Perspectives

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove

R. A. Fisher’s 1943 Unravelling of the Rhesus Blood-Group System

A. W. F. Edwards1 Gonville and Caius College, Cambridge CB2 1TA, United Kingdom

VEN if R. A. Fisher’s elucidation of the human blood- binatorial skills and his relations with A. S. Wiener, and E group system Rhesus in terms of the three linked to add a personal coda. It is best to take first the diagram loci C, D, and E had not proved to be substantially (Figure 1) from Fisher (1947) because it explains the correct, it would still have been an outstanding example relationship between the original Rhesus notation and of the power of analytical thought to unravel a complex that proposed by Fisher. As Fisher writes, ‘‘We may repre- array of genetical data. In fact, as a recent review relates sent the eight heritable antigen complexes geometrically (Avent et al. 2006), D is one gene (carrying the D anti- as the corners of a cube, while the six elementary antigens gen) and C and E are different splicing forms of another are represented by the faces; each allelomorphic pair of (CE carrying the C or c antigens and the E or e antigens). antigens is then a pair of opposite faces, and the three Fisher’s solution is recognizable beneath the modern faces meeting in any point specify the antigens in each molecular detail. complex.’’ (Ry) and (CdE) are in parentheses because The story of the unravelling of the Rhesus puzzle is the anti-d antibody had not yet been discovered (nor has told in chapter 13 of Joan Fisher Box’s R. A. Fisher: The it to this date); it was part of the brilliance of the hypothe- Life of a Scientist (1978). As well as describing the serol- sis that this haplotype and some of the missing antibodies ogy, Box gives a comprehensive description of the war- that the model predicted were later found. This is one of time circumstances, which in 1943 reunited in Cambridge the very few diagrams in the whole of Fisher’s work, Fisher and his former London colleague R. R. Race. Box surprising in view of Fisher’s geometric way of thinking. was able to draw on Race’s unpublished 1968 Fisher Next is Fisher’s solution as he wrote it out on the back Memorial Lecture ‘‘Blood Groups in Human Genetics’’ of a piece of Caius College notepaper (Figure 2), prob- in which he described the meetings with Fisher in The ably after dinner in College (where he lived) on the day Bun Shop, a Cambridge public house, where the inter- that Race had told him of his latest results when they pretation of the Rhesus reactions was discussed over met in The Bun Shop. It was presented to the Fisher pints of beer. Much later, he and his wife Ruth Sanger Memorial Trust by J. J. van Loghem and is reproduced told the story (Race and Sanger 1982), and on the with the permission of the Trust. The ‘‘CDE’’ notation occasion of the centenary of Fisher’s birth it was re- has not yet put in an appearance, but the tables may be peated by Clarke (1990) and Bodmer (1990), both of readily interpreted by reference to Figure 1. Note in whom also knew Fisher. Bodmer (1992) gave a fuller particular the two faces of a cube at the top of Figure 2 account still to the Eighth Congress of Human Genetics and the list of some of the genotypes and their reactions in 1991. Fisher himself described the purely scientific on the right, divided into ‘‘5 common,’’ ‘‘5 rare,’’ and ‘‘2 development in a lecture that he gave at Woods Hole, not observed.’’ Of this occasion Race and Sanger Massachusetts, in 1946: ‘‘The Rhesus Factor: A Study in (1982) wrote, ‘‘The immediate reaction was puzzlement Scientific Method’’ (Fisher 1947). and lack of understanding, especially on the part of The purpose of this article is simply to bring together Race; colours and even music were tried but did not in one place for the first time five illustrations that help; eventually unaided understanding alone came to belong to the story, to remark briefly on Fisher’s com- the rescue, and thereafter it was impossible to believe anything else.’’ Figure 3 is the beer-stained piece of paper on which, 1Address for correspondence: Gonville and Caius College, Trinity St., in The Bun Shop the following year, Fisher outlined his Cambridge CB2 1TA, United Kingdom. E-mail: [email protected] crossing-over hypothesis explaining the occurrence of

Genetics 175: 471–476 (February 2007) 472 A. W. F. Edwards

Figure 1.—The two notations for the Rhesus antigens from Fisher (1947).

