Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.
Director, National Institute of Neuroscience, Tokyo, Japan
The name "Nambyou" means intractable, and many neurological diseases are intractable and are caused by a genetic defect. However, for many intractable neurological diseases we lack direct biological information, and their primary cause is not known. Therefore in the past genetic counseling has been made principally based on family pedigree studies. Only recently have we moved onto molecular study of these diseases. This was a result of the remarkable and unprecedented advances in molecular genetic techniques. Positional cloning has opened a new window for gene identification of diseases, even when direct information about the biological defect is unknown. Using this process, accurate clinical DNA diagnosis has become possible and DNA testing for carrier status and prenatal diagnosis is now widely accepted.
But this progress does not necessarily solve all our problems, because the relationship between the clinical phenotype and genetic abnormality is not always a one to one correlation. As we heard from Prof. McKusick there are many genetic diseases where we can diagnose a disease but are unable to cure it. In the case of late onset diseases, there are a number of problems about what the real meaning of early diagnosis is.
All of the people in this session are clinical researchers, not especially molecular biologists, and hence are participating in diagnosis and counseling of patients, with all those associated dilemmas, while progressing with their research. We will hear from them about their work and experience, and hopefully have active discussion from the floor.
Sugita: Thank you Prof. Suzuki. Prof. Suzuki has been researching primarily on gangliosidoses and the relationship between phenotype and genotype where the mutation has appeared, and has discovered to a certain degree whether they will be of early or late onset. He also touched on the diversity of this disease, and even the same mutation has a different phenotype in Japanese and non-Japanese. Do you have any questions or comments.
Takebe: The I51T mutation, is it in the second exon? If it is in the middle of this, then is there no difference in the splicing of mRNA?
Suzuki: This is a mutation due to a single amino acid substitution. Most of the mutations of §-galactosidosis we have studied express mRNA.
Takebe: So this 8% that you showed on your slide was the residual protein activity?
Suzuki: Yes, that is correct.
Sugita: Is there anybody else? I'd like to make a simple question, if the mutation is different then the structure of the protein product is also different. Does this have some physical effect and so lead to the difference in onset of the disease?
Suzuki: To the extent to which we have looked at this, we have found mutations which prevent the glucosylation at the Golgi body or phosphorylation, so the enzyme cannot be transferred to the lysosome. There are some which are transferred but are destroyed when they get there. There are other mutations which react with the substrate, but the affinity is different, so they are not as effective. So there are different mutations giving rise to different proteins, and these mutant enzymes are expressed in different amounts, according to the mutant. Another type of mutation affects the quality.
Sugita: Thank you very much. Dr Arahata has carried out a lot of important research on muscular dystrophy using positional cloning, and he has also talked about clinical treatment. We have time for one or two questions.
Tsuji: When there is no deletion in the case of Duschenne muscular dystrophy (DMD) how do you carry out the carrier diagnosis? You have described the use of microsatellites. We've done this a little as well, but if you don't analyse the haplotype as well there is the problem of double recombinations or gene conversion. The mutation may not be inherited, so when we apply these results to counseling, how do you explain these results to clients. I think this is a very serious problem. When you have carried out this kind of analysis how do you explain to patients? If you can tell us this would be very useful to us.
Arahata: In genetic diagnosis, if the patients have no deletion, we are testing normal microsatellite probes to get accurate diagnosis. When we cannot be sure of the results just from the genetic analysis we also carry out protein analysis. The problem you have just raised is also something we are scratching our heads over too, but we tried to explain this in terms of what the chance of being a carrier the person is (e.g. how many percent risk). I give the data to the patient or family, and leave the judgement to the good sense of the patient themselves.
Tsuji: If you don't take into account the double recombinations you won't have 100% certainty in the diagnosis.
Arahata: One cannot expect 100% certainty in the genetic diagnosis, because the recombination rate in the huge dystrophin gene is estimated to be 7-9%.
Lo: I was very interested to see the work on DMD because we see quite a number of patients in China too. The determination of the carrier status of the mother is very important in this disease. We have also tried to see whether PCR will be able to help us. Now we find that even if the proband has a deletion revealed by multiplex PCR, the obligate mother does not necessarily show a diminution of the band, so in that case you still are unable to know whether the mother is a carrier or not, even though from pedigree analysis the mother is an obligate carrier. Do you find the same experience?
Arahata: Yes, exactly. Actually the PCR analysis is not a quantitative analysis. So other methods should also be employed, no single technique is sufficient to detect the carrier status of the Duschenne patient.
Lo: I have another question, that is if you use anti-dystrophin to try to see if the mother has dystrophin in the muscle fibres, what is the reliability of this test.
