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female with pyruvate (Ela subunit) deficiency the enzyme is present in some granules) and not in others. Hresult confirmed that the gene is on chromosome. The project is discussed detail in the report.

The Murdoch Institute is remarkable interna tionally among Institutes working on genetic diseases for an even balance between fun damental research and clinical practice covering genetic disease in all age groups in the whole State of Victoria. This gives the Institute a an particular style of which we are very proud innovative approach to clinical work and a practical start to basic research. It also provides special opportunities for training free-thinking young clinicians and research workers. The Murdoch Institute is the major Australian centre for research on genetic diseases and for training clinical geneticists.

Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052.

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The high standing of the Institute within Australia is shown by its inclusion among the five Research Institutes supported by Block Grants from the National Health and Medical Research Council. Internationally its scientists and clini cians are in demand as speakers at major scientific meetings, on editorial boards of scien tific journals and as authors of chapters in important books. Birth defects are very important to the Austra lian community. Each year 5000 Australian couples feel the anguish of the birth of a baby with a serious defect. Australian taxpayers provide over $2 billion annually to care for the survivors and still we cannot give all the care they and their families would like. Surely we all have a responsibility to reduce this personal and commu nal burden next decade. Only research and prompt implementation of the results of research can achieve this.

How you can support the work of the Institute. At the Murdoch Institute we are doing our best to bring about this reduction in birth defects — we need your understanding of our efforts, your voice to persuade the Government to continue and increase its support and your personal financial assistance. To maintain our research we need to raise $1 million every year — help us now with a donation and help us in the future by remembering us in your will. DONATIONS ARE TAX DEDUCTIBLE. The Murdoch Institute for Research into Birth Defects Royal Children’s Hospital Flemington Road PARKVILLE Victoria 3052 Telephone (03) 345 5045

MAJOR DONORS TO THE MURDOCH INSTITUTE

During 1988 the Board decided that it was not sufficient to acknowledge donors by making them members of the Institute. The following special categories of membership were created.

FOUNDERS — Donors of $1 million or more The Murdoch Family: Dame Elisabeth Murdoch, Mr. Rupert Murdoch, Mrs. Helen Handbury, Mrs. Anne Kantor, Mrs. Janet Calvert-Jones. The late Sir Jack Brockhoflf, The Brockhoff Foundation, The Scobie and Claire Mackinnon Trust.

BENEFACTORS — Donors of $250,000 or more The Miller Foundation, The Helen M. Schutt Trust.

TRUSTEES — Donors of $25,000 or more Mrs. M. L. Griffin, J. B. Were & Son Charitable Foundation, National Australia Bank, The Percy Baxter Charitable Trust, The Ian Potter Foundation, The late Mr. Clive Roxburgh, Mrs. Joan Roxburgh, H. & L. Hecht Trust, The late Mrs. L. B. Quayle.

DONATIONS TO THE MURDOCH INSTITUTE

January — December 1988 The Board and Staff gratefully acknowledge the following donations received during the year. The Brockhoff Foundation Cruden Investments (Murdoch Family) Helen M. Schutt Trust Scobie & Claire Mackinnon Trust The Miller Foundation J. B. Were & Son Charitable Fund Mrs. M. L. Griffin Pacific Dunlop Limited Percy Baxter Charitable Trust Estate of F. H. Pennifold Repco Rothschild Australia Limited Uncle Bob’s Club Arthur Andersen & Co. Foundation The Morris Family Trust Coles/Myer Limited In memory of Emma Ritchie Mrs. D. Simpson Petra Foundation Comalco CRA Services Limited Sportscraft Pty Ltd William Angliss Charitable Fund Westpac

$250,000 250,000 125,000 100,000 50,000 15,000 10,000 10,000 5,000 5,000 5,000 5,000 4.500 4,000 4,000 3,750 3,539 3,000 2,622 2.500 2,500 2,500 2,000 1,500

In memory of Stephanie Waldron Dr. J. M. Gooch ICI Australia Mayne Nickless Limited The Shell Company of Australia Limited Mr. F. D. Ryan Australian Guarantee Corporation Mr. J. Bailleu Mr. and Mrs. S. F. Gooley W. G. Grace Australia Limited McMullin Unit Trust Rotaract Club ofCasterton National Australia Bank In memory of Christopher Oakes Mrs. G. Grimwade Mr. B. R. Redpath Mr. and Mrs. L. Barbieri Mr. T. M. Bryant Mr. and Mrs. 1. M. Cole Cadbury Schweppes Pty Ltd Cystic Fibrosis Association of Victoria Dame Patricia Mackinnon Mr. and Mrs. Thomas E. H. Tan & Co.

1,220 1,000 1,000 1,000 1,000 600

500 500 500 500 400 254 250 214 200 200 150 150 150 125 100 100 50 20

Equipment Donations Flow Laboratories — Multistepper

$900

CHAIRMAN’S REPORT

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INDEX Chairman’s Report..............................................................

Dame Elisabeth Murdoch..................................................

Board of The Murdoch Institute.....................................

Finance Committee.............................................................. Director’s Report.................................................................

6 ....7

Research in Progress...........................................................

Post Doctoral Fellows.........................................................

Fellows in Clinical Genetics..............................................

Visiting Scientists and Clinicians....................................

Stafflist of The Murdoch Institute..................................

Murdoch Institute Lecture Series — 1988....................

Staff Involvement in Australian and International Scientific Community Activities...................................

Postgraduate Degrees Awards..........................................

22 22

Overseas and Interstate Visits, Lectures and Seminars by Institute Staff.......................................... .

Research Collaborations....................................................

The Hot Method of Detection of Mutations................. . Pyruvate Dehydrogenase..................................................

25 28

Possum Progress.................................................................

The Board of the Victorian Clinical Genetics Services

Staff List of the Victorian Clinical Genetics Services..

Victorian Clinical Genetics Services..............................

Studies in Transgenic Mice..............................................

Detailed Project Reports...................................................

List of Publications — 1988..............................................

Editorial Boards..................................................................

Mr Neil Watford

During 1988 the Murdoch Institute made sound progress with its major research programmes. The year was also marked by some special events both in the areas of research and of general operations. Details about research matters are contained in the Director’s Report and in other sections of this Annual Report. In the 1987 Annual Report we reported our plans to establish the Victorian Clinical Genetics Service as a wholly owned subsidiary of the Murdoch Institute. Its role would be to co-ordinate and administer the provision of a wide range of genetic services available to Victorian hospitals, including the screening for certain diseases of all newborn babies in Victoria. I am delighted to report that the VCGS is now in operation and I particularly wish to express our gratitude to the Minister of Health, Mr. David White, for his personal support and co-operation. Without the additio nal financial support made available by the Victorian Government the setting up of the VCGS would not have been possible. It is, I believe, also very proper that we should congratulate the Victorian Government on the progress being made with AMRAD, the organisation which is sponsored to develop and commercialize discoveries made by the major medical research institutes. A significant event is the recent AMRAD joint agreement with Merck Sharp and Dohme (USA), the world’s biggest phar maceutical company. This promises to be a source of great strength for the effective marketing of new products. The Murdoch Institute is a beneficial shareholder in AMRAD. Medical research institutes do indeed discover products which can be commercially marketed. The Murdoch Institute in recent years has developed two such products — one as recently as 1988. They are known by the acronyms POSSUM and HOT — the first is our computerized system to assist clinicians in diagnosing birth defect syndromes, and the second about methods of identifying mutations in genes. Over the years from these and other original developments we hope to earn steady revenue. Perhaps the main responsibility of the Board of Directors is to ensure the financial stability of the Institute. Our major sources of revenue are government grants and investment income. We depend on donations from corporations, trusts and individuals to close a gap which, apart from any large capital works, amounts to about $800,000 a year. It is now an accepted part of the social ethics in 1

OUR PATRON ELISABETH MURDOCH D.B.E. AT 80 YEARS

Australia as it is also in the United Kingdom, United States and Canada, that both corporations and indi viduals should accept some responsibility for a range of community activities including the arts, welfare and medical research. Only through such private sector support will many of these things happen and it is a very important consideration that such support means a significant degree of independence from government control in these matters. The Murdoch Institute has greatly benefited from generous private giving. The successful 1985-86 Appeal that launched the Institute bears witness. That success was underwritten not only by large corporate donations but by major gifts from the Murdoch family, the Jack Brockhoff Foundation and others. In the future we urgently need regular annual support. Over the years we hope that bequests will become an important source of income. We are working on this but of course it will take time. We well understand that major gifts by corporations cannot always be divorced from commercial considera tions. There are various ways in which we can meet this need including naming rights in respect of certain of our operations which happen to have a high public visibility. An exciting event which for us marked the advent of 1989 was a 80th birthday party for our Patron, Dame Elisabeth Murdoch. We at the Institute are very proud of her, of her high standards of community service and personal endeavour which are an inspiration to us all. Regrettably, during the year Dame Patricia Mackinnon resigned as a member of the Institute’s Board. Dame Patricia has been associated with the Murdoch Institute throughout its development and had been a Board member since incorporation. She will be sadly missed. We welcomed in her place her daughter, Mrs. Ann McFarling who continues into the fourth generation the association of the Mackinnon family and the Royal Ghildren’s Hospital. Finally, I thank sincerely my colleagues on the Board and on the Finance Committee and congratulate our Director and his staff on the excellent progress of the Institute in 1988.

Dame Elisabeth Murdoch

(Photograph courtesy of New Idea)

Although not strictly an event of the year covered by this report, the 80th birthday of our Patron, on February 8 1989, was an event of such importance to us to need reporting immediately. We were delighted when Dame Elisabeth agreed to join us in celebrating her birthday at a party at the Danks’ home on February 12. It was a very happy get-together of the Institute family — staff. Board Members, Members of the Finance Committee along with Dame Elisabeth and her immediate family (unfortunately without Rupert and Anna). As is always the case when Murdochs are present, the evening had a special sparkle. We all found ourselves wishing that we could have, even at our present ages, the zest for life and the breadth of interest of Dame Elisabeth. How lucky we are that genetics has always been high on her list of special interests. It was this special belief in the importance of genetics within child health which caused her, as President of the Hospital in 1960, to join with Dr Howard Williams in persuading the Committee of Management to pay the salary of a young paediatrician named Danks who wished to stay longer at the Hospital for Sick Children to be trained in genetics. This was a novel, adventurous step for the Hospital to take at that time. Her interest in genetics and in the development of a genetics research group within the Hospital has continued ever since that time and it was the same special interest which eventually made this the Murdoch Institute. Back in 1982, when the idea of establishing a separate institute of genetic research was in its infancy. Dame Elisabeth, no longer on the Hospital committees con cerned, heard of this idea and gave a substantial donation towards the objective “to help you get it started”. In previous reports we have recorded our gratitude for the major generosity of the Murdoch family which has finally made the establishment of the Institute possible. The special thing about Dame Elisabeth is that she never just gives money to a cause. She always backs up her financial support with enthusiasm and personal interest. We have been especially fortunate as recipients of this enthusiasm and interest, as have her other special causes — the Victorian Tapestry Workshop, the National Gallery of Victoria, the education of the deaf, the subject of landscape architecture, the McClelland Gallery at Langwarrin and many others. It may sound presumptuous to suggest we at the Institute feel almost part of an extended Murdoch family, but we are sure that the people who work in the other organisations have a similar, very delightful feeling. We all want to use this opportunity to wish Dame Elisabeth many, many more years of happy and active life. 3

BOARD OF THE MURDOCH INSTITUTE Mrs. I. McFarling, Mr. N. Walford, B.Com., F.C.A. Chairman

Dr. Bsr R; Catchlo^sp, M B., B.S., F.R.A.C.P., F.R.A.C.M.A., F.H.A.

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Mr. L. G. Cox, B.Com., A.A.S.A., F.S.I.A. ^ Vice Chairman

Professor Ms 6. Clack, B.Sk' Ph.D., F.I.Biol.

Dr. G. L. Barnes,

Dr. R. G. H. Cotton, " B.Ag.Sci., Ph.D., D.Sc.

: M.D., Ch.B., F.R.A.C.P.

Professor G. J. Fraenkel > < A.M., M.A., B.M., M.Ch., Hon. M.D., F.R.C.S., F.R.A.C.S., F.R.A.C.M.A., Hon. F.F.A.R.A.C.S.

Mr. J. S. Guest, A.M., O.B.E., V.R-d,

Mr. W. H. Hodgson

Professpr P, D, Phelan, M.D., bTs", B.Sc.,

B.Sc., M.B., B.S., F.R.C.S •3 F.R.A.C.S.

F.R.A.C.P.

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Mrs. J. Calvert-Jones

Mrs. J. Lewisohn, DfA.

Professor D. M. Banks, M.D., B.S., F.R.A.C.P.

Professat-G».-.R^,Ryan, M.D., B.S., Ph.D., F.R.C.P.A., F.R.A.C.P.

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DIRECTOR’S REPORT

FINANCE COMMITTEE Mr. L. G. Cox, B.Com., A.A.S.A., F.S.I.A. I Chairman

Mr. G. E. Heeley, B.Ec., F.A.S.A.

Mr. C. P. Abbott, L.L.B., C.P.A.

j Mr. D. E. Meiklejohn, f B.Com., F.A.S.A., C.P.A.

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Mr. D. T. Craig, A.C.A.(N.Z.), A.A.I.B.

Mr. P. J. Griffin, B.Com., A.S.I.A.

Mr. F. D. Ryan, F.C.S.

Professor David Banks

Our Annual Report has to serve a number of different functions. It provides an opportunity for us to tell our many generous supporters about the work that we are doing. We also hope that the contents of the Report may encourage some readers to become supporters. The Report is our means of giving an overall description of our research to our scientific and medical colleagues. It must also fulfil the requirements of the Companies Code as the Report of the Board of Directors to the Members of the Institute. It was natural for the first two Annual Reports to be dominated by the events involved in the establishment of the Institute and by the wonderful generosity of donors who made it all possible. This year we report another step in the development of the Institute — the establishment of the Victorian Clinical Genetics Services. We hope that over the next two or three years we will be able to tell readers about the progressive expansion of the space available for the Institute and the new research this will allow. Each year we have chosen to highlight two or three areas of our research and to tell something about the staff members concerned with these projects. This practice is continued in this Report. It is easy for this style of reporting to ignore the bulk of the research that is going on in the Institute at any time. To ensure that this does not happen, I have introduced a new section entitled, “Research in Progress” in which I have tried to give a simple description of the full range of the research that is going on in the Institute, albeit described very briefly. Of course, we always cover the full range of our research in the Detailed Research Reports, but these are perhaps too detailed to interest most of our lay readers. In Research in Progress I have arranged the brief reports so as to display the roles played by the senior scientists and clinicians who make up the real driving force of this Institute. They are the people who have the new ideas which become research projects and the skills needed to test these ideas. Without them, there would be no Institute. The last few years have been trying ones for them. Plans for expansion have been frustrated by lack of space. There has been space for the senior scientists themselves and for the essential research assistants, but not enough for the bright young postdoctoral fellows and PhD students who provide much of the sparkle and enthusiasm of good research institutes. During 1987 we decided that we would just have to put up with even more cramped working space in order to add some postdoctoral fellows during 1988 and the arrival of six of these bright young people has made a great difference to the Institute. 7

At last it does seem likely that some additional space will become available to expand our laboratories in 1990 and we hope we will then be able to recruit a number of PhD students. The establishment of the Victorian Clinical Genetics Service is important formally and functionally. Formally the Victorian Clinical Genetics Services Ltd is a subsidiary company of the Murdoch Institute for Re search into Birth Defects Ltd and this Report will therefore include a description of its operations. The VCGS Ltd is financed by the Health Department of Victoria which operates on a July 1 to June 30 year and its balance sheet and accounts will be reported separately at the time of its own Annual Meeting. Functionally the establishment of the new Service is of great importance to the Institute. Close integration of clinical activity and basic research has been a special characteristic of our research group since its inception, long before the establishment of the Institute, and the new arrangement offers great advantages which are spelt out in detail later in the Report. Many of our best research projects have begun with observations made in patients by our clinicians. Our basic scientists have often been able to contribute ideas which have improved the diagnosis and treatment of individual patients. We are very keen to sustain this particular style and will always be watching out to recruit clinicians who are interested in basic science and scientists who are willing to think about clinical problems. This close relationship between clinical work and research does bring with it some false expectations. Many people expect that because we are so heavily involved in clinical work we should direct most of our research effort towards those genetic diseases which seem to be of greatest practical importance to the community i.e. those which are frequent, serious and poorly understood. We are often asked why we have not had major research projects on cystic fibrosis. This is because we have not had any bright new ideas about this disease. In choosing new research projects, we look for topics which pose a significant question (significant for basic importance or for practical value) to which we think we may be able to find the correct answer because of the background and skills of our scientists or because of some new idea which one of us has developed. We make no apology for concentrating much of our effort on rare diseases because we are confident that by defining the basic defects in these diseases we will provide important new insights into the normal processes of human physiology and biochemistry and a new basis for understanding many of the more common diseases. Further, new techniques developed to study rare diseases are often applicable to common diseases. This statement of general philosophy is made because it arises naturally in talking about the relationship between 8

our clinical activities and our research and because it needs to be stated and restated at a time when it is under strong attack by the Australian Government. Certain Ministers seem to believe that they, or the committees that they establish, can identify those research projects which are most important to the future of Australia and that the progress of our nation will be augmented by channelling research funds to these projects. The whole history of science and discovery argues that they are wrong. Most important practical advances have been based upon fundamental discoveries by researchers whose objectives had nothing to do with the practical advance that eventually occurred, generally by researchers pursuing fundamental knowledge for its own sake. History shows clearly that good progress is achieved when highly skilled scientists are given a considerable degree of freedom to choose their own fields of work and ways of going about their research. Australia will prosper best if more money is put into basic research and if this money is allocated according to the abilities of the research workers and the nov elty of their projects as judged by panels of research workers of acknowledged excellence. Another event of considerable importance to the Institute is described earlier in this Report even though it took place in early February 1989. I refer to the 80th birthday of our Patron, Dame Elisabeth Murdoch. We were very proud indeed that she and her immediate family could join with us to celebrate this event. As you will see from Research in Progress it has been a year of pleasing achievement across a wide range of projects. The two projects which are chosen for a detailed presentation in this report made particularly spectacular progress, but I know that the scientists concerned would agree with me that there is an element of good fortune involved in such special bursts of progress. They are not frequent during a research career and must be treasured and remembered during the years of more ordinary progress which precede such bursts and may come again in the following years. Some of the projects which have made good, but less spectacular, progress in 1988 will blossom into one of these special bursts of progress in some future year. Pyruvate dehydrogenase (PDH) is a key enzyme in the production of energy in cells and is a very complex structure made of many copies of multiple protein subunits. When Garry Brown chose it as his main research project several years ago he was attracted partly by the challenge of its inherent complexity. To have been attracted by this complexity alone might have indicated pigheadedness or masochism. The combination of the complexity and the prospect that the new techniques of molecular genetics might be able to unravel this complexity made Garry’s choice a sound one. For many years we have wanted a universal method of identifying the subtle mutations that underlie most genetic

diseases. In a few patients the mutation is a crude one like the deletion of a large part of the gene; these mutations are relatively easy to identify. However, most genetic defects are more subtle and involve a change in just one of the thousands of code letters which make up the message of a gene. Techniques for identifying this type of mutation have been very cumbersome and quite impractical for use in individual patients. During his recent sabbatical year in Oxford Dick Cotton developed an elegant and simple method which seems to identify all of these subtle mutations. It has been called the HOT method, an acronym based upon the names of the chemicals used. The method is described in more detail later in this Report. Since Dick returned to Melbourne, Henrik Dahl has joined in assessing the method and also John Bateman and Shereen Lamande from the Orthopaedic Research Unit. Together they have been able to identify a number of mutations in patients with osteogenesis imperfecta, making progress in a few weeks which would previously have taken a year or two. Dick has been busy improving and extending the range of applications of the technique and has also collaborated with virologists to demonstrate the power of the method in this discipline. Since publication of Dick’s results in the Proceedings of the National Academy of Sciences of the United States a number of overseas groups have applied the method to their research and are reporting success. We were sorry to see Dame Patricia Mackinnon retire from the Board, but were pleased that her daughter, Mrs Ann McFarling, agreed to fill the vacancy. Dame Patricia has been a very staunch friend as President of the Hospital and as Chairman of the Research Foundation during the early phase of expansion of genetic research and through all the negotiations which led up to the establishment of the Institute. We thank her sincerely for all her assistance. There were a number of staff changes during 1988 and at the beginning of 1989, many caused by an epidemic of pregnancy among the female staff! My secretary, Mar garet Turnbull, was the first to leave after eight years of very competent and cheerful service in this role. Margaret and Wendy Russell, who followed her a few months later, were particularly notable for the extra bit that they put into their roles in the Institute, always being among those to volunteer to organise a social event or to assist in the little extra activities which give an Institute a personality. Wendy had worked with Dick Cotton in the Protein Chemistry laboratory in a most successful way for eight years, initially as Wendy McAdam. Robyn McCaskill (Scholem) joined the Institute on the same day as Wendy working with Garry Brown in the Enzymology/Metabolism laboratory. They formed a very expert and efficient team. Margaret Turnbull’s retirement is probably perma nent, but we may see Wendy or Robyn back in the Institute at a later date, although neitherwishes to specify

a date of return. On the other hand, Pam Dry and Sue Dale have retired only temporarily and will return after periods of maternity leave. Pam has been with us for only a short time and has been in charge of the DNA diagnostic work in the VCGS. In her absence Peter Colley and then Susan Forrest have supervised. Sue Dale has been a member of the cytogenetics laboratory for eight years. Kerry Fowler had been one of the long stayers of the Institute staff, coming to us from the Hall Institute in 1976 when we decided to set up a tissue culture laboratory. She superintended this activity with great skill and tact over the intervening years, training dozens of scientists m the mysteries of tissue culture, chastising firmly, yet gently, those who sought to depart from the well trodden paths of tissue culture technique and giving a number of young laboratory technicians a very sound grounding m these important methods. In addition, she undertook valuable research studies in collaboration with Dick Cotton and other scientists. We were sorry when she found the stress of her very busy laboratory too great and decided to move to a less onerous and more research orientated position at the Howard Florey Institute. We are delighted to learn that she is enjoying this new role. We were fortunate to attract Marjorie Crawford to come from the Hall Institute to take over Kerry’s role. The Institute is the sum of the activities of a large number of people and each of them is important to the end result. Senior scientists and clinicians play a very obvious critical role. These roles have been described already but I now want to express my gratitude to all of these people for the way they have coped with difficult circumstances during 1988. I am sure that the prospects of additional space which are now visible to us at last will sustain them through 1989. These pressures have affected similarly our excellent team of research assistants, . secretaries and technicians — I thank them all. Of course, all our activities are dependent on adequate financial support and we thank all those wonderful people whose donations keep the Institute going. Our fonvard budgeting shows how much we need their continued generosity and indicate that we must find further supporters. Indeed, we need to raise at least $1 million each year. I am grateful to Ms Davina Hanson who joined us during 1988 in a voluntary capacity to assist in achieving this target. All who work in the Institute are very grateful to the members of the Board and the Finance Committee for their guidance and support. I depend particularly upon the wise advice and encouragement of Neil Walford (Chairman of Board) and Laurie Cox (Chairman, Finance Committee). Anne Ellis (Business Manager) and Barry Holt (Laboratory Manager) play very special roles in the maintenance of the Institute. Without them, most of the day to day activities would grind to a halt. 9

HF.SEARCH IN PROGRESS

The intent of this section, which will become a regular feature of our Annual Reports, is to give brief overviews, intended for our lay readers, of the progress of research in the Institute over the last 12 months. Our research is determined by the particular skills and interests of our key scientists and clinicians. Therefore it is the most logical to take these people one by one and talk about the work they are doing, grouping those who are collaborating together. Some of these collaborations are long term, as with the three senior scientists involved in work on trace elements. Other collaborations may be more transient as with the joint work of Garry Brown and Henrik Dahl on pyruvate dehydrogenase or of Dick Cotton and Henrik Dahl on the HOT method of detection of mutations. There is no particular significance to the sequence in which the work of the various scientists is reported except for starting with Dick Cotton, Deputy Director. I have found it useful to make a few comments about the background of each of the senior scientists because this has generally influenced their current research.