the rare antigens. He uses the CDE notation. The piece allelomorphic antigens are arbitrarily denoted by C, c, of paper was presented to the Fisher Memorial Trust by D, d, E, e, chosen to avoid confusion with any symbols so Ruth Sanger with the annotation, ‘‘This was the first far used’’ (presumably the allusion is to A and B of the writing down by Professor Sir Ronald Fisher of his very ABO system); the letter is dated May 17 and Race would elegant idea that the less frequent Rh chromosomes of course have seen the proofs of it by the time of the might have arisen by crossing-over in heterozygotes for meeting in The Bun Shop on June 22. Fisher and Race [sic] the more frequent chromosomes. He wrote it in (1946) record that the notation was Fisher’s idea, and ‘The Bun Shop,’ a ‘pub’ in Cambridge, on 22nd June Taylor and Race (1944) wrote, ‘‘The terminology 1944. The Professor is very short sighted and was not used in the present account of the Rh system [that is, aware of a good deal of beer on the table—the cause of the original terminology] will certainly not be perma- the marks on the lower part of the paper.’’ The figure is nent. Race (1944) has described a most ingenious and reproduced with the permission of the Trust. attractive scheme, formulated by R. A. Fisher, for the Rh In the telling of the story, the two sessions in The Bun genes, antigens and antibodies.’’ Shop have tended to be confused or at least conflated Race submitted his Ph.D. thesis ‘‘The Rh Blood (e.g.,Clarke 1990) but the evidence is that the first was Groups’’ (Race 1948) on March 24, 1948. It should be in late 1943 (as Race once said) and the second was a valuable source for any historian of serology. (On indeed in the summer of 1944. The date of the latter is examining the copy in Cambridge University Library, I significant, for Race’s letter to Nature, ‘‘An ‘incomplete’ find that I am the only reader ever to have consulted it.) antibody in human serum,’’ was published on June 24 Fisher noted that cDe could be produced by three (Race 1944). With his characteristic generosity toward different crossovers among the three commoner types his students (Race was registered for a Cambridge and that this was more common than the three rarer Ph.D.) Fisher did not co-author the letter in which Race types. From this, he suggested the chromosomal se- wrote, ‘‘The research arose out of a suggestion by quence DCE, with C and E being close together Professor R. A. Fisher,’’ adding, ‘‘The three forms of and separated from D by a longer distance (Box 1978,

Figure 2.—Fisher’s 1943 solution to the Rhesus complex. Perspectives 473

Figure 3.—Fisher’s 1944 explanation of the rare antigens. p. 365). At the time, the evidence was not very strong, but it now looks as if, once more, Fisher was on the right track because, as mentioned earlier, it is now known that D is one locus, whereas C and E are alternative splice forms of a single gene. Next is a picture of The Bun Shop itself (Figure 4), Figure 5.—R. A. Fisher about 1943. kindly supplied by Sir Walter Bodmer and already larke published in C (1990). Sir Walter and I joined Because Fisher was reelected to a Fellowship at his old the Cambridge Department of Genetics at the start of college, Gonville and Caius, on assuming the Univer- Fisher’s last academic year as professor (1956–1957) sity’s Arthur Balfour Professorship of Genetics in 1943, and we both remember visiting The Bun Shop with he supplied a fresh photograph for the Fellows Photo- Fisher after his elementary lectures the previous aca- graph Album (Figure 5), contemporary with the eluci- demic year. It was pulled down not long afterward dation of Rhesus. It is reproduced by courtesy of the during the redevelopment of that part of Cambridge. Master and Fellows. (The redevelopment has itself since been pulled down and the site is currently undergoing another cycle of building; visitors to Cambridge should not be misled by THE COMBINATORIAL BACKGROUND the fact that there is now another Bun Shop elsewhere in That Fisher was an ace combinatorialist is obvious the city.) from even a cursory examination of his writings. His memorial window in the Hall of Caius College indeed reﬂects this by depicting a Latin Square (Figure 6, from the dust jacket of his book The Design of Experiments; Fisher 1935). This talent was deployed to great effect in the unravelling of genetic systems. In section 16 of his path-breaking paper, ‘‘On the correlation between relatives on the supposition of Mendelian inheritance,’’ Fisher (1918) considered the effect of ‘‘coupling’’ between two loci (that is, linkage) and, in particular, the two extreme cases of no linkage and complete linkage. ‘‘The above analysis of polymorphic factors enables us to compare these two extreme cases; for there are 9 phase combinations of a pair of dimorphic factors, or, if we separate the two kinds of double heterozygote, 10, which ... can be interpreted as the 4 homozygous and the 6 heterozygous phases of a tetramorphic factor.’’ In their commentary Moran and Smith (1966) write, ‘‘Thismapping of a system with two factors at each of two Figure 4.—The Bun Shop public house in Cambridge. loci on to a system with four factors at a single locus is 474 A. W. F. Edwards

