Phenylthiocarbamide: A 75-Year Adventure in Genetics and Natural Selection

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove VARIATION in taste sensitivity to the bitter compound phenylthiocarbamide (PTC) is one of the best known Mendelian traits in human populations, ranking alongside eye color and blood types in the canon of classic examples. Much of PTC's appeal arises from the fact that it is nearly impossible to guess one's phenotype until explicitly tested, yet, when tested, the phenotype is so striking as to be amusing. This property is important, particularly in education, because it can spice up lessons on inheritance. Less obvious, especially today, is PTC's appeal as an easily typed yet highly informative genetic marker. It was this aspect of the trait that made PTC an important instrument in the earliest efforts to dissect the human genome. Variation in PTC sensitivity has also had long-standing appeal from an evolutionary standpoint. The connection of variable taste sensitivity with an aspect of behavior so obviously connected to fitness—diet choice—has long raised a basic question: Has natural selection been acting on this trait? It was this question that motivated the great statistician, R. A. Fisher, and his friends E. B. Ford and Julian Huxley, to perform what appears to be the first test for natural selection in a specific human gene. In an often-overlooked article published in 1939 (listed by Science Citation Index as having been cited <50 times in the ensuing 65 years), Fisher et al. (1939) reported that chimpanzees, like humans, show variable sensitivity to PTC. This, they argued, is strong evidence that balancing natural selection must have actively maintained variation at the locus from a time prior to the human–chimp divergence. While little-known today, this article set the stage for what has become an industry in the field of genetics. Variation in PTC sensitivity was first discovered in the early 1930s. In a laboratory incident that would curl the toes of the most stoic OSHA officer, Arthur L. Fox was pouring some PTC (a white powder) into a bottle when some “flew around in the air” (Figure 1) (Fox 1932). A co-worker nearby, C. R. Noller, complained that the dust tasted bitter, but Fox insisted that he could taste nothing. The two then took turns tasting the powder and found that they really did differ dramatically in sensitivity. Determined to find out whether he and Noller were unique, Fox set about testing “a large number” of people and found that distinct variation was common regardless of age, sex, and ethnicity. Most interestingly, Fox found that most people fall into just two categories: those able to taste the compound even at very low concentrations, whom he referred to as “tasters,” and those unable to taste the compound except at very high concentrations, whom he referred to as “nontasters” or “taste blind.” Facsimile of the heading and first paragraph of Arthur Fox's first article on PTC sensitivity in the Proceedings of the National Academy of Sciences (Fox 1932). This article was accompanied by Albert Blakeslee's article on the inheritance of PTC sensitivity (Blakeslee 1932b). Fox's finding received immediate attention, appearing in brief news stories in both Science and The Scientific News Letter (Anonymous 1931a,b). These stories focused on the curiosity of the finding; however, they also caught the attention of geneticists, who were beginning to explore the organization of the human genome. T. H. Morgan's earlier work with Drosophila, which would earn him the Nobel Prize in 1933, had shown that Mendelian markers could inform genomic organization: studies of these markers in flies had led to the discovery of “linkage groups.” The availability of a similar set of markers in humans would be similarly useful; however, they were difficult to find. Further, while novel Mendelian markers in flies could be driven rapidly to usefully high frequencies through selective breeding, human variants had to be used at their natural frequencies. In mid-1931, Fox's work came to the attention of L. H. Snyder, who had been working on Mendelian markers in human populations. At the time, the number of such traits was small. Snyder (1931b) listed just six, including dubious entries like the direction of the whorl of hair on the back of the head and the presence of hair on the second joint of the fingers and toes. Primed to recognize more such traits, Snyder siezed upon the utility of Fox's finding right away, reporting later that on learning of Fox's results, he “immediately wrote Dr. Fox asking for some of the compound with which to investigate the possible inheritance of this taste deficiency” (Snyder 1931a, p. 151 ). Snyder confirmed Fox's basic results and also tested a number of families, which led him to the conclusion that nontaster status is conferred by the recessive allele at a single locus (Snyder 1931a). Fox's findings also caught the attention of Albert F. Blakeslee. Blakeslee had achieved fame for his work on the genetics of plants, but his interest in Fox's work seemed to stem more from the fact that it involved variation in the human senses. Blakeslee had long been interested in sensory variation. In 1918, for instance, he had reported that humans vary in sensitivity to the scent of certain flowers, with some