A worldwide population study of the Ag-system haplotypes, a genetic polymorphism of human low-density lipoprotein

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Article

Reference

BREGUET, Georges, et al.

Abstract

The aim of this investigation is to examine the distribution of the Ag immunological polymorphism in human populations on a worldwide scale and to look for possible explanations of this distribution in the field of modern human peopling history and Ag-system evolution. Extensive Ag-antigene typings were carried out on 13 human population samples, including sub-Saharan African, European, west and east Asiatic, Melanesian, Australian aborigine, and Amerindian groups. Complete Ag-haplotype frequencies were estimated by maximum-likelihood-score procedures, and the data were analyzed by genetic distance computations and principal coordinate projections. With the exception of the Amerindian sample, the Ag polymorphism is shown to be highly polymorphic in all the populations tested.

Their genetic relationships appear to be closely correlated to their geographical distribution.

This suggests that the Ag system has evolved as a neutral or nearly neutral polymorphism and that it is highly informative for modern human peopling history studies. From the worldwide Ag haplotypic distributions, a model for the Ag molecular structure is [...]

BREGUET, Georges, et al. A worldwide population study of the Ag-system haplotypes, a genetic polymorphism of human low-density lipoprotein. American Journal of Human Genetics, 1990, vol. 46, no. 3, p. 502-17

PMID : 1689953

Available at:

http://archive-ouverte.unige.ch/unige:14331

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Am. J. Hum. Genet. 46:502-517, 1990

A Worldwide Population Study of the AgSystem Haplotypes,

^a

Genetic Polymorphism of Human Low-Density Lipoprotein

G. Breguet,* R. Butlert E. Butler-Brunnert and A. Sanchez-Mazas*

*Laboratoire deGenetique etBiometrie, DepartmentofAnthropology, UniversityofGeneva, Geneva; and tCentralLaboratory ofthe Blood Transfusion Service, SwissRedCross, Berne

Summary

The aim of this investigation is to examine the distribution of the Ag immunological polymorphism in hu-

man populations on a worldwide scale and to look for possible explanations of this distribution in the field of modern human peoplinghistory and Ag-system evolution. Extensive Ag-antigene typings were car-

ried out on 13 human population samples, including sub-Saharan African, European, west and east

Asiatic, Melanesian, Australian aborigine, and Amerindian groups. Complete Ag-haplotype frequencies

were estimated by maximum-likelihood-score procedures, and the data were analyzed by genetic distance computations and principal coordinate projections. With the exception of the Amerindian sample, the Ag polymorphism is shown to be highly polymorphic in all the populations tested. Their genetic relationships

appear to be closely correlated to their geographical distribution. This suggests that the Ag system has evolved as a neutral ornearly neutral polymorphism and that itis highly informative for modern human peopling history studies. Fromthe worldwide Ag haplotypic distributions, a model for the Agmolecular

structure is derived. According to this model and to the most recent results obtained from molecular data, the establishment of theAg polymorphism could be explained by several mutations and recombination

events between the haplotypes mostfrequently found in human populations today. As aconclusion, genetic and paleontological data ^suggest that the genetic structure of caucasoid populations (located from North Africa ^to India) may be the least differentiated from an ancestralgenetic stock. Worldwide genetic differentiations are properly explained as the results of westward and eastward human migrations from a

Near East-centered but undefined geographical areawhere modern humans may have originated. The im-

portance of Agpolymorphism analyses for the reconstruction of human settlement history and origins is discussed in the light ofthe main conclusions of the ^most recent genetic polymorphism studies.

Introduction

Fromthediscoveryof the ABO blood groups up to some recent estimations of the molecular variability at the DNAlevel, studiesonhuman geneticpolymorphisms havebecomeanimportantfieldof investigation improv- ing ourunderstanding of the microevolution of the mod- ernHomosapiens sapiensindistinct populations. Ge-

Received May 17, 1989; revision receivedOctober 24, 1989.

Address for correspondence and reprints: A. Sanchez-Mazas, Laboratoirede GenetiqueetBiometrie, DepartmentofAnthropol- ogy, University of Geneva, 12 Rue Gustave-Revilliod, CH-1227 Geneva, Switzerland.

0002-9297/90/4603-0013$02.00

neticpolymorphismswerefoundto occurfor the great majorityofhumanbloodproteins. Onecanevenspec- ulate thatgeneticallycontrolledvariants, which could be revealedbytheapplicationof appropriatemethods, existfor all knownserumproteins.It is notsurprising that geneticpolymorphismswerealsoobserved within the familyof human (and also animal) lipoproteins.

The lipoproteins represent one of themost complex groupsof human serum proteins. Theirclassification isbasedmainlyontheirbehaviorintheultracentrifu- galsedimentationanalysis.Accordingtothedensities, a distinction ismade between chylo- orlipomicrons, very-low-densityproteins(VLDL),low-densitylipoproteins(LDL), andhigh-densitylipoproteins(HDL).LDLs are more orless identical to the ^serumprotein class 502

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Ag-System Haplotypes

which, on the basisof its electrophoretical properties, was designated as

0-lipoprotein.

Variousstudies reportedintheliterature demonstrate the existence of genetic lipoprotein polymorphisms (Mourant etal. 1976). Oneof themostimportant and mostthoroughlyinvestigated systems is the Ag groups discoveredin1961 byAllison andBlumberg (1961). In theserumofamultitransfused patient (C. de B.), these authorsdemonstrated the presence of an isoprecipitin that reacted with 57%ofseraamongwhiteAmericans.