incidentally, a footnote acknowledges the help of R. R. Race with Bernstein’s original papers. Bodmer (1990, 1992) recollects that Fisher told him that his Rhesus hypothesis was also influenced ‘‘by the Swiss geneticist Ernst’s interpretation of the Primula incompatibility system as a complex of closely linked loci,’’ as confirmed in a letter Fisher wrote to Ernst in 1957 (Bennett 1983): ‘‘It is a pleasure to send you one of my remaining copies of the paper I gave at Woods Hole in 1946 [Fisher 1947]. I was, indeed, influenced in forming my ideas about the Rhesus complex by the system you had first proposed for the factor in Primula determining dimorphism.’’ A reference to A. Ernst’s 1933 article may be found in Bodmer (1960). There were still some copies of Fisher’s article in his offprint boxes in 1957. I possess one of them. A. S. Wiener: Wiener, one of the co-discoverers of the Rhesus system, was a strong advocate of the original notation, which portrayed a single multiallelic locus. He was never reconciled to the CDE notation, even after this had become the norm. A prolific writer, he took every opportunity to criticize it. Fisher, although of course always recording the role of Wiener as one of the discoverers of the system, limited himself to oblique references such as, ‘‘The efforts to force a notation on the system which does not recognize its genetic charac- ter, has been, and perhaps still is, a source of contro- versy’’ (BBC radio talk, July 17, 1958; Fisher 1958). I am sorry to have to report that when Wiener started to give his invited article to the Blood Groups session of the igure F 6.—Commemorative window to Fellows in Caius Second International Conference of Human Genetics College, Cambridge, UK. Clockwise from top left: C. S. iener Sherrington, John Venn, George Green, James Chadwick, in Rome in 1961 (W 1961), we witnessed Fisher, R. A. Fisher, and F. H. C. Crick. who seemed to have occupied a front seat deliber- ately, rise from his seat and, slowly picking up his hat and stick, ostentatiously walk out. particularly interesting and can be illustrated as fol- In earlier times Wiener and Fisher probably had a lows.’’ The reader is referred to Moran and Smith for friendly relationship. There are articles co-authored by the further details. Wiener in the Annals of Eugenics during Fisher’s Here we have, of course, the two-locus precursor of editorship. One entire box in Fisher’s offprint collec- the three-locus CDE scheme, in which the genetical tion is devoted to Wiener, and several of the articles that situation can be mapped either onto a single multiallelic Wiener sent are autographed. Of particular interest to locus (A. S. Wiener’s preferred solution; see below) or me is ‘‘Method of measuring linkage in human genet- onto three fully linked diallelic loci (Fisher’s solution). ics’’ (Wiener 1932), autographed ‘‘To R. A. Fisher, With Fisher was keen to learn of any crossovers that would sincere appreciation,’’ an important paper that I missed have confirmed a physical basis for his hypothesis, but in my history of linkage estimation (Edwards 2005). even without them the three-locus analysis offers an There is even a typescript of ‘‘Genetic theory of the Rh enormous clarification of thought by comparison with blood types’’ later published as Wiener (1943), which its rival, as Race observed (‘‘thereafter it was impossible appears to be the copy to which Race et al. (1944) refer to believe anything else’’). when they say, ‘‘This was the state of our work when a Fisher will of course have later been perfectly familiar letter, dated October 11, 1943, came from Wiener with F. Bernstein’s 1924 conclusion that the ABO blood- enclosing the typescript of a paper then in the press, group reactions could better be explained by a single in which is described the behaviour of six allelomorphs triallelic locus than by two unlinked diallelic loci, the of the Rh gene.’’ This typescript was certainly seen by reverse of the Rhesus hypothesis. The grounds in this Fisher because on the back is a note in his hand giving case were the population frequencies of the genotypes. details of a mating in mouse genetics taken from The most accessible reference for this is the worked Gru¨neberg (1943), The Genetics of the Mouse (Mary Lyon analysis in my book Likelihood (Edwards 1992) where, kindly identified this for me). Perspectives 475

haplotype accurately reﬂects the proportions that would obtain if there were no pairwise associations or three- fold interactions between the loci. When the haplotype frequencies are added in the form of dots randomly placed in the corresponding areas, the departures from the equilibrium state are readily perceived (Figure 7). ‘‘Thediagram clearly displays what Fisher himself noted, that the chromosomes are not in linkage equilibrium and that they fall into three groups, common, rare, and absent (i.e., extremely rare). The extremely rare chromosome, CdE, is entirely surrounded by three rare chromosomes, Cde, cdE, and CDE, these being the only three from which it could be derived by the mutation of a single gene...If CdE is deleterious but is maintained by mutation this may explain its rarity, though Fisher actually suggested crossing-over rather than mutation.’’ Note: The account related here involves only Fisher andRaceinthediscussionsintheBunShopin1943and 1944. It is often assumed that Ruth Sanger was there, too, but she is not recorded as having arrived from Australia before 1946. The impression that she was present can easily arise from the use of ‘‘we’’ by Race and Sanger (1982), but this can be interpreted as meaning Race and a colleague or colleagues from the Blood Group Laboratory, perhaps G. L. Taylor. I should be glad to be corrected on this point.