Dr Dick Cotton Dick Cotton has had a very broad background in biology — agricultural science, microbial genetics, protein chemistry, immunology, human inborn errors of metabolism cell biology, aspects of embryology and now recombinant DNA techniques. He leads the Olive Miller Protein Chemistry Research Group. His long term special interest in the enzymes at fault in phenylketonuria (PKU) has as an ultimate objective understanding the structure and function of these enzymes and the genes which control them in sufficient detail to be able to offer gene therapy (correction of the gene defect in body cells) for PKU. He is also interested in developing new techniques to use to achieve these goals. Although this objective is still far away we do see it as achievable some day in the future. Actually, the two major components of future gene therapy for PKU are under study in different groups within the Institute. Dick Cotton is working out exactly which piece of DNA should be inserted into the defective cells to correct the disease and Choo is trying to work out how one can direct a piece of DNA into the appropriate place in the chromosome so that it may replace the faulty gene. In both instances the immediate goals are more modest compared with these grand ultimate objectives. Enzymes like phenylalanine hydroxylase (PAH) are large proteins in which quite a small proportion of the amino acid subunits are critical to the function, the

remaining amino acids serving to provide a general structure which holds the key active amino acids together in the right relationship to one another. This cluster of active amino acids is called the active site of the enzyme. Much of Dick’s effort is directed to identifying amino acids which constitute the active site of PAH. One approach involves the use of a specific type of antibody which mimics the shape of the reacting molecules. This work is making steady progress in the hands of Ian Jennings, who has worked with Dick for many years. The second approach is based on the argument that mutations which cause PKU by inactivating the enzyme generally have this effect because the mutation has damaged the active site. Identifying the site of the mutation in each of a number of different patients should point us to the amino acids which are involved in the active site. Previously this would have been a mammoth undertaking involving about a year’s work to identify the mutation in each patient. The new HOT method should allow us to identify these mutations much more rapidly. This work is being undertaken by Susan Forrest, a postdoctoral fellow who joined Dick’s group in the middle of the year. A second postdoctoral fellow working with Dick (David Howells) is taking a similar approach to the enzyme dihydropteridine reductase (DHPR), defects in which can also cause PKU. Dick’s other major project has arisen from the work that he did on his sabbatical leave and involves the HOT method for detection of mutations. This is the subject of one of the two major presentations in this report. Anthony Urban is assisting Dick in modifying the actual chemical reactions used in the work and Dick is also collaborating with Dr W. Armarego in Canberra. Henrik Dahl, together with John Bateman and Shereen Lamande of the Orthopaedic Research Unit, have been applying the technique to mutations in patients with osteogenesis imperfecta. Dick is collaborating with Peter Wright of Monash University using the method to study the mutational changes in Dengue virus. Dick has collaborated over many years with the Depart ment of Gastroenterology in producing monoclonal antibodies for use in diagnosing rotavirus infection in babies with gastroenteritis. Diagnostic kits were launched by Silenus Laboratories during 1988. Garry Brown is another scientist with a very broad background — medical degree, PhD in enzymology, postdoctoral experience in organic chemistry and mass spectometry and in molecular aspects of enzymology, work here on many different inborn errors of metabolism and a period of work with nuclear magnetic resonance spectroscopy during sabbatical leave. His work involves close collaboration with clinicians and the Metabolic Laboratory in the Department of Clinical Biochemistry in the Hospital to provide a very high quality service for the diagnosis of inborn errors of metabolism in patients throughout Victoria and in patients in other parts of Australia and New Zealand. The emphasis of the diagnostic services is upon disorders of amino acid and organic acid metabolism and Garry’s laboratory serves as

a reference centre for the whole of Australia for many of these conditions. For diagnosis of two other classes of metabolic defects (lysosomal and peroxisomal defects) we rely on the expertise available in the Department of Chemical Pathology at the Adelaide Children’s Hospital, referring to them samples from all our cases of these conditions.

Dr Henrik Dahl

'i Dr Garry Brown Garry’s role in this diagnostic work is mentioned in this research report because many of his smaller, but very successful, projects have grown out of the need to reach a diagnosis in a patient with an unusual metabolic disturbance. John Christodoulou’s project on malonyl CoA decarboxylase is a case in point. Two patients from two different families have been found to suffer from deficiency of this enzyme, a disease which had never been described previously. Features shown by these patients suggested this enzyme is more important in human metabolism than had previously been realised. John’s work will shed important new light on the function of this enzyme and is giving him an experience of laboratory science which is important in his training for a career as a clinical geneticist with a special interest in metabolic diseases. Recognition of two babies with sulphite oxidase deficiency has made us realise that this condition is probably more common than we and others had believed and that there are reasons why its diagnosis may have been missed in the past. It should be sought by specific tests in all young babies with very severe neurological damage. New approaches to investigation of young babies born with profound neurological damage forms part of the story behind Garry’s principal project on pyruvate dehyd rogenase (PDH) which is one of the major topics discussed in this report. Over a number of years we recognized a series of babies born with profound neurological abnorma lities, and often with structural defects in the brain revealed by CT scans, who had PDH deficiency without the generalised lactic acidosis which is usually expected in this condition. We learnt that one must measure the lactic acid levels in the cerebrospinal fluid if one is to recognize this condition and not be satisfied with measuring this compound in urine and blood. Others around the world have now accepted our published advice on this point and are recognizing similar patients.

Henrik Dahl has been collaborating very closely with Garry Brown in the work on PDH and much of his contribution is dealt with in that special report. He came to us after training in biochemistry in Denmark and extensive experience in molecular genetics in one of the top European groups, initially in Holland and then in Britain. During his five years in the Institute he has played a very important role in establishing molecular genetics and especially in training a number of our staff and staff of other research groups. It is dangerous to’be a good teacher because too many people want you to teach them. Although training others has occupied a substantial part of Henrik’s time over these years, it has also brought him into some valued collaborations, with Steve Goodman an American visitor on sabbatical leave, with John Bateman, Shereen Lamande and Bill Cole of the Orthopaedic Research Unit and most recently with Garry Brown and with Dick Cotton. It is no coincidence that Henrik is a major collaborator in both of the projects which have been chosen for detailed presentation this year. Henrik’s collaborations with the Orthopaedic Research Unit have come to fruition during 1988 in a series of papers describing different and very interesting mutations in patients with severe lethal forms of osteogenesis imperfecta (brittle bone disease). Bill Cole and John Bateman brought to the study of these conditions an expertise in protein chemistry and in cell culture, developing some very elegant methods of defining the approximate location of mutations in collagen molecules in these patients. Henrik was able to help them establish the additional molecular techniques needed to pinpoint the precise site and nature of the mutation in each case. Shereen Lamande proved a very competent pupil and has rapidly become an expert in the analysis of these cases. Finally, Dick Cotton’s HOT method came along at just the right time to speed up the whole process, enabling the team to define more mutations in the last 12 months than in the previous five years. Conversely, the availability of appropriate cases, already partially worked out, was very fortunate for Dick, allowing rapid demonstration of the power of his technique. It is this type of interaction between research groups which make it worth building up substantial research institutes and having a number of research institutes and groups in one Hospital. 11

Dr K. H. Choo Choo did his PhD and three years post doctoral work with us before spending three years overseas. Here he learnt about cell biology and some aspects of enzymology and protein chemistry. In Oxford and San Francisco he learnt molecular genetics from two real experts in the field — George Brownlee and Y.W. Kan. Since his return four years ago Choo has taken a particular interest in a number of important technical aspects of molecular genetics and in some very fundamental questions about human genetic material. One of Choo’s current projects involves the analysis of the various types of repetitive DNA which make up a large proportion of the entire human genetic material. We tend to imagine that the functional genes controlling individual characteristics make up most of the genetic material of humans and other organisms. In fact these genes with relatively easily defined functions constitute only about 10% of the whole genetic material. Much of the remainder is made up of thousands, or millions, or tens of millions, of relatively short DNA sequences with no currently known function. It is unclear whether these represent the accumulated waste materials of evolution, whether they serve a function by creating spaces between the functional genes or whether they have really important roles in organising the structure of chromosomes and the orderly sorting of chromosomes and genes during cell division. Choo and a PhD student, Bryce Vissel, have been studying particularly those repeated sequences which are the found in the centromeres of chromosomes specialised regions by which the chromosomes are attached to the spindle which pulls them apart into daughter cells during cell division. These are now called alfa satellite repeats and Choo and Bryce have obtained some evidence that they play a functional role in the centromere. Certain sequences present on each of chromosomes 13, 14 and 21 may explain why these chromosomes are more often subject to errors of cell division, leading to diseases like trisomy 21 (Down’s Syndrome) or trisomy 13 (a more serious birth defect). Another of Choo’s projects has the ultimate aim of understanding more about the factors which influence where pieces of DNA, which are introduced into cells, incorporate into the chromosomes. Molecular geneticists were very surprised when they found that pieces of DNA which are introduced into cells can find their way into the nucleus and become incorporated into a chromosome. At 12 1,

first it seemed that the process of the incorporation into chromosomes was entirely random and uncontrollable, but some recent findings offer hope that it may be possible to direct some pieces of DNA to insert at chosen sites in the chromosomes. For gene therapy we would like to be able to insert a normal gene into the correct site within the chromosomes replacing the defective gene which is causing the disease. Another use of this site-directed insertion is to destroy a specific gene in an experimental animal like a mouse in order to produce a model of a human genetic disease. This latter approach lends itself better to experimentation and is a major project currently occupying Choo, Neil Fraser and Anna Michalska, postdoctoral fellows with skills in molecular genetics and in mouse embryology respectively. Together they are making good progress towards the production of a mouse which is unable to produce the copper protein, caerulo plasmin. Once produced, this will bring about a collaboration with the trace element group because such a mouse would enable many important questions to be asked about the function of this copper protein.

Dr Jim Camakaris Jim Camakaris, Julian Mercer and Harry McArdle collaborate together in our research on trace elements, especially copper, making up the Scobie and Claire Mackinnon Research Group. Each comes from a different background and brings special skills which determines his particular approach to their common interest in the processes by which copper is transported to cells and within cells. Jim Camakaris has a background in microbial genetics and in cell biology and has concen trated on studying copper transport in cultured cells making extensive use of naturally occurring and induced mutations. Julian Mercer trained in the chemistry and biochemistry of nucleic acids and joined us to establish molecular genetics in our group. For various reasons he started by cloning the gene which encodes metallothio nein, an interesting metal binding protein. He became more and more interested in the problems of copper transport and now concentrates his effort on the structure and functional control of various genes which are important in this process. Harry McArdle was trained as a physiologist with a special interest in the transport of molecules across cell membranes, working most recently with the transport of iron. His approach is a physiological one with particular emphasis upon the use of liver cells in culture.

It is very good to have people with these different backgrounds collaborating together and it is also pleasing to observe the way in which each is coming to recognize the strengths of the other’s approach and to devise experiments which combine the best of each approach. Work on trace elements was described in detail in the 1987 Report and only an outline will be given here. Our research on copper transport began because of a particular interest in the two main genetically determined diseases of copper transport — Wilson’s disease, a disease of copper accumulation which damages liver and brain, and Menkes’ disease, a disease of copper deficiency which causes rapid fatal brain degeneration and arterial degeneration. The copper defect in Menkes’ disease was discovered in our group in 1971 and work has gone on from that point. We are still trying to identify the basic disturbance in copper transport in these two diseases partly because this knowledge would help us to develop more rational methods of treatment of the conditions and particularly because these discoveries would give us a great deal of new knowledge about the normal processes of copper transport. As we have argued elsewhere in this Report, the analysis of rare genetic diseases is a very powerful method of learning about normal bodily processes. Our efforts have focused more on Menkes’ disease because the disturbance of copper transport in this condition can be studied in cultured skin fibroblasts and cultured white blood cells and because there are a series of mutant mice which have defects very similar to that in Menkes’ disease. These advantages have not been available in Wilson’s disease until recently, but we do now have a collaboration with Dr Harold Rauch from the United States who discovered a strain of mouse with a defect similar to that in Wilson’s disease. Julian Mercer has been collaborating with him in studying the mice in his laboratory during 1988 and we now have some mice in our own laboratory. Dr Rauch is planning to spend a sabbatical year with us commencing late in 1989.

Dr Harry McArdle In the last 18 months Harry McArdle has been doing some studies which are relevant to Wilson’s disease. starting with an evaluation of some new copper binding chemicals called sar and diamsar which were developed by Professor Sargeson in Canberra. These proved very powerful in their ability to prevent the uptake of copper by liver cells and in removing copper which was already in the liver cells. This led on to investigation of penicilla mine, the standard drug used in the treatment of Wilson’s

disease, and tetrathiomolybdate, an agent which has been used extensively in treating copper poisoning in sheep and is showing considerable promise in our own clinical work, as an agent with more rapid effects than penicillamine. Tetrathiomolybdate had very similar effects on liver cells to the two new synthetic drugs, but was not quite as potent. On the other hand penicillamine did not alter the uptake or release of copper from liver cells in culture. In one way this is a very surprising finding, but it does fit in with some recent observations from overseas indicating that although long term treatment with penicillamine is effective in keeping patients with Wilson’s disease healthy, it does not reduce the burden of copper in their liver to a great extent. We plan to use the toxic milk mice to explore these findings further. We have always wanted to track the movement of copper from protein to protein within the cell and have found it extremely difficult to do this, partly because the radioactive isotope of copper is very short-lived and partly because it is difficult to know whether the distribution of copper between different cellular proteins observed after disrupting the cells is the same as existed before the cells were disrupted. Jim Camakaris and Rohan Farrell have now shown very clearly that the copper tends to move off other proteins on to metallothionein during the process of disruption of the cells and analysis in the laboratory. This problem is particularly serious when cells have high levels of metallothionein, as in patients with Menkes’ disease. Fortunately, we have a cell line which does not make metallothionein and we are now starting to study the distribution of copper on proteins in these cells. Jim and Rohan have also developed a new approach to analysing copper binding proteins — separating the proteins from one another in the laboratory and then adding copper isotope to identify those which bind copper specifically. Others have tried to do this, but found that the tendency for all proteins to bind copper loosely obscured the specific binding properties of the copper transport proteins. Jim and Rohan have overcome these problems by keeping the level of copper in all their solutions very low. Chinese hamster ovary cells have been used by Jim and Janet Patton and now by Jasmine Georgiou (two successive Ph.D. students) to identify mutations which alter copper transport. Two distinct and different muta tions have now been defined and we hope to identify the proteins and genes concerned in future work. These are mammalian cells and it is likely that the copper transport proteins in them will be similar to those in humans. One would expect bacteria to have copper transport systems which may have similarities to those in the mammalian cells, but may not be identical. Genetic systems can be analysed much more rapidly in bacteria like E. coli and Jim Camakaris, Barry Lee (Senior Lecturer, Department of Genetics, University of Mel bourne), and a postdoctoral fellow, Suzanne Rogers, have made rapid progress in defining a number of the steps involved in copper transport in this bacterium and have already isolated two of the genes concerned. We are starting to test these systems in mammalian cells. 13

• ' 1

lie 111 Dr Julian Mercer **: Julian Mercer’s collaboration with Harold Rauch m the United States has told us that the defect in coppp transport in the toxic milk mice is not a defect in metallothionein or caeruloplasmin or in their control. It is unfortunate that these careful experiments did not prove a defect in one of these two known copper proteins, but at least it narrows the field of possible mutations. Julian’s collaboration with veterinary researchers in Western Australia has provided further evidence that variations m zinc nutrition are primarily responsible for the hup variations in metallothionein levels we have observed in sheep. It is still not clear whether an excessive response to a relatively high zinc intake is responsible for the tendency of sheep to accumulate large amounts of copper in the liver. Julian and Andrew Grimes have been collaborating with Choo and his colleagues in the projects which are aimed at manipulating the function of caeruloplasmin and later metallothionein in transgenic mice. This has necessitated isolation and analysis of the mouse caerulo plasmin gene.

'2-

■1 Dr Malgorzata Schmidt Malgorzata Schmidt came to lead our research in Gytogenetics and to collaborate with Margaret Leversha in supervising the Diagnostic Gytogenetics Laboratory. She was trained in medicine and then in Gytogenetics in her native Poland and directed a clinical cytogenetics laboratory there before moving to the United States where she worked at Johns Hopkins Hospital for three years before joining us. In Baltimore she became ve^ interested

also has an interest in a condition called the fragile X-syndrome. This is the second most common cause ol mental retardation, after Down’s syndrome, causing moderate to severe retardation in affected males and mild to moderate retardation in a proportion of carrier females. The name denotes a stretched out appearance of the lower end of the X-chromosome which is called a fragile site. This change is visible only when cells are grown m media which have low levels of certain nutrients. Many group around the world have been trying very hard to find the genetic explanation of this disease, with little success to date. Shortly before Malgorzata arrived in Melbourne, a sharp-eyed cytogeneticist in our laboratory recognized a deletion of that region of the X-chromosome whichp abnormal in the fragile X-syndrome from op Xchromosome of a mentally retarded girl. When Malgorza ta studied this cell she found some surprising changes in the pattern of inactivation of X-chromosomes in cells suggesting that this deletion had somehow affected this process . Consequently the cells of this patient offer the prospect of adding significantly to the understanding of both Malgorzata’s principal interests. With a great deal of skill and a great deal of luck it should be possible to isolate from a normal Xchromosome the stretches of DNA which reside m this region and to find out something about their peculiar features which are the cause of fragile X-syndrome. More simple and traditional approaches to the identification of the DNA in this region have failed in the past. This is an ambitious and difficult project and we are not expecting quick results, but they may be very exciting when they come. , , , vu At the same time Malgorzata is involved, along with colleagues in the cytogenetics laboratory, in extracting the maximum new knowledge from unusual chromosome findings which occur in some of our patients. Modern molecular genetic skills enable us to find out much more about the actual DNA changes in these chrornosomal defects than was previously possible. Gonversely these defects constitute an important resource for working out the physical sequence of genes on human chromosomes. In some cases we study the molecular defect in our own laboratories, but in most cases best use of a particular chromosome finding can be made m some overseas laboratory specializing in that type of research. We are quick to establish collaborations with the overseas scientists concerned. Our clinical geneticists are all very interested in research, but naturally they have less time available to devote to research than our scientists. This has been the especially true in the last few years . when „ u load u ot clinical work has increased substantially. Although we have additional funds in the Victorian Glmical Genetics Service to recruit another senior clinical geneticist, it has been difficult to find the ^ appropriate person.

Dr Les Sheffield consumed far more of his time than. , was• planned. Nonetheless, he has been able to get on with an interesting project on the classification of patients with chondrodys plasia punctata (GDP), an interesting and unusual bone disorder which has a moderate effect upon growth and produces a very characteristic facial appearance m all the affected children. Jane Halliday has been assisting him in this study which is making good progress. We have oyer 100 patients with this disorder in Melbourne, by far the biggest collection of such cases in the world. The large number is the consequence of recognition of a mild and relatively frequent form of the condition which seems to have been ignored elsewhere. Les has also been busy, along with Judy Dodge, m setting up the statistical analysis necessary for the DINA diagnostic tests. . One of his longer term interests is in developing methods of identifying further chemicals or drugs which are able to cause birth defects. In fact his study on GDP has turned out to be quite relevant to this objective because the features produced in the foetus by maternal exposure to an anti-convulsant drug called Dilantin or to an anti coagulant drug called Warfarin mimic the features of GDP. A biochemical explanation of the overlap ol these conditions has emerged in the last 12 months and we are excited about the possibility that the common type of GUP may be a genetic defect of the same biochemical process. In the pursuit of his longer term goal, Les has started a study at the Royal Women’s Hospital monitoring the use of drugs in pregnancy. He has also established a close liaison with Dr Judith Lumley who is in charge of the Victorian Birth Defect Register at the Health Depart-

Dr Agnes Bankier Agnes Bankier’s research interest has been in POSSUM. This very successful computerised system for the diagnosis of birth defects has been discussed in previous annual reports and is mentioned separately again in this report. It is part research project, part diagnostic tooHor our own use, and part commercial venture. On all three fronts it is moving along satisfactorily.

ment. John Rogers spends most of his time in clinical work and has little time free for personal research work. However, he has always made a valuable contribution to our research because of his skills in clinical diagnosis and because of his good “sense of smell” for the unusual findings which may be of interest for research projects. Over recent years John has developed a particular interest in the counselling aspect of genetic work, particularly the support of couples who have lost babies or who are grieving the loss of the normal baby that they had hoped to produce when in fact they produced a baby with a severe defect. 15

FF^T.T.0WS in clinical genetics

POST DOCTORAL FELLOWS

Dr Susan Forrest

It is customary for young scientists to spend two or three periods of two or three years each in different laboratories after completing a PhD and before taking up a career position. It is fortunate for Australian science that there has long been a tradition of moving internationally for this added experience and indeed this may be one of the factors that has contributed to Australia’s strong achievements in basic science over the decades. (In many other countries it is more usual for postdoctoral fellows to merely move between different Institutions within their own country). We are particularly pleased that one of this year’s postdoctoral fellows. Dr Susan Forrest, is the first holder of the Helen McPherson Schutt Fellowship. This Fellow ship was established by a gift of $250,000 from the Helen McPherson Schutt Trust (see 1987 Report). We are hopeful that other Trusts and Foundations and some corporations may follow the excellent example of the Schutt Trust in establishing a named Post-doctoral Fellowship within the Institute. The availability of such Fellowships in perpetuity is a wonderful boost to our research. Susan Forrest is working with Dick Cotton, studying the mutations in the enzyme phenylalanine hydroxylase (PAH) which is most frequently at fault in phenylketonur ia (PKU). She is applying the HOT method of detection of mutations to this enzyme so that her results will have double importance, extending the range of application of the new mutation detection method as well as giving us the knowledge that we want about PAH. Sue is a Melbourne graduate originally who undertook her PhD studies in the Nuffield Department of Medicine at Oxford University where she worked on muscular dystrophy. Also working in Dick Cotton’s laboratory is Dr David

Dr David Howells

Dr Connie Maragos

Howells, who undertook his PhD studies in the Institute of Child Health in London. He will be studying another of the enzymes that can be at fault in PKU, dihydropteridine reductase (DHPR), identifying the individual mutations in different patients and learning more about how the different parts of the enzyme molecule relate to one another in determining the function of a molecule. He is also using the HOT method. Dr Connie Maragos completed her PhD in microbial molecular genetics at LaTrobe University in 1987 and has been working throughout 1988 with Henrik Dahl in the analysis of PDH gene. She has moved quickly and efficiently into the study of mammalian genes which differ in quite a number of ways from bacterial genes and is showing a great enthusiasm for her work. Dr Phil Kearney did his PhD at Monash University in microbial genetics and then moved into human genetics during a Post-doctoral Fellowship at the Galton Institute in London. Since he joined us in March he has been developing several different approaches to the cloning of the gene at fault in Menkes’ disease. Dr Neil Fraser joined us after completing his PhD in the Department of Genetics at Oxford University, but was no stranger because he had spent three months in Melbourne during the course of his PhD studies when his supervisor. Dr Ian Craig, came for a short sabbatical here. Neil is working with Choo and Dr Anna Michalska to produce transgenic mice with defective function of the gene coding for caeruloplasmin, a protein involved in copper trans port. Dr Anna Michalska came to us from the University of Adelaide where she did her PhD on production of transgenic pigs. Prior to this she had been trained in science in Poland where she obtained an MSc in embryology.

Dr Neil Fraser

Dr John Christodoulou

For the last 15 years we have trained most of the clinical geneticists who have received their training in Australia. Since 1980 we have had a more formal training programme which was the model used by the Human Genetics Society of Australasia when it formulated its policy for a three year training scheme. In 1988 we had three Trainee Fellows undertaking their whole program me with us and one who came for just the final year of a programme commenced in Sydney. Those who do their whole training here undertake a major research project, attend formal lectures along with science students at Melbourne University, work in clinics for two years and are exposed to all aspects of human and clinical genetics. They play important roles in our clinical service work and in the weekly routine of the Institute. We regard it as very important to help each trainee to find a good post overseas for further experience before taking up a consultant post. Jim McGill completed his formal training at the end of 1987, but worked on until the moment of his departure to Montreal in July 1988 to complete the collection of the results of his important genetic study of congenital deafness. It was unfortunate that his willingness to accept clinical responsibility and his skill as a clinician lead us to let him curtail too greatly the time available to analyse his results. His determination is such that we are quite confident that this analysis will soon be completed. We eagerly await the full results of this interesting study. He is an excellent clinical geneticist and we feel very proud of the glowing reports coming back from his Canadian mentor. Professor Charles Scriver, one of the world’s best known medical geneticists. Jim will return to a consultant post in Brisbane late in 1989. We were fortunate that his wife, Jenny, also worked with us as a part-time medical fellow, helping with the development of POSSUM. John Christodoulou was trained in paediatrics in Sydney and joined us in 1986 to train in metabolic diseases. We insisted that this be a special aspect to an overall training in medical genetics. He has found this

Dr David Ravine

combination to suit him and we are impressed by his competence in both aspects. A large part of his time has been devoted to his project on malonyl CoA decarboxylase for which he has been supported by an NH & MRC Medical Research Scholarship. He took leave from this Scholarship for part of 1988 for a period of intensive clinical work, but has returned to the laboratory to complete the project in 1989. David Ravine joined us in 1988, also supported by an NH & MRC Medical Research Scholarship to carry out a large project on polycystic kidney disease, using DNA linkage studies to define those who have inherited the defective gene and then assessing the reliability of ultrasound scanning as a method of diagnosis. David trained in paediatrics in Perth and Melbourne before returning to the career in medical genetics which had been his goal for a number of years. Like Jim McGill and John Christodoulou before him, he will take leave from his Scholarship for a period of intensive clinical experience, probably in 1989. The combination of research and clinical work here allows these young people to maintain contact with the subsidiary activity of a particular year while pursuing energetically the major activity (research or clinical) of that year. We hope that this will establish a pattern of practice which they can sustain in their careers. Alison Colley spent just one year here, the third year of her training, after two years in Sydney during which her particular focus was on the fragile X-syndrome and other X-chromosomal syndromes causing mental retardation. The major part of her time was spent gaining the further and wider clinical experience necessary for certification in clinical genetics, but she still found time to carry out several small clinical projects, especially ones associated with cytogenetic cases. We were able to arrange positions for Alison and her husband, Peter, (who worked in our DNA diagnostic group) in Manchester and they departed in January 1989. 17

STAFF LIST MURDOCH INSTITUTE

VISITING SCIENTISTS AND CLINICIANS Administration: David Danks, M.D., B.S., F.R.A.C.P. Scientific Director, 1962Dick Cotton, B.Ag.Sci., Ph.D., D.Sc. Deputy Scientific Director, 1968-70; 1973ii?if

Anne Ellis, B.Sc., B.Bus.(Acc.) Business Manager, 1975-

f h

Barry Holt, B.App.Sci.(M.T.), A.A.I.M.L.S. Laboratory Manager, 1972-

' Dr Kiyoshi Hayasaka

Kiyoshi Hayasaka worked in the Institute for a little over two years, from May 1986 until August 1988. He came to us from the Department of Paediatrics in Sendai after a very thorough training in both paediatrics and biochemistry and having already conducted some very elegant research on the enzyme defect in a disease called non ketotic hyperglycinaemia. He worked with Garry Brown, studying the enzyme defect in patients with cytochrome oxidase deficiency, a cause of brain and muscle disease. Later he joined in the project on pyruvate dehydrogenase which was expanding rapidly in the latter part of 1987. Working with Henrik Dahl, he played an important part in identifying the part of the PDH gene which is involved in the control of the gene function. This switch of work to the DNA laboratory suited Kiyoshi very well because he was returning to Japan to a position in which he would be expected to supervise others undertak ing molecular genetic studies as well as using enzyme techniques. We all enjoyed having Kiyoshi with us and we respected very greatly his knowledge, his ability and his sincerity. We also enjoyed getting to know his delightful wife Junko and his four children. Dr Tomiko Hokama is a paediatrician/geneticist from Okinawa. We had come to know her a little some years ago when her husband was training in paediatric surgery in Melbourne and she visited our clinics each week. At that time her children were very young and she was not able to spend much time with us. In May 1987 she returned in her own right on a scholarship awarded to her in Japan for a nine month period of intensive training and experience in clinical genetics. She joined in all of our clinical activities with remarkable enthusiasm and made good progress with three clinical projects. On her former visit her command of English had been meagre, but she had made a great effort in Japan and during the early months in Melbourne and became fluent in English. We enjoyed her delightful and friendly personality and particularly admired the way in which she managed to work very hard and yet to look after her three happy children aged 6-10 years who accompanied her.

