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Q: What do you mean "blood dyscrasias?"
Beutler: Any blood disorder. And by blood dyscrasia one means, for example, low white count, or low platelet count, or low hemoglobin. Those are all blood dyscrasias Wintrobe had become interested in organizing this committee because there'd been a sort of tragedy involving the drug chloramphenicol. We now realize that chloramphenicol (or chloromycetin) is a dangerous drug which can produce fatal aplastic anemia. It turned out that there'd been a fair number of cases of aplastic anemia associated with chloramphenicol before the cause-and-effect relationship was recognized, simply perhaps because people hadn't communicated with each other. It's a rare event, so if somebody had one case and somebody else had another case, and unless they talked about it they wouldn't realize that there was an association. He felt that if some kind of registry were established that this kind of situation might be avoided in the future. So he organized a committee on blood dyscrasias under the auspices of the AMA. Arno Motulsky and I were on that committee and Arno told me about a very bright young geneticist at the City of Hope by the name of Susumo Ohno. Now, I'd only been there for a few months and I thought I'd heard the name but I'd really hadn't met him, though there were quite a few people I hadn't met there. I didn't really meet Ohno until I attended the International Congress of Hematology in Tokyo, Japan in August-September 1960.
Q: In other words you were working in the same -‑
Beutler: -- Institute but we met in Japan. We may have met briefly at the City of Hope, but there we had a chance to visit a little bit. But more important I had a chance to hear him give a lecture about what he was doing. He had done studies in rodents in which he had shown that the two X chromosomes of the female were morphologically different. One was tightly condensed and the other one looked like the other chromosomes. And this gave me the idea that the two X chromosomes might not both be active in females, that only one might be active. It led me to formulate the X-inactivation hypotheses. The same hypothesis, based also on Ohno's work, was proposed by Mary Lyon and it turned out as events unfolded that she published her work before I published mine, although I presented mine earlier. She's very often credited with what's called lyonization, which I prefer to call the X-inactivation hypothesis. Basically that proved to be a rather fundamental discovery in biology and genetics and although Mary Lyon ended up getting a very large share of credit for this, I don't think it escaped the scientific community that I made this contribution also. It played a role in my later election to the National Academy of Science, because I think some of my colleagues have recognized that I also had the same insight.
Q: Was she a hematologist?
Beutler: No. She was a mouse geneticist. Well, you know, in retrospect, many great ideas are obvious and that one is and she had a couple of major advantages over me and one was that being a geneticist, perhaps she was a little closer to the whole situation. But basically what she did is this. She knew that there were mutant genes in mice that effected coat color and so she reasoned that Motulsky only one or the other x was active in each cell that mice who were heterozygous for these coat colored genes would have a doppled, patchwork appearance. And they did. And that was her evidence. I didn't know about these genes. As a matter of fact I asked our geneticist at the City of Hope about such genes but he didn't know about them either because he was a drosophila geneticist. And this phenomenon doesn't occur in drosophila. So my approach was quite characteristically hematologic. I reasoned that if only one X was active in each cell and different cells had different X's active, that if we examined a heterozygote for G6PD deficiency, that she should have one population of normal cells and one population of deficient cells, rather than what had been thought heretofore as just an intermediate population. It was much harder to measure the activity in individual red cells than it was to observe the coat color of a mouse. And actually it was very difficult for me to solve that problem. I solved it in I think what was a reasonably elegant way, but it took me more than a year to solve it.
Q: How did you solve that problem of distinguishing two different --?
Beutler: Well, the way that I tried to solve it initially was to see whether there was some way in which I could histochemically measure individual red cell enzyme activity. But I failed doing that. And then one day I got the idea that I could approach the problem by measuring glutathione stability. As I mentioned earlier if when one incubates deficient cells with acetylphenylhydrazine the glutathione disappears. If one measures it in normal cells, it's stable. It suddenly occurred to me that if one had a mixture of normal cells and mutant cells, that one would observe a rapid fall as the glutathione disappeared from the deficient cells, but then, after that, the remaining glutathione being in normal cells would be stable. If, on the other hand, the enzyme activity were intermediate, then one might have a fall in concentration, but it would be intermediate in severity between being mutant and being normal. And one could test this model by taking blood from a person who was G6PD deficient, a male, and mixing it with blood from a male who was not deficient, seeing how the glutathione disappeared. Indeed it disappeared in components, and then studying a female, it disappeared exactly.
Q: I see. So there are certain kinds of interesting costs and benefits to looking at the blood level as opposed to the mouse coat.
Beutler: That's right.