LITERATURE CITED Avent, N. D., T. E. Madgett,Z.E.Lee,D.J.Head,D.G.Maddocks et al., 2006 Molecular biology of Rh proteins and relevance to molecular medicine. Expert Rev. Mol. Med. 8: 1–30. Bennett, J. H. (Editor), 1983 Natural Selection, Heredity, and Eugen- ics. Including Selected Correspondence of R. A. Fisher With Leonard Dar- Figure 7.—(a) The Rhesus haplotype frequencies (see the win and Others. Clarendon Press, Oxford. text for explanation). (b) The key to a. Bodmer, W., 1960 The genetics of homostyly in populations of Primula vulgaris. Philos. Trans. R. Soc. Lond. Ser. B 242: 517–549. The arrival of this typescript and the Cambridge Bodmer, W., 1990 Genetic sequences. Proc. R. Soc. Lond. Ser. B ace anger 241: 85–92. discovery of the fourth antiserum (R and S Bodmer, W., 1992 Early British discoveries in human genetics: con- 1982) were the stimuli for Fisher’s ‘‘Bun Shop’’ proposal tributions of R. A. Fisher and J. B. S. Haldane to the development of the three-locus complex, which must indeed have of blood groups. Am. J. Hum. Genet. 50: 671–676. Box, J. F., 1978 R. A. Fisher, The Life of a Scientist. John Wiley & Sons, taken place some time after mid-October 1943. New York. A personal coda: As a graduate student I was sent by Clarke, C., 1990 Professor Sir Ronald Fisher, FRS. Brit. Med. J. 301: Fisher to Race and Sanger’s laboratory, by then in 1446–1448. Edwards, A. W. F., 1992 Likelihood, expanded edition. Johns Hopkins London again, to learn simple blood-grouping tech- University Press, Baltimore. niques, and I kept in touch with them thereafter. As Edwards, A. W. F., 2005 Linkage methods in human genetics be- Race mentions in his Fisher Memorial Lecture, he was fore the computer. Hum. Genet. 118: 515–530. Edwards, A. W. F., and J. H. Edwards, 1992 Metrical Venn dia- also in touch with J. H. Edwards, my brother, both in grams. Ann. Hum. Gen. 56: 71–75 (reprinted in 2004 in Edwards, connection with one of John’s patients with an ab- A. W. F., Cogwheels of the Mind. Johns Hopkins University Press, normal chromosome 18, and his computer program Baltimore). Fisher, R. A., 1918 The correlation between relatives on the sup- for determining the origin of the X chromosomes position of Mendelian inheritance. Trans. R. Soc. Edinb. 52: in Klinefelter’s syndrome. Many years later John and 399–433. I published a joint article (Edwards and Edwards Fisher, R. A., 1935 The Design of Experiments. Oliver & Boyd, Edinburgh. 1992), which happily reﬂected these links with the Races Fisher, R. A., 1947 The Rhesus factor: a study in scientiﬁc method. and can serve as a coda to this story. Am. Sci. 35: 95–102, 113. We showed how the Rhesus frequencies published Fisher, R. A., 1958 The discontinuous inheritance. Listener 60: isher ace 85–87. by F and R (1946) could be represented in a Fisher, R. A., and R. R. Race, 1946 Rh frequencies in Great Britain. square diagram in which the area designated for each Nature 157: 48–49. 476 A. W. F. Edwards

Gru¨neberg, H., 1943 The Genetics of the Mouse. Cambridge Univer- Race, R. R., G. L. Taylor,D.F.Cappell and M. N. McFarlane, sity Press, Cambridge, UK/London/New York. 1944 Recognition of a further common Rh genotype in man. Moran,P.A.P.,andC.A.B.Smith,1966 CommentaryonR.A.Fisher’s Nature 153: 52–53. paper ‘‘The correlation between relatives on the supposition of Taylor, G. L., and R. R. Race, 1944 Human blood groups. Br. Med. Mendelian inheritance,’’ in Eugenics Laboratory Memoirs XLI.Galton Bull. 2: 160–164. Laboratory, University College London. Wiener, A. S., 1932 Method of measuring linkage in human genet- Race, R. R., 1944 An ‘incomplete’ antibody in human serum. ics; with special reference to blood groups. Genetics 17: 335–350. Nature 153: 771–772. Wiener, A. S., 1943 Genetic theory of the Rh blood types. Proc. Soc. Race, R. R., 1948 The Rh blood groups. Ph.D. Thesis, Cambridge Exp. Biol. Med. 54: 316–319. University Library, Cambridge, UK. Wiener, A. S., 1961 Principles of immunogenetics and the blood Race, R. R., and R. Sanger, 1982 Fisher’s contribution to Rh. Vox groups, pp. E27–E28 in Second International Conference on Human Sang. 43: 354–356. Genetics. Excerpta Medica, Amsterdam.