people being “blind” to the smell of some strains of verbena (Blakeslee 1918, 1935b). Blakeslee, like Snyder (1931a), immediately replicated Fox's basic results, along with the finding that PTC blindness appears to be a Mendelian recessive (Blakeslee and Salmon 1931). Blakeslee's interest in the chemosenses would persist for some decades, during which he would publish a number of articles on human variation in taste and smell (Blakeslee and Salmon 1931, 1935; Blakeslee 1932a,b, 1935a,b; Blakeslee and Fox 1932; Salmon and Blakeslee 1935; Blakeslee and Campbell 1948). In 1932, Fox published the definitive description of the PTC sensitivity polymorphism in the Proceedings of the National Academy of Sciences (most likely at the invitation of Blakeslee, who was a member) (Fox 1932). In this article, Fox described his initial discovery of the polymorphism along with his early efforts to determine whether variation in PTC sensitivity is rare or common. The article went on to show that sensitivity to PTC is correlated with sensitivity to a variety of related compounds characterized by the presence of a distinctive N=S moiety. Further, Fox found that the bitterness of many of these compounds could be eliminated by substituting the sulfur (S) with an oxygen. One of these compounds (para-ethoxy-phenylthiocarbamide) differed from Dulcin, an artificial sweetener, by this simple substitution. However, Dulcin does not show variation in taste as PTC does (Fox 1932). In an article immediately following Fox's in the Proceedings of the National Academy of Sciences, Blakeslee described the first large-scale study of PTC inheritance in families and also explored the threshold of PTC sensitivity (the minimum concentration at which PTC can be detected), discovering the almost incredible fact that sensitivity can vary by almost five orders of magnitude (Blakeslee 1932b). This study supported earlier results, but it also suggested that while the vast majority of variance in PTC sensitivity must be accounted for by a single locus, other genes are likely involved as well. Thus, PTC sensitivity is not a simple Mendelian trait. It is (in today's parlance) complex. Nonetheless, inheritance of PTC sensitivity is so close to simple Mendelian that it rightly retained its use as a marker for decades. The identification of variable PTC sensitivity, its (nearly) simple Mendelian pattern of inheritance, and the relative ease of PTC phenotyping using treated blotter paper led to an explosion of studies of PTC sensitivity in human populations. In the 10 years following Fox's initial findings, sample sizes reached the thousands, with estimates of the nontaster allele frequency ranging from 13 to 63% with an average of ∼50% (Blakeslee and Fox 1932; Fernberger 1932; Levine and Anderson 1932; Blakeslee 1935a,b; Blakeslee and Salmon 1935; Salmon and Blakeslee 1935; Rikimaru 1936; Strandskov 1941). This number would eventually grow to include tens of thousands of subjects in hundreds of studies (Cavalli-Sforza et al. 1994; Guo and Reed 2001) (Figure 2). (a) Frequency distribution of “nontaster” allele frequencies reported by Guo and Reed (2001). The mean frequency across the 344 samples examined is 0.48 (modified from Wooding et al. 2004). (b) Geographical distribution of PTC allele frequencies (modified from Cavalli-Sforza et al. 1994). In August 1939, the Seventh International Congress of Genetics convened in Edinburgh, Scotland (Crew 1939). This meeting had suffered an inauspicious start, with its location having been moved from Moscow due to disputes with the Soviet government, which was under a strong Lysenkoist influence (Soyfer 2003). To make matters worse, Europe stood on the brink of war. The Congress started as planned, but the war was a major disruption. First the German delegation, and then the Dutch, were forced to leave prematurely. Immediately following the meeting, on September 3, several American participants trying to return home were among those on board the Athenia, the first ship sunk in the war by a German submarine (Crew 1939; Caulfield 1959). Among the Athenia survivors were Charles Cotterman and Bronson Price, who were rescued by the City of Flint—which happened to be transporting James Neel and George Beadle (Opitz 1989; Schull 2002; Berg and Singer 2003, pp. 127–128). This meeting was later described as being “of no great significance” scientifically (Punnett 1941); however, from this meeting emerged a remarkable experiment involving a remarkable cast of characters: R. A. Fisher, E. B. Ford, and Julian Huxley (Yates and Mather 1963; Baker 1976; Clarke 1995) (Figure 3). (Left to right) R. A. Fisher, E. B. Ford, and Julian Huxley. Portraits are from Yates and Mather (1963), Clarke (1995), and Baker (1976). At the time of the Seventh Congress, Fisher, Ford, and Huxley had been friends for many years. Ford recalled that he and Fisher had first been introduced by Huxley in 1923, when Fisher came to see him at Oxford to learn more about his ideas on genetics and evolution, in spite of Ford's being a mere undergraduate at the time (Ford 2005). By Ford's account, the three were united in their interest in the possibility that natural selection, which was viewed as being “of minor importance, only capable, at most, of bringing about small adaptations,” could be an important