22), althoughthere is a single report of individuals who synthesize approxi-mately 20% Hb G (18). Genetic studies suggest that in mostsubjects the aG locus is the only functional a locus on the af-fected chromosome (16, 22, 23). Whether inheritance of theaG-bearing chromosome confers a-thalassemia has been thesubject of debate. Rieder et al. (23) and Milner and Huisman(22) have described Black subjects with Hb H disease in asso-ciation with Hb G, supporting the linked inheritance ofa-thalassemia and the aG locus. However, normal erythrocyteindices in subjects synthesizing 30% Hb G have been observedby several investigators (17, 18, 22), and Baine et al. (18) havereported balanced globin synthesis in individuals with 20%,30%, and 50% Hb G. We have performed restriction endonu-clease mapping studies on subjects from four families in whichHb G Philadelphia is segregating. The results indicate that theproportion of Hb G synthesized by heterozygotes is the resultof interaction with a gene deletions cis and trans to the aGlocus.

MATERIALS AND METHODSHematological Studies. Routine hematological values were

determined by using a Coulter Counter model S. Hemoglobinswere analyzed by starch gel electrophoresis at pH 8.5; thepresence of Hb H was confirmed by electrophoresis at pH 7.1(24). The proportion of Hb G Philadelphia was determined bycolumn chromatography on DE-52 DEAE-cellulose (What-man) (25) or DEAE-Sephadex (Pharmacia) (26). Globin wasprepared from the purified abnormal hemoglobin, and the

Abbreviations: MCV, mean corpuscular volume; MCH, mean cor-puscular hemoglobin; kb, kilobase(s).tTo whom reprint requests should be addressed.

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constituent chains were separated on CM-,cellulose columns;fingerprint and amino acid analyses were performed as de-scribed (27).

Globin chain biosynthesis was studied by in vitro incubationof peripheral blood with [3R]leucine (28) for 1-2 hr. Globin wasprepared from the entire hemolysate, including membranes.After separation by CM-cellulose chromatography (27), theradioactivity incorporated into each globin chain was deter-mined by liquid scintillation counting.

Molecular Techniques. Preparation of DNA from periph-eral blood leukocytes and from liver and spleen tissues of a fetuswith Hb Bart's hydrops fetalis syndrome, purification of totalglobin mRNA, and synthesis of [32P]cDNA were performed asdescribed (29). DNA was digested with restriction endonu-cleases, using the conditions recommended by the supplier, atan enzyme-to-DNA ratio of 2-3:1 (EcoRI, Miles; BamHI, BglII, and HindIII, Bethesda Research Laboratories, Rockville,MD). After ethanol precipitation, 30 jig of digested DNA wasapplied to a 0.8% or 0.85% agarose gel in 40 mM Tris-HCI/5mM sodium acetate/i mM EDTA, pH 7.8, and the restrictionfragments were separated by electrophoresis at 60 mA, 4VC,for 16-24 hr. Molecular weight markers of phage X DNA di-gested with EcoRI, BamHI, or HindIII were included in eachgel and were identified by ethidium bromide staining. Transferof DNA to nitrocellulose paper, hybridization to [32P]cDNA,and autoradiography were done as described by Kan and Dozy(30).

RESULTSHematological Studies. Fig. 1 shows the pedigree of each

study subject, and Table 1 presents the relevant hematologicdata. The presence of Hb G Philadelphia within each familywas confirmed by peptide mapping and amino acid analysis.In family A, II-5 had 94% Hb G, the remainder being Hb H,Hb G2, and Hb G/F (23). III-2 was heterozygous for Hb S andpossessed 23% Hb G, 13% Hb G/S, 25% Hb S, and 39% Hb A.Families B, C, and D each contained one subject with ap-proximately 30% Hb G and one subject with approximately 50%Hb G.

a-Thalassemia also was evident within each family. TheMCV and MCH values of subjects synthesizing 50% Hb G were