Dr. Nigel Brown spent a productive 10 months as a Visiting Fellow in the Department of Genetics, University of Melbourne, collaborating with Dr. Lee and Dr. Camakaris on the E.coli copper transport project. Dr. Brown has returned to the U.K. to take up the Chair of Microbiology at Birmingham University and we look forward to the continuing collaboration which has been established.

Scientists (Senior): Dick Cotton, B.Ag.Sci., Ph.D., D.Sc. 1968-70; 1973-, Protein Chemistry Jim Camakaris, B.Sc.(Hons.), Ph.D., 1975-, Trace Element K.H. Cboo, B.Sc.(Hons.), Ph.D., 1979-1980; 1984-, DNA Julian Mercer, B.Sc.(Hons.), Ph.D., 1979-, DNA/Trace Element Garry Brown, M.B., B.S., Ph.D., 1980-, Enzymology Henrik Dahl, Ph.D., 1984-, DNA r"’.

Harry McArdle, B.Sc.(Hons.), Ph.D., 1985-, Trace Element Malgorzata Schmidt, M.D., Ph.D., 1987-, Cytogenetics !J

Clinical Scientists:

ril 3

Kerry Fowler, B.App.Sci.(M.L.T.), 1976-88, Cell Culture Leigh Ackland, M.Sc. 1978-, Trace Element Ruth Brown, M.Sc., 1980-, Enzymology Wendy Russell, B.App.Sci.(M.L.T.), 1981-, Protein Chemistry Elizabeth Earle, A.A.I.M.L.S., 1982-, Cytogenetics Gay Filby, B.Sc., B.A., 1982-, DNA Jane Halliday, B.Sc.(Hons.), 1981-, Clinical Robyn McCaskill, B.App.Sci.(M.L.T.), 1981-, Enzymology Wendy McGarry, B.App.Sci. (Applied Biology), 1984-, DNA Sharon Gross, B.Sc., Grad.Dip.Diet., 1986-, Trace Element Judy Dodge, B.Sc.(Hons.), M.Sc., 1985-, Clinical Haley Vogel, B.App.Sci., 1986-, Trace Element/DNA Rohan Farrell, B.Sc.(Hons.), 1987-, Trace Element Leigh Faulds, B.App.Sci., 1987-, Cell Culture

John Rogers, M.B., B.S., D.C.H., F.R.A.C.P., 1976-

Peter Kyriakou, B.Sc.(Hons.), 1987-, Trace Element

Les Sheffield, B.Med.Sci., M.B., B.S., M.Sc., D.C.H., F.R.A.C.P., 1985-

Jenny Paynter, B.Sc.(Hons.), 1987-, DNA

Agnes Bankier, M.B., B.S., F.R.A.C.P., 1983-

Rosa De Fazio, 1987-, Cell Culture

Scientific Officers and Research Assistants:

Andrea Certoma, B.Sc.(Hons.), 1988-, Cytogenetics

Ian Jennings, B.Sc., 1975-, Protein Chemistry

Tina Colgan, S.R.N., 1988-, Epidemiology

Andrew Grimes, B.App.Sci., 1975-, DNA

Marjorie Crawford, A.R.M.I.T., 1988-, Cell Culture

Denise Kirby, B.Sc.(Hons.), 1976-, Enzymology

Anthony Urban, B.Sc.(Hons.), 1988-, DNA

i: V

18 : i

MURDOCH INSTITUTE LECTURE SERIES — 1988 Dr. M. Dunlop, Department of Medicine, Royal Melbourne Hospital Phospholipids as signal transducing molecules.

Fiona Wakefield, 1988-, DNA Helen McNeil, M.I.Biol., 1988-, Epidemiology

Dietitian:

Dr. P. Hudson, Department of Protein Chemistry, C.S.I.R.O. The analysis of some novel and interesting genes including viruses , homeoboxes and a new PTH-like calcium regulating hormone.

Betty Lynch, D.LM.(Nutr.), Cert.Dietetics, 1988-, Clinical Studies

Dr. N. Gough, Ludwig Institute, Melbourne. Haemopoietic growth and differentiation factors. Dr. W. L. F. Armarego, Department of Biochemistry, John Curtin School of Medical Research, A.N.U., Canberra. Dihydropteridine reductase from E.coli: A trifunctional enzyme. Dr. L. Kelly, Department of Genetics, University of Melbourne. Genetics of calcium metabolism in the Drosophilia nervous system. Dr. L. Macaulay, Department of Medicine, Royal Melbourne Hospital. Insulin stimulates breakdown of a novel phospholipid: A role in signal transduction.

Postdoctoral Fellows: Kiyoshi Hayasaka, M.D., Ph.D., 1986-1988, Enzymology Neil Fraser, B.Sc.(Hons.), D.Phil., 1988-, DNA David Howells', B.Sc.(Hons.), Ph.D., 1988-, Protein Chemistry Phillip Kearney, B.Sc.(Hons.), Ph.D., 1988-, DNA

Dr. A. Poulos, Department of Chemical Pathology, Adelaide Children’s Hospital. Peroxisomal disorders. Dr. C. Bernard, Department of Psychology, Latrobe University. Immunogenetics of multiple sclerosis. Dr. M. Renfree, Department of Anatomy, Monash University. Sex differentiation in a marsupial. Dr. J. Hamilton, Department of Medicine, Royal Melbourne Hospital. Monocytes, cytokines and arthritic disease. Professor I. F. C. McKenzie, Research Centre for Cancer & Transplantation, Melbourne University. Monoclonal antibodies for diagnosis and treatment of cancer.

Constantina Maragos, B.Sc.(Hons.), Ph.D., 1988-, DNA Anna Michalska, M.Sc., Ph.D., 1988-, DNA,

I' j

STAFF INVOLVEMENT IN AUSTRALIAN AND INTFBNATIONAL scientific community activities

Sue Forrest, B.Sc.(Hons.), D.Phil., 1988-, Protein Chemistry

Secretaries: Lorraine White, 1971-77; 1984-1988 (P/T) Margaret Turnbull, 1981-1988 Debbie Davis, 1987Julie-Ann Goding, 1988Jane Humphreys, 1988-

Photography/Design: Anneke Veenstra, Cert.App.Sci.(M.Lab.), Ass.Dip.App.Sci. (Media Production), 1985-1988 Kati Bromly, 1988-

Professor D. M. Danks

Dr. J. G. Rogers

Deputy Chairman — Genetic Manipulation Advisory Committee, Australian Government Member — Scientific Program Committee, 8th In ternational Congress of Human Genetics Member — International Organising Committee, Vth International Congress on Inborn Errors of Metabolism Member — Scientific Council and Research Grants Committee, Adelaide Children’s Hospital Member — Appointment Committee, Chair of Human Genetics, John Curtin School of Medical Research, Canberra Chairman — Expert Co-ordinating Committee on Gene tic Services, Health Department, Victoria Member — Congenital Malformations Subcommittee, Consultative Council on Obstetric and Paediatric Mortal ity and Morbidity, Health Department, Victoria Chairman — Neonatal Metabolic Screening JointCommittee, Human Genetics Society of Australasia and Australian College of Paediatrics Member — Board of Censors in Clinical Genetics, Human Genetics Society of Australasia

Member — Paediatric Examination Committee, Royal Australasian College of Physicians Member — Congenital Malformations Subcommittee, Australian Drug Evaluation Committee Secretary — Board of Censors in Clinical Genetics, Human Genetics Society of Australasia Member — Expert Co-ordinating Committee on Genetics Services, Health Department, Victoria Member — Committee of National Association of Loss and Grief, Victoria

Dr. R. G. H. Cotton Member — NH&MRC Assigners Committee Member — NH&MRC Regional Grants Committee

Dr. L. J. Sheffield Member — Australian Ionizing Radiation Advisory Council Member — Expert Co-ordinating Committee on Genetic Services, Health Department, Victoria Member — Congenital Malformations Subcommittee, Consultative Council on Obstetric and Paediatric Mortality and Morbidity, Health Department, Victoria Chairperson — Prenatal Diagnosis Committee, Human Genetics Society of Australasia Convenor — Working Party on Genetic Counselling, Human Genetics Society of Australasia Member of Council — Human Genetics Society of Australasia 21

20 b

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EDITORIAL BOARDS Professor D. M. Danks American Journal of Medical Genetics Birth Defects Encyclopedia Brain Dysfunction European Journal of Pediatrics Genomics Journal of Trace Elements in Medicine Journal of Trace Elements and Electrolytes in Health and Disease Molecular Biology and Medicine (Associate Editor) Prenatal Diagnosis

Dr. R. G. H. Cotton Pteridines

POSTGRADUATE DEGREES AWARDED Doctor of Philosophy Janet Patton — Copper-resistant CHO cells

OVERSEAS AND INTERSTATE VISITS lectures and seminars by INSTITUTE STAFF Dr. A. Bankier March of Dimes, Baltimore, U.S.A. Conference on Methodology in Syndrome Identification, Instituto Giannina Gaslim, Genova, Italy. Invited Speaker.

Symposium — Metal Homeostasis Molecular Biology and Chemistry, Colorado, U.S.A. — Invited Speaker. University of California at Fullerton — Invited lecturer. California Institute of Technology

Dr. G. K. Brown

Dr. K. H. Choo

International Conference on ‘-ketoacid dehydrogenase complexes organised by the New York Academy of Sciences to honour Lester Reed, Austin, Texas. Invited Speaker.

International Symposium on Fatty Acid Oxidation, Philadelphia, U.S.A. Mrs. R. Brown

Master of Biotechnology Course, Monash University — Hereditary disease gene replacement therapy. Human Genetics Society of Australasia, Brisbane — a) Molecular mechanism of Robertsonian translocations and b) Satellite DNA amplification in large 14p chromosomes. Peter MacCallum Cancer Institute — Molecular studies on Down syndrome and Robertsonian transloca tions. Montech Group, Monash University — Genetic man ipulation of animals. Microbiology Department, Monash University — Evolution of centromeric satellite DNA on acrocentric chromosomes. The American Society of Human Genetics, New Orleans, U.S.A.

Seminars:

Dr. R. G. H. Cotton

Genetics Department, Oxford MRC Radiobiology Unit, Harwell, United Kingdom

Society for the Study of Inherited Metabolic Disease, Asilomar, U.S.A., March — A simple chemical method to

Seminars: Department of Biochemistry, Oxford, United Kingdom Genetics Laboratory, Oxford, United Kingdom MRC Radiobiology Unit, Harwell, United Kingdom Royal Alexandra Hospital for Children, Sydney

Visits:

!'■

Li,

Dr. J. Camakaris

detect all mutations in DNA — Invited speaker. Pteridine Workshop, Snowbird, U.S.A., March — An antiidiotype monoclonal antibody which binds to a range of pterin binding enzymes. American Society of Human Genetics, New Orleans, U.S.A., October — A simple chemical method to detect point mutations in DNA — Invited speaker. Australian Biochemical Society, Adelaide — Study of an enzyme with monoclonal antibodies — phenylalanine hydroxylase — Invited speaker. London, Oxford, Cambridge, Baltimore, Toledo, Snow bird and Asilomar — Six lectures on the “HOT” method in various departments. Toledo, Houston, New Orleans and Mobile — Three lectures on the “HOT” method in various departments. Australian Biochemical Society — Chair of session, poster and lecture. Six lectures on the “HOT” method: CSIRO Protein Chemistry Human Genetics Society of Australasia De partment of Endocrinology, Royal Children’s HospitalLudwig Institute Royal Children’s Hospital Research Foundation Department of Genetics, University of Mel bourne.

Dr. H. H. M. Dahl Human Genetics Society of Australasia, Brisbane — Detection of point mutations in DNA and RNA using chemical cleavage of mismatched base pairs. University of Sydney — Detection of point mutations in DNA and RNA using a combination of PCR and the chemical cleavage method. Queensland Institute for Medical Research, Brisbane — The polymerase chain reaction. University of Queensland, Brisbane — The polymerase chain reaction and its application to molecular genetics. Queensland Institute for Medical Research, Brisbane — Detection of mutations in DNA and RNA using the chemical cleavage reaction. The Royal College of Pathologists of Australia, A.S.M., Hobart — The use of DNA techniques in clinical analysis. Australia Association of Clinical Biochemists, Mel bourne - Recent advances in DNA methodology and their clinical application. Perkin Elmer PCR Seminar, Melbourne — Experience using PCR techniques with a robotic sampling system.

Latrobe University — The polymerase chain reaction; Detection of point mutations in DNA and RNA using a chemical cleavage method.

Professor D. M. Danks Clavering Fison Visiting Professor, Institute of Child Health, London, United Kingdom, March-April, 1988. Windermere Lecturer, British Paediatric Association, York, April 1988 — Molecular genetics and medicine American Society of Human Genetics, Annual Meeting, New Orleans, October 1988 — Deletion of Xp27 in a mentally retarded girl. Bicentennial Meeting, Royal Australasian College of Physicians, Sydney, May 1988 - DNA Technology and Medicine ANZAAS Conference, Sydney, May 1988 — Molecular genetics and diagnosis and treatment of genetic disease.

Dr. Anna Michalska CSIRO Division of Animal Health — Transgenics: super mice and giant pigs. CSL Transgenic meeting — Transgenic mice and pigs — growth regulation by porcine growth hormone cDNA.

Dr. N. Fraser Genetics Department, Monash University — Evolutionarily recent exchanges between the human sex chromosomes.

Dr. J. F. B. Mercer UCLA Symposium — Metal Homeostasis Molecular Biology and Chemistry, Colorado, U.S.A. — Speaker. Biochemistry Department, Latrobe University and Physiology Department, Melbourne University. Second year veterinary science.

Dr. L. J. Sheffield Australasian Mutagenesis Society — Keynote Speaker. Australian Laboratory Scientists’ Association — Keynote Speaker. Cleft Palate Conference, Melbourne — Invited speaker. WHO Meeting on Assessment of Perinatal Technology — Invited speaker 23

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RESEARCH COLLABORATIONS

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Protein Laboratory

Copper Projects

John Curtin School of Medical Research, A.N.U., Canberra — Expression of DHPR in E.coli. MRC Unit of Immunochemistry, Oxford, United Kingdom — HOT method to detect mutations. Department of Biochemistry, Toledo, Ohio, U.S.A. — Pterin antiidiotype antibodies, DHFR and DHPR. Institute of Medical Genetics, Moscow, U.S.S.R. — Study of an inactive form of human PAH. University of Valencia, Valencia, Spain — Drosophilia aromatic amino acid hydroxylases. Hammersmith Hospital, London, United Kingdom — Staining of the peripheral nervous system with antibody PH8. Department of Medicine, University of Sydney, Sydney — Study of carotid body cells with antibody PH8. New Jersey Medical School, Newark, U.S.A. — Evolution of neuro transmitter systems. Institute for Toxicology and Biochemistry,Munich, — Biopterin receptors. University of Michigan, Ann Arbor, U.S.A. — Pterin antiidiotype antibodies and methylene tetrahydro-folate reductase. St. Vincent’s Medical Research Institute, Melbourne — Crystals of antibody PH7. Department of Biochemistry, Monash University — Chemical fingerprinting of Dengue virus. Lady Davis Institute for Medical Research, Montreal, Canada — PAH inhibitory antibody. Flinders Medical Centre, S.A. — Serotonergic neurons in human pons and mid brain using PH8 antibody. Institute of Clinical Pediatrics, East Berlin — PKU diagnosis and mutations. Institute of Clinical Paediatrics, Turin, Italy — Studies of PKU and DHPR deficiency. Melbourne University — Drosophilia phenylalanine hydroxylase.

Department of Zoology, University of Massachusetts, Amherst, Mass., U.S.A. — Regulation of Metallothionein and Caeruloplasmin Gene Expression in the Toxic Milk Mouse. CSIRO, Floriet Park, W.A. — Metallothionein Gene Expression in Zinc Deficient Sheep. Murdoch University, W.A. — Metallothionein and Metallothionein mRNA in sheep suffering from Copper and Heliotrope Alkaloid Toxicosis. Genetics Department, Melbourne University — Cop per transport and homeostasis in Escherichia coli. Plasmid-mediated copper resistance in Escherichia coli. Regulation of copper-inducible promoters in E.coli.

Back row: Dr David Howells, Dr Dick Cotton Front row: Mr Ian Jennings, Dr Susan Forrest (Absent: Mr Anthony Urban)

Molecular Genetics Projects Howard Florey Institute, Melbourne University — Role of the natural 5’ and 3’ noncoding regions of factor IX gene expression in transgenic mice. Orthopaedic Research Unit, Department of Paediat rics, Melbourne University — Detection of base changes in RNA. Characterisations of mutations in collagen in patients with osteogenesis imperfecta type 11.

Clinical/Epidemiology Projects Adelaide Children’s Hospital — Chondrodysplasia punctata. Garvan Institute, Sydney — Chondrodysplasia punctata. Pharmacy Department, Royal Women’s Hospital — Study of the effects of drugs on the fetus during pregnancy. Royal Adelaide Hospital — Genetics of Haemophilia A. Atlanta, Georgia, U.S.A. — Genetics of Haemophilia A. Department of Nephrology, Royal Melbourne Hospital — DNA linkage study of adult polycystic kidney disease.

A gene is a long segment of the chemical molecule called DNA. DNA is made up of two very long strands which are bonded together as a duplex because they are complimentary to one another. One of the strands encodes a message which instructs a cell to make a specific protein. The other strand which is complimentary, stabilises the molecule and provides a method of making new copies when the cell divides. The alphabet used in the long coded message is a very simple one made up of just four letters (A,T,G and C), which are read as words which are always three letters (bases) long. Each three base word instructs the cell to insert a specific amino acid building block into the protein that is being made (Figure 1). Generally the mutation (change) which produces a disease involves an alteration in just one base in a particular three base word. This is sufficient to cause the cell to insert a wrong amino acid which in turn disrupts the function of the protein. It is quite difficult to identify such a subtle change in a gene which contains 1000 to 2000 bases. (Occasionally the mutation causing a disease

may be much more gross, for instance, the loss of 100 or more bases from the gene; it is much simpler to identify this type of mutation, but unfortunately they are the exception). To set a gene in perspective one must point out that humans have about 100,000 genes arranged on 23 chromosomes. The entire genetic complement can be likened to a 23 volume manual containing all the instructions needed to make and run a human, each volume (chromosome) having about 5000 chapters (genes) each devoted to one component of the task. Chapters contain between 100 and 1000 words of coded instruction (in 3 letter words using an alphabet of just 4 letters) arranged in paragraphs. Between these para graphs which we can decipher are other long paragraphs which we cannot decipher and we think that these are much less important functionally. The conventional method of detecting genetic diseases, used over the last two or three decades, has been to measure the function of the protein which the gene is

Figure I

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Normal gene

Mutant gene

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Mutant protein

Normal function

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Loss of function 25

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producing. However, the development of molecular genetics, with its ability to isolate and analyse individual genes, has offered the possibility of direct identification of mutations in the genes as a method of diagnosing genetic diseases. Unfortunately the methods available to identify the usual subtle change in a single base have been very laborious indeed, requiring about one year’s work for an experienced scientist to identify the mutation in one patient. Any test that is so laborious is quite impractical for clinical use. A simple method was needed. To understand the HOT method it is necessary to look a little more carefully at the matter of complimentarity between the two strands of the DNA duplex. An A in one strand can only form a satisfactory chemical bond with a T in the other strand and vice versa. Likewise, a G can bond only with a C. This is the rule which is referred to as complimentarity. If a mutation changes an A in the coding sequence to a G, then the opposite position in the other strand will become a C instead of a T (Figure 1). If a duplex were formed with a G actually opposite a T, then the two strands would be held together less strongly at this point than elsewhere in the molecule where perfect complimentarity is maintained. This circumstance does not actually occur in nature, but can be created in the laboratory by hybridizing together a coding strand from a mutant gene with the second strand from a normal gene. (Hybrid molecules can be produced by mixing together double stranded normal molecules and double stranded mutant molecules, subjecting the mixture to heat treat ment which causes the strands to separate then cooling to favour formation of duplexes. While some of the molecules will reform perfectly paired normal duplexes or perfectly paired mutant duplexes, one will also obtain a proportion of duplexes with one mutant strand and one normal strand hybridized together. (Figure 2 describes this experiment). During his sabbatical leave in Oxford in 1987-88, Dr. Dick Cotton identified some chemicals which will cut DNA easily at sites where there is a mismatch of the bases in two strands. One particular chemical called hydroxyla mine weakens bonds at which a C is falsely paired with another C, a T or an A. Another chemical called Osmium Tetroxide has a similar effect on the hybrid molecules wherever a T is mismatched with another T, a G or a C. The result of a typical experiment is shown in Figure 3 in which different DNA samples are run in parallel vertical tracks. In each track one can see a very faint ladder of horizontal grey lines. These locate the series of normally matched base pairs which are cut to a very small extent by the chemicals. The dense black line occurs at the site of the mismatch. One experiment like that shown in this Figure can analyse a piece of DNA about 500 to 1000 base pairs long and takes about three or four days’ work for an experienced scientist. To survey a whole gene for mutations, a scientist would need to cut the gene into several pieces and analyse each of these. Actually all of the fragments can be processed at the same time so that the whole analysis can still be conducted in three or four days. 26

To obtain the same information by standard DNA methods would have taken 6-12 months. This method of rapid detection of DNA base changes has been called the HOT method, the letters of this acronym being derived from the names of the chemicals that are used — Hydroxylamine and Osmium Tetroxide. When described in this way it sounds as though Dick Cotton’s discovery is absolutely revolutionary. This exaggerates the situation somewhat because the standard DNA methods have been speeded up over the last two years and other short-cut methods have also been described. The standard method that had been used for a number of years involved isolating the whole gene of interest from each patient (a process which takes several months) and then analysing the entire sequence of bases that makes up the gene, finally comparing the sequence determined in the patient with that which had previously been determined in a gene isolated from a normal person. By contrast, Dick’s method is able to compare directly the base sequence in the normal gene and mutant gene ignoring the regions of identity and identifying immediate ly the one point at which the two genes differ. As mentioned, it depends upon the greater ease of cutting the DNA at the point where the two genes differ. Several other methods of achieving this objective have been described over the last four years, but none of these has proved to recognize all mutations. Each has tended to miss certain types of mutation and/or to work well in one part of the gene and badly in another part. So far Dick’s method has worked well in all regions of all the genes that have been tested and has recognized all single base changes. It is also simpler and quicker than the other methods. The other important change during the last two years has been the development of a method of amplifying a particular piece of a gene very rapidly in the laboratory. This technique, which is called the polymerase chain reaction, or PCR, can allow a scientist to make millions of copies of a particular piece of a gene in a few hours, instead of a few months. Consequently it is no longer really fair to compare the four or five days it takes to identify a mutation in a gene by Dick’s method with the six or 12 months that it used to take to isolate a gene and work out its base sequence. This latter process can now be accomplished in just a few weeks. However, we believe that the HOT method can scan long sections of DNA (e.g. 10,000 bases) much more quickly. It is also less prone to error than sequencing. It is not yet clear whether Dick’s method will prove to be more efficient than the sequencing of a gene after PCR amplification as a means of finding mutations in the genes of individual patients. In fact, it may be best to combine the two methods using the PCR reaction to amplify the fragments of the gene and then applying Dick’s technique to find the mutation rather than sequencing all the bases and comparing the completed sequence. Henrik Dahl and our colleagues in the Orthopaedic Research Unit (John Bateman and Shereen Lamande) have had great success with identifying mutations in the collagen gene in patients with lethal osteogenesis imperfecta (brittle bone disease)

