Q: And this is the tradition of the research -‑

Beutler: That I inherited maybe -- yes, from Leon Jacobson. Of course you never know for sure. Maybe I would have done things just the same. Maybe I would have had the wisdom to do it that way even if I'd never been exposed to him. But I doubt it. I think one tends to emulate one's chiefs. That's the way his program was and while I never consciously said to myself, "I want to do this the way that Jacobson did," that's the way it evolved. Now when I look back I realize that many of the things I'm doing are done the same way that Jacobson did them.

Q: Can you give us an example of how the organizational structure that you just pointed to was parleyed into a particular insight that came out of your laboratory? In other words, the benefits of training technicians, as opposed to collaborating with others?

Beutler: OK. Well, insights -- major insights usually don't come from technical personnel. They usually come from the investigator who has a broader and deeper understanding. But it's not enough to have an insight; you've got to develop the data. That's where the technicians are just invaluable. I'll give you one example. One of the topics that I've been concerned with for the longest period of time is glucose-6-phosphate dehydrogenase deficiency, and I'm probably better known for my work with that enzyme than anything else. That's work that I started when I was in the army and has been ongoing for about 35 years. By the late 70s that area was pretty well played out in terms of what one could do because the technology was really limiting. One of my colleagues, Lucio Luzzato, worked very hard and very effectively in the early 80s with a collaborator, a molecular biologist by the name of Graziella Persico, to clone the G6PD gene. Once that gene had been cloned and the sequence published, it was possible to begin to study mutations in G6PD at the DNA level. Now we’re able to actually sequence the DNA to see where the mutations were. I actually started this work in our laboratory with a postdoc, with Dr. Akira Hirono, who had been sent to me by Shiro Miwa in Japan. I put him on the project of finding the mutation in the most common African deficient variant G6PD A-. He worked on this project for nearly two years. He didn't know any molecular biology to start with, but Wanda and the others in our laboratory were efficient and taught him the methods, and he found those mutations. After Hirono left, or shortly before, I put Wanda Kuhl on the project. Over the preceding 10 years or so we had accumulated, DNA from many patients with variants. Now the task was to find out what the mutation was in these individuals. I worked with Wanda for two years working out a simplified technology to find these mutations rapidly. Now we can find the mutation in a G6PD variant in a week; it took two years to find the first one. We have now basically opened up this entire field, shown where the mutations are in about 20 different variants, and developed some insights in that we now realize that many of the mutations that were thought to be different are really the same. For example, with respect to G6PD A-, which was thought to have been an African mutation, it turned out that that mutation is not uncommon in southern Europe but was thought to be different mutations under different names. But when we perform DNA analysis we find that they're the very same mutation. While these are not major insights that have changed the course of medicine and biology, they have been important in this area of the genetics of a very important blood disorder. And I couldn't have done it very well with a postdoctoral fellow. It took two years just to work out the technology and if a fellow worked out that technology in two years and then moved on and then tried to hand it over to somebody else, and somebody else tried to perform these studies, it wouldn't have worked out nearly as well. Moreover, after the first 10 or 15 mutants had been studied, perhaps a fellow wouldn't be interested in investigating any more. But I wanted to get more done because I'm interest in population genetics, and in working with a technician and particularly with a very excellent and loyal one, well, there's no problem. We just sit down and decide which ones we were going to study, and study them.

Q: So, this technology really has to do with sort of increasing the speed with which you can do hemoglobin analysis?

Beutler: This is an enzyme.

Q: Right. An enzyme.