I

I:[

m

FAMILY A

II 2

II 44.7 32.11 2

FAMILY B

[JTO OT94~~~~~~3.529. 2 2

3.6 30.01 1

FAMILY C FAMILY D

Table 1. Hematological values for Hb G subjects andselected relatives

MCV, MCH, Synthesis ratiosSubject fi Pg al3 aG/laG + aA

Family AI-2*II-1II-2*II-5III-1III-2

Family BII-111-2

Family CI-1I-2II-1

Family DI-2II-1

72 21.067 20.666 20.461 17.361 20.477 25.9

ND0.820.760.630.730.87

1.tt

1.0t

0.31

75 24.3 0.75 0.5082 26.9 0.78 0.31

78 27.1 0.89 0.3081 27.7 0.82 t65 21.8 0.82 0.49

66 24.0 0.90 0.5076 28.0 0.96 0.34

MCV, mean corpuscular volume; MCH, mean corpuscular hemo-globin; ND, not determined.* These data are taken from ref. 22.t Subjects without Hb G.

within the range reported for a-thalassemia trait, whereas insubjects synthesizing 30% Hb G the hematologic indices werenormal or slightly decreased, as is frequently observed ina-thalassemia 2 heterozygotes or silent carriers (31). Except for1-2 of family D, deficient a-chain synthesis was observed forall 50% Hb G subjects and for 30% Hb G subjects in families Aand B. Subjects synthesizing 30% Hb G in families C and D hada/:f synthesis ratios within normal range. II-5 of family Asynthesizes 100% aG, has markedly decreased MCV and MCH,deficient a-globin chain synthesis, and 5-8% Hb H, consistentwith the diagnosis of Hb G-Hb H disease (23). Homozygosityfor Hb G was ruled out by the absence of Hb G in her mother(1-2) and one child (III-1). These data indicate that subject II-5has a single functional a-globin gene and this gene bears theG-Philadelphia mutation.

Restriction Endonuclease Mapping. A map of some of therestriction endonuclease sites around the human a-globin lociis shown in Fig. 2. Restriction endonuclease EcoRI cleaves DNAon each side of the linked a-globin genes, producing in hema-tologically normal individuals a 23-kilobase (kb) fragmentcontaining both a loci (refs. 4, 32, 33; Figs. 2 and 3). DNA fromHb G subjects of families A and B contained a 19-kb a-specificEcoRI fragment (Fig. 3). This fragment must bear the aG locusbecause DNA from II-5 of family A, whose single functionala locus bears the aG mutation, displayed only the 19-kba-fragment. DNA from III-2 of family A contained both a23-kb EcoRI fragment inherited from II-6 and a 19-kb frag-ment inherited from II-5. IH-2 synthesized 31% aG and appears

EH BgB_.1 ,5'. 4

a-thal 2 I Hb H disease f-J not studied

a-thal I [j Hb G Philadelphia ID deceasedFIG. 1. Pedigrees of four American Black families in which

a-thalassemia and Hb G Philadelphia were segregating. The per-centage of the total circulating hemoglobin that was Hb G is indi-cated.

6 10 20 30kb

FIG. 2. Map of some of the restriction endonuclease cleavage sitessurrounding the human a-globin genes. The relative positions of thesesites were taken from refs. 4,32, and 33; the exact positions have beenplaced to correspond with the fragments reported here. E, EcoRI; H,HindIII; Bg, Bgl II; B, BamHI.

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2:3 kb-

139 kb'

Ni A A 13Jj.;'7 III-2 11-2

13lI-i

FIG. 3. Autoradiogram of a-specific fragments obtained afterEcoRI digestion ofDNA from a hematologically normal subject (lane1), and Hb G subjects (lanes 2-5). All ,B-, y-, and b-specific fragmentswere of identical size in all subjects (not shown).

to have the silent carrier form-of a-thalassemia. The simplestinterpretation of these observations is that III-2 has two normala genes on the 23-kb EcoRI fragment and a single aG locus onthe 19-kb EcoRI fragment. Similarly, EcoRI digestion of DNAfrom II-2 of family B, who also synthesizes 31% aG, revealedboth 23-kb and 19-kb a-specific fragments, whereas DNA from11-1, who synthesized 50% aG, contained only the smallerfragment. Thus in family B the aG locus is also carried on a19-kb EcoRI fragment. The 19-kb band seen in II-1 mustcontain two fragments of similar size, one bearing an aA locusfrom one chromosome and the other the aG locus from thehomologous chromosome.