Normal gene

AGCT t|g CCA CGGT TCG

Mutant gene

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using this combination of methods, and we are applying the combination to a number of other projects in our own laboratories. Several major research laboratories in Britain and the U.S. are also using the HOT method with or without PCR. Throughout this description we have referred to analysis of “a gene”. In actual practice, analyses performed to date have been of the messenger RNA molecule which genes produce in the cells in which they are actually functioning. There would be advantages in analysing genes in cells that are most easily obtained from patients (e.g. white blood cells) whether the gene is functioning there or not. We believe that the HOT method can be adapted to analysing DNA directly, but have not yet completed this adaptation. In addition to detecting mutations causing genetic disease in patients, the method has many other potential applications. Important among these is the rapid com parison of different isolates of viruses obtained in different outbreaks of a disease. Viruses keep mutating and this

Figure 3 may mean that immunity developed during one attack of infection is no longer effective against a new strain of the virus in another year or in another place. We are all familiar with the way our immunity fails us when a new strain of influenza virus crops up. This process is extremely important in viruses which are more lethal than influenza, such as the Dengue virus and the AIDS virus. Scientists studying these diseases need rapid methods of comparing different virus isolates and already Dr. Peter Wright at Monash University has applied the HOT method to the Dengue virus with great success. We foresee wide application of the technique in this field of research which is known as viral epidemiology. Because the range of possible applications is very wide and some applications have commercial potential, we have patented the method in Britain, Europe, Japan and the U.S. It will take some years to determine the commercial potential and also the scientific importance of Dr. Cotton’s discovery, but we are confident that the world will rate it highly. 27

PYRUVATE DEHYDROGENASE plucose Stores M„< Glycogen)

Dietary Carbohydrate

Glucose ENERGY 2 UMTS Lactate

Pyruvate

I-

i Dietary Fat

PDH Acetyl CoAj?=i^ Fatty Acids

ENERGY 36 UNITS

Tat Stores

Krebs cycle

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Figure I Pyruvate dehydrogenase (PDH) is a large and complex enzyme (biological catalyst) controlling one of the key metabolic (chemical conversion) reactions which allow cells to utilize carbohydrates, fats and proteins to produce the energy they need. In our clinical work we encounter a number of babies with life threatening metabolic illness caused by PDH deficiency and also older children with more chronic forms of this condition. They present some puzzling features which are discussed later. The desire to understand fully the exact basis of their disease formed part of the motivation for a major study of PDH. An even stronger reason for studying PDH lay in the realisation that its crucial role in energy production must make a complete understanding of its function and control necessary if we are to combat major health problems like diabetes and obesity. We know that insulin acts on PDH — indeed it is the only definitely established point of action of insulin inside cells. PDH activity is also essential for balancing the amount of energy received from sugars and fats. One could give many other reasons for the claim that PDH must be important in these common diseases. One might imagine that we should therefore begin by studying PDH in patients with diabetes or obesity. However, we know from past experience the great power of using rare genetic diseases as “experiments of nature” to learn the details of the control of enzymes and then bringing this detailed knowledge to bear upon the more common conditions. One must gain basic knowledge first, then apply it. Although all cells contain PDH and need energy to carry out their functions, the relationship of energy to function is most easily understood in muscle cells. It is obvious that the contraction of a muscle must utilize

energy, as does the working of any machine. If a short burst of intensive movement is required, as when a sprinter runs 100 metres, energy must be made available very rapidly, but not for long. The energy consumed in running a 10,000 metre race is much greater and its supply must be sustained over a long period. Muscles store energy in glucose molecules joined together into a large conglomerate (polymer) called glycogen (similar to the starch found in cereals) from which glucose molecules can be released very quickly. This fuel is used for short bursts of activity and for the initial phase of more sustained muscular effort. As well as using only glucose in these phases of activity, the muscle also “burns” the glucose incompletely, breaking it down only as far as pyruvate (Figure 1) and not bothering to start up the more complex, and more effective, cycle of reactions (Krebs’ cycle) that lie beyond PDH in this metabolic pathway. All readers will be familiar with the sequence of feeling lively with plenty of strength in the first few minutes of running or walking, then starting to tire, and later gaining “second wind” and being able to go on provided the pace is not too fast. This sequence corresponds to using glucose only, accumulating the partly burned fuel products (pyruvate and lactate), then getting the Krebs cycle going via PDH, allowing the cells to extract much more energy from each molecule of glucose (actually 8 times more), and to use fats as fuels (Figure 2). Fat is much more abundant in the body than glycogen, but is not stored in the muscle cells themselves. It takes a little time to mobilize the more distant reserve stores of fat. This description has deliberately simplified the whole story of energy production by using a cell in which the major factors controlling energy requirement are obvious. In other body cells the circumstances which determine the Figure 2

Glucose Stores! (Glycogen) .g

Dietary Carbohydrate

Glucose ENERGY 2 UMTS Lactate

Pyruvate

iDietary Fat

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ENERGY 36 UNITS

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Back left to right: Mrs Wendy McGarry, Dr Henrik Dahl, Dr Garry Brown, Mrs Ruth Brown Front left to right: Ms Denise Kirby, Dr Connie Maragos, Ms Effie Tsotsis activation of PDH are less clear, but no less critical. Indeed, tissues like heart, kidney and brain have a very high energy requirement and must use the highly efficient Krebs’ cycle. Heart and kidney are able to use both glucose and fatty acids, but the brain can use only glucose as fuel, so it is particularly dependent on PDH. This explains the predominance of neurological effects among the symptoms of PDH deficiency. It is clear that PDH, the gateway to the Krebs’ cycle, must be under very fine control by many different factors, including the need for energy and the availability of carbohydrate and fats. Its action must be integrated with the release of fats from distant stores, and many other bodily functions. One can appreciate what a challenge a complex enzyme like PDH represents. The PDH molecule is no ordinary, simple enzyme composed of a single protein made by a single gene, but a giant molecule composed of at least 200 protein subunits of eight different types encoded by six different genes. Ultimately one would like to work out how the proteins are assembled — why they occur in particular proportions and how they are integrated into the cells. For the present, Garry Brown and Henrik Dahl have been concentrating upon the Ela subunit because it is the component which is mainly responsible for turning on and off the activity of PDH as a whole. Also most patients with PDH deficiency seem to have defects in this subunit. They have isolated the gene which encodes the Ela subunit and have analysed a number of aspects of the structure and function of the gene. The findings have been very interesting and have included two major surprises — that the gene is on the X-chromosome and that a different form of the enzyme is found in sperm. In this work Garry and Henrik have been assisted by Ruth Brown, Connie

Maragos, Kiyoshi Hayasaka, Wendy McGarry and Robin McCaskill. There are two different genes producing two different forms of the subunit, one used in most body cells and the other used only in sperm, and the principal gene is located on the X-chromosome. The clinical effects of PDH deficiency are remarkably variable between patients. The most severe effect is an overwhelming illness accompanied by the build up of massive amounts of lactic acid in the blood and cerebro-spinal fluid, and presumably in all body tissues. The babies become acutely ill within a few days after birth with vomiting, drowsiness and circulatory failure; gener ally they die despite intensive treatment. They cannot generate enough energy to stay alive. It is particularly difficult to measure the activity of PDH accurately, but the levels found in the cells in these patients are very low. The inaccuracy of the assay is a greater problem in those patients with less severe illness and only partial loss of enzyme function. These patients may suffer recurrent episodes of acidosis with less severe symptoms of the type seen in the severe cases, or they may merely suffer recurrent episodes of muscle weakness, and unsteadiness of movement. These episodes may begin only later in childhood, or even in adult life. One special effect of PDH deficiency was first recog nised in our group and has been called “cerebral lactic acidosis” to draw attention to the occurrence of severe brain damage in babies who do not have any build up of lactic acid in the blood, but do have high levels of lactate in the cerebro-spinal fluid. Many of these babies have severe anatomical brain abnormalities present already at birth. The forms of PDH deficiency which have been recognized for many years are seen in both males and

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females and occasionally in brothers or sisters. All doctors assumed that PDH deficiency, like most other inborn errors of metabolism, was caused by a double dose of a defective gen,e — autosomal recessive inheritance is the technical name for this situation. When Henrik Dahl cloned the gene for the Ela subunit, Ruth Brown undertook to search for the chromosomal location of the gene using the technique of in situ hybridization of a gene probe. Humans have 22 pairs of chromosomes plus two X-chromosomes (in females) or an X and a Y chromo some (in males). Each gene has a specific location on one of these chromosomes in all humans. If a copy of a gene isolated in the laboratory is added to the chromosome of a cell, treated in a particular way, it will find the corresponding gene in the chromosomes and bind to it. If the isolated gene is labelled with a radioactive element, the scientist can see where it is binding to a chromosome by coating the cells with a photographic emulsion on which a dark grain can be seen, signalling the site of the gene. Using this method Ruth found, to our great surprise, that the strongest signal was on the X-chromosome at Xp22.1, to be precise (Figure 3). When she analysed a large number of cells to be certain that this was right, she also found a weaker signal on chromosome 4. The next problem was to determine which of these two genes was the one at fault in patients with PDH deficiency and why there are two genes. Several different methods were used to establish beyond doubt that the Xchromosomal gene controls the PDH Ela subunit in most body cells. One of the most elegant tests used an antibody that binds to the Ela subunit to stain cultured cells from normal and affected individuals, picking patients who had been shown by other methods to make very little of this subunit (rather than making normal amounts of a functionally inactive subunit, as seen in some patients). As expected, the normal cells showed granular staining and cells from male patients showed no staining (Figure 4). The findings in female patients were more critical. If the gene were really on the X-chromosome, they expected to see normal staining in some cells and no staining in other cells. This is because females use only one of their two X chromosomes in each cell. If the gene were on an autosome one would find uniform, but reduced, staining of all cells. Their findings (Cover picture) confirmed the X chromosomal locations of the genes. 30

Dr Agnes Bankier demonstrates POSSUM Normal Armed with the new knowledge of the X-chromosomal location of the main Ela gene, it was interesting to look again at the effects of enzyme deficiency in male and female patients. At last the extreme variability of the disease began to make sense. Most of the babies who die of overwhelming generalised illness in the newborn period are male. Most of the very mild, late onset cases are female, as are all of our cases of cerebral lactic acidosis. The structural abnormalities seen in the brain of these patients are probably caused by the death of those brain cells which used the X-chromosome which contained the defective gene. The new knowledge is very important for laboratories which have been undertaking prenatal diagnostic tests for PDH deficiency. We now know that it could be very difficult to interpret a test result in a female foetus — although low activity would truly show the foetus affected, but normal activity could not reliably prove the foetus normal. Fortunately, the results also show that the risk of disease in new babies in the affected families is low because nearly all babies with the disease (male or female) are the result of new mutations (error in copying the gene during cell division) and very few have inherited the mutant gene from the mother as one usually expects for X-chromosomal genes. It seems to be very rare for a female with the gene to escape its devastating effects. Next, attention was turned to the second gene on chromosome 4. In the male this gene has been found to be expressed only in sperm and the cells from which sperm are formed. This finding shows promise of leading Garry, Henrik and their colleagues into an intriguing series of studies of a special set of genes which are expressed only in germ cells. Already they hold a leading position interna tionally in this work.

POSSUM — “Pictures Of Standard Syndromes and Unknown Malformations” — is a computerised system developed to help paediatricians and geneticists to diagnose malformation syndromes. The principles of the system were established by David Danks in 1972 and a series of young geneticists have collected photographs and coded information about patients over the last 16 years. A major upgrading of the system for commercial distribution commenced in 1984 led by Agnes Bankier, and it was first put on the market in April 1987. The system is now in use in 32 countries worldwide with a total of 125 systems sold. The clinician who is faced with a baby or child who has several abnormal features must try to decide whether those abnormal features belong together, forming a pattern which has been seen repeatedly in other children in the past, or whether the array is unique in the particular child. Arrays of features which are seen together repeatedly constitute syndromes. Over 1500 syndromes are accepted internationally and given specific names. To make the matter more complicated, there are often several different names used for the same syndrome.

-S

Recognition of a syndrome with which a clinician is familiar occurs instantly just as one can recognize immediately a friend or relation amongst a crowd of other people. There is no conscious process of analysis, merely instant recognition. No one clinician has seen patients with all 1500 syndromes and it is extremely difficult to retain in the mind, ready for use in instant recognition, the verbal and visual components of the patterns that make up syndromes. POSSUM is intended to help the clinician with this memory task, but not to displace him from his proper role of integrating information and judging

whether the correspondence between the features ex pected in a particular syndrome and those observed in a particular patient is adequate. It is clear that this type of help can be provided only by a system which has a mixture of verbal and visual content. In POSSUM, the verbal information is stored in the memory of a personal computer and 24,000 frames of photographs are stored on a single 12 inch video disc. All of the verbal data and every one of the 24,000 photographs is instantly accessible to the operating program. The normal mode of operation of POSSUM as a diagnostic aid involves entering the abnormal features seen in the patient and having the computer match these against the features of all 1500 syndromes so that it displays a list of those syndromes which match on all features or the majority of features, according to the operator’s instructions. The whole system works so rapidly that it is easy for the operator to modify his instructions several times in order to retrieve an appropri ate amount of information. (Some search strategies might result in a list of 400 syndromes which is not very helpful; a strategy which gives only two possible diagnoses may be equally unhelpful). At any point in the process, the operator can switch to viewing photographs of patients with the various possible syndromes rather than reviewing verbal information on the computer screen. Again, the retrieval of photographs is very rapid and very useful shortcuts are available, for instance one can flip through photographs of just the facial appearance of a large number of syndromes without looking at photographs of other parts of the body. Dr Bankier, who has been in charge of POSSUM ever 31

THE BOARD OF THE VICTORIAN CLINICAL, GENETICS SERVICES

■■

since the escalation of the systems commenced in 1984, is continually reviewing the system along with Mr John Marquet, the very skilled programmer who developed the POSSUM software and with other clinicians to improve the system in many little and some big ways. These improvements are accumulated until they reach a sufficient number to warrant a reissue of a new version of POSSUM. Version 1.5 was distributed 12 months after the original release of Version 1 and Version 2 followed in December 1988. Version 1.5 differed from Version 1 only by the addition of some verbal data about some additional syndromes and by some small modifications to the operating program. Version 2 encompassed more substan tial changes to the operating program and the production of a new video disc containing an extra 6000 frames, especially many additional x-rays. Overseas sales of POSSUM began slowly. It was natural enough for doctors in North America and Europe to be suspicious of this antipodean device and to wonder whether we would have the capacity to sustain and keep on building the’ system. POSSUM was displayed at an International Paediatric Congress and at an International Human Genetics Congress in 1986 and attracted consider able interest, but few orders. We probably made a tactical error in not displaying it at the American Society for Human Genetics later that year and soon we were hearing rumours that our system had collapsed and was no longer on the market. Now that it has been displayed at least twice at most relevant major international meetings and our updates have appeared exactly at the times promised, our credibility is rising rapidly. There was a surge of additional orders after Version 1.5 was seen and we are just starting to experience the next surge after the release of Version 2. There are other systems on the market which offer a similar verbal search capacity, but none of these has photographs. Initially one of these systems was a little more complete than POSSUM in its coverage of

syndromes, but there is now no practical difference between the data bases in regard to the verbal content — just possum’s advantage through having pictures. We are proud that the system that we have developed has achieved such international acceptance. We are enjoying the scientific interchange which occurs as colleagues write in with criticisms or suggestions, or with new information to include, and we look forward with some optimism to the possibility that POSSUM may one day become the world accepted system for cataloguing birth defect syndromes. One byproduct of the move towards a universal data base is constructive discussion between different groups interested in the syndromes about more uniform use of the words used to describe the various features which make up a syndrome. Meetings of experts interested in this terminology are planned during 1989. We are particularly looking forward to closer collaboration with the London based group whose system is similar to ours (without pictures), but designed more for research specialists in the field, whereas POSSUM is directed towards a wide range of paediatricians and geneticists who wish to make a correct diagnosis rather than to undertake original research on the conditions. We can see an ongoing place for both systems, provided we make them compatible. Financially the Murdoch Institute is receiving royalties on possum’s sales, but it will be some time before these catch up with expenditure we have incurred. Nonetheless we do see the possibility of reaching a break even point or even having a little surplus to use in supporting other research. Even a break even point would be a great achievement because it is very unusual for research projects to be self funding. POSSUM has always really been a research project. We were attempting to determine whether one could produce a computerised system which would be of real practical value in diagnosing birth defects. This question has certainly been answered in the affirmative.

Mr. N. Walford, B.Com., F.C.A. Chairman

i Mr. L. G. Cox, B.Com., A.A.S.A., F.S.I.A. Vice-Chairman

Mr. J. S. Guest, A.M., O.B.E., V.R.D., B.Sc., M.B., B.S., F.R.C.S., F.R.A.C.S.

Dr. G. L. Barnes, M.D., Ch.B., F.R.A.C.P.

Dr. J. G. Rogers, M.B., B.S., D.C.H. F.R.A.C.P.

Dr. B. R. Catchlove, M.B., B.S., F.R.A.C.P., F.R.A.C.M.A., F.H.A.

Mr. G. E. Heeley, B.Ec., F.A.S.A.

Professor D. M. Banks, M.D., B.S., F.R.A.C.P.

VHJTtlKlAIN UJLirNlUAL.

STAFF LIST yiCTORIAN CLINICAL GENETICS SERVICES Clinical Geneticists: David Banks, M.D., B.S., F.R.A.C.P. — Executive Director John Rogers, M.B., B.S., D.C.H., F.R.A.C.P. — Director, RCH Clinic Agnes Bankier, M.B., B.S., F.R.A.C.P. Les Sheffield, B.Med.Sci., M.B., B.S., M.Sci., D.C.H., F.R.A.C.P.

Clinical Fellows: John Christodoulou, M.B., B.S. (NHMRC Medical Postgraduate Scholar) Alison Colley, M.B., B.S., F.R.A.C.P. Tomiko Holama, M.D. Jenny McGill, M.B., B.S. Jim McGill, M.B., B.S., F.R.A.C.P. (NHMRC Medical Postgraduate Scholar) David Ravine, B.Med.Sci., M.B., B.S. (NHMRC Medical Postgraduate Scholar)

Scientists — DNA Diagnosis: Pam Dry, B.Sc.(Hons.), Dip.Ed., Ph.D. Peter Colley, B.Sc.(Hons.) Steven Nasioulas, B.Sc.(Hons.)

Co-ordinator — Royal Children’s Hospital Clinic: Ann Glynn, B.S.W.

Co-ordinator — Royal Women’s Hospital Clinic:

Ann Robertson, S.R.N. V

Monash Medical Co-ordinator Centre Genetics Services:

I:? ll

Mary van den Berk, B.S.W.(Hons)

Social Worker:

Hi]

Margaret Sahhar, B.A., Dip.Soc.Studies

Business Manager: Anne Ellis, B.Sc., B.Bus.(Acc.) Dr David Ravine and Mr Ivan Francis, members of the team involved in newborn screening for cystic fibrosis.

Secretaries: Sue Tomkins Sharon Grosvenor

Administrative Assistant: Jo Wells

Cytogeneticists: Margaret Leversha, B.Sc.(Hons.) Sue Dale, B.Sc.(Hons.) Desiree Dusart, B.App.Sci. Rhqnda Hutchinson, M.Sc. Vida Petrovic, B.Sc. Lucille Voullaire, M.Sc. Julie Roberts, B.Sc. Dean Foster, B.Sc. Michael O’Rourke, B.Sc. Louise Hills, B.Sc. Melissa Grey, B.Sc. Ralph Oertel, B.Sc.

The Victorian Clinical Genetics Service was formally established, as a subsidiary of the Murdoch Institute for Research into Birth Defects on July 14 1988 and took over the conduct of genetic services in Victoria from July 1 1988. Although formally a subsidiary of the Murdoch Institute which operates on a calendar year basis, the VCGS will operate on a July 1 to June 30 basis to comply with the reporting intervals required by the Health Department of Victoria which finances the Services. Because of the difference in reporting dates, this Report will describe the operational aspects of the VCGS and its financial statements will be reported separately.

Neonatal Screening Laboratory: Ivan Francis, B.Sc., Dip.Comp.Sci. Leonard Bonaquisto, B.Sc.(Hons.) Nick Tzanakos, B.App.Chem. Maureen Ryan

On July 1 1988 the Health Department transferred to the Service the funds previously budgeted for genetic services through the Royal Children’s Hospital (RCH) (Clinical Genetics and Cytogenetics) and the Office of Psychiatric Services (Newborn Screening Laboratory) plus additional funds allocated in the 1988-89 budget. Staff previously employed through the RCH were transferred to the VCGS payroll. The present staff of VGGS is listed above.

The existing services which were brought together in the VCGS comprise — Clinical diagnosis and counselling, provided by clinical geneticists through clinics conducted at the Royal Children’s Hospital (weekly), Royal Women’s Hospital (weekly). Monash Medical Centre (weekly), Geelong Hospital (monthly). Royal Victorian Eye and Ear Hospital (monthly). Royal Hobart Hospital and hospitals in Launceston, Devonport and Burnie (quarterly); Long

term management of patients with inborn errors of metabolism and some other groups of patients with selected genetic diseases through a weekly clinic at the Royal Children’s Hospital; Emergency clinical diagnosis and investigation of patients with metabolic diseases or other birth defects in all hospitals in Melbourne; Cytogenetic tests for patients born with birth defects through the cytogenetics laboratory located at the Royal Children’s Hospital; Diagnosis of inborn errors of metabolism and genetic diseases, through close collabora tion with the Metabolic Screening Laboratory of the Royal Children’s Hospital and with other laboratories in that hospital; Newborn screening for metabolic diseases conducted through the laboratory located at the Mont Park Pathology Centre; Provision of prenatal diagnostic tests for a wide range of genetic diseases and other birth defects, through close collaboration with the cytogenetics laboratories at the Royal Women’s Hospital and Monash Medical Centre, with specialist obstetricians and obstetric ultrasonographers at these two hospitals, and with the laboratories of the Department of Chemical Pathology at the Adelaide Children’s Hospital and of the Murdoch Institute. The new funds in the 1988-89 budget were allocated to appoint genetic co-ordinators at the Royal Women’s Hospital and Monash Medical Centre, to increase the number of clinical geneticists so that new clinics can be opened in adult teaching hospitals and in rural centres and to establish DNA diagnostic tests for the prenatal diagnosis of serious early onset genetic diseases and in the presymptomatic diagnosis of late onset diseases. 35

Diagnostic and Counselling Services The central purpose of a clinical genetics service is to give accurate advice about the magnitude of their risk to couples who have a special risk of having children with a genetic disease or some other type of birth defect, and, when possible, to reduce the risk. It is equally important to reassure couples who fear that they may have a high risk of producing an abnormal baby, but do not in fact have much more risk than average couples. We believe that advice should be easily available to those who want it, but should not be thrust upon those who would prefer not to discuss this matter. Whenever a baby with a serious birth defect is delivered, the risk of recurrence of the problem in a future pregnancy and the probable cause of the problem should be discussed with the parents by the doctor concerned, or referral to a clinical geneticist should be offered. This practice should be followed whether the doctor regards the risk of recurrence as high or low because parents in this situation often imagine the risk to be greater than it really is and may be scared to ask questions. Advice is also needed by patients themselves or by other relevant family members when a genetic disease is diagnosed later in childhood or during adult life. Adults with «birth defects should generally be encouraged to discuss the risks of passing their defect on to their offspring before they start a family, but here one has to be careful because this discussion can be quite threatening if handled in the wrong way. Some people have suggested that all young couples should receive genetic counselling before marrying. This would be an extraordinarily unproductive intrusion upon their lives and we would certainly not recommend such an approach. In all genetic counselling the crucial first step is to make an accurate diagnosis of the condition present in the family. Consequently, a clinical geneticist spends more of his time determining the correct diagnosis than in counselling family members. Extensive clinical experience is essential in clinical genetics and a network of collaborations with various medical specialists is needed. Chromosome analysis (cytogenetics) and a wide range of very sophisticated biochemical analyses must be avail able. Some of these tests are available through routine diagnostic laboratories, but others are more specialised and are available only within our own research laborator ies or in the laboratories of other similar groups interstate or overseas. Recently it has become possible to analyse the DNA of genes directly and these tests can be used for some genetic diseases.