Q: -- As an indicator of -‑
Beutler: Well, you know, if I'd known about that mouse mutation that would have been very easy. I would have gotten a mouse. I would have looked at the heterozygotes and I could have published the same paper that Mary Lyon did. As it was I worked for a year or more trying to work out a technique -- trying to think of a technique in which I could demonstrate there were two populations and that took time. You know, when I took the chairmanship of the Department of Medicine at the City of Hope, people assumed that I was giving up research. That was the end of my research career. Even then people considered chairmanships to be so demanding that somebody really couldn't continue to do research when they were chairman. I moved to the City of Hope at the very end of '59, so basically I started at 1960 and I did this work at the end of '60, the beginning of '61. So actually not only was it a somewhat a difficult problem, but also I was in a new place. I had a lot of responsibilities that slowed me up a little bit. And then, another thing that slowed me up is that I didn't want to publish something that was wrong and even though I had the data quite a bit earlier, I was hesitant to make a fool of myself, and I re-repeated the work numerous times. So the sum and substance of it was that her paper came out a little bit before mine did.
But I continued to work on the genetics of G6PD deficiency. It became evident that this was a heterogeneous disorder. When we first studied it we studied it mostly among black American males. When I had worked out the methods for detecting this defect in red cells, investigators in different parts of the world began to look for this defect, too, and it was found I think almost simultaneously in Israel by Chaim Sheba and Ari Szeinberg and by Sansone in Italy. At first we thought that G6PD deficiency was the same wherever it was found. But then it became apparent that that the G6PD deficiency in the Mediterranean area was much more severe than the African kind of G6PD deficiency and that the residual enzyme, the little bit that there was, was really very different in its properties. Then I participated in standardizing methods which would have allowed people all over the world to study enzymes and to compare the enzymes. The result of this as I told you yesterday was that over 400 variants have been described. In the last few years we've been able to study these at the DNA level and we find that there aren't really that many different variants. But there are still quite a few. And that's basically the thread that has run through my G6PD work.
Q: Now you mentioned yesterday that by the late 70s the technology had become limiting for exploring for that area of research. Can you talk a little bit more about that and particularly about what the technology had been?
Beutler: All one could do until this last decade is to purify the enzyme from red cells and determine its characteristics. One couldn't even determine its structure because one couldn't obtain enough enzyme to sequence it, except from normal blood; with these deficient mutants one simply couldn't get enough. There wasn't really very much one could do except to study additional cases, to characterize them, and then to publish a paper about them. Really what I think it was, from a point of view of acquiring Knowledge, it was really kind of spinning wheels. That was really true through most of the 70s. Most of the excitement went out of G6PD deficiency for me after the 60s, and actually it led me into other areas that were perhaps equally fruitful.
One of these was the study of galactosemia which is a hereditary defect that is relatively uncommon but extremely important to diagnose early because if one detects galactosemia at the time of birth or within a few days thereafter, a lactose free diet will salvage that child. But if one doesn't detect it the child will usually die, or if one detects it before it dies, the child will be severely damaged in terms of blindness, mental retardation and so forth. I'd learned quite a lot about the metabolism of red cells in my work with G6PD and so I decided to see whether one could use red cells to screen for galactosemia. There was a complex assay that one could carry out, but could one do a simple test on every newborn? And to make a long story short we developed such a test and that test is still being performed all over the world and many millions of infants have been tested with it. And this also got us interested in matters such as the frequency of galactosemia. We found that there was another very common mutation that caused lowering of transferase, which is the enzyme which is defective in glactosemia; lowering of transferase activity, but not a clinical disease. We worked on galactosemia for some 10 years or so, but then I moved on to other areas. One of the areas that we moved into was red cell storage, because I thought that one of the really practical applications of understanding red cell metabolism was in the storage of blood and blood banking. That's an area in which we've worked intermittently probably for the last 25 years and we're still working on it to some extent. We've been involved in developing better blood preservatives. Another major area that we have been studying for quite a long time is in the glycolipid storage diseases. I became interested in the glycolipid storage diseases through a patient whom I first saw in about 1965 or 66 with very severe Gaucher's disease. She was a girl of 17 or so at that time. As it happened 10 years previously when I was taking care of patients with sickle cell disease, I felt very frustrated by the idea that here is a disease that we actually understood to some extent, but we really couldn't do anything about. And it was about that time that the enzyme defect that caused Gaucher's disease had been discovered and I began to wonder whether there was something that I could do to correct the enzyme deficiency. I've worked on Gaucher's disease intermittently now for about 25 years and actually a treatment is just beginning to emerge from some of this work, and there may be better treatments in the future. But we also studied two other diseases in that general group. One is called Fabry disease. It's a rather rare disease. And the other one, more common, is Tay Sachs disease. There we were quite successful in actually determining the structure of the enzyme that is deficient in the disease. We showed that it was made up of two different kinds of subunits. We showed that one of these subunits was missing in Tay Sachs disease and the other subunit was missing in a disease called Sandhoff's disease, which is very similar to Tay Sachs disease. So we worked in that area as well.
Q: Did you think that your later work then has shifted from studying deficiencies, the mechanism for deficiencies to studying diseases and possible therapies based on your knowledge of the deficiencies?