These results were confirmed by using EcoRI + BamHIdigestion of DNA. In normal individuals a single a-specific14.5-kb fragment is produced by a BamHI cut on the 5' sideand an EcoRI cut on the 3' side of the a-globin genes (ref. 4 andFigs. 2 and 4). The resulting fragment contains both linked aloci. A new 9.8-kb a-specific fragment was observed in DNAfrom all subjects who synthesized Hb G. Synthesis of 30% HbG was associated in every case with the presence of both the14.5-kb fragment and the 9.8-kb fragment, whereas all subjectssynthesizing 50% Hb G exhibited only the 9.8-kb fragment. Inaddition, DNA from II-5 of family A, with Hb G-Hb H disease,contained only the 9.8-kb a-specific fragment.The presence of a 9.8-kb fragment plus a 14.5-kb fragment

in III-1 of family A was unexpected. This pattern is consistentwith the presence of three a genes in spite of III-1 having thetwo-gene (a-thal trait) phenotype. His mother, II-5, does notpossess a 14.5-kb fragment, and therefore must have been thesource of the 9.8-kb fragment in III-1. However, because 111-1does not synthesize aG, his 9.8-kb fragment cannot be the

1--) kh *4 .4 k

3 kh

Fani- A 13Faor'! 2g 4

FIG. 4. Autoradiograms of DNA fragments containing globingenes following digestion with restriction endonucleases EcoRI andBamHI. The 14.5-kb and 9.8-kb bands are a-specific, as indicated bythe absence of these fragments in the DNA from a fetus with HbBart's hydrops fetalis syndrome. The 3.9-kb band is from the # locus(34). The origin of the 6.8-kb band seen in some hybridizations isunknown, although it may correspond to the J,#1 gene recentlyidentified by Fritsch et al. (35). In each experiment in which the6.8-kb band appears, it is present inDNA from all subjects, includingnormal and hydrops samples.

aG-bearing 9.8-kb fragment in II-5. We conclude that both II-5and 111-1 of family A carry a nonfunctional a locus on a 9.8-kbEcoRI + BamHI fragment that segregates independently ofthe aG locus. A more detailed analysis of the segregation of thislocus will be published elsewhere.The results of EcoRI and EcoRI + BamHI digestions indicate

that the chromosome carrying the aG locus has a deletion of 4-5kb between the BamHI site 5' to the a-globin genes and theEcoRI site 3' to these loci. Hematologic studies on 11-5 of familyA indicate that the aG locus is the sole functional a gene on theaffected chromosome. Thus it appears possible that part or allof one a locus is included in the deletion carried by the aGchromosome. To test this hypothesis, DNA from Hb G subjectswas digested with restriction endonuclease Bgl II, which cutsbetween the linked a loci. DNA from a normal subject yieldstwo a-specific fragments of 12.5 and 7.7 kb (Figs. 2 and 5), theformer containing the 5' a-globin gene and the latter the 3'a-globin gene (33). A new 15.8-kb a fragment was observedin all Hb G subjects (Fig. 5). In addition to the 15.8-kb fragment,the normal 7.7-kb a fragment also was present in DNA ob-tained from 30% aG subjects, but was absent from subjectssynthesizing 50% and 100% aG. Although the 12.5-kb a frag-ment was not completely resolved from the 13.3-kb y fragment(Fig. 5), comparison of the width, intensity, and migration ofthe bands in this region with these aspects of the normal andhydrops controls suggests that the 12.5-kb band is absent insubjects synthesizing 100% and 50% aG-Philadelphia, and ispresent in 30% aG subjects. The appearance of only a 15.8-kba fragment in DNA from subjects synthesizing 100% or 50%aG indicates that the intergenic Bgl II site has been lost fromthe chromosome bearing the aG locus through either mutationor deletion. Mutation of the Bgl II site would produce a frag-ment of 20.2 kb (12.5 kb + 7.7 kb), while a deletion of 4-5 kb