Beutler: Yes. Right. And as a matter of fact, a really interesting example of how much better this system works is that just last week, using Reference Update I found that Lucio Luzzato had a paper -- that had just come out in Biochemical Journal, which is sort of the British equivalent of our Journal of Biological Chemistry in the United States. And this proposed a -- I could tell from the title and from the abstract -- a rapid technique for sequences of G6PD variants. They had done one variant. Their technique is not nearly as simple as ours. We have a paper in press in the Journal of Biological Chemistry and in that we did ten variants and presented a method which was really much better than theirs. That's the kind of work I can do with Wanda. The other thing about technical people like Wanda, or like Carol West, who's the other person who's been with me for a great length of time, is they themselves have really very good insights into what's happening. It's very common for them to come in to see me and to tell me that they've encountered a problem. Then they show me the problem and they wait respectfully for me to tell them what the solution is. I usually don't know. And then after giving me my opportunity to speak, they say, "Well, I think I know what it might be, Dr. Beutler." And then they tell me. And usually their answer's right. They really have very good insight into problems, particularly methodological problems; sometimes theoretical problems too. I'll give you another example, however. One of the most important genes to clone right now, or a very important one, is a gene for idiopathic hemochromatosis, iron storage disease. It's a very common disease. Not many people in molecular biology know about it. I know about it because I'm a physician and a hematologist, and patients with this disorder are usually taken care of by hematologists. I had a long shot idea of how to clone that gene. And with a long shot idea like that it probably won't work. But if it does, it'll be as important as cloning the cystic fibrosis gene, which as you know has gotten a tremendous amount of publicity recently. Well, I put Carol West on the project. Carol's been working on it for nearly a year. And actually I sent her to France for a week or so to learn some techniques that we needed and I have to say that we probably only have a 5 or 10 percent chance of cloning that gene using the approach I’ve chosen. But if we don't succeed, it won't be Carol's career or my career. It will be a disappointment but science is made up of a succession of disappointments and triumphs, I guess, and you have to take them as they come.

Q: So that's a really wonderful way of managing the risks.

Beutler: That's right. I have another fellow. She's a clinical fellow, really, who's spending a year in the laboratory, and she wants to do something in molecular biology to help her get a future job. I've given her a very circumscribed topic, that is going to give an answer one way or another. It has to do with linkage analysis of the G6PD A mutation. I was able to obtain the DNA from colleagues, and she's learned from one of my technicians how to perform the needed experiments. A paper will come out of that, and it will be an interesting one. But that kind of work is never going to be as important as cloning a-gene for an important disease. We can achieve a balance in our laboratory. There are some laboratories now that have 10 or even 20 fellows working in them. I don't understand how the head of that laboratory can give those people training or guidance or any of his or her time. You can do that with two or three fellows, but I don't think you can accomplish it with twenty. When you use fellows as technicians, as many investigators do, you're subverting the education process, and I don't think the outcome of the research is as good as either.

Q: Can you talk a little bit about the status of hematology when you first entered the field? The sorts of research problems that Dr. Jacobson was interested in, or the sorts of clinical reasons for pursuing hematology?

Beutler: Yes. Well, first off, when I entered hematology, it was a relatively small specialty. When I came to the City of Hope in 1959 to become chairman of the Department of Medicine there, there were probably a total of 6 or 8 hematologists in all of the Los Angeles area, and 5 of those were at UCLA. So, I knew everybody in hematology. Now there are probably 300 in the Los Angeles area. So first off, it was a much smaller field. The kind of problems that were being addressed then are really not all so different from the kind of problems that are being approached now but they're being approached at a different level. Jacobson had two major interests. First of all was protection against radiation damage. He tried very hard to isolate a humoral factor, which protected against radiation. It turned out that that humoral factor probably didn't exist, and that what he was really observing was the transplantation of cells. That is why I said earlier that his work really led quite directly to bone marrow transplantation. The second area in which he was interested was the regulation of red cell formation. He was one of the pioneers in the study of erythropoietin and there are still a large number of investigators studying erythropoietin. Only now they are studying the receptor for erythropoietin. They're studying the transduction of the signal that erythropoietin creates. They're studying the production of messenger RNA when cells are stimulated by erythropoietin and questions of that sort. My own interests in those days had to do with iron metabolism -- what regulated iron absorption. We didn't figure it out and there are still hematologists working on it today. I was working on defects in red cells that produce hemolytic anemia, and there are still hematologists working on that today, and sickle cell disease, and there's still many hematologists working on that today. So I would say that in the environment in which I worked, hematology was not that different from what it is now, except that technology has moved ahead. Investigators are looking at the same problems with different tools. For example, in the late 50s and early 60s, I conceived the idea that one could treat sickle cell disease by switching hemoglobin production from sickle hemoglobin to fetal hemoglobin. This involved switching production from the beta chain to the gamma chain. Well, we actually worked on this for-quite a while, but with the tools at hand we couldn't understand the switch and we couldn't influence it. People are working on it today and they can't understand it either, but they're getting closer because now they can study at the DNA and messenger RNA. I believe that within a few years they'll get there. But the problems are the same. If I looked back at the generation of hematologists before mine, who were still senior at that time, there were some like William A. Castle, who was very physiologically oriented and he was doing the same kind of work. He worked on iron. He worked on what turned out be vitamin B12. But there were also a large number of hematologists whose orientation was very largely morphological.