Family studies showed that the antigen, designated Ag(a), was inherited inanautosomal dominant fash- ion. Later, the serum protein carryingthe Ag(a) epitope was identified as LDL.

Two periods can be distinguished in the discovery of the Ag system. Between 1961 and1966the detection of Ag antigens was exclusively basedonimmunodif- fusiontechniques using humanserawithprecipitating antibodies. In 1966, Butler and Brunner (1966) de- scribeda passivehemagglutination assay to study Ag antibodies which would also detect nonprecipitating Agantibodies.Afirst exampleof suchanantibody was discoveredin1967byButleretal.(1967), whobymeans of thismethod were then able to detectaseriesof new Agspecificities. In 1974the 10 Agfactors depictedin table 1 wereknown, andno additional Agspecificity hasbeen observed since that date. Inour table 1, the absenceof the first observed Agfactor, namely,Ag(a) factorof Allison andBlumberg (1961),isimmediately apparent. Ina reexaminationof the anti-Ag(a) serum (C. de B.) with the aid of absorption experiments, Hirschfeldet al. (1964) showed that this serum con- tained isoprecipitins reacting against three different Ag specificitieswhichthey calledAg(ai),Ag(x)and Ag(z).

Extended family and population studiesconducted byButler and alsosomeother authors revealed thefol- lowingfacts(reviewedinButleretal. 1974, 1978): All Agfactors are inheritedaccordingto a dominant au- tosomal modeoftransmission.The genesdefiningthe Agantigens areallclosely linked. If the Ag factors of defined individualsare arranged inpairsoftwo antigens,accordingtothe fourphenotypicalcombinations (Ag(++),Ag(+-),Ag(- +), and Ag(- -),one canob- serve that certain Ag(--) combinations, namely, Ag(x-y-),

Ag(ai

-d-), Ag(c-g-), Ag(t-z-), and Ag(h-i-), never occur, whereas practically all other phenotypicalcombinations have been encountered. This means that the Ag antigens behave correspondingto thefollowingfive pairs of antithetical factors: Ag(x)/

Ag(y), Ag(al)/Ag(d), Ag(c)/Ag(g), Ag(t)/Ag(z), and Ag(h)/Ag(i).

Table I

Ten Antigensof the Agsystem Yearof

Accepted Notation Detection Reference

Ag(x) ... 1964 Hirschfeld andBlomback 1964 Ag(a1) ... 1964 Hirschfeld et al. 1964 Ag(z) ... 1964 Hirschfeld et al. 1964 Ag(y) ... 1966 Contu 1966; Okochi 1967 Ag(t) ... 1966 Hirschfeld et al. 1966 Ag(c) ... 1967 Butleret al. 1967 Ag(d) ... 1970 Butler et al. 1970b Ag(g) ... 1970 Butler et al. 1970a Ag(h) ... 1973 Butler et al. 1973 Ag(i) ... 1974 Butler andBrunner 1974

Interpreting the geneticdata,Butler and co-workers haveproposed a chromosome model for the Ag system by assuming the existence offive closely linked loci, each with twocodominant alleles. The sequence of the five loci is of course entirely arbitrary. Apoprotein B100 (apo B)isthe major proteinof the cholesterol-richLDL.

Thesuccessful cloning, by several researchers (Deeb et al. 1985; Huang et al. 1985; Lusisetal. 1985; Black- hart et al. 1986;Cladaras et al. 1986; Knott etal. 1986;

Law et al. 1986),of the apo B gene has made possible the studyof the genetic variations at the apo B gene locus, which is located in the p24 region of chromosome 2 (Knott et al. 1985; Law et al. 1985).

Recently, the discoveries of completecorrelations between some RFLPand some Ag-epitopepolymorphisms have led several authors (Berg et al. 1986; Ma et al.

1987; Dunning et al. 1988; Huang et al. 1988; Wang etal. 1988; Xuetal. 1989) to proposeamore accurate topology for the Ag genetic model. Themaincharac- teristicof thistopologyisanintermediate(Ag(al )/(d)- Ag(t)/(z) position for locus Ag(i/h) (fig. 1).

In the pastfew years several groups of researchers haveprepared monoclonal antibodiestohumanLDLs produced by mouse/mouse hybridoma cells. Schumaker et al. (1984) recorded binding profiles for individual LDLs obtained with a battery of such monoclonals.

In further studies, one of the monoclonals of this set turned out tohave anti-Ag(c) specificity(Robinsonet al. 1986; Tikkanen et al. 1986).Schlapferetal. (1987) wereabletoproducemonoclonals which detect the epi- topesAg(c) andAg(d). In conclusion,onthe basisof the reagents availablefor thetimebeing,typingof three of the 10 Ag factors-Ag(c), Ag(g), and Ag(d)-by means of monoclonal antibodies is now possible.

503

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Breguet et al.

