The Frequency of " Rare " Protein Variants in Marshall Islanders and Other Micronesians

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1. Research supported by the Atomic Energy Comniasion 2. Department of Numan Genetics; University of Michigan, 3. Brookhaven National Laboratory, Associated Universities, Inc., Upton, L. I. .

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l~.~c:eritly,i n connecti.on with ail oiigoing medical study of certain :.I it-sli.il.1 Lslanders prompted by the Uikinj. incident (1) , we have had the opl)ort*.iiiity t o examine i i s<>riesof blood sm plc s from these Islanders : UT tile uccvrence of both polymorphisms and tare variants of serum p:-ot-eins arid cry thro cyte enzymes. The results, when treated in tlia ~ rsun lfas!iiorl and combined vith the findings of others on Micronesians, su,qgest/a lover frequency of rare variants i n this group than in such F,roups as Japanese, Cmicasians , o r South American Indians. There are scme bothersome problems in comparisons across groups sampled in different ways ,however, which make the usual statistical contrasts impossi-ble; some ol' tlieuz prohlemq are aired. The study population is composed of persons now residing on Ebeye, Rongelap, and Majuro Islands, for the most part related to one another as i7rcrnbers of nuclear fsrnilies. The number of independent genomes i n t h e sample is thus considerably less than the number of persons. lmlf of the children in the sample were born to parents inadvertently radiated as a result of fall-out from a nuclear explosion at the time of the E il dni test, i n 1954. the clues tion of a radiation effect will not arise i n any substantial However, as will be apparent under RESULTS,

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The sarnplcs were shipped by air, on ice, from \<.dshed red I ~ luo dc ells and plasma were stored The conditions for electrophoresis and typing o f systems I, 2,8,9, 10, 17, 13, 15, 16, 18 and 20-24 in Table 1 were carried out as described 1’1 cniployed the method of Spencer, Hopkinson and Harris (3) , and sta € ning for system 19 employed the positive staining method as reported by Peters, Electrophoresis of systems 14 and 1 9 and st‘nining o f Hopkinson and Harris ( 4 ) . System 3 was determined by the method of Chclrlesworth (5) , system 4 by the method of Tashian ( 6 ) , system 5 by thc i,ietlioJ of C lien, Anderson and Gi bl c tt(7), system 6 by the method o f Wei t kr imp(8), sys tem 11 by the method of Edwards , Hopkinson and Harris (3),and cyst om 20 also by the methods of We it km pet a1 (10) and Tanis of the s y s terns: haptoglobin, phosphoglucomutase-I, adenosine deaminases component.. Phenotype and allele frequencies are given i n Tab€e 2.

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Vary in!; miatbsrs of rleterminntions for the systems lisi - cdin Tables 1 artd p.itr.crn o r ~2.11 cwlcar pattern (even after repetition) was occasionally clswiiere i n Xicrones ia (see below) ,we tabulate it with the polymorphisms. Tiiese three individuals were related as siblings, one of whose parents 2. cart? variants.--One rare variant was observed in a total o f-- /;,047 cicCerininations. This was afast albumin variant detected by only one of the three screenings ys terns in use for albumins, namely, the pH 5.0 sorlium acetate buffer system of Weitkamp e t a1 (10). The variant is illustrated in Fig. I. Its electro ph ore tic behavior appears similar to that o f all~urnin14ecl:n as reported by Weitkamp e t a1 (10). occnrred inagi.rl, aged 5 , whose mother was normal (and not exposed to fall - out) and €ather not available for study. The fact that the vari-ant has heen demonstrated in only a single person with only one technique leaves its identification somewhat unsatisfactory,but the situation cannot b e improved upon a t preeent. encountered a11 fell within the rather ron side rab lc range reported Fn o th cr studies of Micronesians (12, 13, 14, 15, 16,17, 18, 19).

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3,. ;!& r a n vari.ants.--In Table 2 we. have stitmmrizsd n o t only oiir varicincs oi 24 systems in Micronesinns which we hrrve been able to 1ocat.r ltr bit ra ryde ci3 ions; no approach is apt to find universal acceptance : i t Lhis the. \<c excluda from this srimmarg any variant which for toia'Li.tv 0 4 the represent z t ives of the_ population studied to date, occurs i n more than 2.07, of the group (one of the conventional definitions of t h e tabtilation the polymorphisms involving types 3 and 7 of t h e PGMl By this definition of rare variant,we exclude from a d tlic polymorphism for the type 2 of phosphoglycerate kina se reported by these Sam authors. This same convention will requite us to eliminate v z ri ant02 nlburnin, a variant thus far encountered in a single tribe, but th-lie. with a gene frequency of 0.08. I n sampling populations where ratlitir close biological relationships between individuals can scarcely bc a-,roided, it is well to remzmber that even i n samples of 1000, a variant limited t o members of a single extended kindred may assume the proportions of 3poly iw rp his m as here defined. lrom the Micronesia count, a variant with the electro ph ore tic mobility Inthe case of the vartan ts excluded of t.ype 7 PGNL has b w n encountered at several local it fes in the Far East, and i t seems likely this is a ''widespread" polymorphism, However, the same cilnliot: be said for tlie variant with the mobility of PGM -3 o r L he v asian twit li the mobility of PGK-2. nlorphisms. Iffur tlier studies in Nicronealo revealed these alleles