Beutler: I don't think it's really been a shift. In every case I've been interested in the deficiency from a clinical point of view. I've been interested in the possibility of doing something about it and I've been interested in understanding the fundamental mechanism. When all we could do is to do protein structure then I was interested in the mechanism from the point of view of protein structure. Now that we can look at DNA structure much more easily, we examine DNA structure.
Q: Getting back for one moment to the G6PD work. I noticed that you were involved in several groups. The WHO group on hemoglobinopathy in the late 60s and the early 70s, the National Research Council Committee on sickle cell disease, CDC hemoglobinopathy task force. Can you explain something about the importance of those committees, or those organizations, and for you at the time? Why did they appear when they did. What kinds of work did you do?
Beutler: The WHO committee was really one that had to do with standardization and perhaps working on methods to bring technology to developing countries -- there was just one meeting. I learned a lot about the hemoglobins I didn't really know. I was the lucky person who was appointed the recorder so I had to write the report. I got to meet some people, some of whom have been lifelong friends. But that was sort of a one shot type of thing. I think the most important of those bodies probably was the NRC committee on sickle cell disease. It was probably in the late 60s when sickle cell disease became very politically prominent, there was a great move to screen everybody for sickling. And I might say that I was opposed to that from the beginning. As a matter of fact I organized a letter that was written for The New England Journal of Medicine, opposing that in the early 70s. I felt that it would do much more harm than good to identify people with sickle trait because there was such a tremendous amount of misunderstanding and also a lack of information about what the trait really meant in terms of a person's health. Here you're dealing with something that nine percent of black people have. Obviously it can't be very bad, but now you have stories of people who developed splenic infarcts at high altitude flights, people who developed blood in their urine, and perhaps most important, people who died suddenly during severe exercise. And then the issue became, "Well, if you identify people with the sickle trait and particularly in the military, which was the organization that sponsored the commission, the NRC study, what do you do with that?" What do you let them do and what don't you let them do? If a young black man wants to be a marine and he has sickle trait, do you exclude him? Or do you let him be a marine? And if you let him be a marine, well what are the chances that he's going to die suddenly when he's exercising in the heat one day? Well, these are very hard questions. Do you let a person with sickle trait be a pilot? Do you let him be a diver? All those kinds of questions. The committee that I was on tried to grapple with those questions and we made recommendations that we thought were reasonable with the state of knowledge at that time, but we strongly recommended that a study be done, which really determined what the risks were. That study was never really done, although fragments of the information have come out since through some partial studies. Our recommendations, as I remember at the time, were that with the uncertainty of what might happen to a pilot, particularly a pilot who was piloting an aircraft that had other people aboard, that one couldn't really let a person with sickle trait be a pilot. But that in terms of any other activities, we just didn't see that there was enough evidence to preclude people from following a career which might be rewarding to them because of sickle trait.
Q: What were –
Beutler: Well, that's just what I was going to say, I think that was really sort of the extent of my involvement in that issue.
Q: What other fields were represented on that committee?
Beutler: Well, the committee as I recall had a few people from the humanities, you know, a professor of philosophy, who I think didn't show up for the meeting. That sort of thing. Then there were several prominent hematologists. I think Clem Finch was on the committee -‑
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Beutler: My memory might not serve me well on this but I think Lou [Louis] Sullivan was on the committee, too, and as you know he's Secretary of Health, Education and Welfare now. But there were participants from hematology, from genetics, from the humanities, and those were really basically the issues that we dealt with.
Q: Is that still the -- the question of the importance of the sickle cell trait, the possible effects that the sickle cell trait at high altitudes is still a problem that -‑
Beutler: Well, I don't hear as much about it anymore. There was a study published two or three years ago in which a large survey was made in the military, which is something that we recommended many years before, that indicated that there was an increase in the incidence of sudden death of people with sickle trait. But the incidence was so low that I'm not sure that one really should be making decisions on that basis. There are too many people, not only on this matter but I think in all matters affecting humankind, who expect absolute answers. That yes it's OK or no it's not OK. So suppose you have a situation -- and here I don't think I stray far from the truth -- let's say that a black recruit with sickle trait has two chances in a million of dying while he's running on the parade ground in the hot sun. But there are also probably 20 chances in a million that people will die from various other misadventures healthwise and probably a thousand in a million that they're going to be run over by a truck or some other accident happening. To what extent do you set policy in terms of such rare risks? You get into a position where you can't say there is no risk and yet if you take the risk and that one thing happens, then you're in the soup. If you're in the army there's a congressional investigation, a lawsuit from the family and so forth. "You should have known, you see there's this paper." These are tough problems. Tougher than sequencing than DNA, I'll tell you.
Q: What was the importance of establishing a hemoglobin standard in this country. I notice also that you were a member of the National Academy of Science, special committee on hemoglobin standards.
Beutler: Can I see what that is? I can't remember what we did [laughter].
Q: I have it written down here.
Beutler: I can't really tell you. It might have had to do with the standardization of hemoglobinopathy, but I really don't know. I just don't remember.
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