Arahata: We don't know the exact percentage of mosaicism, because not all the carrier females were biopsied yet. Particularly if the mother has no muscle weakness, it is very hard to get a muscle biopsy.
Lo: Have you ever had a false negative result using the anti-dystrophin test?
Arahata: Always you have to add the appropriate control at the same time. It prevents a false negative. However, if the antibody is raised against the deleted portion you will get a negative result, so you have to use at least more than three antibodies at the same time.
Verma: I would like to know from you if you have data on the frequency of DMD at birth in Japan? Secondly, in your case you had more cases of DMD, is it an artefact that you saw more Becker type muscular dystrophy in your analysis.
Arahata: The frequency of DMD in Japan is the same as Western countries or India. The incidence of DMD in Japan is calculated as 14 to 27 per 100,000 population. About one tenth of DMD is Becker-type. We have a biased population in our Institute because of the selection of the referrals by doctors.
Billings: I just wanted to ask you about what therapeutic approach you are applying. Duschenne's is a multisystem's disease, not only skeletal muscle but also smooth muscle of the gut and a variety of other sites that can be involved, particularly as patients survive longer. What approaches have you been applying to this problem?
Arahata: Before starting gene therapy, we need more basic research. If you want to have the normal gene in all muscles, you will have to inject vector(s) into a vein or have the patient inhale through the airway.
Sugita: I think you may have other questions, but there is no time now, so please ask in the general discussion. We thank Prof. Nonaka who is also at the National Institute of Neuroscience. He has talked to us about maternally inherited diseases, and in particular about MELAS which is due to a point mutation. In this disease the wild and the mutant types of DNA coexist heteroplasmically in the cell, and according to their relative amounts the severity of the disease is different. But this ratio differs between organs, so it is very difficult to predict what kind of phenotype the patient will express. We have time for some discussion or questions.
Sobue: Thank you for your very clear presentation. For the maternally derived cases you looked at the epithelial blood vessel cells, do you think this has any relationship to organ specificity?
Nonaka: We have microdissected some of these veins and found that over 90% of the DNA in them was mutant. So we think that it is not just at organ level but also at the tissue level, but we will have to look at the detailed distribution in order to make prognosis of the patients. I'd like to look at this problem at the cellular level in my future research.
Takebe: In this tRNA leucine abnormality, what kind of protein is affected in the mitochondria. Can this be supplemented by mRNA from the nucleus?
Nonaka: We don't know at the moment what kind of protein is affected. If we look at the enzymes in the MELAS patients we can see that Complex 1 activity is dropping, but we don't know what protein is lacking, and which function is missing. We have also tried to culture cells to find out what protein is missing, but we have not yet succeeded in doing so. So we know what gene mutation is responsible, but we don't yet know what protein is affected.
Nakanishi: If some normal mitochondria exist in these patients, is there any way of activating these mitochondria, for example can you use a new treatment like high pressure oxygen to treat them?
Nonaka: At the moment we haven't found any effect from this kind of treatment. As the disease progresses the mutant DNA increases and the wild type normally decreases. But there are some patients, where exactly the reverse happens, where wild type increases, so that the symptoms lessen. If we could find out why the wild type increases, then perhaps we could apply this to treatment. I think something in the cell nucleus is controlling this but we don't really know what is going on.
Billings: I thought it was a very interesting and clear talk. In one slide you showed a pedigree in which a carrier mother had short stature, hearing loss and diabetes. Do you imply that this was the result of the mutation in that woman?
Billings: Do you have other pedigrees that show similar findings?
Nonaka: Yes, recently we have been analysing many pedigrees, and the mothers of the patients usually have the mutation. Most of them have short stature and occasionally diabetes mellitus.
Leavitt: I am neither a biologist nor a physician, so I am going to phrase my question in amateurist terms. When you find a heterogeneity, or a lack of correlation between genetic data and clinical symptoms, is the proper response always to assume that you need to find more genetic data and to keep looking for more genetic information, or is there room here to consider non-genetic co-factors, such as nutrition or environment or psychosomatic factors, or something of those sorts.
Nonaka: At the present we don't have enough time to look at other factors that might be involved, so as a first approach we are looking at the amount of the DNA that wild and mutant mitochondria may contribute. We are starting our research from that point. Of course as you have said we may have to develop our research to include other factors.