Ag EPITOPE PAIRS:

kb: 0 2 4 6 8 10 12 14

|c a h t |

J I

NUCLEOTIDE: 421? 1'981

AMINO ACID SUBSTITUTION: Val/Ala

CODON: 591

Figure I TheapoB genemodel anditscorrespondence with th

Material and Methods

Thepopulation samplesaredescribedintable2.As quoted, we have used already published data (Swiss, Tibetan, Senegalese, Bantu, Indian, and Amerindian samples),aswellas new results ofAg typings on sera

from China, Indonesia, Australia, and western New Guinea. In these different countries, the collection of blood specimen was done in the field. The sera were

frozen andsentunder refrigerationtothe Central Lab-

oratoryof theBlood Transfusion Serviceof the Swiss RedCross in Berne.The blood collection of Turks and Tamils was done directly in Switzerland.

The notation used, which has been adopted by themajorityof authors in thisfield, isthe following:

(1)single Agfactors: Ag(x), Ag(y),etc.; (2) Ag ^genes

i

II

tz

1 '041 12'669 13816 Arg/Gln Glu/Lys

3611 4154

Agx, AgY,etc.; (3)partialAgphenotypes:Ag(x+y+), Ag(ai +d-), etc.; (4) complete Ag phenotypes: Ag (x-y+al-d+c+g+t+z-h-i+)^orphenotype^no.222,

etc.(the complete phenotype numbersusedcorrespond

toourinternal code listingall of the 243possiblecom-

binations);(5)partial haplotypes: Agxdz, etc.;(6)com-

plete haplotypes: AgYdrti, etc. A total of32 complete haplotypesarepredictableontheoretical grounds. The

occurrence of 14 of them has been either deduced directly ^or postulated from the set of phenotypes recorded thus far.

Theseraweretypedusing thepassivehemagglutina- tion/inhibitionassay(PHAIA)describedbyButler and Brunner (1966). Since then the technique has been modified andimproved. Thetwomainchangesare(1)

Table 2

Ag-System Polymorphism: Populations Studied,Presented inChronological Order ofLaboratoryTests(1973-88) No. of

Population Individuals Reference

Swiss: blooddonors, Berne (Switzerland)... ... 362 Butleretal. 1974 Tibetans: refugees livingin Switzerland ... ... 86 Butleretal. 1974 Senegalese: residents of Dakar (Senegal)... ... 100 Butleretal. 1974 Bantu: individuals from various ethnic groups of South Africa . 94 Hitzerothetal. 1980 Indians: immigrants livinginTransvaal(South Africa) ... 70 Hitzerothetal. 1980

Kraho: Amerindians from Brazil ... ... 66 Salzanoetal. 1981 and presentstudy Chinese: individuals livinginShanghaior inIndonesia ... 141 Presentstudy

Balinese: inhabitants of Bali (Indonesia) ... ... 152 Presentstudy EastIndonesians: individuals from variousethnic groups of

EasternIndonesia (Sulawesi-Flores-Timor) ... ... 80 Presentstudy Mowanjum: aboriginesfrom northwestern Australia ... 86 Presentstudy Papuans: individualsfrom various ethnic groups of central

westernNewGuinea (IrianJaya) ... ... 76 Presentstudy Turks: individuals from TurkeylivinginSwitzerland ... 80 Presentstudy Tamils: Dravidian Indians from SriLanka... ... 71 Presentstudy

Totalno. of individuals sampled ... 1,464 504

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thereplacement ofbisdiazoted benzidine by chromium chloride for thefixation of LDLs to the red cells and (2) the use ofmicrotiter plates. Details of the full new procedure have been described by Butler (in press).

All the sera weretested on nine common Ag specificities i.e., Ag(x), Ag(y), Ag(ai), Ag(d), Ag(c), Ag(g), Ag(t), Ag(z), and Ag(h), almost the complete panel.

We also tested the Ag(i) specificity both on all Swiss, Amerindian, Papuan, Turk, and Tamil blood samples and on the Ag(h+) samples for the other populations [Senegalese, Australian Mowanjum, Bantu and Indians, as Tibetans, Chinese, Balinese, and east Indonesians were allAg(h-)]. Thefew cases showing unspecific ag- glutination orambiguous diagnosis have not been taken into consideration in the results.

Phenotypedistributions for the 13 population samples are reported in table 3. They have been explained byaminimalnumber of haplotypes. For a few populations,different models were possible (see table legends).

Haplotype frequencies (tables4and 5) were calculated using the maximum-likelihood-scores method basedontheHardy-Weinberg equilibrium assumption.

The computer program used, GENEF 2, is Lalouel's adaptation of Yasuda's ALL-TYPE program.Allresults fit with the assumptionofHardy-Weinbergequilibrium.

Geneticdistanceswerecomputed using theformula Dxy = 1/2 Z(i=ltom) lxi

-yil,

where m is thetotal numberof haplotypes and wherexiandyiarethefrequen- ciesof the ithhaplotypeinpopulationsxand y, respectively.Thisis avariantof Manhattan distancedescribed by Nei (1987). For a discussion of the advantagesof using this distance, see Sanchez-Mazas et al. (1986).

These data were represented by means of principal coordinate analysis.

Results

Continental Genetic Structures

The results includedinthissectionrefertothecom- plete and partialhaplotypedistributionslisted,respectively, in tables 4 and 5.