END OF SIDE 1 TAPE 1; BEGINNING OF SIDE 2 TAPE 1

Beutler: -- And it's still true today that there are those who were trained by the previous generation of hematologists who believe that morphology is the beginning and end of hematologic diagnosis. They were trained to believe that you have to look personally at every blood film of every patient that you see. And I don't think they do it, but they feel guilty about not doing so. I don't personally study each blood film and I don't feel guilty about it. So I believe that at that time, perhaps, the strong morphologic roots of hematology were more heavily represented in research. But in the environment in which I was, that wasn't really nearly as true. We were studying physiology and biochemistry, and the main change has been that one can now extend this by investigating the biochemistry of the DNA and the RNA, which we couldn't do earlier because the technology hadn't been developed.

Q: One of the things I asked you to do with your curriculum vitae was to highlight what you felt were the most important research contributions, and one of the things you did, you highlighted a series of articles on primaquine, if that's the correct pronunciation, and I was wondering why you chose to highlight those and how that fits into the themes that you're talking about now.

Beutler: Primaquine sensitivity was a phenomenon in which certain individuals, usually black, sometimes of Mediterranean origin, develop severe anemia every time they took the drug primaquine. Our work showed that this was due to a hereditary enzyme deficiency, glucose 6 phosphate dehydrogenase deficiency. There are about 200 million people in the world who have this deficiency. So it's pretty important, and basically my studies were instrumental in leading to that discovery.

Q: What was that drug used primarily for?

Beutler: For malaria. It was actually developed during World War II when the supply of quinine was cut off from Southeast Asia, and it was an analogue of a drug that was used earlier, actually introduced in the 1920s. It was called pamaquinine or plasmoquin. And pamaquinine or plasmoquin had fairly favorable anti-malarial properties, but it produced severe and even fatal anemia in some susceptible individuals. When primaquine was developed as an analogue of plasmoquin, it had the same properties. It was a good anti-malarial --probably better than plasmoquin but it also caused hemolytic anemia. When I entered the army in 1953 after my second year of residency at the University of Chicago, I was assigned to the Army Malaria Research Project in Joliet. This was not a coincidence, because that project was run by the University of Chicago, I'd been a resident at the University of Chicago in hematology, and one of the problems in which they were interested was this kind of anemia. And so, my major projects during my year there was to investigate the cause of that anemia. It was those investigations led to the discovery that the defect was in the enzyme glucose 6 phosphate dehydrogenase. That's why I consider those articles to be important.

Q: How were those studies constructed? In other words I notice that many of them were published in the Journal for Laboratory and, Clinical-Medicine.

Beutler: That's right.

Q: And then you published a summary somewhere, some years later in Blood. I'm wondering about your co-authors. How those studies were put together, how the clinical studies were actually -‑

Beutler: The senior person in charge of those studies was Alf Alving. Alf Alving was not a hematologist. He was a nephrologist and quite frankly he really didn't understand very much about the studies. His name appears in the all the papers and it did use to wound us a little bit in our younger days when the studies were referred to "That fine work that Alving did", because Alving really hadn't done any of it. There were basically two people who did the work. One was Raymond J. Dern (deceased 2001) and the other myself. Ray Dern was also assigned to Joliet as a military officer. He had been a resident also at the University of Chicago. But he was some years senior to me and also had a Ph.D. in physiology, from the University of Rochester. Ray Dern has retired recently. He lives in Redondo Beach. He and I utilized what was then a quite new technique of chromium-51 survival of red cells. We took red cells from primaquine sensitive individuals, labeled them with chromium-51, transfused them into non-sensitive individuals, and then showed that those cells were destroyed when primaquine was given to non-sensitive individuals.

Q: Could you describe that technology?