( )ritsil - * " '

Vskh

II.. . MOkbk:.:i1.4 :

Ni; 1k-\) 13

drj, II-.. 11-2

~./;A.

a'6..i

11-1 1-2 2' 1i4j1tim -

Z,-41 )t cx

FIG. 5. Autoradiograms of DNA fragments containing globingenes after digestion with restriction endonuclease Bgl H. The 12.5-kband 7.7-kb fragments seen in a normal subject (lane 1) and the 15.8-kbfragment seen in Hb G and thalassemic subjects (lanes 3-7) area-specific as indicated by their absence in DNA from a fetus with HbBart's hydrops fetalis syndrome (lane 2). The 13.3-, 8.5-, and 5.1-kbfragments are derived from they, 6, and B loci, respectively (36). Al-though the 12.5-kb and 13.0-kb fragments did not separate well in thisexperiment, comparison of the width and intensity of these bands inhydrops DNA and DNA from subjects AII-5, DI-2, and an a-thalas-semia 2 homozygote with those in DNA from a normal subject andsubjects BII-2 and DII-1 indicates the presence ofboth bands in theformer group and the absence of the 12.5-kb band in the lattergroup.

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that included the intergenic Bgl II site would produce a 15- to16-kb fragment (20.2 kb - 4 or 5 kb) as observed in the Hb Gsubjects. This deletion must include either one of the two a locior a portion of both. The results of Bgl II + HindIII digestionof DNA from subjects synthesizing 50% Hb G confirm this in-terpretation. HindIII + Bgl II digestion of normal DNA pro-duces a-specific fragments 10.5, 2.0, 1.7, and 4.7 kb in lengththat contain, respectively, the 5' half of the 5' a gene, the 3' halfof the 5' gene, the 5' half of the 3' gene, and the 3' half of the3' a gene (ref. 34 and Fig. 2). In Hb G subjects the 1.7-kb and2.0-kb fragments are missing and no new a-specific fragmentsappear (data not shown). Thus the equivalent of a complete alocus has been deleted from the chromosome carrying the acGlocus. Furthermore, Bgl II (Fig. 5) and Bgl II + HindIII re-striction patterns obtained from subjects synthesizing 50% or100% Hb G are identical to those observed for American Blackshomozygous for a-thalassemia 2.

DISCUSSIONThe results reported here demonstrate that.in subjects synthe-sizing either 30% or 50% Hb G Philadelphia the aG gene resideson a chromosome that lacks any other a locus. Several lines ofevidence indicate that in Blacks the aG chromosome and thea-thalassemia 2 chromosome (-a) are functionally equivalent:(i) 11-5 of family A, whose only functional a locus is a single aGgene, has the clinical stigmata of Hb H disease, while 111-2 hasinherited both the aG locus and the silent carrier form ofa-thalassemia from II-5; (ii) in II-I of family C, the interactionof an aG chromosome and an a-thalassemia 2 chromosomeproduces the clinical stigmata of a-thalassemia trait, indicatingthe functional equivalent of 2 a genes; (iii) most subjects syn-thesizing 50% Hb G have reduced MCV, MCH, and a/l syn-thesis ratios and all possess one a locus on each of two homol-ogous chromosomes, whereas subjects synthesizing 30% Hb Gappear to have mild a-thalassemia trait and bear two a locitrans to the aG locus. Thus the proportions of Hb G synthesizedby heterozygotes are the result of interactions with a deletionalform of a-thalassemia carried cis and trans to the aG locus.Ohene-Frempong et al. (37) have reached similar conclusionson the basis of restriction endonuclease mapping of membersof two Black families in which the aG gene was segregating.The conclusions reached here are entirely compatible with