driving force in evolution (Ford 2005, p. 416). While theoretical work on the relationship between genetics, natural selection, and evolution by then was well developed (Haldane 1924; Fisher 1930), the genetic effects of selection in humans were yet to be demonstrated empirically. However, Fisher et al. (1939, p. 750) later related, “in the course of discussions on the possibility that the blood-group frequencies found in man were determined by a balance of selective influences, it occurred to one of the authors that evidence on the parallel possibility for taste-[sensitivity] could be obtained by testing the anthropoid apes.” Thus, the three recognized that PTC sensitivity represented a unique opportunity to test the hypothesis that natural selection has acted on a specific human gene. With the aid of a “Dr. Riddell” from Glasgow, Fisher was able to procure graduated solutions composed of “2% sugar in all, and either none, 6 1/4, 50, or 400 parts per million P.T.C.” (Fisher 1939a). Then, with the assistance of F. A. E. Crew and a “Dr. Gillespie,” these solutions were presented to eight chimpanzees and one orangutan at the Edinburgh Zoo, apparently as a drink (Figure 4). These tests were not without incident. David Finney was in attendance at the Seventh Congress when Fisher et al. made an expedition to the Edinburgh Zoo to test the animals there. Finney did not go along but the back with the that one of the had a to Fisher, at him or even to Cavalli-Sforza the which was related to him by either Mather or E. B. was some initial about whether it would be possible to determine from an whether it was a or however, first they tested took out of because it at Fisher in his and at A possible who is in Fisher et (1939) article on the first tests of PTC sensitivity. from The results of the Edinburgh experiment were of the eight tested chimpanzees to be and two to be the that PTC sensitivity is conferred by the allele in a these results that the and nontaster were at frequencies similar to those in humans (Fisher Fisher et al. 1939). Fisher immediately the of the to Ford, to to be that be polymorphism with a apparently for of years in a which had of as without selective Fisher argued, variation in PTC sensitivity is to have just then the presence of polymorphism both humans and chimpanzees can be only by a single of PTC and nontaster prior to the evolutionary of these two by the of both (Figure for the of PTC and nontaster Fisher et (1939) this the and nontaster prior to the divergence. both were maintained in up to the The of both for such an that it must be million or million is balancing selection has not been because one allele or the other would be to go to Wooding et this nontaster were from in the divergence. This finding was of such that Fisher, Ford, and Huxley immediately to it as a to (Fisher Ford however, they to the to include animals at the Zoo, tests were on September and the Zoo, on (Fisher A of among Fisher, Ford, and Huxley in the course of these (Fisher Ford Huxley 1939). about the Edinburgh results, Ford wrote to Fisher, it be possible to to make a of the of and in (Ford Fisher that Huxley on to test of other in the of this came as for its with were to the war at this time and Fisher, at was In a September to Ford, Fisher is in a of having been without for are they to their work it be as a of the (Fisher In spite of these the experiment Ford find it to (Ford Fisher and Huxley the possibility of more work on sensitivity (Fisher but the of war Fisher was forced out of the and the to a its early the results of the PTC were Fisher, Ford, and Huxley could have and their article on the tests was published in the 1939, of (Fisher et al. 1939). PTC sensitivity was in the of one which was (Fisher et al. 1939, p. sizes for of the tested were and the two to be two of the three were the sample had two and two but this was by the fact that two were and one of these was represented by two However, the results in chimpanzees were of the tested, were and were allele frequencies of and for the and nontaster nearly to several studies in humans 1941). Fisher et al. (1939, p. 750) the of it is that the have the million or more which have the of the anthropoid and The remarkable that this the for this apparently have a selective both the and both in the of the chimpanzees and in that of the selective it would at be to but of the of a and polymorphism determined by this can be no for of results of PTC tests in by Fisher et al. (1939) two of results of PTC tests in by Fisher et al. (1939) two in their article, Fisher, Ford, and Huxley had sensitivity be driven by natural However, as early as 1931, variation in sensitivity had been suggested as a possible of in humans and that these into variation in 1931). It was also recognized that a common of bitter compounds in was found in plants, which a of bitter to et al. of such as and are The use of by would to a that be driving the evolution of the PTC however, PTC is and no compounds found were known to with PTC sensitivity. The first connection between and PTC sensitivity was made in when William reported that taste sensitivity to nearly with taste sensitivity to PTC This which is found in common like and was recognized as a of et al. Thus, a through which natural selection on the PTC was able to taste such compounds