Beutler: Yes. It's a technique that's still used today. What you do is you take blood, you add chromium-51, radioactive chromium, wash the red cells and then you inject those red cells into the recipient. One can perform such studies with a person's own cells or one can with those of somebody else. Nowadays with concern about AIDS we wouldn't use another person's cells, but in those days there was no AIDS and while were concerned about hepatitis it was not that much of a problem. And so we were able to perform those studies on volunteers. Blood samples are taken every two days or so, and their radioactivity measured. Normally the radioactivity declines very slowly, maybe two percent or so a day. But if the red cells are being destroyed rapidly because they're abnormal or they're being challenged by primaquine, then there's a drop off of radioactivity. So if you start out by asking the question, "Are individuals who are sensitive to the hemolytic effect of primaquine sensitive because their red cells are different, or do they metabolize the drug in some different fashion?", one can obtain an answer by performing this kind of cross transfusion experiment. If the defect resides with the red cells then red cells from a primaquine sensitive person will still be sensitive in the circulation of a non-sensitive person. Coversely, if you take red cells from a non-sensitive person and transfuse them into a sensitive person, even when that sensitive person has hemolysis, the radioactivity of the transfused cells will remain unchanged because non-sensitive cells will not be destroyed. Those studies, by the way, were done by Raymond Dern and Irwin Weinstein. In this way it was established that primaquine sensitivity was due to a defect of the, red blood cells. Over the next year I performed many studies trying to determine what was wrong with those red blood cells. One of the measurements that I made, for reasons that probably are not worth going into now, was the content of glutathione in red blood cells. And that turned out to be low. Then I showed that not only was the content of glutathione low, but the glutathione stability was low. That finding led my colleague Paul Carson to measure the enzymes that maintained glutathione in the reduced state and he found that the defect was in glucose-6-phosphate dehydrogenase. Thus, primaquine was the drug that opened the door to the finding this defect. Now the defect itself could be identified without administering primaquine or performing red cell survival studies. One could measure the activity of the enzyme. One could measure the properties of the enzyme. To leap frog to where we are now with this defect, it was shown over the subsequent 20 or 25 years that glucose-6-phosphate dehydrogenase deficiency was common all over the world, but the properties of the residual enzyme in the deficient subjects differ from population to population. Among Africans, for example, the enzyme moved fast and therefore it was called "A minus" because its mobility was faster and it was deficient. In the Mediterranean areas the defect was much more severe than it was in Africa. There was much less residual enzyme and the properties of the enzyme were different. There was a bi-modal pH optimum curve and the affinity for substrates was different. This enzyme was called G6PD Mediterranean. In the Orient there were different variants, yet. Eventually a list of more than 400 different variants emerged. What we've been able to do just in the last 2 or 3 years is to examine many of these variants and find out that many of them that were assigned different names are actually the same. So there aren't 400 variants. Probably there are one hundred, or 80 or a number in that range. We don't know. We'll never know for sure, because some of them we'll never be able to examine again. But we found out, for example, that in the Mediterranean area, where many different variants had been described and claimed to be different from one another, many are actually the same. They have mutations in nucleotide 563. Another interesting feature about these variants is that they also have a nucleotide substitution in a different place, nucleotide 1311, which does not produce a coding change. Very recently we found that the 1311 mutation occurs in about 10 or 20 percent of the population at large in the Mediterranean region, not just in those with G6PD deficiency. But in the G6PD deficient subjects almost all of them had this mutation. What that means is that G6PD deficiency in the Mediterranean area probably arose in one individual and that the millions of people who have it now are descendants of that one individual: he happened to have that 1311 mutation. We found the same Mediterranean mutation in India. We've now examined three such subjects from India and they don't have the 1311 mutation. What that implies is that this mutation arose independently in India, so that we have the same mutation at 563, rose in one guy in the Mediterranean area, one guy in the Indian subcontinent, and now there are millions of people who have inherited that chromosome, presumably because they had some kind of survival advantage, probably defense against malaria. And the same thing is true of G6PDA-. There's a nearby polymorphism, and all the subjects who have G6PDA- have that polymorphism.

So G6PDA- probably arose only once too. And the individuals in Europe, too, who have the A- mutation, all carry the additional polymorphism. Well, I find these discoveries very interesting. It's one of the things that I enjoy about doing science -- We can answer questions that we didn't even dare ask, let's say 10 or 15 years ago, because there just wouldn't have been any way to explore the problem. How would one even have approached the question of whether all the G6PD deficient people in Europe arose from one founder, or rather they were all a bunch of different mutations that occurred repeatedly?

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