Sub-SaharanAfricanpopulations.-ThetwoAfricansam- ples usedinthis survey havealready been discussed and themaindata havebeenpublished(Butleretal. 1974;

Hitzerothetal. 1980), but thecompletephenotypedis- tributions haveneverbefore been used for computing complete haplotypefrequencies. IntheSenegaleseand Bantu samples representing the sub-Saharan African populationsinour survey, we foundonly twohaplo- types linked with the geneAgx, namely, Agxalgti and Agxdgti,whosefrequencies(.040and .095,respectively,

for Senegalese and .020 and .039, respectively, for Bantu) areparticularly low compared with thefrequen- ciesobserved in other parts of the world. This observa- tion hasalready been brought to light for other African populations tested for the Ag(x) specificity alone - i.e., Senegalese, Angolans (Muller et al. 1974), or various ethnic groups from Nigeria(Heikenetal. 1974)- and for various ethnic groups from South Africatested for the Ag(x) andAg(y)specificities(Hitzerothetal. 1980).

To ouractual knowledge, Agx frequencies seem to be invariably low in sub-Saharan African populations, never exceeding .16.

Among the Senegalese sample, the most frequent complete haplotype, Agydgti, accounts for more than half (.58), and it accounts for more than two-thirds (.70)among the Bantusample. Such high frequencies are also found in Kraho Amerindians (see below).

Anotherstriking result is the presence of Agydgzi, a haplotype not yet found outside Africa. Whether this haplotype is widely distributed in this continent and absentinother parts of the worldshouldbe confirmed bymore extensivedata. Thiswouldbringamajorcon- tributiontotheunderstanding ofpeopling history and Ag-system evolution.

European populations.-Europeanpopulationsarerep- resentedby a singleSwisssample, whichislarge enough (N=362),however,togivegoodhaplotype frequency estimates. Twelveof the14haplotypesrecordedinhu- manpopulationsareobserved. The Europeanpopula- tiongeneticstructure isslightlydeviantfrom whatcan be observedinthepopulationsfrom theNear-Eastand India, withhigherAgya"9zi andAgydcti frequencies and lowerAgYdgti andAgxalrtg frequencies. Ifwecompare ourresults with thoseof other Europeanpopulations, wemust limit ourworktopartial haplotypefrequencies, such asthose obtained fromtests done with five Agspecificitiesonly: Ag(x), Ag(y), Ag(ai), Ag(t), and Ag(z). Table5reportspartialhaplotypefrequenciesfor bothourSwissandaSwedishsample(Hirschfeld1968):

thereis no striking difference between thetworesults.

The presence of thepartial haplotypeAgxalzamong the Swedesisprobablyanindicationof the presence of the uncommoncomplete haplotype Agxalgzl. Inthe Swiss sample, the Agx genefrequencyis .21,aresult similar to those forneighboring populations (Germans, .20 [Mulleretal. 1974];Italians,.23[Morgantietal.1967];

andBulgarians, .26[Mulleretal. 1974]).AGreeksam- ple(Mulleretal. 1974) forms theonlyexceptionwith a higher Agxfrequency (.40), a result which iscloser to those for the Turk (.41) and Indian (.47) samples thantothefrequenciesof the other Europeanpopula- 505

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Ag-System Haplotypes Table 5

Distribution of Partial Ag-Haplotype Frequencies among 10 Populations

POPULATION (no.of individuals)

Australian Australian Amer- Amer-

Swiss Swedes Chinese Japanese Balinese Aboriginesa Aboriginesb indiansc indiansd Senegalese

(362) (456) (141) (67) (152) (86) (132) (66) (121) (100)

Hirschfeld Johnston

Present Hirschfeld Present and Okochi Present Present Hirschfeld Present etal. Present

Reference ... study 1968 study 1973 study study 1973 study 1969 study

Haplotype:

Agxait . .193193 .195 .695 .603 .570 .587 .605 ... .080 .040

Agxaz ^.. 009 .011 ^. ^... .. .. ... ... .017 ...

Agxdt. .013013 .020 .095 .045 .079 .012 .012 ... .128 .095

Agxdz... ... ^... ^... ^... ^... ^... ^... .023 ...

Agyait... .079 .097 .114 .169 .206 .035 .112 . . . .024 .037

Agyaiz . .201201 .155 .028 .055 .033 .006 ... ... ... .098

AgYdt ^.505 .522 .068 .123 .112 .360 .271 1.000 .718 .663

Agydz.... ^... ... ... ... ... ... .010 .067

aMowanjum, from the north coast, south of the town of Derby (western Australia).

b Fromthe western desert, eastof the town of Kalgoorlie (western Australia).

cKrah6, fromthe Brazilian stateof Goias.

dCashinahua, from Amazonian Peru.

tions tested. TheAga, gene frequencies arealso close tothosefor the fewEuropeanpopulationstested: the Swissfrequencyis .48,comparedwith the Italian fre- quencyof.43(Morganti et al. 1968) and the German frequency of.45 (HirschfeldandRittner 1969); the same remark canbemade fortheAgz genefrequencies: the Swissfrequencyis .21,comparedwith the Italian frequency of .14 (Vierucci et al. 1967), the German frequency of .18 (Hirschfeld and Rittner 1969) and the white Americanfrequencyof.13(Vierucci et al. 1967).

Continental Asiaticpopulations.-The haplotypeAgxalit isclearly the most frequent haplotyperevealed among the five populationstested. Its frequencyvaries from .37 in theTurksample to .68 in the Chinesesamples, and we can observe an evidentcline spreading from west to east.