Prenatal Diagnosis Over the last 15 years, methods of diagnosing a number of genetic diseases during pregnancy have been developed.

These tests may involve ultrasound scanning, chromo some analysis, biochemical assays or DNA tests. Most of these tests are performed in VCGS laboratories, in the Murdoch Institute or in other specialised laboratories. Ultrasound scanning can detect a number of major physical malformations, some of which are hereditary or at least more likely to occur in future pregnancies of couples who already have one affected child. Spina bifida is the most frequent example of this last situation. Several obstetricians associated with the Royal Women’s Hospital and the Monash Medical Centre are very skilled in the detecting of fetal abnormalities by ultrasound. Scanning is most accurate in detection of abnormalities when applied at about 18 weeks gestation. Chromosomes can be examined in fetal cells obtained at 16 weeks gestation by amniocentesis (sampling of the fluid around the fetus by insertion of a fine needle through the abdominal wall of the mother), or at 10 weeks gestation by chorion villus sampling (sampling of the tissue which will later become the placenta by the passage of a needle through the mother’s abdominal wall). Abnormalities of chromosomes are responsible for Down’s syndrome and for the fragile-X syndrome, the two most frequent causes of mental retardation. There are also a number of other less frequent abnormalities which have even more severe effects on the fetus. Fragile-X syndrome is hereditary. Couples who might benefit from testing for this condition are recognized in the course of genetic counselling. So too are a few exceptional families with a hereditary reason for having babies with Down’s syndrome. In the majority of instances, Down’s syndrome does not have a hereditary cause, but it is well known that the risk of having a Down’s syndrome baby increases considerably and progressively in older women beyond the mid thirties. Prenatal chromosome tests have been made available to all women in Victoria over the age of 37 years for the last decade and these tests have proved very acceptable, having been requested by over 70% of women aged 40 years and over, and over 50% of women aged 37-39 years. When one considers that even the women who are over 40 years of age are facing only a 1-2% risk of having a baby with Down’s syndrome, it is clear that the majority of Victorian couples find acceptable the general strategy of diagnosing fetal abnormalities early in pregnancy and terminating affected pregnancies. The minority view that this approach is not morally acceptable gains a great deal of media attention, but it is important that all couples should be able to exercise the option of using these procedures. Of course, it is also very important to ensure that those couples who find termination of an abnormal pregnancy unacceptable are not put under pressure to use prenatal diagnostic tests. Down’s syndrome, neural tube defects and fragile-X

syndrome are all relatively common conditions for which prenatal diagnosis is available. There are also specific diagnostic tests for well over 200 rarer genetic diseases. Although rare in the whole community, these diseases are very important to the couples concerned who are generally facing 1 in 4, or 1 in 2, risks of having children affected by severely disabling diseases. Most of the tests for these genetic diseases are what we would call conventional tests using assays of enzymes or other tests of a type which have been in use in biochemistry laboratories for many years. Each test is specific for the disease concerned and considerable expertise in needed to perform each test. It is therefore most efficient for the performance of these tests to be rationalised between laboratories throughout Aus tralia. The more recently developed DNA diagnostic tests have attracted more public attention even though they are available for only 15 or 20 diseases. This is partly because of the novelty of molecular genetic techniques and partly because some of the diseases concerned are better known to the general public — e.g. cystic fibrosis, muscular dystrophy and the haemophilias. There are also a number of novel aspects to the DNA tests which are causing a considerable amount of extra work in genetic counselling. Most of the tests are not specifically directed at the faulty gene, but use a marker gene close to this gene to track it through a family. They therefore depend upon the availability of certain key family members and may be inapplicable in some families, a fact which is very hard to explain to a couple. They also have a built-in error rate of 1 or 2% whereas conventional tests are less error prone. All of this has considerably increased the work of our genetic counsellors.

Presymptomatic diagnosis The new DNA tests are also making it possible to take a new look at some of the genetic diseases which cause symptoms only in mid adult life, but are then progressive ly disabling. Huntington’s disease, which causes progres sive dementia and loss of control of movements starting at 30 — 50 years of age and progressing to complete disability and death over 5-10 years, is the best known of these condition. Others include myotonic dystrophy (a severe late onset muscle disease) and polycystic kidney disease (one of the most frequent reasons for kidney transplant). These conditions are inherited from parent to child and the healthy 25 year old offspring of an affected individual finds it very difficult to decide what to do about having children. If he or she has inherited the normal gene from the parent, then there is no risk of having affected children, but those who have inherited the defective gene will have a 50% chance of passing it on to their children and will certainly develop the disease themselves later in life. We now have DNA tests which can distinguish those

offspring who have inherited the normal gene from those who have inherited the defective gene. The availability of this test provides a serious dilemma for the individuals concerned. Nearly all of them would like to know whether or not they are capable of passing on the disease to their children, but some find that they could not handle the knowledge that they will certainly develop the disease themselves at a later stage. Counselling these couples is a very protracted business, both before testing and after testing of those whose result is abnormal. It is the advent of these tests for this particular group of diseases which created an urgent need to establish Genetic Clinics in the various adult teaching hospital in Mel bourne where most of these patients are diagnosed and treated. Although the Victorian Government has provided the money necessary to employ additional trained Clinical Geneticists to set up these clinics, it has proved very difficult to recruit appropriate people because there is a world-wide shortage. Fortunately, we have persuaded a senior and very experienced Canadian Geneticist to join us for a year or two in the latter part of his career commencing June 1989. We have a number of very able young people in training who will be able to take up the positions in due course.

Future trends in diagnostic tests While the indirect procedures involved with the current DNA diagnostic tests have been valuable in providing prenatal diagnosis for a number of important genetic diseases, we regard these tests as transient ones which we hope will be replaced by tests directed specifically to the fault in the gene which is causing the disease. The HOT method of detecting mutations described elsewhere in this Report is an important step in this direction and we hope that we will soon be able to identify the mutation responsible for a disease in a particular family so quickly that we can go on to develop a specific test for that mutation for future cases of the disease in the same family.

Benefits of Research to Clinical Service Elsewhere in the Report we have spoken about the advantage which an involvement in clinical service work brings to the research of the Murdoch Institute. Several of the matters just discussed provide good examples of the converse benefit of the close liaison between service and research — i.e. the ability of the research group to come up with new practical solutions to the problems that have to be dealt with in the clinic. It is also useful to have research workers who are able to assess critically claimed new advances in laboratory tests or treatments, many of which are not as good as initially claimed. Expert assessment of procedures before they are introduced into clinical use can save millions of dollars. 37

STUDIES IN TRANSGENIC MICE AND OTHER MOLECULAR GENETIC STUDIES

EARLY

REMOVE

GROW

INTRODUCE

SELECT

EMBRYO

INNER

ES CELLS

MUTATED

FOR

CELL MASS

IN A

GENES

CELLS

For many years we have wished that mice were known with mutations which corresponded precisely to most human diseases. Despite the enormous amount of work put into the production of catalogues of mouse mutations on the one hand, and human mutations on the other, only a very small proportion of mutations can be matched up between the two species. Over the last seven or eight years many different human gene functions have been intro duced into mice by injection of the appropriate piece of DNA into the nucleus of the fertilized egg. The resulting animals are known as transgenic mice. Initially these studies involved adding the function of a specific human gene. More recently several methods of disabling specific murine genes have been discovered and these have allowed the production of a few models of human genetic diseases. The method which interests us most is the targeted introduction of a disabling error into a specifical ly chosen mouse gene. This method appeals because it produces stable mutants which can be bred through many generations and because the process of targeting the specific gene is of great interest in itself as a component of future methods of gene therapy.

CARRYING

CULTURE DISH

MUTATION

Although all three of our senior molecular geneticists have a strong interest in this type of work, it seemed inefficient for more than one to become heavily involved and Dr Choo chose to develop this project. We were fortunate to recruit from Adelaide an embryologist (Dr Anna Michalska) who had just completed a PhD adapting the murine transgenic mouse technique to use in pigs. It was obvious that the gene chosen for first experiment should be one of particular interest to one or other of the research groups in the Institute. After some discussion Caeruloplasmin was chosen.

MICROINJECT

Production of transgenic, mice carrying specific mutations A Selection ofembryonic stem (ES) cells carrying the desired mutation due to homologous recombination B Microinjection of the selected ES cells into a mouse blastocyst. C Reimplantation of the blastocyst into foster mother leading to the production of a chimeric mouse (some cells normal, some mutated) from which fully mutated mice can be bred.

For several years Dr Choo, and Bryce Vissel, a PhD student working with him, have been studying various classes of repetitive DNA found in the genome. One class of repetitive DNA is particularly concentrated in centro meres- and is likely to play some important role in the function of this specialised region of the chromosome. Further progress of this work is reported.

Production of transgenic mice using embryonic stem (ES) cells: Culture of ES cells and gene transfer by lipofection A. E. Michalska and K. H. Choo

REIMPLANT

CHIMERIC

MOUSE

Mouse embryonic stem cells are derived from the inner cell mass of the early embryo at the blastocyst stage. These cells are grown under culture conditions which suppress differentiation of the cells while allowing them to remain fully pluripotent. When reintroduced into mouse blastocysts, they will form chimaeric animals and colonise the germline tissue. A pure transgenic animal can then be produced by breeding. Use of ES cells for transgenic mouse production offers the advantage of in vitro selection or other manipulations prior to implantation. After testing a number of ES cell lines from different overseas laboratories and different feeder systems we have chosen to maintain our cultures on STO cells except when

MUTATED MOUSE

certain experimental steps necessitate the use of condi tioned medium. We have investigated a new way of introducing cloned DNA into ES cells by lipofection, or lipid-mediated DNA transfection. This procedure is extremely simple and involves encapsulating the DNA in lipofectin by mixing the two components together (Feigner et al, Proc. Natl. Acad. Sci. USA, 84:7413) and layering the DNA/ lipofectin complex onto ES cells to allow transmembrane absorption of DNA and integration into the genome. Using a pMClneoPA selectable marker (see below), we have observed a transformation frequency of approx imately 10-4, 15-fold higher than we obtained with the standard calcium phosphate precipitation method. This frequency is high enough to be used in gene targeting experiments (following section). We are currently assessing electroporation and mic roinjection as alternative methods of introducing DNA into ES cells. Conditions for establishing chimaeric animals are also being tested.

Targeted mutation of the caeruloplasmin gene in mouse embryonic stem cells. Assay for homologous recombination using the polymerase chain reaction N. J. Fraser, A. E. Michalska and K. H. Choo We aim to mutate, by gene targeting, the endogenous caeruloplasmin gene in mouse embryo-derived stem (ES) cells. Caeruloplasmin is a serum a2-glycoprotein which contains more than 95% of plasma copper. The functions of caeruloplasmin are not well defined but may include 1) transport and donation of copper; 2) ferroxidase, amine oxidase, and superoxide dismutase-like activities; 3) deaminase activity, and 4) tissue angiogenesis. By creating a mutant mouse deficient in caeruloplasmin, we hope to learn which of these possible roles are important. We have isolated a series of overlapping genomic clones in phage lambda corresponding to the mouse caeruloplas min gene (see separate section). To achieve an optimal gene-targeting frequency it is necessary to use extensive homology between the homing sequence and the target sequence. Our construct is therefore based on a large, 11.5kb, fragment present in one of the lambda clones. The most 5’ exon in this fragment has been mutated by introducing a 20bp oligonucleotide containing several in-frame stop codons. The neo"' gene from pMClNeoPA has been inserted into the 11.5kb fragment at a position 3’ to the site of the mutation to allow in vitro selection of transformed ES cells. This neo" gene has been previously shown to be efficiently expressed in ES cells and, because of its small size (1.1 kb), keeps the region of non-homology in the construct to a minimum. Our assay for targeted mutation is based on the polymerase chain reaction (PCR), using a pair of primers derived from i) a sequence present in the endogenous gene, but 5’ of the region used in our construct; ii) one of the complementary 20bp oligonucleotides used to create the mutation in our construct. A productive PCR reaction 39

38 1

DETAILED PROJECT REPORTS will only occur when the construct has been correctly targeted into the endogenous gene. We have demonstrated that this assay can detect one successful recombination event in a complex pool of more than 1000 transformed Experiments are now underway to introduce the above construct into mouse ES cells.

Role of the natural 5* and 3’ noncoding regions of factor IX gene in expression in transgenic mice

N. J. Fraser, I. G. Lyons*, K. Fowler*, R. Richards*, and K. H. Choo (* Howard Florey Institute, University of Melbourne) We have previously constructed a human factor IX cDNA with complete mRNA sequence and successfully expressed this in transgenic mice. However, Richard Palmiter and Ralph Brinster reported nonexpression of their factor IX constructs in many transgenic mice despite demonstration that the constructs were functional in tissue culture cells. Direct comparison of constructs revealed that Palmiter and Brinster had removed most of the 5’ and 3’ noncoding regions of the natural human factor IX mRNA. We therefore carried out further experiments to test the possibility that inclusion of critical 5’ and 3’ noncoding regions was responsible for our successful expression. Three new constructs were prepared by modifying the original full-length cDNA. In these, one lacked all the 5’ noncoding region, whereas the other two lacked greater than 80% ofthe 3’noncoding region. The deleted portions were re placed by sheep metallothionein noncoding sequences. These constructs were used for the production of transgenic mice. In each case, more than one convincing expressor was found. This result suggested that the noncoding regions which have been tested did not carry any vital information uniquely essential for factor IX expression in transgenic mice. It is possible the expression assay used by Palmiter and Brinster was not sufficiently sensitive.

Evolution and structure of human centromeric repetitive DNAs and their role in Robertsonian translocation

If ,:ili

K. H. Choo, E. Earle, B. Vissel, R. Brown and G. Filby Human alpha satellite DNA constitutes up to 10% of the genome. This DNA is present in all the centromeres, with chromosome-specific subfamilies being found for most of the chromosomes. However, the acrocentric chromosomes are exceptional, several chromosomes shar ing a common alphoid subfamily with each chromosome, at the same time, carrying more than one identifiable subfamily. Resolution of the complex structural organisa tion of these DNAs may shed light on the evolution of the acrocentric chromosomes, and on the aetiology of Robertsonian translocations. 40

We have identified an alphoid subfamily which is shared by chromosomes 13, 14 and 21. These chromosomes are particularly prone to Robertsonian translocations involving recombination of whole acrocentric chromosome arms. Other subfamilies which have been identified by ourselves and others include ones with the following distributions:— (a) chromosomes 13 and 21; (b) chromosomes 14 and 22; (c) chromosome 14; (d) chromosome 22; and (e) shared by all five acrocentric chromosomes. Based on these observations, we have proposed a model of evolution and recombination exchange of centromeric DNA between different acrocentric chromosomes which explains all available genetic data, as well as the high prevalence of 13/14 and 14/21 Robertsonian translocations. Parallel studies have been carried out on the mouse centromeric satellite DNA. Direct sequencing of 30 different monomeric repeating units (totalling over 7,000bp) revealed a very high degree of conservation amongst the monomers. These monomeric units were further shown to be arranged into long tandem arrays of 250kb to greater than 2000kb using pulsed field gel electrophoresis. Interestingly, mouse chromosomes are all acrocentric in nature, and are also frequently involved in Robertsonian translocations. These observations led us to propose a model similar to that for the human acrocentric chromosomes, in which regular recombination exchange of centromeric DNA between nonhomologous chromo somes may be responsible for sequence homogenisation and Robertsonian translocations. Publications: 22, 69 ChromOSOmC “painting” aS an i in proved method of detecting

trisomy 21 K. H. Choo, G. Filby, R. Brown and E. Earle DNA techniques may offer an alternative to the laborious and costly cytogenetic methods used to diagnose trisomy 21. A particularly appealing approach involves the preparation of a mixture of chromosome 21-specific probes which would allow the “painting” of this chromosome in interphase cells. Such a method eliminates a culturing step (which takes 7-10 days), and requires counting of two signal spots in normals versus three spots in trisomy 21. We previously isolated 50 independent unique sequ ences from a human chromosome 21 library, each individually assigned to chromosome 21 using a mousehuman somatic hybrid cell line. A mixture of these probes was used in a dual labelling method and for in situ hybridisation of human metaphase chromosome which gave strong signals on chromosome 21, but the signal was too weak to see in interphase cell nuclei. We have modified our colony screening procedure and have isolated over 10,000 new clones from the chromosome 21 library after 6 rounds of hybridisation to remove any moderate to highly repetitive sequences. In situ hybridisation, with DNADNA competition to block residual low repeats, is currently underway. Publications: 21, 30

Over the last two or three years DNA techniques have been used increasingly in our research. This has led to an increase in collaborative projects involving our molecular geneticists and scientists from other laboratories and makes it more appropriate to group our project reports according to subject rather than by the laboratories in which they are conducted. Those visiting our laboratories will still find rooms devoted to Enzymology/Metabolism (Garry Brown), Protein Chemistry (Dick Cotton) and DNA (Henrik Dahl/Julian Mercer/Choo), but the work done in these rooms is grouped differently in this report. Already in 1987 we were listing most of Julian Mercer’s

work along with other trace element research, so this is just a further evolution in our presentation. It is important that we do not lose track of the identities and special personal contributions of our senior scientists — these have been mentioned in the Director’s Report and in Research in Progress. Nor do we wish to obscure the important relationships of our major supporters to particular research groups — the Olive Miller Protein Chemistry Research Group headed by Dick Cotton and the Scobie and Claire Mackinnon Trace Element Re search Group which includes Jim Camakaris, Harry McArdle, Julian Mercer and their associates.

STUDIES ON PYRUVATE DEHYDROGENASE (PDH) G. K. Brown, H. H. -M. Dahl

Research in the enzymology and metabolism laboratory is centered on inborn errors of energy metabolism, and, in particular, defects in the enzymes of pyruvate oxidation. In the past year, significant progress has been made in our studies of the human pyruvate dehydrogenase (PDH) complex. Isolation of cDNA clones for the Elasubunit of the complex has led to a detailed analysis of the structure of the gene for this subunit and its localisation to the short arm of the X chromosome. This unexpected mapping result has major implications for our understanding of PDH deficiency. We have been able to utilise our large collection of patient material and our previously deve loped techniques to exploit this finding and provide explanations for much of the clinical and biochemical heterogeneity of the disorder. Dr. Brown was invited to present our work on pyruvate dehydrogenase at an international symposium on alphaketoacid dehydrogenases in Austin, Texas to honour one of the leading workers in this field, Lester Reed. Discussions there led to an invitation to join an international collaborative study of treatment of PDH deficiency.

The structure of the PDH Ela gene C. Maragos, W. D. Hutchison, K. Hayasaka, G. K. Brown and H. D. H. M. Dahl Clones containing the human X-linked PDH Ela gene have been isolated from a genomic cosmid library. This has enabled us to determine the structural organisation of this gene by restriction endonuclease mapping and DNA sequence analysis. It was shown that the gene is approximately 17 kb long, it contains 11 exons ranging from 21 basepairs to 174 basepairs and introns ranging

from 600 basepairs to nearly 6 kilobases. This analysis also enabled us to look at the regulatory region for this gene. The features of the regulatory region is characteris tic of a house-keeping gene. It also contains regions characteristic of hormone responsive elements and it might be speculated that these hormones would have an effect on the expression of the PDH Ela gene. Having characterised the normal gene for the human PDH Ela subunit, further studies on the expression and regulation of this gene are underway. These studies will provide the necessary information for analysis of mutations and their effects in patients with PDH Ela deficiency.

Mapping the PDH Ela gene R. M. Brown, H. -H. M. Dahl and G. K. Brown The PDH Ela cDNA clones isolated previously have been used to map the gene for this subunit by in situ hybridisation to human metaphase chromosomes and Southern blot analysis of DNA from humanDmouse somatic cell hybrids with various X chromosome rearrangements. Two loci with sequence homology to the PDH Ela cDNA have been detected in the human genome. The major locus is on the X chromosome in the region Xp22.13-22.2. A weaker signal was detected from an autosomal locus and this has been localised to the long arm of chromosome 4. Studies of PDH Ela expression in cells and tissues from female patients with PDH deficiency confirm that the X chromosome locus determines the synthesis of this subunit in somatic cells. Heterozygous females with PDH deficien cy and reduced levels of PDH Ela immunoreactive protein are mosaics, with two populations of cells, one with the normal level of PDH Ela protein, the other deficient (see cover picture). Publication: 19 41

STUDIES RELATED TO PHENYLKETONURIA (PKU) R. G. H. Cotton (Olive Miller Protein Chemistry Group)

Isolation of a testis specific PDH Ela gene. H. H -M. Dahl, W. D. Hutchinson, R. Brown, c' Maragos and G. K. Brown Energy production in sperm is absolutely dependent on aerobic metabolism. Pyruvate dehydrogenase is an essential enzyme in this metabolic pathway. We had shown that in the somatic tissues analysed, the functional gene for the Ela subunit was located on the X chromosome. An interesting question was therefore: How do those sperm that lack an X-chromosome (that is, Y bearing sperm) synthesise this essential subunit? We have therefore isolated cDNA clones from a human testis library. These clones were examined by DNA sequence analysis and it was shown that the pyruvate dehydrogenase Ela subunit in sperm is actually different from the corresponding subunit in other tissues. The PDH Ela subunit expressed in sperm is approximately 90% homologous to that coded for by the X chromosome. So far, sperm is the only tissue in which we have shown it expressed. It has similar properties with regard to phosphorylation as the X-linked PDH Ela subunit. Identification of this autosomal gene for PDH Ela is not only of considerable interest with regard to expression and regulation of this gene, it also enables us to study X chromosome inactivation in more detail.

The clinical and biochemical spectrum of human PDH Ela deficiency. R. D. Scbolem, D. M. Kirby, R. M. Brown and G. K. Brown. Localisation of the gene for the Ela subunit of the PDH complex to the X chromosome has necessitated a com plete reinterpretation of the clinical, genetic and biochemical features of PDH deficiency. As defects in the Ela subunit are by far the most common, this involves almost all patients with PDH deficiency. Over the past years, we have assembled a large collection of fibroblast lines and tissue samples from patients with this disorder. At the same time, we have been developing methods for analysing the basic defects in these patients. Assays of PDH activity have been considerably improved, we have prepared antibodies to both the intact complex and the individual subunits for structural studies and we have isolated cDNA and genomic clones for the Ela subunit for investigation of the basic genetic defects. With these techniques, and the knowledge of X-linkage of the PDH Ela gene, we have been able to investigate a number of different aspects of PDH deficiency. In female patients, it is now apparent that all are manifesting heterozygotes, with one normal and one mutant PDH Ela gene. We have considerable evidence that the presence of the mutant gene does not affect the normal random pattern of X-inactivation during early embryogenesis as all tissues in these females show a mosaic pattern of PDH Ela expression. Variations in the

pattern of X-inactivation account for-the difference in PDH activity in different tissues and the poor correlation between activity in cultured fibroblasts and clinical severity. The problem of obtaining a representative cell sample for diagnosis in these female patients has been highlighted by gross deviations in activity in different samples from the one individual. Expression of severe PDH deficiency in a proportion of cells may also account for the significant structural abnormalities in the brain of female patients. The central nervous system is particularly vulnerable to PDH deficiency as it has an obligatory requirement for aerobic glucose oxidation under normal conditions. Cells which completely lack PDH activity may degenerate and die leading to the gross cerebral atrophy often seen in these patients. Male patients with PDH Ela deficiency do not present such a problem for diagnosis as all cells in the body are equally affected. However, this may also mean that the most severe forms of the disease are not seen in male babies because the functional impairment would be so great that it would lead to early foetal death. The most important question in male patients is whether or not their mothers are carriers of the condition. We have not yet identified any obligate female carriers of PDH Ela deficiency, nor have we found any evidence for carrier status in any of the mothers of our patients. At present it appears that most heterozygous females manifest the condition and most patients, males and females, are the result of new mutations, however, this will only be established by more extensive family studies.

Susan Forrest and David Howells joined the laboratory this year as postdoctoral workers. Wendy Russell left this year after 7 years of much appreciated assistance. She was replaced by Anthony Urban. The main thrust of the Olive Miller Protein Laboratory is to understand completely the structure and function of two enzymes affected in phenylketonuria (PKU) — phenylalanine hydroxylase (PAH) and dihydropteridine reductase (DHPR). The current objective is the identifica tion of the amino acids which constitute the active sites of these enzymes. We are particularly attempting to improve the methods of obtaining this information. One approach involves the use of antiidiotype monoclonal antibodies to identify the amino acids which comprise the active sites. Another approach uses a new rapid method of detecting mutations in patients with PKU arguing that the mutations which cause disease must change those aminoacids which are important for the function of the enzyme.

Structure-function of phenylalanine hydroxylase

Publications: 1, 2

Basic genetic studies using the PDH Ela gene as a defined XD linked marker. R. M. Brown, N. Fraser, R. D. Scbolem, H D H. M. Dabl and G. K. Brown. The localisation of the PDH Ela gene to the p22.2 region of the X chromosome makes it the most distal locus on the short arm of the human X chromosome with a defined protein product whose expression is subject to random inactivation. Comparative mapping of the mouse PDH Ela gene is currently in progress and is of great interest in view of the close homology between the X chromosomes in a wide variety of species. The location of the PDH Ela gene and its high degree of conservation means that it is an ideal marker for studies of X chromosome inactivation and the boundaries of the pseudo-autosomal region in different species. With samples from a number of female patients with PDH deficiency whose mutant cells fail to express any Ela protein we are also able to analyse patterns of X chromosome inactivation in different tissues directly. In these studies, we can correlate enzyme activity, the amount of immunoreactive Ela protein, the proportion of normal and mutant cells and the proportion of normal X chromosomes which are active.