Matsunaga: I am also very interested in your pedigree data showing the heterogeneous expression of symptoms among different members. From the standpoint of classical genetics could you please estimate the penetrance, focusing on the specific symptoms. These may be quite different from the expected ratios. Theoretically, all siblings from the same mother should possess the same mitochondrial DNA, but can you estimate what fraction of these offspring show for example short stature. These may be quite stochastic problems, so we need to add much more classical studies. What is the probability of showing some symptoms. We might expect that all the children have the same amount of mitochondrial DNA from their mother but we see that symptoms are different. This should be stochastic. A women will have mitochondrial DNA mutants, so what type of counseling do you do if she is worried about having children? You may say at least there is some probability of affected offspring.
Nonaka: The examples I showed there are very exceptional ones, usually when the mother has mutant DNA then more than 90% of the children show some symptoms, though it varies in severity and less than 10% have no symptoms.
McKusick: I have a comment and a question. I think that historically pointed out that mitochondrial disease, it was called cytoplasmic inheritance, was first suggested for a human disorder, namely Leber's optic atropy by a Japanese, I think in 1937, a very long time ago. Prof. Matsunaga, what was the name of the Japanese worker?
Matsunaga: Prof. Imai and Moriaki, in 1936.
McKusick: The question was, I find it very surprising that endothelial cell function should be so sensitive to a mitochondrial gene defect. I wonder if this isn't telling us something, I don't know what, about the nature of the protein that is affected by this mutation. It must be something very critical to mitochondrial function.
Nonaka: So far we do not know the tissue specificity, why we have selected blood vessel involvement, for example in CPEO the mitochondrial DNA deletion has only eye (optical) symptoms, and in other mitochondrial disorders DNA mutations can produce epilepsy, so far we don't know why we have such tissue specific involvement in these mitochondrial disorders.
Sugita: So thank you again. Prof. Tsuji has given us a good description of Gaucher's disease, a monogenic disease, showing the phenotypes that exist in different populations, and showing us the reasons why we have to be rather careful in our counseling of patients because of the exceptions that occur in the correlations between genotypes and phenotypes. He has also explained how we cannot really hope for much from protein replacement therapy as it currently stands, and furthermore this therapy is extremely expensive. Do you have any questions or comments?
Lo: We have been working with phenylketonuria, and it seems that the phenotype is pretty well correlated with the level of residual protein activity, so I just wonder if you have measured the residual activity of your mutant proteins?
Tsuji: It was clearly demonstrated in Gaucher's disease that there is no correlation between the residual enzyme activity and clinical phenotypes. So the idea is that another mechanism may be involved in the presentation of clinical phenotypes, and one possibility is the psychozinel (glucosyl-sphinyzosine) hypothesis, because the glucocerebrosidase can hydrolyse glucocerebroside as well as psychosine, which is highly toxic to neuronal cells. So, there is a hypothesis that the residual activity towards psychosine might be related to the presentation of clinical phenotypes.
Sugita: Other questions or comments?
Arahata: First you talked about genetic analysis or genetic diagnosis, how do you tell if it is positive or negative?
Tsuji: The first problem is a technical matter, that the pseudogenes will be amplified at the same time if we do gene amplification by PCR. As far as I understand, there is a possibility that the same mutation may be found in the pseudogenes, therefore more than one mutation may be transferred to the genotype by crossover or other mechanisms. So we have to look at the whole of the genotype, for example sequencing in order to discover all of the mutations that are present in it.
Sugita: I'd like to move onto the next lecture. Prof. Araki is a world acknowledged leader in this field, and we thank him for his wide-ranging presentation. We then had a further talk on amyloidosis by Dr. Imaizumi. Are there any questions.
Matsunaga: I would like to pay great respect to Prof. Araki and your group for having achieved such an excellent scientific work. I have been wondering why I have heard nothing about the prevention, the prenatal diagnosis combined with selective abortion, but now today you said you have formed a patient's group. So do you educate these patients or their families especially emphasising that even the patient could have a normal child by utilising the selective abortion. If we could utilise selective abortion for this disease, among the 200 or so patients, perhaps this mutation could be resolved within one or two generations.
Araki: Thank you for your comment. As you told us, we are educating the patients and the patient's families, and every year I am giving lectures to them, what is pathogenesis research and what kind of treatment we can give them, internationally, including the use of liver transplantation. Now they are trying to reduce or eliminate child birth, I don't know why, but they don't want to have many children. But unfortunately in Japan they never ask about prenatal diagnosis as yet, so we are hoping to have such opportunity in the future. I am sure two or three generations later we will eliminate FAP from our areas. We believe so, thank you.
Billings: I want to congratulate you on a lovely presentation. In the United States transplantation for FAP has recently been given a boost since a prominent politician underwent transplantation for this disorder, so more attention has been paid to this. I wonder whether the availability and effect of liver transplantation will have an impact both on the question of prenatal diagnosis and presymptomatic diagnosis in children. How that might play out in the Japanese situation?