the results of hematologic studies on a large number of Hb Gsubjects reported by Milner and Huisman (22) and Rucknageland Rising (17). In every case the erythrocytes of subjects syn-thesizing 40-50% Hb G, as well as those of a Hb G homozygoteand a Hb G-Hb H subject, were hypochromic, microcytic, orboth and, where synthetic studies were performed, a decreasein a-chain synthesis was observed. In contrast, the observationsof Baine et al. (18) are difficult to interpret in the context of thea-thalassemia model. As expected, these investigators founda negative correlation between the proportion of Hb G andMCV values in heterozygotes. However, two families in whichall members synthesized 20% Hb G were detected, and globinchain synthesis was balanced in all heterozygotes with 20%,30%, or 50% Hb G. It is possible that the subjects who synthe-sized 20% Hb G were heterozygous for a chromosome bearingthree aA loci, of the type described by ourselves (29) and others(33). Alternatively, it may be that in a small subpopulation ofAmerican Blacks the acG locus is adjacent to an aA locus, as theresult of an independent mutation or of a rare crossover event.Balanced globin chain synthesis in subjects synthesizing 30%Hb G is not incompatible with the a-thalassemia model becausethese individuals are predicted to be silent carriers; chain syn-thesis ratios of such subjects frequently fall within the rangeobserved for nonthalassemic subjects (ref. 31; I-1 of family C

and II-1 of family D in this study). In addition, the length ofexposure of erythrocytes to labeled amino acid and removal ofstroma prior to globin preparation in the studies of Baine et al.(18) may have produced an overestimate of a-chain synthesisdue to the loss of free fl-globin chains. Our own observations(unpublished) indicate that, at least in IT-5 of family A, the ratioof radioactivity incorporated into a chains relative to (3 chainsincreases during the first hour of incubation, apparently dueto the rapid degradation of excess (3chains. This effect may alsoaccount for the synthesis ratio observed in 11-1 of family D.The trans arrangement of a gene deletions responsible for

the a-thalassemia trait among American Blacks has been re-ported previously (29, 38, 39). Dozy et al. (38) have estimatedthat the frequency of the a-thalassemia 2 chromosome (-a)approaches 16% in this racial group. The loss of intergenic HpaI, Sac I, and Bgl II restriction endonuclease sites has been in-terpreted by Goossens et al. (33) as suggesting a gene deletionvia an unequal crossover, although simple deletion of an a locusand the neighboring intergenic sites is not ruled out. Whateverthe mechanism involved, the observation that EcoRI, EcoRI+ BamHI, Bgl TI, and Bgl II + HindIII digestions producesimilar restriction patterns in subjects synthesizing 50% Hb Gand subjects homozygous for a-thalassemia 2 (ref. 29 and un-published observations) suggests that the mutation that pro-duced the aG locus took place on an a-thalassemia 2 chromo-some. The relatively high frequency of Hb G in African andAmerican Blacks may then be due to the same selective pres-sures that have maintained the a-thalassemia 2 chromosomewithin the population.

Recently, Lie-Injo et al. (40) reported restriction endonu-clease mapping studies which indicate that the gene coding foranother "high expression" a-variant, Hb Q Thai, is also linkedto an a-globin gene deletion that confers a-thalassemia.Whether most or all high expression variants may be explainedon this basis awaits further investigation.

We thank Dr. Prawase Wasi, Siriraj Hospital, University of MedicalSciences, Bangkok, Thailand, for providing liver and spleen tissues froma hydropic newborn. This work was supported by National Institutesof Health Grant AM-12401 (to R.F.R.) and Grant 3-252 from the Di-vision of Hematology-Oncology of the Long Island Jewish-HillsideMedical Center (to B.T.).

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