Adecreasing gradientis also visibleconcerning the haplotypeAgydgti, present with a frequency of .27 in theTurksample butvery rare (.006 frequency)in our Chinesesample.Wehave found traces of thehaplotype Agxalctiinthe Swissbutmuch more so in some of the continental Asiatic samples, where this haplotype is present at apolymorphic level, with a peakfrequency of.11 inthe Tibetans. Thehaplotype AgYdcth hasnot been observedinoureastAsiaticsamples(Tibetansand Chinese) and is always present at lowfrequencies in other regions. It is alsolackinginourIndonesiansam- ples (Balinese and east Indonesians).

Other Ag-system data for Asiaticpopulations are presently scarce. The most relevant comparisons are thepartialAg-haplotypefrequencies presentedintable 5, which takes into consideration the available Ag- haplotype data relativetothe Japanese, on thebasis offiveAgspecificities:Ag(x),Ag(y),Ag(ai),Ag(t), and Ag(z) (Hirschfeld and Okochi 1973). We can observe that thepartialAg-haplotype frequenciesfoundinthe Japanesesamplearevery similar to those foundinthe Chinese and Balinese populations.

Results concerning the Agx gene frequencies only addlimitedinformation. Among east Asiaticpopula- tions, Agxfrequenciesarealways dominantinrelation toAgYfrequencies.Theyvaryfrom.69 (atthelowest) among the Ainu (Misawa et al. 1971) and the Thais (HirschfeldandOkochi1967) to .89inone Vietnamese sample (Hung 1967).

Sunda,Wallacea,and Sahulpopulations.-Thevariouseth- nicgroupsoftheIndonesianarchipelago,NewGuinea, and Australia areofspecialinterest totheanthropolo- gistsstudyingthe transition areasituatedbetween the Southeast Asian continental shelf(theSunda), theis- landsofWallacea, and the Australo-New Guinean shelf (Sahul).The Balinese live onanisland which forms the easterntop of the Sundashelf.Asalreadydemonstrated withother geneticdata(Breguetetal. 1982),theybe- longto the cluster of SoutheastAsiatics, an observa- tionconfirmedbytheirAg-haplotype frequencyprofile.

509

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Breguet et al.

Forinstance, the Balinese and the Chinese samples pre- sentthe same eight complete Ag haplotypes. The two main particularities of the Balinese Ag results are the relatively high frequency of the Agyalgtihaplotype, around .20, and the absence-asinthe other east Asiatic samples (Chinese and Tibetans)-of the haplotypes linkedtotheAg(h) specificity. Anextensive sampling of different Balinesesubgroupsandvillages tested only with the Ag(x) and the Ag(y) specificities has shown that thefrequenciesof Agx varyfrom .60 to .75,which is not a largevariability compared with those in other genetic systems (Scherzetal. 1988). However, agood exampleof thepossible influence of genetic drift and founder effect of the Agx genefrequency- and,conse- quently, onthe Agcomplete haplotype frequencies- canbefound inBali. Indeed the isolated community ofTenganan showsanAgx genefrequencyof.878(193 individuals tested; original data)compared with .674 (497 Balinese tested; Scherz et al. 1988).

Oursamplefrom Wallacea (eastIndonesians)iscom- posed ofindividuals showingsome Melanesianmor- pho-anthropological characteristics. Compared with ourBalinese sample genetic structure, wecannote a reductioninthe numberof haplotypes necessary to ex- plainthe observed phenotypes, but this may be due to lowsample sizes; for example, the Agxalctihaplotype isabsent in our Wallacea sample, asinboth the Papuan and theAustraliansamples, whereasit isobserved (at alow frequency of .06) in the Balinese.

OurPapuansampleiscomposed ofindividuals orig- inatingfromvariousisolated ethnicgroupsofwestern New Guinea (Irian Jaya). The Agxalgti haplotype is overdominant,with afrequency higherthan .75. It is interestingthatwefound theAgYdcthhaplotype, which isabsentinoureast Asiaticsamples. On theotherhand, no Agyalgzi haplotype is observed.

Inourspecific sample of Australian aborigines, the Mowanjum tribe, the most common haplotype was

Agxalgti, as in the Asiatic and Papuan populations, withafrequencyof .59; the othercommonhaplotypes wereAgydctiandAgydcth, whose frequencies are .22 and .12,respectively.Anestimationofthe Ag-systemvari- abilitybetween the different Australianaboriginetribes isdifficulttomakeintheactualstateofresearch.Only incompleteinformationLavailableinthe Agx genefrequencies,calculated byMulleretal. (1974),concerning two aborigine tribes, namely, the Awa (.47) and the Yo (.53).

Table5showsacomparisonofour owndata,based onpartial haplotype frequencies,witharesultofHirsch- feld's laboratory (Hirschfeld 1973), whose tests were

done with only five Ag specificities-Ag(x), Ag(y), Ag(ai),Ag(t),and Ag(z)-on asampleof central west- ernaborigines. There isgoodcorrespondence between the two results.

Amerindian populations.-With our improved PHAIA technique, we wereable to retest the material collected by Salzano among the Kraho Indians livingin Brazil (Salzano et al. 1981). We tested 66 individuals for the complete set of antigenes, compared with only 13 in thefirstattempt. The main change registered was the presence of Ag(h) in 20 individuals. The presence of only threedifferent haplotypes in the Kraho sample is the lowest result observed among the 13 populations tested and is probably due in part to the low sample size but also to founder effects, whose consequences are often visible in the genetic structure of isolated Amerindian populations.