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to have S ~ Ircstrlcterl a (listcibutioa that their f rcqiiency inthe r-ilciL*c smp?.(? dropped below 2 perccnr, then they woiilcl of coiirse ciiicr the list of rare variants. 'Vie final column of 7'a1)2c 2 presents the frequency per 1000 of \*,<riaots for cnch syst an r and the .In Gtiweighted aver qe is employed frequency with which variants are unwei:;hted average o f all the systems. to prevent an extensive sturiy of a picture. By this approach, the nverag* encountered i n the sys terns under Some of the Elicronesian Islands have had extensive contact with nth:xr ethnic grvups , and there has in addition been active exchange i n ~s'ir.d thata rare variant may have been introduced from the outside, as pointed out: by Blake e t a1 (12) i n connection with the LDH variant t1ic.y ciicountered on Faraulep Atoll, which is similar to the Calcutta - 1 varia nk wi.despread iu India. We note in this connection that the cA I variant ot Tashian e t a1 (20) encountered i n the Charnorros of Guam and Sa I pan isclt ? c troph ure tic ~ l l yRlmilar to a variant encountered in have 'wel.l-doci~mentcdh is tori cal contacts with these arcas. On the other liaml, we restate the well-rccognizcd caveat that electro ph ore tic ident ep oT two variants is not synonymous with biochemical identity. Therc israp 131y accuriiulnting an extensive literature on the. frequency o l rare variiints L no variety of popiiIntions. He mentioned earlier the

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hio svries which originate in this1 ahoratory and involve essentially the sane pio te in s and techniques as the original material here in reported, pli ioa third composttc sz ric s for Caucasians. We will again use an uiiweightcd average, based on as many o f the systems listed in Table 2 comparison, w e will coiitent ourselves with reference to ;I!: arc coverzd inthe references cited. By this convention, tlie variant irc!qui?ncy for South Amprican Indians, based on systems 1, 2 , 8-10, 11-16, 18 and 20-24 o f Table 2 , i s 1.7/1000 (2, and unpublislwd). The lnver F‘rcc;cicncy than i n our last publication on this subject (2) i s Tlie frcquzncy i n West European Caucasians, based on systems 1, 2, 8-16, ant1 tI#qrri.s, ftopkinsnn, and Robson (33) is 2.7/1000, and f o r Japanese z:luI.ts, hctsed on systcins 1, 2 , 4 , 8-10, 1 2 , 1 3 , 1 5 , 16, 1 8 , and 2 0 - 2 4 , As nnt.cd, there are obvious difficulties in statistical compartsvns between tiwsc series. extent that of others,contains closely related individuals, as does our data on Amerindians, Our own Micronesian data, and we suspect to some By contrast, the data on Cauca s lms and Japanese

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in t:' 'st: dj.ffcrcn: fashions, but not the number of different variants (2), and we maintain that position here. considerable sl ruff li up, of tribal yopiilations , such 23 occurred inthz dc tribal iz ation oE the ancestors of inodern Japanese o r Europeans, shortld not alter the total number of variants present. However, conventional statistical contrasts ol: 1otal frequencies i n these various 'populations seen inappropriate. In particular, estimates of frequencies from studies o € pol ml nt iii w such a s Nicronesians o r Amerindians are quite susceptible The logic is that arat hz r to a " j ~ vkpot " cffcct--one island with a high frequency variant could innr1:eclly alter the picture. However, some 27 islands have been sampled t o d;t k,coatpri:;iiiy a rather representative group. 'I'dkcn a t face value, there is a 3-fold range among ethnic groups i n c??e frequency of rdre variants as definad, these variants occrirring i n EiicronP3ians with appro.yimately half the frequency i n which they have bzen encountered i n s,-veral other ethnic groups. However, this apparent diffcrencw hinges, €11 part, on the definition of rare variant, which in turn i.p intimately related to the size and nature of the sample. Thus, i T iiq the study of Ilicronesians is extended i t becomes clear that the PCN -3-1 ike variant encountered in 49 persons (12) and the PGK-2-like variant cnc-ooltntered i n 39 persons (12) are sharply localized and no o r icw addir:ioii,i.t exainples of the variant are encountered, then either or both of (lies€*might drop below the arbitrary2 percent frequency level and liose t o be c1;issed as rare variants, with a rnarlted inpact on the average