Araki: Well, the patients are very much afraid to have children that might be affected later, so they have fear of DNA diagnosis. But from the academic point of view we are doing some studies about which child is affected and which child is not affected, but we never tell to the patients or their families. Usually they don't ask about the result of DNA studies, so we are very much interested in their future attitudes. But we cannot give out any treatment in Japan as yet.
Billings: When the child grows up, and is an adult, will you offer them the opportunity to have the information?
Araki: Yes, if they ask us. During the last three years we have had only three persons who asked us, but these were all doctors, related to the families. Fortunately they were all negative in the DNA study so they were very happy when I told them.
Takebe: Regarding the question of liver transplantation I have to point out that in Japan liver transplantation from brain dead patients has not been performed or accepted yet. At Kyoto University for example, we have been doing liver transplantation only for children, so this is not applicable for this particular disease. Because of this, if this kind of treatment became available, we can discuss whether prevention could be achieved by this. Would the function of the liver continue after it had been transplanted.
Araki: These transplants have been done since 1990, so internationally they have an interest in what is going on, but some followup is necessary to evaluate the treatment. I talked to Prof. Sugimachi of Kyushu University about liver transplantation. He didn't know about this metabolic disease so I explained to him that if in the future liver transplantation can be performed, we want to contact with him. But they said that it is very expensive and we need a surgical team and 10 hour operation, with blood transfusion of about 3000ml. So it is very expensive and we have to wait for the future.
Kanazawa: We will touch on this later in the case of Huntington's disease. You just mentioned that three people asked about the results of their tests, and they were negative. Would the revealing of the information affect the relatives?
Araki: Three relatives of patients affected with FAP knew about FAP, but one doctor was thinking about marriage so he asked us about the results of the DNA analysis. He asked, that is why we told him. If they don't ask we never tell.
McKusick: It seems to me, perhaps this is obvious, that here we have a good illustration of how preventive medicine saves money. If one were more provocative in pushing advice for prenatal diagnosis, one could avoid the necessity of liver transplantation for the patients, a very expensive process later in life. I wonder whether you shouldn't be more of a salesman, and as we say in an English proverb, "An once of prevention is worth a pound of cure".
Sugita: You may have more questions but we are a little over time now, so let's go on to the next topic. Prof. Kanazawa talked about genetic linkage studies of Huntington's disease. We then had an explanation of the geographical distribution of Huntington's disease in Japan by Dr Imaizumi. Do you have any comments or questions?
Arahata: Prof. Kanazawa showed very interesting work on twins, that the age of onset of the disease was also the same. In FSH dystrophy even for twins the age of onset of the disease is completely different. Is there any particular reason why twins with Huntington's disease tend to show the same age of onset?
Kanazawa: Something which is at present puzzling us researchers into Huntington's disease is that Huntington's disease seems to be an example of a rigid classically transmitted disease, but when we look at the molecular level it seems to be very unstable. There seems to be some contradiction. I think that this problem will be solved sometime, but even Prof. Gusella may have some doubts as to whether this gene that he has discovered is actually the causative gene. There are still many things we don't know about this disease.
Sugita: Are there any more comments or questions?
Bodurtha: Thank you very much for your interesting talk. You and several other speakers have mentioned that ethical committees have some involvement. I wonder whether you could elaborate on the possible role of informed consent. I am more familiar with the role of committees in the USA in human investigations in an Institutional Review Board. How do informed consent documents, and work with patients and families influence your work?
Kanazawa: Our committee not only includes Professors and Assistant Professors from the Medical School but also leading people, such as a person from a broadcasting organisation. It is very open.
Sugita: Thank you very much, now we would like Dr Cook-Deegan's comments.
Cook-Deegan: In my paper I try to tease out some of the ethical issues that underlie the scientific presentations. We have seen a lot of beautiful science in the talks, so I would like to pull out some of the ethical issues. First, before I do that I would like to thank everyone. I work for the National Academy of Sciences in Washington D.C., and what we do is hold meetings for a living, so I certainly appreciate the amount of work that has gone into the venue here, and we all owe a debt of gratitude especially to the translators who are having to wrestle with highly abstruse vocabulary and highly complex areas, and we should all thank them for their efforts in what has been a long day. And we thank Profs' Sugita and Fujiki for putting it all together.
Sugita: We've just heard from Dr Cook-Deegan, the problems of pedigree study and the problems that occur in genetics when we treat research as a horse race. But in these diseases international cooperation is very important. I think we have really no time left, we have a reception from six o'clock. I want to thank all for your heated discussion this afternoon and for the speakers for giving their talks today.