No haplotype including the Ag(x) specificity is ob- servedintheKraho, contrarytothe situationinall other populations. The most frequent haplotype, Agydgti (.61), is also presentin all Asiatic-Pacificpopulations tested in this survey but occurs at lower frequencies (.01-.20). On the otherhand, as in Oceania, wefind the AgYdcth haplotype, which is absent in our east Asiatic (Tibetan, Chinese, and Indonesian) samples.

This haplotype may be present, however, inthe north- east Asiatic (Japanese) sample tested for a few Ag specificities(table5),assuggestedby theAgydtpartial haplotype,whosefrequency is higher than thatinthe othereastAsiaticsamples.This result mayindicate the presence ofAgYdcth in the Far East in spite of its apparent rarity and may explain its penetration into America. Another explanation couldbe the presence ofa foreigngene amongtheKraho as aresult of200 yearsofcontactwithnon-Indians,but thishypothesis isnotsupported by othergenetic data(Salzanoetal.

1981).

Otherinformation concerning thedistributionof the Ag system is veryscarce forAmerindians.Johnstonet al. (1969)reportedsomedatafortheCashinahuafrom Amazonian Peru;thissamplewasonlytestedforAg(x), Ag(y),

Ag(ai),

Ag(t),andAg(z).Fortunately, wewere able to work with the original data (F. E. Johnston, personalcommunication) andtocomputepartial haplotypefrequencies (table 5). Themost frequentpartial haplotype, AgYdt (.72), isthe same partial haplotype thatis mostfrequentamongtheKraho (1.00).Butthis timethehaplotypes includingthespecificity Ag(x)are found (.25), as are haplotypes linked to the Ag(z) specificity (.05), also unobserved among the Kraho.

AmongtheCashinahua,Ag(z) ispresentnotonly on 510

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Agydz, asinthe Africanpopulations testedsofar, and

on Agxaiz, ^as in the European populations (possibly

two indications of foreign gene admixture), but also

on Agxdz, never before postulated. Ifthe presence of

a newpartialhaplotype Agxdzcould beconfirmed,we

would have ^an example ofmicroevolution ofthe Ag

system in Amerindian populations.

Earlier studies with theC.deB.serummade by Blum- bergand thathavebeen quotedbyMourantetal.(1976)

aredifficulttousebecausethis^serumis^nowregarded

as amixtureofantibodies[Ag(x),Ag(al),andAg(z)].

However, these data areinteresting because the nega-

tive^tests with these sera, coupled withthe useof the Bernstein formula1 ^- 1- f provided^afrequency approximationforthepartialhaplotype Agydt-which is non-Ag(x), non-Ag(ai), and non-Ag(z)-in the population tested.

Thefrequency of the partial haplotype Agydtvaries from.17amongtheNaskapi-Montagnais from Canada,

to.30amongtheSioux from the UnitedStates,to .55

among the Quechua from Peru to .93 among the Yanomanafrom Venezuela. Thesefacts areofimpor-

tance, given the frequency uniformity (1.00) found

among the Kraho from Brazil, because they demon-

strate the heterogeneity of the Agsystem among the Amerindians.

Worldwide Differentiations

The genetic relationships between all the human populationgroupshave beenanalyzedonthe basis of computedgenetic distances (see Material andMethods) plotted on two different kinds ofexplicit representa-

tions(figs.2and3).Worldwide Ag-haplotypefrequencies areplottedinfigure2. Theorder ofpopulations,

onthehorizontal axis,isdefinedbyapermutational- gorithm clusteringthesamples accordingtohighper- centagesofcommongeneticpools (see fig. 2,legend).

This order resembles therelative geographic settle-

mentsofthe populations. The overallpicture isfollowed by an important Agxalgti frequency dine spreading from eastern to western populations, thusextending whatwehave already observedatthe local levelofcon-

tinental Asia. ForAgydgti, whichis themost frequent Ag haplotypeinsub-SaharanAfrica,agradientisalso

,' ~~~~EUROPE .--

/ /S r e ss I| XO C EA NI A

SUB-S HARAN NEAR-EAST EAST AND

1AFRICA EUROPE INDIA SOUTH-EAST ASIA OCEANIA

Ag

g .6- l 1 1 ID: F ~~~-s

Ag

e v

Figure 2 Ag-haplotype frequencies (cumulated) for12popu-

lation samples from allcontinentsexceptthe Americas. The Amerin- dian Krahoarenotrepresented,fortheirhaplotype distribution is the result of^a specificmicrodifferentiation, compared withother Amerindiangroups(see table5 foracomparisonwith theCashina- huapartialhaplotype frequencies, andseefig. 2fortheirrelative distance from the othergroups).Onthe horizontalaxis,thesamples

areordered according to a permutation algorithm (described by Sanchez-Mazasetal. 1986):thesumofgeneticdistances between adjacent population samplesisclosetoaminimal value.Rarehaplo-

types(AgxaLzi, AgyaIczi Agyalgth,andAgYdgth)^arepooled (residualfre-

quencyis atthetop ofthefigure).