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I. Jennings and R. G. H. Cotton An anti-idiotypic monoclonal antibody isolated from a mouse immunized with the phenylalanine hydroxylase pterin cofactor (bound to a carrier protein) has been found to react at the phenylalanine hydroxylase cofactor binding site within the enzyme active site. This antibody has demonstrated 3-dimensional structural homology in the pterin binding site of a diverse range of pterin requiring enzymes through its immunoreactivity with the aromatic aminoacid hydroxylases, dihydrofolate reductases, dihyd ropteridine reductases and sepiapterin reductase. Structu ral localisation of the epitope to a highly conserved region of phenylalanine hydroxylase has been performed using limited proteolysis and chemical cleavage of phenylalaI nine hydroxylase. The localisation was further confirmed by the antibody’s immunoreactivity with a 27 amino acid synthetic peptide found within this region of the phenylalanine hydroxylase sequence. Future work will involve studying the amino acids within this peptide necessary for antibody immunoreactiv ity and cofactor binding. In parallel, affinity labelling of the enzyme active site with reactive pterins and in vitro mutagenesis of the enzyme may confirm this region’s role in cofactor binding and enzyme activity. In collaboration with Michael Parniak (Montreal) a monoclonal antibody to phenylalanine hydroxylase, PHI, has been shown to bind at or near the regulatory binding site for phenylalanine on phenylalanine hydroxylase. This

site has been postulated to exist at a subunit to subunit interface and further work is proceeding in this direction. Publication: 59

Dihydropteridine reductase (DHPR) and mutations causing tetrahydrobiopterin deficiency D. Howells, H. Dahl, W. Armarego and R. Cotton This year saw an increase in the effort towards the understanding of DHPR structure and function. Dr. Armarego achieved expression of human DHPR in E.coli in good yield paving the way for ready study of its properties. Dr. Howells initiated work on the analysis of mutations in DHPR deficiency. The system for the study of these mutations has been established. This involves amplification of the mRNA, followed by localization of the mutation using the chemical (HOT) method of mutation detection. Publications: 5, 12, 31, 71

Use of monoclonal antibodies to phenylalanine hydroxylase in various systems R. G. H. Cotton and I. Jennings In the past we have isolated a series of monoclonal antibodies for the study of phenylalanine hydroxylase (PH). Detailed characterisation of the properties of these antibodies indicated that some were useful for our purposes (e.g. enzyme purification) and others would be of interest to others studying PH in different ways or in immunohistochemical staining. There has been an in creasing interest in these latter antibodies. Some are interested in the antibody (PH7) which recognises the phosphorylated form of enzyme so that the action of effectors outside the liver cell can be studied by measurement of PH phosphorylation. Dr. Isaacs is working toward defining the structure of the binding site of this antibody with and without the phosphopeptide epitope bound. Dr. Doskeland is defining the effect of a whole series of the antibodies on ligand binding to PH. However the antibody which is continuing to generate the most interest is PH8. Dr. Tork first showed its utility in mapping serotonergic neurones in human post mortem brain stems and more recently Dr. Halliday working with Professor GefiFen and Dr. Blessing have published a series of papers using the antibodies for localizing more amine containing neurones in the human medulla oblongata. Publications: 27, 48, 49, 50 43

DETECTION OF POINT MUTATIONS R. G. H. Cotton

This section brings together several quite separate projects which have in common the objective of improving the techniques that we have available for analysing gene mutations or genetic defects and other studies which are of a more basic nature exploring the normal construction of the gene in ways which may shed light on important genetic defects. The reason for seeking a method of detecting point mutations in human genes has been discussed at some length in the earlier part of this Report. During his sabbatical leave in Oxford Dr Cotton developed a method which is able to identify directly all single base interchanges which introduce falsely a T or C and can be used indirectly to recognize A or G introductions. During 1988 Dr Cotton has been busy perfecting this basic technique using cDNA molecules. Dr Dahl has applied the technique to messenger RNA, both a very abundant message (for collagen in fibroblasts) and a less abundant message (for phenylalanine hydroxylase in mouse liver).

Mutations of phenylalanine hydroxylase (PAH) in PKU S. Forrest, H. Dahl, R. Cotton Our attempts to identify mutations in PKU started some years ago when Julian Mercer and Andrew Grimes purified mRNA from a PKU liver (prepared on incom plete cDNA labelling) with the aim of attempting one of the short cut methods to identify rnutations. This line of work lapsed due to the perceived and actual limitations of the methods available at the time. This program has been revived now that the HOT method has been developed. Dr. Susan Forrest is undertaking this program and already has exciting preliminary results. Details of the mutations detected and their affect on the phenylalanine hydroxylase molecule should be known in the next 1 — 2 years.

Chemical (HOT) method of mutation detection

S. Forrest, A. Urban, W. Russell, P. Wright (Monash University), R. D. Campbell (Oxford University), H. H. -M. Dahl, R. G. H. Cotton The HOT (Hydroxylamine Osmium Tetroxide) method of detecting point mutations analyses double stranded nucleic acid molecules (DNA/DNA, DNA/ mRNA) made by hybridizing a mutant strand with a normal strand. Treatment with hydroxylamine or with osmium tetroxide, followed by piperidine cleaves nucleic acid duplexes at C or T residues, respectively. Duplexes with a mismatched C (CA, CG, CC) or T (TT, TC, TG) are much more sensitive to cleavage at the sites of mismatch than perfect duplexes. These reagents allow detection of all point mutations which lead to the introduction of a T or C base by mutation. Errors which introduce a A or G can be detected by making a complimentary strand and analysing it for the resulting T or C. The main effort during the year was directed to development of new reagents. We wish to identify an alternative reagent to osmium tetroxide for detection of mismatched T bases. Osmium tetroxide works well but is toxic and must be used in a fume hood. Potassium permanganate shows promise as a useful non-toxic reagent and full authentication is underway. We also assessed a series of reagents for identification of mismatch ed A or G bases. Addition of such reagents would allow one step detection of all mutations in mRNA without need to make cDNA and would provide more convenient detection of mutations in DNA using only one probe and four reagents. A number of promising reagents have been identified. A systematic analysis of the reactivity of all C and T bases in a complex heteroduplex has been completed. This heteroduplex of 363 bases was made from two portions of

Analysis of the difference between two Thai Dengue Virus strains by the HOT method. Tracks 1 to 4 can be used to derive the sequence oforu of the strains. Track 5 represents reactive T bases generated by differences be tween the strains and 6 represents reactive C bases. The viral RNA used was unpuri fied RNA isolated directly from cells which represents considerable con venience. 45

44 V.,

the 21 hydroxylase gene with differences in 35 base reactions with probes labelled appropriately allows the positions. These studies indicated that the HOT reagents detection of all possible base changes, could detect insertions and deletions readily and also that To detect RNA mutations in low abundance the distortion produced by mismatches could be transmitspecies, cDNA was synthesized from total or poly(A) ted at least to a distance three bases away from the RNA and the polymerase chain reaction used to mismatch and detected by enhanced C or T reactivity. specifically amplify the wanted cDNA regions. Mutations Both these observations have important practical implicawere then demonstrated in the amplified cDNA strands, tions as they allow indirect detection of many mismatches So far we have detected and localized all expected base involving mismatched A or G in the probe, and also changes. These changes include point mutations, deledetection of insertions and deletions. Applications of the tions and insertions. Several separate base changes can be method have been mentioned elsewhere. detected and mapped simultaneously. An advantage of Sue Forrest is applying the method to the analysis of this technique is observed when analysing de etions mutations in PKU and also for the detection of covered by the probe. In the heteroduplex these deletions polymorphisms in genes which might be useful in gene create a loop in the probe Lcking in prenatal diagnosis. David Howells is applying unmatched bases and % , audioradiogram. The bands not only define the size, but It to DhirK mutat o . boundaries of the deletion. In our experiments we In collaboration with Dr. P. Wright, Monash Univerhave used probes up to approximately 2 kb in size. sity, we have demonstrated the utility of the HOT However there is no apparent reason why longer probes chemicals in fingerprinting Dengue virus isolates. We cannot be used. The chemical cleavage reaction in showed that a distinctive pattern of difference could be combination with the polymerase chain reaction provides shown between two isolates not only at the DNA level but a simple reliable method for the rapid detection of also by direct analysis of RNA isolated from infected cells. mutations in RNA. We believe this simple method of obtaining precise information about sequence differences will allow an . . j. j. a

enormous saving of effort and speed up the accumulation of epidemiological data; it may even allow identification of areas of genes/proteins suitable as vaccine targets. Publication: 28

Detection of base changes in RNA H. H. -M. Dahl, R.G.H. Cotton, S.R. Lamande and J. F. Bateman Department of Paediatrics, University of Melbourne The detection of base changes in DNA and RNA is of central importance in genetic research. Until recently identification of mutations and polymorphisms have involved slow, labour intensive and tedious procedures. However, many of these problems were overcome by the description by Dr. Cotton and collaborators of the rapid and simple chemical cleavage method for identification and localization of mismatched base pairs in DNA:DNA heteroduplexes using cloned DNA fragments. We have adapted the chemical cleavage reaction in order to detect and localize base changes in RNA either directly, or in the case of low abundance mRNA’s, after specific amplifica tion. To establish the RNA mutation detection method and test its fidelity, defined mutations in genes for type I collagen and allelic sequence differences in the rat phenylalanine hydroxylase gene were studied in detail. The method involves formation of an RNA/DNA heter oduplex which is then treated with either hydroxylamine or osmium tetroxide followed by cleavage by piperidine. However, only those mutations resulting in a mismatch C or T in the DNA probe can be located by forming a direct RNA/DNA heteroduplex. This problem can be overcome by synthesizing the complementary cDNA strand and using this instead of the RNA. Analysis of RNA and cDNA using both hydroxylamine and osmium tetroxide 46

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perinatal form (OI type 2) is characterised by abnormali ties of type I collagen. We have analysed the collagen gene from a number of patients with OI type II in order to define and characterise the molecular lesions leading to this disorder. In one patient 51 collagen al(l) cDNA clones were isolated and analysed by DNA sequencing. The clone was identified with an A14 mutation which led to the substitution of arginine for glycine in the triple helical region of a al(l) chain. This mutation was shown to be responsible for the peptide map abnormality by in vitro transcription and translation of the mutant clone. Modification of the chemical cleavage reaction enabled us to detect mutations directly in the RNA. Having located the mutations the regions containing the mismatches were amplified by the polymerase chain reaction and sequ-

enced. In one patient the mutation is a frameshift mutation caused by a single base insertion in the COL lAl gene. The frameshift resulted in a truncated proal(l) carboxy terminal propeptide affecting chain assembly and helix provocation. Five other patients all had heterozy gous single base mutations which lead to substitution of glycine residues in the helical region of the pro alfa 1 chain. In four of these patients the mutations were detected in the proa(l) RNA and in the fifth patient the mismatch was detected in the proci2(l) RNA. Determina tion of these collagen mutations is of fundamental importance in defining sequence domains involving helix assembly and stability, collagen secretion and the correct formation of a functional extra cellular matrix. Publication: 16

TRACE ELEMENT STUDIES (SCOBIE & CLAIRE MACKINNON RESEARCH GROUP) J. Camakaris, H. McArdle, J. F. B. Mercer, D. M. Banks

Characterisation OI mUtatlOnS in collagen in patients with osteOgenesis imperfecta type II H. H. -M. Dahl with S. R. Lamande, W. G. Cole and J. F. Bateman Department of Paediatrics, University of Melbourne Osteogenesis imperfecta is a group of biochemically and clinically heterogeneous connective tissue disorders in which bone fragility is the main clinical feature. The lethal

0 I 60

^649 bp

^312bp

The objectives and approaches of our work on trace elements were described in detail in the 1987 report. Briefly, our interest in two human genetic diseases of copper metabolism — Menkes’ disease and Wilson’s disease — has led to an objective of discovering the various steps involved in the normal intracellular and intercellular transport of copper, and later of zinc. Both these trace elements are essential for human health. Copper is highly toxic if accumulated in excess. Orderly and gene controlled methods of transport must exist. Three senior scientists are involved in this work and each has a small group working with him. All these people collaborate although they work physically in different laboratories (or even in different institutions, for Dr Camakaris works in the Department of Genetics at the University of Melbourne).

Detection of a mutation causing lethal osteogenesis imperfecta. The mutation has occurred in the gene for collagen a(l). The probe was a radioactively labelled 649 base pair collagen DNA fragment. After annealing to RNA from a normal person and from the patient (0151) the mixture was treated with hydroxylamine and piperidine. A 312 base pair band appears in the 0151 RNA lanes. This band localises and to some extent defines the mutation in this patient.

Dr Camakaris trained as a microbial geneticist then worked extensively with human cell culture so his approach has focused upon the analysis of cells in culture as models of the in vivo situation and has placed particular emphasis upon the use of natural mutants or the discovery of artificial mutants selected in cell culture.

Recently he has collaborated with Dr Barry Lee and Dr Suzanne Rogers in the Genetics Department in analysing the microbial pathways of copper transport believing that they will at least parallel those found in mammalian cells if they do not prove identical. Dr Mercer is a molecular geneticist who chose to isolate the gene coding for metallothionein as a practise step towards the cloning of less abundant genes like phenylala nine hydroxylase. He became so interested in metallothio nein that he has made extensive studies of the factors which control his production and function and went on to clone the caeruloplasmin gene and study its control. He continues to study many aspects of copper transport using a molecular genetic approach. Dr McArdle is a physiolog ist who has always worked with transport of molecules across cell membranes and through organs, initially amino acids, subsequently iron, and, for the last four years, copper and zinc. Naturally his approaches are physiological. As one might expect these scientists have each learned special skills from one another and all tend to use an amalgam of the various skills that each has brought to this work.

Regulation of metallothionein and caeruloplasmin gene expression in the toxic milk mouse J. Mercer, A. Grimes and H. Rauch (University of Massachusetts, U.S.A). The toxic milk mouse is an animal model of the human genetic disorder of copper metabolism, Wilson’s disease. We are fortunate to be able to collaborate with Dr. Harold Rauch, the discoverer of this mutant. The pups of the mutant females are born copper deficient and have to be

Mouse caeruloplasmin gene structure

copper concentrations were very high and the zinc concentrations were also elevated compared with the normal controls. At this age MT mRNA seemed to be responding to both hepatic copper and zinc. This is in contrast with the copper loaded sheep in which MT mRNA is not related to the concentration of hepatic copper. The concentration of CP mRNA in the late foetal animals was 50% of the adult, in agreement with our results with the developing rat, which showed that CP mRNA began to increase in the late foetal period. There was no difference between the Tx animals and normals, despite the copper deficient state of the pups of the Tx dams, showing that the activation of the CP gene in the developing liver is not copper dependent. Neither copper nor zinc administration stimulated CP mRNA production in the neonatal animals. The adult Tx animals had normal levels of CP mRNA despite a copper loaded condition. We concluded that the Tx mutation does not directly or indirectly result in alterations of CP gene expression, and that the level of copper in the liver has no influence over the expression of the CP gene.

K. H. Choo, A. Grimes, P. Kearney, G. Filby, N. Fraser and J. Mercer We used rat and human caeruloplasmin clones to isolate cDNA clones encoding mouse caeruloplasmin. The mouse cDNA clones were used to isolate genomic clones. We are currently sequencing the mouse cDNA and have obtained most of the primary sequence of mouse caeruloplasmin. The sequence is highly homologous to the rat and human protein. The cDNA clones have been used in the Toxic Milk study discussed above. The genomic clones are being analysed and some of the exons have been identified and sequenced. The gene appears to exceed 30kb in length so complete analysis of its structure will entail a considerable amount of work. Preliminary data suggests the gene consists of greater than 15 exons. These will be sequenced and the position of splice junctions will be determined. Dr. Choo and Dr. Fraser are using this gene in their experiments of targeted mutagenesis, which are discussed in another section.

Metallothionein gene expression in zinc deficient sheep

Towards isolation of the Menkes’ disease gene

J. Mercer, J. Paynter and C. White (CSIRO Floreit Park, W.A.) As part of our investigation of the expression of metallothionein genes in sheep, we analysed some samples from zinc deficient animals. The concentrations of metallothionein mRNA were quite low in the deficient animals, in contrast to the high concentrations of MT mRNA found in previous analysis of zinc replete animals. These results support the conclusion that zinc is the major regulator of metallothionein gene expression in the sheep.

Toxic milk mice fostered onto normal dams if they are to survive. Following this transient period of deficiency, the survivors begin to accumulate hepatic copper in a manner reminiscent of Wilson’s disease patients. We have conducted an extensive analysis of the concentrations of metallothionein (MT) and caeruloplasmin (CP) mRNA in the livers of these mice, to assess whether the mutation results in any abnormality of expression of these genes. As anticipated, we found no evidence of a primary defect in expression of MT genes. In the late foetal period the pups of the mutant mothers have normal hepatic concentrations of metallothionein mRNA despite very low copper concentrations and irrespective of the genotype of the animal, indicating that MT gene expression IS ' not being regulated by copper in the developing liver. These conclusions are supported by analysis of the induction of MT genes by copper and zinc in the neonates. No difference was observed between the Tx and normals. The adult Tx had high concentrations of MT mRNA, the 48

Metallothionein and metallothionein mRNA in sheep suffering from copper and heliotrope alkaloid toxicosis. J. Mercer, J. Paynter and J. McC Howell (Murdoch Institute, W.A.) We are continuing our collaborative study of copper toxicosis in sheep which is now funded in part with a grant from the Australian Wool Corporation. The first part of the study involves an assessment of the effect of copper in combination with hepatotoxic alkaloids found in certain plants, upon the concentrations of MT and MT mRNA. Subsequent experiments will explore the effect of zinc upon the development of toxicosis and expression of MT genes. In our preliminary analysis of the first batch of samples, we have found no effect of copper and alkaloid ingestion on MT gene expression, even when considerable liver damage was evident. Indeed the concentrations of MT mRNA are quite low, and this is probably related to the rather low zinc concentrations in the livers.

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P. Kearney, J. Mercer and D. M. Danks We are using a number of approaches to try to isolate the gene at fault in Menkes’ disease. The first entails analysis of the region of the X-chromosome which is thought to contain the gene. Various probes have been obtained around the putative locus, Xql3, and used to analyse an extensive Menkes’ disease family by pulsedfield gel electrophoresis. We are searching for gross changes in the banding pattern that are associated with the disorder. This approach is also being applied to the brindled mouse analogue of Menkes, thus giving a comparative map for the human and mouse X-chromo some. An alternative approach is based on developing a regime to culture selectively Menkes fibroblast cells. Normal and Menkes cells are cultured in various concentrations of copper, zinc and mercury and tested for their viability at different time intervals. Preliminary results suggest that it may be possible to culture cells under conditions which fully inhibit normal cells but allow growth of the Menkes cells.

the available copper isotopes decay too rapidly to allow really long term experiments. However, the data do suggest that the copper taken into the long term pool is not very readily exchangeable with the rapidly labelled pool. We thought that the pools could be filled at different rates by incubating cells with different concentrations of copper. This seems not to be the case, however. It now seems more likely that copper has to follow a very clearly defined path through the cell. Definition of this path remains our major objective. Concurrently, we have been examining how copper uptake is controlled by the cell. We expected that copper levels inside the cell would control the amount of copper taken up by the cell; as the concentration inside increased, we thought that the cell would decrease the amount it takes up. This turns out not to be the case; increasing the intracellular copper content does not affect the rate of copper uptake. On reflection, this is probably not so surprising in the liver, which is the most important organ in copper homeostasis. Probably the liver cells have to be able to continue to take up copper irrespective of how much is already inside the cell, in order to protect other organs from copper excess. We anticipate that other cell types may behave differently. The hormonal control of copper uptake has been examined, drenalin and phenylephrine have no effect on copper uptake. Neither do compounds altering the intracellular second messenger systems. Again, this is not surprising, considering that the hepatocyte has a fixed capacity for copper uptake. Towards the end of pregnan cy, caeruloplasmin levels in maternal plasma increase. In the mouse, copper levels in the maternal liver are not significantly altered and we are examining oestrogens to see if we can see a compensatory effect on copper uptake in hepatocytes. We have also studied how copper and other factors alter the expression of copper proteins inside hepatocytes. In these cells, cyclic AMP, a second messenger, decreases the amount of caeruloplasmin mRNA. Otherwise the amount of mRNA produced by the cell for caeruloplasmin is kept within a very narrow range, and even when copper levels are reduced markedly, the synthetic machinery is still kept at the same level. What effect this has on the protein remains to be discovered. Does the cell produce caerulo plasmin without copper in it, as some people have suggested, or is the apoceruloplasmin unstable? We are presently making antibodies to the different proteins to try and find the answers to these questions.

Metallothionein (MT) seems to follow a very different pattern. It is induced by copper and zinc, much as has been shown in the intact animal, but it is interesting that The metabolism of copper by the very act of putting the cells into culture results in an mouse hepatocytes increase in MT mRNA. This is related to free copper levels in the medium, rather than removal of an inhibitory H. McArdle, S. Gross, P. Kyriakou substance in the animal, and levels can be kept low by In the previous report we discussed briefly the use of adding diamsar, the chelator produced by Professor chelators to identify rapidly labelled and long term copper Sargeson’s group. By manipulation of the system, pools within cells. During this year, we have examined therefore, we are able to control the copper levels in the these pools more closely. They are not easily studied, since cell over a very wide range.

The effect of chelators used to treat Wilson’s disease on copper metabolism in mouse hepatocytes H. McArdle, S. Gross We have been examining more closely the effect of penicillamine, tetrathiomolybdate and other copper chela tors on copper uptake and metabolism. Tetrathiomolybdate is an important new chelator which has proved very effective in copper poisoning in sheep and has shown promise in treatment of humans. In hepatocytes in culture, TTM decreases the uptake of copper and stimulates the release of the metal. It is not as efficacious as sar or diamsar (two new chelators synthe sized by Professor Alan Sargeson in Canberra), but our data does show clearly how it exerts its copper depleting effect in vivo and its extensive use in sheep provides a background knowledge of its safety which is not yet available for sar and diamsar. We have been trying to find out more about the effect of these agents and have shown that the effect is temperature dependent. Before we can draw any conclusions from this, however, we need to know whether the chelators are actually taken up by the cells. Eventually, we hope that use of radioactively labelled chelators will answer this question. Penicillamine is the compound used most widely in the treatment of Wilson’s disease. It has been thought to act as a “decoppering” agent, but our experiments show no effect on the uptake or release of copper in cultured hepatocytes. It does stimulate the production of metallothionein, however, probably by mobilising in tracellular copper, since diamsar can block the penicilla mine effect. We plan to extend our studies of chelators in toxic milk mice now that we have these animals available as models of Wilson’s disease.

Wilson’s disease when in acute liver failure. At first we were very tentative in the use of this new agent, giving it only in a very late stage in the disease. Biochemically the drug had the effect we were seeking, but the patients did not recover. The drug was given at a somewhat earlier stage in the last two cases that were treated and in each of these we came very close to achieving recovery. We feel that the experience gained in treating these patients may enable us to succeed on the next occasion that we encounter this circumstance. Liver transplantation is the other method of treatment which sometimes has been successful in this type of patient, but no suitable donor liver became available during the period of treatment of our patients. Survival without liver transplantation has only been reported once in such a patient, but we do feel that we are close to having a form of treatment which may achieve survival more frequently. We have also used TTM in a 20 year old girl who developed severe neurological disorders similar to those seen in Wilson’s disease as a consequence of copper accumulation secondary to cholestatic liver disease. an Penicillamine made her symptoms much worse experience also described in some patients with Wilson’s disease. TTM has controlled her disease very satisfac torily and she is making an excellent recovery.

Liver disease in middle aged adults

D. M. Danks, H. Vogel For many years it has been recognized that Wilson’s disease may produce serious or even lethal liver damage in children, adolescents or even young adults without having any effect upon the brain. However, doctors have believed that adults over the age of 25 or 30 years will always have symptoms of neurological damage as well as liver damage. It is accepted that one cannot always rely upon the classical diagnostic signs and tests for Wilson’s disease (Kayser-Fleischer rings in the eyes and low serum copper Publication: 56 and caeruloplasmin) in children with the disease, but doctors have believed these tests to be quite reliable in Tetrathiomolybdate treatment of older adults. Wilson’s disease and other forms We have long suspected that there may be some adult of copper toxicity patients with Wilson’s disease who have liver disease D. M. Danks, H. Vogel alone and whose diagnosis may be missed if doctors rely Over the last 25 years penicillamine has been the completely upon the classic findings and tests. This belief principal drug used to treat Wilson’s disease. It has has been vindicated over the last 12 months during which proved very effective in long term treatment of patients we have joined with various gastroenterologists from adult who are not acutely ill at the time of diagnosis. However, teaching hospitals to diagnose Wilson’s disease m four it generally takes three to six months for penicillamine to adults aged 38-58 years presenting with liver disease alone start having a beneficial effect. Some children and young which proved rapidly fatal in two of the four patients, adults who present with acute liver failure cannot be kept Three of the four patients were diagnosed only by using alive this long by other means. There is a need for some more specific tests for Wilson’s disease such as meacopper binding drug which can remove toxic copper from surement of the copper content of a liver biopsy or the blood stream and liver cells very quickly. Tetassessment of copper utilisation in the body by admmisrathiomolybdate (TTM) can bind copper very tightly and tration of copper isotope. We now advocate firmly that, as quickly and has proved effective in sheep suffering acute in childhood, adult patients with liver disease without copper toxicosis, a situation quite similar to the acute liver another clearly demonstrated cause should be regarded as crises in Wilson’s disease. having Wilson’s disease until this diagnosis has been Over the last five or six years we have used TTM in four dismissed, preferably by the demonstration of a normal young patients who were first diagnosed as having level of copper in a liver biopsy.