and This was a but it to the of both and in chimpanzees and humans, for the to taste such compounds were then the allele rapidly have reached In spite of in Fisher et (1939) the of PTC sensitivity in a of focused on the of along with its and In and the that was to become in of sensitivity and and a large of work tested about the relationship between taste sensitivity and specific such as and et al. 1963; et al. et al. et al. et al. and 2002; and 2002; et al. et al. et al. 2005). A of and more for taste sensitivity were developed during this time and and the of variable PTC sensitivity were explored as well However, in these studies was a of the genetics of sensitivity, almost years of the of genetic variation in sensitivity were in when et al. two at the of taste work that one of these as a for bitter and that it is just one of many such found in et al. et al. et al. 2003). These small (most in are by the in the of the are to the through a small the taste et al. they are to into with the many compounds that the of these compounds a of to a and These are of to a of et al. et al. et al. et al. et al. 2005). While some early studies had that PTC sensitivity was correlated with variation in the blood was made that in a PTC locus et al. 1976; et al. However, the discovery of the as a led immediately to a of studies of the inheritance of PTC sensitivity. early by Reed et al. with a related phenotype to results, a on However, definitive results were obtained by et al. in an and by et al. in a of the These that variation at the locus for of variance in PTC sensitivity and that most of this variance is accounted for by the presence of just two common a and a “nontaster” Further, the frequencies of these in human well to frequencies from phenotype (Cavalli-Sforza et al. 1994; Guo and Reed Wooding et al. 2004). The identification of a major locus for PTC sensitivity for human sensitivity to this compound in tests for between and the between and PTC sensitivity, are the et al. et al. et al. 2005). One of the most important of the of has been the in on the nontaster with the very earliest findings, PTC sensitivity has been described in of and “nontaster” with to the the between The has been that the nontaster allele is or However, studies of variation at that this could be The major and nontaster differ from other by just three no or other obviously are et al. et al. Wooding et al. et al. 2005). Further, while to the and nontaster show to is not et al. 2005). Thus, the human nontaster allele be a for some of compounds that does not include PTC. specific for the PTC nontaster allele has yet been however, two studies have reported that the of the taste bitter to PTC but to PTC the possibility that it such a and et al. 2005). The of et al. likely to this that the nontaster allele is an immediate through which at this the allele sensitivity to PTC and its and the nontaster allele sensitivity to some other set of then be able to taste both of Thus, they a by being able to the of a of bitter compounds can The identification of the as a major of PTC sensitivity has the long-standing hypothesis of Fisher, Ford, and Huxley to be using genetic not at the time of their These on the basic as those used by Fisher et al. but are able to on of variation at the and Wooding 2003). Wooding et al. of variation at in a sample of and This that at is the number of variable when human to relative is into In of at this locus are average for humans among that the high be by among populations. These were by Wooding et al. as evidence that balancing natural selection as first suggested by Fisher et al. actively maintained two distinct in human populations. However, of variation in chimpanzees that Fisher et al. (1939) were not in their of this To determine whether PTC sensitivity in chimpanzees as in humans, by two major at Wooding et al. of variation in a in this sample that chimpanzees two common at and an experiment to the that these are with PTC sensitivity. However, the common human and nontaster which differ by three the and nontaster were found to differ by a single in the second of the it from to This with of by the which the nontaster Further, a that the and nontaster in chimpanzees are more similar to other either is to the found in the findings of Wooding et al. Fisher et (1939) finding that both humans and chimpanzees and nontaster and that these are found at similar frequencies in however, they the hypothesis that these were prior to the divergence. the nontaster which their effects through to have The of the selective this more a of Arthur Fox could not have in 1931, the that his discovery would have the years. the decades, his was as a in as as genetics, evolution, and even The of this trait in R. A. on natural selection, and his of the test for the effects of natural selection in a specific human is to the of the trait in to the of genetic variation in In many the started by Fox and Fisher et al. (1939) is just as the discovery of variable PTC sensitivity by Fox the for Fisher, Ford, and Huxley to early the evolutionary of genetic the discovery of more such genes has many such et al. et al. 2005). are the by the availability of from which can on the of the genes et al. et al. et al. et al. et al. 2005). One has that the early would be with the that have made and by F. and and