-.. , NE ARE AST'| , In'NDONESI^

I.~~~~~~~~~~~~~~~~~~~~~~~~~~

IINDIA -->, BT~I

|EASTASIA

EUROPE,' a-

~~~~~~~~~~~~~~~~~~~I. TI*E N9X

' TURKS

_T

K

U~~~~~~~~~~~~~~~~~~~~~

nol^5

INE5E

L~~~~~EASTASIA l > r ,, .,, * N E ARE AS T DIA'

/' NTU|

_~~~~~~~~~~~~~~~~~~~~~~~_CE ANI A--| ,

Figure3 Principalcoordinateanalysisforthe 13population samplestested forthecomplete panelof theAgspecificities(seetext foradescriptionofthegeneticdistanceused). (a),Firstand second

axes(61%and 15%of the totalinformation,respectively). (b),First and thirdaxes(61%and 9% of the totalinformation,respectively).

511

SUB-SAHARAN AFRICA ,'

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Breguet et al.

visible along a longitudinal axis, for its frequency is still highinEurope andwesternAsia and is much lower in east Asia.

According to most haplotype frequencies, Asiatic populations can bedifferentiated into (a) a western cluster including Turks, Indians, and to a lesser extent, Tamils and (b) an eastern clusterincluding the other groups.Thepopulationsintheformer cluster are ge- neticallyclose to Europeans. The cluster including these populations can thus be defined as a caucasoid one.

This term iscommonly used to designate the popula- tionsfrom northAfrica, Europe, the Near East, and India, as their genetic closeness,confirmed by the present study, has already been emphasized.

Oceanianpopulations, represented by Papuans and Mowanjum, show some genetic heterogeneity dueto

AgYdg4, AgYti, andAgxaiOtfrequency differences. How- ever,bothsamplesareclosely relatedtotheeastAsiatic populations, according to the worldwide frequency ranges for these haplotypes.

TheAmerindianKrahoarenotrepresentedinfigure 2,for their haplotypic distribution seems to be the result ofastrongspecificgeneticmicrodifferentiation,as compared with thecasefor all other human (even other Amerindian) tribes (see next section).

The13populations ofthisstudyarealsorepresented bymeansofaprincipalcoordinate analysisplottedin figure 3. The great advantage ofitstwo-dimensional scaleistoallowa moredirect comparison withageo- graphical space.

Except for the Amerindian Kraho sample, the Ag worldwide geneticdifferentiations appear to be strongly correlated with geography. The Turk and Indian samples areprojectedclose to the center of each plane, ow- ingto intermediatehaplotypic frequencies within the entiresetof worldwide populations.Ontheother hand, all Oriental (east Asiatic and Oceanian)populations clustertogether according to the most informativeaxis (61%of totalinformation).Thesecondandthirdaxes (15%and 9%oftotal information,respectively), however,make prominentOceaniandifferentiationswithin the Oriental cluster. They separate, alternatively, the Mowanjum andthe Papuan from the eastAsiatic samples. Onthe otherhand,the absence of direct genetic links between Amerindianand east Asiatic populations was fully predictable onthe basisoftheir lowdegree ofpolymorphism.The Krah6sample sharesaveryhigh Agydgtifrequencywith thesub-Saharan African group, but this resultmustbeseen as arandom genetic con- vergence. As already mentioned, amongAmerindian tribes the frequency of the partial haplotype

Agydt

variesin anexceptionally broad (.17-1.00) range, a variability which emphasizes the frequent drastic effects of genetic drifts on the small and isolated populations' geneticstructures. The Agpolymorphism still proves tobe highlyinformative at more accurate local genetic differentiation levels. The third axis differentiates the westAfrican sample from the Bantu, the former ap- pearingcloser to caucasoids. This result agrees with sub-Saharanpeopling history reconstruction based on the highlyinformative Rh,Gm, andHLApopulation studies and supported by linguistic peopling theories (Excoffier et al. 1987). The good agreement between these analyses lets uspredictaneven morecaucasoid- likegenetic structure for east African populations, as expected from the extra-African extension of the lin- guisticAfro-Asiaticfamily.Wehopetobe abletoconfirm such a hypothesis in the future.

Atthelocal levelof the Indian area, the Ag polymor- phismisconsistentwith human historical relationships as well. It is worth noticing, from figure 3b, that the principal coordinate projections for the Indian, Tamil, and Tibetansamples correspond,in avery accuratefash- ion, totherelative geographical locations of these populations.

Discussion

The presentworldwide population study reveals a strikinganalogy betweenthe human geneticdifferenti- ationsexpressed bytheAgpolymorphismand theresults ofpopulation analyses obtainedfrom otherimmuno- logical systems (Rh, Gm, and HLA) (Sanchez-Mazas and Langaney 1988). Closerelationshipsareevidenced between (a) Rh, Gm, Ag, and HLA haplotype- or allele- frequency distributions and(b) the presentgeographi- cal settlementplacesand/orlinguisticsubdivisions of humanpopulations, at either a global or more local levels(Excoffieretal. 1987;Sanchez-Mazas,inpress).

ThefewexceptionsaccountforsomeisolatedAmerin- dian orPapuan populations, whosegenetic structure hasbeensubmitted torapid andspecificchanges. These facts, togetherwith the completeabsence ofphysical linkage of Ag, Rh, Gm, andHLAclustersontheDNA (chromosomes 2, 1, 14, and 6, respectively), indicate thatthe major forcesdrivingthe evolution of thesepoly- morphisms across human generations are the main stochastic components of human peopling history (migrations,demographicevents,andsocioculturalfac- tors). Whenever they can be demonstrated, selective effectsmayaccountforonlysomeweak differencesin the way that each system presently expresses itself,