Copper transport during pregnancy H. McArdle, R. Erlich Earlier observations by Jeff Mann at this Institute showed that, after a single injection of copper, transport across the placenta of a pregnant mouse was linear over 56 hours. This observation aroused our interest, because it implied that the copper was being held somewhere in the dam prior to being transported across the placenta. Consequently, we set out to identify the store and the mechanism of transport across the placenta. Our data suggests that there are two possible stores for the copper — the plasma or the liver. The liver is the more likely of these two candidates. This implies that caerulo plasmin may be the transporter of copper to the placenta, but there are a number of disconcerting features about the data. We are resolving these problems, however, and feel that this system may soon provide clear evidence in the argument about the role of caeruloplasmin in copper transport.

Effect of L-histidine on copper uptake by continuous lymphoid cell lines (CLC’s) and fibroblasts J. Camakaris, A. Bruzanniti, H. McArdle Low concentrations of L-histidine (his) stimulated Cu uptake by hepatocytes whilst uptake by fibroblasts was inhibited (McArdle and Gross). Studies using cultured CLC’s showed that the ligand histidine stimulated Cu uptake according to the Cu concentration and the histidine:Cu ratio. Histidine:Cu was varied in Hanks balanced salt solution containing 0.1 uM Cu or 2uM Cu and 64Cu uptake estimated. At 0.1 uM Cu, maximal stimulation of uptake was observed at a 1000 molar excess of histidine for both normal and Menkes CLC’s. At 2uM Cu a slight stimulation was observed at a histidine: Cu ratio of 10, whilst inhibition was observed when the ratio reached 1000. These results indicated a system for Cu transport which operates at low Cu concentration which is stimulated by the ligand histidine, whilst a second system may exist for high copper concentrations which is inhibited by excess histidine. It is noteworthy that maximal stimulation by histidine occurs at physiological concentrations of “free” Cu and histidine. Fibroblasts showed a different pattern — at low concentrations, histidine had no effect and at high concentration it inhibited Cu uptake. 64Cu uptake was also investigated in CLC’s and fibroblasts as a function of Cu and Cu(His)2 concentra tion (Cu(His)2 is a well characterised complex). Whereas with fibroblasts an apparent saturation was observed at about lOuM Cu(His)2, saturation occurred at 60-80uM Cu or Cu(His)2 in CLCs. Both normal and Menkes lymphocytes behaved in a similar manner. These data indicate that different cell types may possess Cu transport systems with different properties.

Effects of metallothionein levels on observed copper distribution observed in cell-free extracts J. Camakaris, R. Farrell Following 64Cu labelling of cells and tissues, cell-free extracts are prepared and 64Cu proteins are fractionated using gel filtration. A significant proportion of 64Cu is usually found to be associated with the metallothionein peak. We investigated the possibility that a portion of this 64Cu redistributed from other Cu-binding proteins onto metallothionein during preparation of cell-free extracts (metallothionein has a very high affinity for Cu). The mouse lymphocytic cell line W7 has low levels of metallothionein due to the methylation of metallothionein genes. Addition of purified metallothionein or of cells containing high levels of metallothionein to 64Cu labelled W7 cells just prior to preparation of cell-free extracts resulted in a marked shift of 64Cu from other peaks to the metallothionein peak. Similar effects were observed in a 64Cu labelled W7 cell line in which expression of metallothionein genes was induced by demethylation. Lymphocytes labelled with 64Cu for short periods of time show high amounts of 64Cu associated with the metallothionein peak when analysed by FPLC gel permeation chromatography. This diminishes rapidly during “chase” periods. One interpretation of this data is that metallothionein has a key role in intracellular Cu transport. An alternative explanation, based on the above findings, is that a significant portion of the 64Cu found in the metallothionein peak originated from 64Cu bound to membranes. The apparent loss of 64Cu from metallothio nein would then be a consequence of reduction of the exchangeable 64Cu on membranes during the period in the “chase” medium. These data show that caution needs to be exercised in interpreting 64Cu protein distribution data in cells with high levels of metallothionein (eg. Menkes’ cells) and that most previously published results need to be reconsidered.

Detection of 64Cu-binding proteins using “Western” blots J. Camakaris, R. Farrell We have searched for satisfactory methods of identify ing cellular proteins which play specific roles in Cu transport. Cu binds non-specifically to all proteins. Electrophoresis strips Cu off most proteins. Gel filtration fractionation of 64Cu binding proteins has now been shown unsatisfactory because of exchange of 64Cu between proteins, especially transfer to metallothionein. We have developed a new approach involving use of 64Cu as a probe of separated proteins. Proteins derived from cells or tissues were separated, according to size, on SDS “stacking” gels. The separated proteins were electrophoretically transferred (’’blotted”) 51

onto nitrocellulose paper. The transferred proteins were probed with 64Cu and 64Cu binding proteins were detected by autoradiography. Filters were stained with amido-black to assess efficiency of transfer. At high Cu concentrations all proteins bind 64Cu and under these conditions the method is a sensitive detection system for

only with high specific-activity 64Cu. Some Zn binding proteins and proteins with haem groups also bind 64Cu under these conditions. As proteins are denatured by SDS, it is possible that only those with Cu binding sites made up of contiguous aminoacids are being detected.

Copper-resistant variants of cultured Chinese Hamster Ovary (CHO) cells J. Georgiou and J. Camakaris Copper-resistant variants of CHO cells had previously been isolated by J. Patton in an attempt to define Cu transport in mammalian cells. One class of resistant variants, isolated by increasing the copper content of growth media in a step-wise fashion proved to comprise a mixture of partially resistant (SqPR) and fully resistant (Sd-CL3) cells. Selection for either cell type is dependent on the Cu concentration in the medium. Maintenance of the population in basal medium, containing no added Cu, results in a high proportion of SdPR cells in the population. S^PR cells grow more rapidly than Sd-CL3 in this medium and this allows them

to predominate the population. S^PR cells contain 20

particular class of regulatory genes which encode proteins which respond to environmental stimuli via membrane sensors. This suggests that the pCoR gene product may interact with a trans-membrane sensor (possibly pCoA gene product). Three novel Cu binding proteins have been identified which have molecular weights of 68KDa, 98KDa and 50KDa. The latter two proteins are inducible by Cu and all three proteins are present in parental and plasmid containing strains. In the presence of detergent a low molecular weight (20-30KDa) Cu-inducible plasmidencoded Cu-binding protein has been identified. Evidence suggests that this may be the product of the pCoC g ene.

Copper transport and homeostasis Escherichia COli Rogers, B. T. O. Lee (Genetics Department, University of Melbourne) and J. Camakaris .^e Cu transport system of a bacterium is

•» -r-*'

""" “

m mammalian cells. Six genes designated cut A-F have previously been identified as involved in Cu transport in E.coh. Following the initial characterisation of copper-sensitive mutants, the next step was to clone the genes invo ve . The strategy used was to “shot-gun” clone genomic DNA into a plasmid vector and select for complementa tion of the copper-sensitive phenotype. Because of gene dosage effects only medium copy number vectors could be successfully used. A copper uptake mutant, GMEl 12, has been partially complemented in this manner by a 2.2kb insert. The storage/carrier mutant GMEl35 has been completely complemented by a 6kb insert. The 6kb insert has been subjected to restriction mapping and a 2.4kb sub-clone also complements GME135. Further sub cloning and DNA sequencing are underway. Gel filtration analysis had previously identified an llkDa copper-inducible copper-binding protein in wildtype E.coli cells. This protein is absent in the mutant GMEl35 but is detectable in the complemented strain. GMEl35 does possess a 6-8kDa Cu-binding protein which is not observed in parental cells. It appears that GMEl35 has a smaller Cu-binding protein with a lower affinity for Cu. This does not seem to be a subunit of the llkDa protein,

Publication: 13

Regulation of copper-inducible promoters in E.Coli A. Bergmann, B. T. O. Lee (Department of Genetics, University of Melbourne) and J. Camakaris In previous work done by Duncan Rouch, copperinducible promoters of pCo (the major copper-resistance determinant on the plasmid pRJ1004) were linked to a reporter gene which codes for p-galactosidase thus facilitating assay of promoter activity. One of the promoter clones, ppA234, was used to isolate mutants with chromosomal gene mutations which affected the level of expression of this copper-inducible promoter. The chromosomal gene was designated cutR. We have used a second promoter clone, ppA223, to identify another chromosomal gene which alters promoter activity. This gene does not appear to be cutR as it does not complement cutR mutations. The gene has been designated cutS. Work in progress is directed towards sequencing cutS, and determining which of the plasmid encoded genes are transcribed from the ppA223 and ppA234 promoters. Evidence that ppA223 and ppA234 are differentially regulated has been gained from studying their induction by other divalent cations. Expression of both promoters is unaffected by the level of Fe2, Mg2 and Ni2 in the medium. However ppA223 is induced at least as efficiently by Zn as it is by Cu, whilst ppA234 is repressed by Zn. These studies suggest that control of Cu metabolism in E.coli results from the complex interaction of a number of regulatory proteins, both chromosomally and plasmid encoded.

plasmid-mediated copper-resistance

chromosomes (normal for CHO cells) and exhibit an increased accumulation of Cu at high media Cu con centrations (20ug/ml) when compared to So-CL3. This increased Cu may be the result of increased levels of a 30,000 molecular weight Cu binding protein in S,PR cells. Maintenance of the population in a high Cu concentration (35ug/ml) favours S„-CL3 cells. These cells accumulate less Cu than So PR cells under these conditions, and this appears to confer Cu resistance. Patton’s earlier results suggested that the reduced Cu accumulation is due .o^r^ucd Cu uptake, S.-CL3 cells possess 21 chrome-

e R T O T.pp N. Brown, M. Aitken, S. D. Rogers, B.T. O. Lee (Genetics Department, University of Melbourne) and J* Camakaris Copper-resistance in E. coli is inediated by a plasmid, PRT1004, which carries a major Cu resistance determinant, designated pCo. pCo contains at least three structural genes (pCoA,B,C) and one regulatory gene (pCoR). Resistance is due to reduced Cu accumulation, "a enhancem*

*The isolation ofOu resistant cells by step-wise selection

experiments were undertaken.

has provided at least two interesting mutations for further study. Completion of their analysis may identify two of the steps in the cellular Cu transport system of mammalian cells The first is associated with the overproduction of a copper-sequestering protein (not metallothionein) and results in low level resistance (S„PR). The second alteration is associated with an extra chromosome which results in reduced uptake by Cu and the high level resistance in Sd-CL3.

A restriction map of pCo was obtained. It proved difficult to clone genes of the resistance determinant into high copy number vectors. This may indicate tha plasmid encoded gene products are detrimental to cells w en present at high concentrations. A complete sequence and reading frame has been identified for pCoR (regulatory gene) and about 95% of pCoA and pCoB has been sequenced, whilst about 50 /o of the pCoA gene has been sequenced. pCoR has a high sequence homology with a

Subsequently, we examined a wide series of other ligands to see if they could be correlated with Zn uptake. The results were somewhat surprising. They suggested clearly that the Zn was being taken up by the cells as the ion, and that ligands had an effect on uptake proportional to their affinity for Zn. This is a very exciting finding, since it suggests a new type of uptake process, and it also lends possible credence to our previous findings suggesting that copper may disassociate from ligands at one stage during the process of uptake into cells. We have been examining the membrane transport protein(s) more closely and there appears to be a “reserve” of protein in the cell. The nature of this protein remains to be elucidated.

in Escherichia coli

Zinc uptake by fibroblasts

I r

L. Ackland, H. McArdle We have extended last years work on the Zn uptake system and have made considerable progress. Initially, we studied a2-macroglobulin as a possible Zn carrier. A2M did bind to the cells, but not in sufficient amounts to explain the amount of Zn taken up by the fibroblasts.

Proton microprobe analysis of copper distribution in tissues of brindled mice B. Kirby, G. Legge (Department of Physics, University of Melbourne), H. McArdle, D.M. Danks For many years now we have known that copper accumulates to abnormally high levels in the intestinal mucosal cells and in the kidneys of the brindled and blotchy mice and in patients with Menkes’ disease. We have long wanted to know just where in the mucosal cells the copper is accumulating and in which cells of the kidney. The proton microprobe, developed in the Physics Department of the University of Melbourne by Dr George Legge, is a very sensitive instrument able to measure quantities of various elements present in small samples of biological tissue. It can be applied to cryostat sections of frozen tissues and has a power of resolution down to a level smaller than an individual cell, however, this resolution is greatest for elements which are abundant and least for elements of low abundance. In studying the intestinal mucosa of a brindled mouse, it proved possible to demonstrate the high levels of copper in the mucosal cells quite satisfactorily, but it was technically very difficult to orientate sections so as to be quite certain of the position of cells on the villus. It is also apparent that the instrument was not able to resolve clearly the copper distribution in different regions of an individual cell. Mr Kirby, who is undertaking this work as a PhD project, therefore concentrated most of his effort upon the kidney in which it was much easier to orientate sections. It was quite clear that the copper was accumulating in the tubules and not in the glomeruli or in other parts of the kidney, as we had expected. He next tried to determine whether the concentration of copper was high in proximal or distal tubule using enzyme markers to differentiate one from the other. These results were not absolutely convincing, but did indicate that the accumulation of copper increased progressively from proximal to distal tubule. 53

CYTOGENETICS M. Schmidt

Progress in medical genetics has depended heavily on cytogenetics findings. Thus, the present task of our cytogenetics groups is not only to provide the routine diagnostic services, but also to ensure that the excep tionally valuable findings will be used for further research. Our present structure seems to meet the above require ments. The diagnostic tests are performed by the routine laboratory (led by Ms. M. Leversha), that functions as a part of the Victorian Clinical Genetics Services. This laboratory is under the scientific supervision of Dr. M. Schmidt who leads the cytogenetic research group of the Murdoch Institute. This way the findings that are particularly important can give rise to long term research projects, usually undertaken in collaboration with the molecular groups of the Murdoch Institute. At present the interests of the cytogenetic group are focused on the fragile X mutation, which causes the most common form of heritable mental retardation. The project aiming at cloning of the responsible DNA sequences has been based on a unique deletion that has been found by our routine laboratory, and then characterized in detail by the cytogenetic research group.

Cloning of sequences from the Xq27 region of the human X chromosome M. Scbmidt, K. H. Cboo and D. Du Sart. A small deletion of the X chromosome (X) (q27.1 q27.3) has been found in a mentally retarded girl, tested in our laboratory. Detailed analysis with 21 DNA probes revealed that the missing piece of the X chromosome corresponds to the area of the fragile site Xq27 (refer adjacent map), and is the smallest deletion known in that region. Thus, the finding provides a unique opportunity to identify DNA fragments involved in the fragile X syndrome. A new X chromosome specific genomic DNA library 54

j^DXS S2 'dxS 159

(pt*r-p11.21)

(q12-q13)

■■M 26 1 26 2 26

27 1 del

__ HPRT + DXS 144 + F9 DXS 152 + DXS 105 + DXS 9S -

ONFl DXS 19 DXS 37 DXS 129 DXS 151 pX 56c pX 71c pX 62d

+ + + + +

(q26>27) (q26-27.3) (q26-27) (q24*27) (q2«-27) <q26-27) (q26-27) (q26-27)

FRAXA F8

^DXS IS SJDXS 52 + _2DXS 157 + oePD

DXS 49 pX 15*

(928) (927-1*1)

X CHROMOSOME Map ofthe deletion (X) (q27.I q27.3) constructed with the available DNA probes indicates a necessity to isolate DNA sequences closer to the defect.

will be constructed and screened against the deleted X chromosome lacking Xq27 to identify fragments from the Xq27 region.

X chromosome inactivation in females with the fragile X syndrome M. Schmidt, D. Du Sart, L. VouIIaire, M. Leversha, R. Hutchinson, V. Petrovic, J. Roberts, D. Foster, L. Hill, M. Gray and R. Oertel. It is not clear why some female carriers of the fragile site Xq27 are clinically affected to the same level as hemizygotic males. Nonrandom inactivation of the X chromosome could account for manifestation of the defect in these cases. To resolve this question fibroblasts from fra-X positive mentally retarded females will be tested for X linked RFLP and then fused with A9 cells. Ten HAT-resistant clones from each female will be tested with informative probes for retention of either of the X chromosomes in these hybrids. This will allow us to estimate whether the fra-X and normal X are active in the

same or different proportion of fibroblasts and whether there are differences in this respect between the mentally retarded and clinically normal fra X carriers.

Y chromosome — derived markers H. Dahl, V. Petrovic, D. Du Sart, L. VouIIaire and M. Schmidt. Female patients bearing Y chromosome — derived material develop malignant gonadal tumors. Location of the responsible sequences is not known. Thus identifica tion and characterization of the deleted Y chromosomes with Y specific DNA probes is important for the proper diagnosis and management of these patients. At the same time the analysis is providing useful mapping data and information on the genetic constitution of gonadoblastoma and dysgerminoma cells. The project involves a collabora tion with the group of Dr Howard Cooke from Edinburgh, which specializes in the human Y chromosome.

Characterisation of three unrelated, enlarged chromosomal variants K. H. Choo, G. Webb*, E. Krumins, S. Eichenbaum, S. Dale, J. Rogers, L. VouIIaire and E. Earle •Department of Human Genetics, John Curtin School of Medical Research, A.N.U., Canberra, Department of Cytogenetics, Royal Women’s Hospital, Melbourne.

Three unusually large chromosomal variants have been found in unrelated, clinically normal individuals. One of these has an expanded short arm of chromosome 9 (9p), whereas the other two carry an apparent 14p chromo some. In each of these cases the chromosome was approximately 30-40% larger than the normal chromo some.

Detailed cytogenetic staining and molecular analyses were performed. The 9p variant proved to be non-Cbanding, and gave the G-banding appearance of a homogeneously staining region (HSR). Attempts at cloning sequences within the enlarged region, including the use of flow-sorted chromosomes, were not successful. Amplification of centromeric alpha satellite sequences, satellite Ill-related sequences, Alu repeats, or ribosomal DNA translocated onto the 9p region were all excluded. Further studies will be required to elucidate the molecular nature of this variant region. The second variant, WSi-var(14)(p-l-) was C-band positive. In situ hybridisation of metaphase cells demons trated a significantly increased amount of centromeric alpha sequences suggesting amplification of some cen tromeric segments. This aberrant event might have pre-disposed the chromosome to instability and transloca tion involving this region, as was detected in one of Wsi’s children who was mentally retarded. The third variant WSm-var(14)(p-l-) was also C-band positive, but no significant increase in centromeric alpha sequence was detected. Instead, however, amplification of satellite III DNA (normally found in small amounts on the short arm of chromosome 14) was shown. The possibility of translocation of heterochromatic segments from Iqh, 9qh and Yqh was excluded. This variant chromosome did not appear to be associated with the abnormal phenotype in WSm’s daughter who was mentally retarded and carried a t(l;?)(q41;?) transloca tion of chromosome 1. The latter two studies suggest that DNA amplification may be a common mechanism for the origin of large size heteromorphisms in chromosomes. Some of these am plification events may destabilise chromosome structures leading to breakage and translocation. These chromoso mal variants should be useful in studying the structural and functional roles of human centromeric and pericentric satellite DNA. 55

EProEMIOLOGICAL AND CLINICAL PROJECTS

Study of the effect of drugs on the fetus during pregnancy L. Sheffield, J. Dodge, H. O’Neill, R. Batagol (Royal Women’s Hospital). The first phase of this study is nearing completion. It involves interviewing pregnant women about their drug intake during pregnancy and comparing this with what is recorded in the Pharmacy computer and in the hospital records at the Royal Women’s Hospital. This study aims to find reliable methods to study the effect of drugs during pregnancy in the production of birth defects.

Clinical, radiological and biochemical features of chondrodysplasia punctata L. Sheffield, J. Halliday, D. Banks, J.

Poulos (Adelaide Children’s Hospital) and N. Mornson (Garvan Institute, Sydney). Over 100 cases of chondrodysplasia punctata (GDP) have been identified. The X-ray review of all patients is com plete and about 50 of the patients have been studied clinically. Much interest has been generated with the findings that high levels of a bone protein, osteocalcin, has been found in several of our patients. The significance of the high level found is being studied in collaboration with the Garvan Institute in Sydney. (The anticoagulant drug. Warfarin, has been shown to act on osteocalcin and fetal exposure to Warfarin causes a form of chondrodysplasia punctata).

The genetics of haemophilia A L. Sheffield, J. Halliday, with J. Lloyd, B. Duncan and J. Braun (Adelaide Children’s Hospital), and S. Sherman (Atlanta, Georgia, U.S.A.). A large amount of data on families and individuals with haemophilia A in South Australia has been previously collected. Where an individual does not have a family history it is possible that a new mutation has taken place either in the affected male or his mother. A collaborative dy with Dr. Sherman (Atlanta, Georgia) is taking place stu

to analyse this data to determine the proportion of cases that represent new mutations and so to further define the genetics of haemophilia A. This analysis uses the computer program “Pointer” which is a program with which Dr. Sherman has had extensive experience. ^ j * • l RandomiSCd COntroUCU trial OI vitamins and folatC in the prevention neural tube defects

individuals with no other family history of the disease who are now aged 30-40 years was studied hoping that many would have children and than an estimate of the frequency of new mutations for autosomal dominant conditions could be made. Careful recording of the family history identified definite indications of either autosomal dominant or autosomal recessive aetiology in 29 of 105 deaf children. Parental audiograms showed autosomal dominant inheritance in a further six. The detailed audiometric assessment of affected children was disappointing as a means of identifying specific categories of deafness. The results of the immunological assessments and of the study of the adult deaf individuals are still being analysed.

i d

T. Colgan, L. Sheffield, A. Robertson. We commenced this study as an Australian contribut ing Centre for the MRC study co-ordinated in Britain. A nurse/co-ordinator was appointed for this study and we have been enrolling patients during the year, The objective is to compare supplementation of folic acid alone with multivitamin supplements and no added vitamins.

^^tiology of congenital deafiiess »

J. J. McGill, H. Paton and E. Keir (Department of Audiology), L. Creati and C. Hosking (Department of Immunology) When a family with two or more individuals affected by congenital deafness presents for genetic counselling this is generally fairly straightforward because the distribution of the cases within the family tells the geneticist the pattern of inheritance. It is much more difficult when confronted with a single individual with congenital deafness whether this be the child of a couple who are seeking guidance about future pregnancies, or a young adult deaf person who is wanting to know about the risk to progeny. The problem is that there are a number of different inherited forms of congenital deafness including autosomal domi nant, autosomal recessive, and X-linked. There are also well documented causes which will have no recurrence risk to either siblings or offspring, such as prenatal infection with rubella virus, and there are probably other environmental causes in this category, This study involved a cohort of deaf children now aged 10-15 years, looking into family history, investigating the value of tests for rubella infection, biochemical investiptions and detailed audiometnc assessments in determining the aetiology. A second cohort of congenitally deal

f i

The 7 surviving patients were noted to have catchup growth and normal growth velocity after infancy (4/7), feeding problems (6/7) most severe in those with significant developmental delay, facial palsy (7/7) and distinctive facial features (down-slanting palpebral fissures, prominent nasal bridge, flattened nose and micrognathia). Of the 10 deceased patients all died by 6 months of age and 7 had a post-mortem examination. Nine had the combination of congenital heart disease and bilateral choanal atresia/stenosis, nine had structural brain defects including absent olfactory tract in all brains examined and minor renal anomalies (6/8).

CHARGE Association: A further reappraisal of clinical features

The burden of genetic disease at the Royal Children’s Hospital, Melbourne

A. S. Harvey, P. M. Leaper (Department of Psycholo gy, Royal Children’s Hospital), A. Bankier Mental retardation and growth failure are generally regarded as integral features of the CHARGE association; Coloboma and microphthalmia, congenital Heart disease, choanal Atresia, Retardation of growth and development/ structural brain abnormality. Genital hypoplasia in males, and Ear abnormalities/deafness. Recent studies have suggested a more favourable outcome for growth and mental development. We reviewed the clinical features and progress of the 17 patients seen during an 11 year period in whom the diagnosis of the CHARGE association could be made in infancy, using 4 of the 7 major diagnostic features (not including mental retardation). Seven patients were alive and had formal psychometric tests. The age range at follow-up was from 2 years 10 months to 15 years 1 month. Two children were functioning in the normal range, the oldest had severe mental deficiency and 4 others had major language delay and variable or no deficits in other areas, significant contributing factors being delay in recognition of deafness and visual deficit, frequent hospitalizations, surgery and poor social circumstances. Two of the deceased patients, who lived to 6 months, were significantly delayed in development.