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t-lic est j mate must have a large variance. The properties o f Pile samples of Pficronesiuns and Amerindians might lit.. rsridercd 1:iorc comparable to thoss of Caucasians and Japanase by eliminating the old and the young from the former two samples, but the 1lasi.c isstie would still remain: the Fndivtduals inthe f-orm,er two samples will be more related than those in the latter fxo. practical way t o meLzt this issue seems an extension of the sample, thu:; rcdiir:i.ng the iinlxict of any one "jackpot". In addition,itis sii~gcstL?(lt hat the definitive treatment of comparative variant frequenci2s which a larger sample will permit must consider a variety of d n * fi n it ions of rarc variant and/or analyze the total heterozygosii-f 'rh? pr u hls n is not a trivial one. The frequency of such variants in natural populations is maintained by a complex balance between scIw.tion, mntation, and population structure. Basic parameters though hey Le, both thz manner of action of selection and the rate and types o f in11 !:ation in higher organi.::ms remain poorly understood. and niu tnt ion may be studi cd directly,i.e., thru surveys followed by the i!:.?ai.prilotLon of: population paraiaeters (35,36) . The former approach i s mrich more laborious than the latter, and it i s tempting to pursue the easier coursc?, but- the latter approach i s only as sound as the estivates of variant frequency (which should be based on as total a

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pro ~ itnl ~l;r pcl-sued b y t+<*i n direct approach, sample size nust b e worth yes tim te of 1-!1e frequency of rare variants in populations like bticronrrsinus a.nd Anie.:inc!ians, and most investigators would probably ilf;i that the ?4icrowshIns do not yet approach that minimuin. Unfuri'rinaf.e'ly, our 1:nowlcdge of the cli iste ringo € specific rare variants i n rvl:ttively untlis LurhPd popillations is still so scRnty that further cxp : . ~itincc: i s necesqriry p r Lor t o setting that reasonablz minimum. liloocl specimens from a sample of 18? Marshall Islanders were stvrclied with referLwc.t?to variants of 22 serum proteins and erythrocyte enzymes. Six of the t rafts studied exhibited genetic polyiiiorpli isms (aclenosine deaminase, phasphoglucomtase acid phosphain 4,047 determinations. These r csu lts on rare variants have been c.oml)ir.ed wi tti those of others on Ificronesians and t h e frequency of rare variants in Micronesians conparetl with the freqttexios i n West

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Ccuetic Structure of R Tribal Population, the Yanomama Erythrocyte Enzyme Systems in the Yaaonama and Five oon-genet ic variation of triose phospha t c isomerase isozymes i n human tis s uz " , Ann. IIum. Genet., Lond, -36, 297. genase., fru c to aldo las e, glyoxylase 11, and sorhital dehydrogenase" Ann. 1Ium. Genet., Lond., 35, 297. Hiwan Erythrocytes", i n Biochemical Methods in Red Cell Genetics, gly cc tnt c iiiutnse: its dmionstration tjy electropliorcsis and

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genetic var im its and their populatioa List r ltu tio ii " , Ann. "Variation i n Red Cell Enlyme Groups anong Populations of "Clood Croups, Serum Cenelic Factors, arid Hemoglobins i n Frequencies o € the Trukese of Micronesia", Acta genet.

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globin s in the F as z cr n Cnrolines", Am. J. Phys. hnthrop., Yrr-quer~cLesu f the Chamorros of Guam", Am. J. Hum. Genet. Yank moto, PI. a d I.. F u 1973 "Red Cell Isozymes In the Eastern Variant o € Erythrocyte Carbonic Anhydrase i n Micronesians frmi Guam And Saipan", Science, 140,5 3 . 8lootl group genetic variations in natives of the Caroline Islands and i n other part5 of Micronesia, with a supplement Ani. J. Hum. Gchnet.. 1 9 . 130. Aff ec t ing bfictones ian anhydrase IC in Filipino e.

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o f the IIicun Ilaptoglobiiis: 126 Norw?giaa Families with 428 "Observations sur l a Repartition des Groupes Senques (I[aptoglobines, Trans ferri nes Groups Gm) clans quelques hacinoglobia J Two new f3-chair variants of Genz tics of t h e Ceruloplasmin Polymorphism, Humanganetik De SCas De Diu Albiminemie Observes En France, Rev. Europ. of rdre ,ille les determining electrophoretic varimts: .data. o n 4 3 ciizyne Loci in man, Ann. Hum. Genet. Lond., 37, 237.

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system. Isr..;ielJ. bled. Sci., 2, 1503. f o r muCatLon rates, Mutation Res., 26, 319.

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Syst an Phano type Total Gene Frequency fk!€!Kl@S ine dinintnase 167 18 0 185 ADA = 0.9'51 d el i yc?r v ge nas t? 164 21 0 185 6-PG$ =I 0.943 Tlie HT() type tias observod in4 individuals. 2 . I'm cxamples of the PGM phenotype 2-7 and a single phenotype 1-7

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6.9. AlbuL:ii.nsoirrples sliown are normaL serum 1, 5 , 7, 8 and 11; l?ap.LshaIia-l, 6 , 12; Naskapi, 10. Normal samples include refcrence serum from o w own laboratory and serum from normal Marshall Is land sainples . All variants, except the presently reported P13rshalL …