512

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within or between human groups. Thefirst study deal- ing with selective valuesfor the different Ag phenotypes (Berg et al. 1976), a study based on thecombined genetic data for10differentpopulations, showsasignificantly higher level of serum cholesterol and triglyceride in Ag(x-)individuals than in Ag (x+)individuals. Such findings are most important for medical purposes but areincapable, for the moment,ofsuggestinglong-term selectiveeffects onthe Ag loci, at the levelof human populations.These resultsemphasizethe usefulness of the Ag polymorphisminanthropological research, with its particular interest in human population early- migration studies.

WithregardtoButler's five-linked-loci genetic model for the Ag system, one ofthe most interesting ques- tions concernsboth (1) the molecular mechanisms which led to the present high levelof complexity of this sys- temand (2) their appearanceinhuman species evolution. Theworldwide distribution and frequencies of the 14Aghaplotypesobservedinhumanpopulations suggest afirst reasonable view that no drastic changes in the genetic structure of the Ag loci have succeeded, from the emergence ofHomosapiens sapiens up to recent times. Commonly found haplotypes show great frequency changesinthecaseswherepopulations are geographically far apart, but when intermediate groups areconsidered they evolve according to frequency clines.

This seems to indicate, for at least six Ag haplotypes (Agxalgti Agxdgti Agyalgt,

AgYalgzi Agydcti

and AgYdrt),

anoriginal common sourcefor all modern populations.

Onthe otherhand, haplotypes which are not observed insomepopulations (such as AgYdcthin east Asiatics) areonly poorly representedinthe others. Sampling bias, well illustratedby the unique occurrence of AgYalci and Agyalgthinthe most numerous (362 Swiss) sample, are always partly responsible for the nondetection of low- frequency haplotypesin somelow-sized samples. But evenwhen this kind of assumption isomitted, lower degrees of polymorphism are mostly revealed in isolated anddemographically underrepresented popula- tionssuch as theAmerindian or Melanesian (Papuan andeastIndonesian) groups. Strong genetic drifts and founder effectsareevendetectableatthe levelof local geneticdifferentiations,asisthecaseforSouthAmeri- can Indians (betweenKraho and Cashinahua) or for Indonesian settlers (between Balinese and east Indone- sians). Thus, a reduction ofpolymorphism seems to be a generalrule applyingto the history of marginal populations,andwe cansupposethateven morethan sixAghaplotypeswerealsopresent atthe timeofour origin. It can be concluded, as it has also been con-

cluded from Rh, Gm, and HLA population studies (Lan- ganey etal. 1988;Sanchez-Mazas,inpress),that ahigh degree of polymorphismmusthavealreadybeen pres- entinthe Ag genetic structureof the earliest modern humans and that the latter must havebeen reduced to a unique population.

Giventhat most structural Ag genetic changes are likely tobeconfined to premodern human times, one canneverthelessspeculate-asdid Fisher(1947) for the Rh system-onthe kind of moleculareventswhichac- cumulatedinthe past and ledtothe firstH. s. sapiens Ag-polymorphismcomplexity. If it is assumed that the presently mostfrequent Ag alleles are more likely to be the oldest ones inpre-sapiens geneticevolution, it isthenpossible to reconstructahypothetical pathway for the emergenceof themorewidely distributed haplotypes:

1. Agg, Agt, and Agi are most likely the earliest alleles at loci Ag(c)/(g), Ag(t)/(z), and Ag(h)/(i), respectively,as they arefound to be the mostfrequentin all human populations. The same kind of conclusion cannot be reached, however, on Agx orAgY, and Agd or Aga, (at the Ag(x)/(y) and Ag(al)/(d) loci, respectively), because allelefrequencies show great variationsbetweenoursamples from the Old World. We can thus assume that four haplotypes carrying Agg, Agt, and Agi were differentiated veryearly onbytwomutations, Ag(y)Ag(x)and Ag(d)*Ag(aj), becoming the widely distributed Agxalgti and AgYdrti but also Agxdgti and Agyalgti haplotypes.

2. Haplotypes including the combinations Agcti and Agrzi could then have resulted if we postulate Ag(g)Ag(c) and Ag(t)- Ag(z) mutations. This wouldexplain the appearance ofAgydcliandAgzl from Agydgti and Agxalgti, respectively.

3. From the partial combination Agcti, a final muta- tion, Ag(i)--Ag(h),may have beenresponsible for the emergence ofAgYdcth from Agydcti.

These major events would have provided, at pre- sapienstimes, both the sixhaplotypes cited above and theadditionalAgydcth haplotype,which are atpresent widely distributedin most human groups.

If we assume Ag loci chromosomal arrangement Ag(c)/(g)-Ag(al)/(d)-Ag(x)/(y)-Ag(t)/(z)-Ag(h)/(i), which agrees with molecular data(fig. 1), it canthen be shown that thesevenhaplotypeswhichareuncom- mon canbe derivedby singlerecombinationeventsbe- tween these sevenwidely distributed Ag haplotypes.

We cansuggest,forexample, thatahaplotypesuchas 513