J. Montgomery, T. Owen, D. McGannon (Medical Student), R. Burnett, J. G. Rogers, A. Bankier Genetic disease and developmental anomalies are an important cause of childhood mortality and morbidity. This study was done to examine the frequency of genetic disease in inpatients during a 2 month period. There were 2317 admissions, including admission of day patients. 13.2% had chromosomal and single gene disorders, 12.4% had multifactorial disorders, 17.8% had disorders that are thought to have a genetic component and 56.6% had non genetic disorders. The large number of autosomal recessive conditions (9.4%) reflects the large number of admissions of some patients with thalassaemia major (101) and cystic fibrosis (59). Patients with autosomal recessive and X-linked disorders had a larger than average number of previous hospital admissions (54.6, 60.5 and 8.3 respectively), the number being a reflection of their intensive treatment regimes. Only 10.5% of patients with simple genetic disorders had received counselling from the Genetics Department, this including 24.1% of autosomal dominant and 7.4% of autosomal recessive conditions. Most of the children with autosomal recessive conditions would have had counselling by staff of the specialist clinics they attend. The average length of stay was 4.4 days, however, 22.3% of patients with autosomal recessive disorders stayed for more than 10 days. 57

LABORATORY STUDIES THAT ARISE FROM CLINICAL CASES

The laboratory investigation of interesting ideas that crop up in the course of clinical care of patients has always been central to our research. This section brings together a group of projects in which the level of basic study varies from very intense in the case of the project on malonyl CoA decarboxylase, and the genetic linkage study of polycystic kidney disease, to much less intensive study in some of the cytogenetic cases and patients with known, but rare, inborn errors of metabolism. At the moment there is a worldwide intensive interest in the mapping of the human genome — that is the locating of each human gene in its proper place on the chromosomes. Patients with a small segment of a chromosome missing or rearranged are valuable in this research. No one group can exploit the research potential of every one of these patients. Consequently the majority of our interesting findings are passed on to other key research groups around the world to whom that particular observation is of special interest and only a small proportion of these unusual findings are worked up in our own laboratory. Those which have fallen into the second category have already been described in section of molecular cytogenetics studies.

Malonyl CoA decarboxylase deficiency J. Christodoulou and G. K. Brown. Attempts to purify the mitochondrial enzyme, malonyl CoA decarboxylase, have been hampered by non-specific aggregation during the purification procedures and instability of the semi-purified product. As a consequ ence sufficient material of suitable quality for raising antibodies has not been obtained. In view of this, characterisation of the enzyme defect in the two patients 58

with malonyl CoA decarboxylase deficiency has been redirected from analysis of the structural defects in the protein to detailed studies of the biological role of the enzyme and the metabolic consequences of its deficiency. In these studies, we will be able to exploit the unique resource of cell lines from the only two known patients with this disorder and their families. As the severity of the defect differs in the two patients, and their parents have intermediate levels of enzyme activity, we have a series of fibroblast lines with a complete spectrum of malonyl CoA decarboxylase activity ranging from normal to almost completely deficient. With this material, we are in a position to characterise all of the metabolic interactions of this enzyme.

Diagnosis, investigation and monitoring of patients with inborn errors of metabolism R. D. Scholem, D. M. Kirby, K. Hayasaka, J. Christodoulou and G. K. Brown. In collaboration with H. B. Croll, J. J. Pitt and P. Vervaart, Department of Biochemistry, Royal Children’s Hospital. We continue to investigate patients with a wide variety of genetic metabolic diseases in conjunction with the metabolic screening laboratory in the Department of Biochemistry of the Royal Children’s Hospital. Each year we perform a large number of assays for enzymes of pyruvate metabolism and mitochondrial electron trans port chain function in cultured fibroblasts sent from all over Australia. When functional defects are demonstrated, in some cases we can analyse the structure of the enzyme involved to confirm the basic defect. In the past year, we have performed detailed studies on

two patients with sulphite oxidase deficiency and have investigated patients with multiple carboxylase deficiency and pyroglutamic aciduria. Publication: 2

DNA linkage study of adult polycystic kidney disease D. Ravine, L. J. Sheffield, P. Kincaid-Smith and R. Walker (Department of Nephrology, Royal Melbourne Hospital). The study aims to clarify the problem of uncertain diagnosis in people from families with polycystic kidney disease. Until now, diagnosis has depended on visualiza tion of kidney cysts by imaging techniques such as ultrasound. There are a proportion of young adults who have inherited the gene but have a normal kidney scan as the cysts have not yet developed. As they have the gene they will ultimately develop renal cysts and their offspring will be at risk of inheriting the gene. The gene causing the disease can be tracked to these individuals by DNA linkage tests, which are being used to check on the reliability of ultrasound. Eventually, ultrasound sensitiv ity and specificity will be determined, allowing a more precise definition of the diagnostic criteria for this condition. Over 100 Victorian families with polycystic kidney disease have been identified, people who have the condition, but who did not previously know they were affected, have been detected. These individuals may benefit in the future from early treatment of complications of the kidney abnormalities. An even larger number of individuals worried by the 1 in 2 risk that they may have inherited the gene, have been reassured by normal ultrasound and DNA test results. The study has enabled us to gather further data about the range and progress of

polycystic kidney disease in many affected people, which will be useful for doctors when advising family members.

Unusual cytogenetic findings M. Leversha, L. Voullaire, S. Dale, D. Dusart, D. Foster, M. Grey, L. Hills, R. Hutchinson, M. Leversha, R. Oertel, V. Petrovic, J. Roberts, C. Vaux and M. Schmidt. In the course of routine cytogenetic analysis chromoso mal variants and abnormalities are encountered that warrant further investigation on either clinical or scientific grounds. Such a finding led to the publication of a variant of chromosome 3 with unusual staining properties. This year a closer liaison between the cytogenetics laboratory and the research scientists of the Murdoch Institute has led to clarification of some of these rare, and cytologically puzzling, chromosomal variants using DNA techniques. Also in conjunction with Murdoch scientific staff we have recently commenced using DNA techniques employing Y chromosomal probes to assist in the identification of female patients with sex chromosome aneuploidy who are at risk for gonadal tumours. The large increase in chromosomal referrals for developmental delay, that has followed the identification of the fragile Xq27 syndrome, prompted analysis of the laboratory data for this group of patients with the interesting conclusion that chromosomal abnormalities other than fragile X are detected in significant numbers amongst this referral group. A child with an unusually complex chromosomal rearrangement was reported to gether with revision of another previously published case using prometaphase banding. The mechanism of meiotic recombination whereby a child acquired a chromosomal duplication from her mother who carried a balanced chromosomal shift constituted another interesting study. Publications: 63, 70 59

TISSUE CULTURE LABORATORY

LIST OF PUBLICATIONS — 1988

R. Cotton, M. Crawford

r In press in previous reports, now published i

1. BROWN, G. K., HAAN, E. A., KIRBY, D. M., SCHOLEM, R. D., WRAITH, J. E., ROGERS, J. G. and DANKS, D. M. — “Cerebral” lactic acidosis: Defects in pyruvate metabolism with profound brain damage and minimal systemic acidosis. Eur. J. Pediat. 147:10-14 (1988).

‘M.

2. BROWN, G. K., SCHOLEM, R. D., HUNT, S. M., HARRISON, J. R. and POLLARD A. C. — Hyperammonaemia and lactic acidosis in a patient with pyruvate dehydrogenase deficiency. J. Inher. Metab. Dis. 10:359-366 (1987).

isai'. Ms Kerry Fowler and Dr Dick Cotton

This year saw a major change as Kerry Fowler, after 13 years of outstanding service running the laboratory, decided to move to a less onerous position at the Howard Florey Institute. We wish her well and thank her. Marjorie Crawford has been running the laboratory with great enthusiasm since May. The major effort for the year has been the preparation of cultures for the team working on pyruvate dehydrogenase.

Tissue culture service M. Crawford, L. Faulds and R. DeFazio March 1988 saw the reintroduction of request forms for cultured cells and for tissue culture media and supplies. The system seems to be working well (there have been over 300 requests of various kinds) and has made the organisation of the tissue culture workload much easier. During the year the Tissue Culture laboratory has received 174 skin biopsies. These included 41 for chromosome analysis, 23 from patients with metabolic disorders, 21 from patients with lactic acidosis, 8 from patients with Leigh’s Syndrome and 40 which were cultured for the Orthopaedic Research Group. We also received 133 skin samples from Anatomical Pathology to be stored (short-term) at -70° C. Of these, 5 were thawed and cultured for further studies. 60

3. CAMAKARIS, J. — Copper absorption, transport and storage. In The Metabolism of Copper in Animals and Man, eds. J. McC. Howell and J. M. Gawthorne, C. R. C. Press Inc. U.S.A. pp63-77 (1987).

Embryonal carcinoma (EC) cells M. Crawford and R. Cotton Studies during the year indicated that leukemia inhibitory factor recently cloned at the Walter & Eliza Hall Institute produced a similar inhibition of differentia tion of EC cells to that obtained by differentiation inhibiting factor discovered in our laboratory several years ago. We have recently shown that the differentiation of single ES cells in culture produces a similar complex pattern in culture to that produced by EC cells. ES cells are pluripotent stem cells similar to EC cells but are closer to derivation from germ cells than EC cells, thus display a normal karyotype, higher percentage chimera formation and more contribution to the germ line of chimeric mice.

Monoclonal antibodies

5. DAHL, H. -H.M., WAKE, S., COTTON, R. G. H. and DANKS, D. M. — The use of restriction fragment length polymorphisms in prenatal diagnosis of dihydropteridine reductase deficiency. J. Med. Genet. 25:25-28 (1988). 6. FUNG, W. -P, THOMAS, T., DICKSON, P. W., ALDRED, A. R., MILLAND, J., DZIADEK, M., POWER, B., HUDSON, P. and SCHREIBER, G. — Structure and expression of the rat transthyretin (Prealbumin) gene. J. Biol. Chem. 263:480-488 (1988).

L. Faulds, I. Jennings and R. Cotton Attempts were made during the year to obtain the antiidiotype antibody as an IgG rather than the less practical and less flexible IgM which we have at present. Strategies have included looking for switch variants and reimmunization. We have also initiated a collaboration with Dr. Robert James to isolate gut cell specific antibodies. Collaboration from some years ago came to fruition this year with the launch of a diagnostic kit for rotavirus by Silenus Laboratories. Dr. Cotton together with members of the Department of Gastroenterology attended the launching. Publications: 7, 8, 44

4. COTTON, R. G. H., HUTCHISON, W. M., WAKE, S., JENNINGS, 1. G., McADAM, W. J., DANKS, D. M. and DAHL, H. -H. M. — Molecular analysis of human dihydropteridine reductase and dihydropteridine reductase deficiency. In Unconju gated Pterins and Related Biogenic Amines, H. -Ch. Curtius and N. Blau, W. de Gruyter & Co., Berlin, New York, pp.275-281 (1987).

7. GEORGIOU, D., ROBERTON, D., PEREIRA, A., SCALETTI, B., KANNOURAKIS, G., HOSKING, C. and FOWLER, K. — A human complement fixing monoclonal antihuman lymphocyte antibody of rat origin. Immunol. & Cell Biol. 66:33-42 (1988).

8. JONES, L.N., FOWLER, K. J., MARSHALL, R. C. and ACKLAND, M. L. — Studies of development of human hair shaft cells in vitro. J. Invest. Dermatol. 90:58-64 (1988).

9. KEITH, C. G., WEBB, G. C. and ROGERS, J. G. — Absence of a lateral rectus muscle associated with duplication of the chromosome segment 7q32-q34. J. Med. Genet. 25:122-127 (1988). 10. McDonald, j. d., cotton, r. g. h., Jen nings, I., McADAM, W., LEDLEY, F., WOO, S. L. C. and BODE, V. C. — The biochemical defect of the hph-1 mouse mutant is a deficiency of GTPcyciohydrolase activity. J. Neurochem. 50:655-657 (1988). 11. MULLEY, J. C., HAAN, E. A., SHEFFIELD, L. J. and SUTHERLAND, G. R. — Recombination frequencies between Duchenne muscular dystrophy and intragenic markers in multigeneration families. Hum. Genet. 78:296-297 (1988). 12. PONZONE, A., GUARDAMAGNA, O., FERRARIS, S., BRACCO, G., NIEDERWIESER, A. and COTTON, R. G. H. — Two mutations of dihydropteridine reductase deficiency. Arch. Dis. Child 63:154157 (1988).

Published and accepted for publication since 1987 report 13. ACKLAND, L. M., DANKS D. M. and McARDLE, H. J. — Studies on the mechanism of zinc uptake by fibroblasts. Dysmorphol. & Clin. Genet. 2:24-50 (1988). 14. BANKIER, A., McGILL, J. J., DANKS, J. J., McGILL, J. A. and DANKS, D. M. — Dysmorphology — problems in nomenclature. Dysmorphology & Clin. Genet. 2:24-50 (1988). 15. BANKIER, A., SHEFFIELD, L. J. and DANKS, D. M. — Renal ultrasound examination of parents in dominantly inherited renal adysplasia — a note of caution. Amer. J. Med. Genet. 29:695-696 (1988). 16. BATEMAN, J. F., LAMANDE, S., DAHL, H. -H. M., CHAN, D. and COLE, W. G. — Substitution of arginine for glycine 664 in the collagen a 1 (1) chain in lethal perinatal osteogenesis imperfecta: Demonstra tion of the peptide defect by in vitro expression of the mutant cDNA. J. Biol. Chem. 263:11627-11630 (1988). 61

17. BEIGHTON, P., de PAEPE, A., DANKS, D., FINIDORI, G., GEDDE-DAHL, T., GOODMAN, R., HALL, J. G., HOLLISTER, D. W., HORTON, W., McKUSICK, V. A., OPITZ, J. M., POPE, F. M., PYERITZ, R. E., RIMOIN, D. L., SILLENCE, D., SPRANGER, J. W., THOMPSON, E., TSIPOURAS, P., VILJOEN, D., WINSHIP, 1. and YOUNG, 1. — International nosology of heritable disorders of connective tissue, Berlin, 1986, Amer. J. Med. Genet. 29:581-594 (1988).

25. CHRISTODOULOU, J., HALL, R. K., MENAHEM, S., HOPKINS, 1. J. and ROGERS, J. — Genetic and clinical features of the syndrome of progressive neurological deterioration and amelogenesis imperfecta. J. Med. Genet, (in press).

18. BROWN, G. K., CROMBY, C. H., MANNING, N. J. and POLLITT, R. J. — Urinary organic acids in succinic semialdehyde dehydrogenase deficiency: Evi dence of a ot-oxidation of 4-hydroxybutyric acid, interaction of succinic semialdehyde with pyruvate dehydrogenase and possible secondary inhibition of mitochondrial P-oxidation. J. Inher. Metab. Dis. 10:367-375 (1987).

27. COTTON, R. G. H., McADAM, W., JENNINGS, 1. and MORGAN, F. J. — A monoclonal antibody to aromatic amino acid hydroxylases: Identification of the epitope. Biochem. J. 255:193-196 (1988).

19. BROWN, R. M., DAHL, H. -H. M. and BROWN, G. K. — X-chromosome localisation of the functional gene for the El alpha subunit of the human pyruvate dehydrogenase complex. Am. J. Hum. Genet. 43:502510 (1988). 20. BROWN, R., FILBY, G., BROWNLEE, G. G., EARLE, E. and CHOO K. H. — Identification and chromosome mapping of a duplicated, X-specific human sequence. Nucl. Acids Res. (in press). 21. CHOO, K. H., FILBY, G., EARLE, E. and BROWN, R. — Isolation of human chromosome 21 sequences and their application to in situ hybridisa tion. Human Genetics, (in press). 22. CHOO, K. H., VISSEL, B., BROWN, R., FILBY, R. G. and EARLE, E. — Homologous alpha satellite sequences of human acrocentric chromosomes with selectivity for chromosomes 13, 14 and 21: Implica tions for recombination between nonhomologues and Robertsonian translocations. Nuc. Acids Res. 16:1273-1284 (1988). 23. CHOW, C. W., HAAN, E. A., GOODMAN, S. L, ANDERSON, R. McD., EVANS, W. A., KLEINSCHMIDT-DeMASTERS, B. K., WISE, G., McGILL, J. J. and DANKS, D. M. — Neuropatholo gy in glutaric acidaemia type 1. Acta Neuropathologica 76:590-594 (1988). 24. CHRISTODOULOU, J., DEWAN, P. A., TAN, H. L. and ROGERS, J. G. — Priapism: A rare complication of Fabry disease. Ped. Surg. (in press) 62

26. COTTON, R. G. H., JENNINGS, 1. G., McADAM, W. J., HUTCHISON, W. M. and DAHL, H. -H. M. — The molecular defect in dihydropteridine reduc tase deficiency. International Days of Paediatrics (in press).

28. COTTON, R. G. H., RODRIGUES, N. R. and CAMPBELL, R. D. — Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc. Natl. Acad. Sci. USA 85:4397-4401 (1988).

i.ii

35. DANKS, D. M. — Disorders of copper metabolism. In The Principles and Practice of Medical Genetics. Second edition, eds. A. E. H. Emery and D. L. Rimoin, Churchill Livingstone, England (in press).

46. GRIMES, A., PETERSON, M. G. and MERCER, J. F. B. — Preparation of RNA by centrifugation through CsCl. In Application Sheets for Cell and Molecular Biology (in press).

36. DANKS, D. M. — Disorders of copper transport. In The Metabolic Basis of Inherited Disease. Sixth edition, eds. C. R. Scriver, A. L. Beaudet, W. L. Sly and D. Valle, McGraw-Hill Company, U.S.A. (in press).

47. HAAN, E. A., MULLEY, J. C., GIDEON, A. K., SHEFFIELD, L. J. and SUTHERLAND, G. R. — Presymptomatic testing for myotonic dystrophy using linked DNA marker Apoc2. Med. J. Aust. (in press).

37. DANKS, D. M. — DNA and clinical medicine, Aust. NZ J. Med. 18:339-348 (1988). 38. DANKS, D. M. — The mild form of Menkes disease: Progress report on the original case. Amer. J. Med. Genet. 30:859-864 (1988). 39. DANKS, D. M. and MERCER, J. F. B. — Metallothionein and caeruloplasmin genes. Trace Element Metabolism in Man and Animals (TEMA 6)(in press).

29. DAHL, H. -H. M. — Prenatal diagnosis of genetic defects using recombinant DNA techniques. Biology in Action, (in press).

40. DANKS, D. M. and WRAITH, J. E. — Prenatal diagnosis of inborn errors. In Fetal and Neonatal Neurology and Neurosurgery, eds. M. 1. Leverne, M. J. Bennett and J. Punt, Churchill Livingstone, London pp.453-458 (1988)

30. DAHL, H. -H. M., CHOO, K. H. and DANKS, D. M. — Application of DNA-DNA hybridization of dual labeled probes to the detection of trisomy 21, monosomy 21, and sex determination. Am. J. Hum. Genet. 43:502-510 (1988).

41. DANKS, D. M., WRAITH, J. E. and BROWN, G. K. — Inborn errors — diagnosis and management. In Fetal and Neonatal Neurology and Neurosurgery, eds. M. 1. Leverne, M. J. Bennett and J. Punt, Churchill Livingstone, London pp.459-468 (1988).

31. DAHL, H. -H. M., HUTCHINSON, W., WAKE, S., McADAM, W., DANKS, D. M. and R. G. H. COTTON — Human dihydropteridine reductase: Isolation of a cDNA clone and studies of patients with defects in this enzyme. Aust. Paediatr. J. 24:81-97 (1988).

42. De JONG, A. P. J. M., HAAN, E. A., MANSON, J. L. WISE, G. A., OUVRIER, R. A. and WADMAN, S. K. — Kinetic study of catecholamine metabolism in hereditary progressive dystonia. Neuropediatrics (in press).

32. DALBOGE, H., CARLSEN, S., JENSEN, E. B., CHRISTENSEN, T. and DAHL, H. -H. M. — Expression of recombinant growth hormone in E.coli: Effect of the region between the Sine & Dalgarno sequence and the initiator ATG codon. DNA 7:399405 (1988).

43. DRY, P. — A quick and easy method for DNA purification from chorionic villus samples. Nucl. Acid Res. 16:7730 (1988).

33. DANKS, D. M. — Birth Defects. In Practical Paediatrics, M. J. Robinson, ed. 2nd edition, Chur chill Livingston, Melbourne (1988). 34. DANKS, D. M. — Copper deficiency in humans. In Annual Review of Nutrition, R. E. Olsen, ed. Annual Reviews Inc. 8:235-257 (1988).

44. DZIADEK, M., CLEMENTS, R., MITRANGAS, K., REITER, H. and FOWLER, K. — Analysis of degradation of the basement membrane protein nidogen, using a specific monoclonal antibody. Eur. J. Biochem. 172:219-225 (1988). 45. GRIMES, A., McARDLE, H. J. and MERCER, J. F. B. — A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells. Anal. Biochem. (in press)

48. HALLIDAY, G. M., LI, Y. W., JOH, T. H., COTTON, R. G. H., HOWE, P. R. C., GEFFEN, L. B. and BLESSING, W. W. — Distribution of monoamine-synthesizing neurons in the human medulla oblongata. J. Comp. Neurol. 273:301-307 (1988) 49. HALLIDAY, G. M., LI, Y. W., JOH, T. H., COTTON, R. G. H., HOWE, P. R. C., GEFFEN, L. B. and BLESSING, W. W. — The distribution of substance P-like immunoreactive neurons in the human medulla oblogata, colocalization with monoamine-synthesizing neurons. Synapse 2:353-370 (1988). 50. HALLIDAY, G. M., LI, Y. W., OLIVER, J. R., JOH, T. H. COTTON, R. G. H., HOWE, P. R. C., GEFFEN, L. B. and BLESSING, W. W. — The distribution of neuropeptide Y-like immunoreactive neurons in the human medulla oblongata. Neurosci ence 26:179-191 (1988). 51. HALLIDAY, J. L., BROWN, G. K. and DANKS, D. M. — Is mild deficiency of mitochondrial malonyl CoA decarboxylase a risk factor for hyperlipidaemia? Biochem. Med. Met. Biol. 39:279-283 (1988). 52. HAYASAKA, K., METOKI, K., KATO, S., CHI BA, T., HIROOKA, M., KIKUCHI, M., KUROBANE, L, NARISAWA, D., and TADA, K. — Partial ornithine transcarbamylase deficiency in females: diagnosis by an immunohistochemical method. Eur. J. Pediatr. 146:370-372 (1987). 53. LANCASTER, P. A. L. and ROGERS, J. G. — Isotretinoin use in pregnancy. Med. J. Aust. (in press). 54. LYNCH, B. C., PITT, D. B., MADDISON, T. G., DANKS, D. M. and WRAITH, J. E. — Maternal phenylketonuria: Successful outcome in four pregnan cies treated prior to conception. Eur. J. Pediat. 148:72-75 (1988). 63

THE MURDOCH INSTITUTE FOR RESEARCH INTO BIRTH DEFECTS LIMITED

Statement of Income and Expenditure For year ended December 31, 1988 55. MANN, J. R. and LOVELL-BADGE, R. G. — Two maternally derived X chromosomes contribute to parthenogenetic inviability. Development 103:129136 (1988). 56. McARDLE, H. J., GROSS, S. M. and DANKS, D. M. — The uptake of copper by mouse hepatocytes. J. Cell Physiol. 136:373-378 (1988). 57. MERCER, J. F. B. — PKU under control. Biology in Action (in press). 58. MERCER, J. F. B., SMITH, J., GRIMES, A., McC. HOWELL, J., GILL, P. and DANKS, D. M. — Zinc, copper and metallothionein mRNA in sheep liver during development. Trace Elements in Man and Animals, TEMA 6 (in press). 59. MULLEY, J. C., GEDEON, A. U., HAAN, E. A., SHEFFIELD, L. J., WHITE, S. J., BATES, L. J., ROBERTSON, E. F. and SUTHERLAND, G. R. — Application of DNA probes to carrier detection and prenatal diagnosis of Duchenne (and Becker) muscu lar dystrophy. Aust. Paed. J. 24:(Suppl.l)92-97 (1988). 60. PARNIAK, M. A., JENNINGS, 1. G. and COT TON, R. G. H. — Interaction with a monoclonal antibody alters the expression of co-operativity by phenylalanine hydroxylase from rat liver. Biochem. J. (in press). 61. PETERSON, M. G. and MERCER, J. F. B. — Differential expression of four linked sheep metallothionein genes. Europ. J. Biochem. 174:425429 (1988). 62. PETERSON, M. G., HANNAN, F. and MERCER, J. F. B. — The sheep metallothionein gene family — structure, sequence and evolutionary relationship of five linked genes. Europ. J. Biochem. 174:417-424 (1988). 63. PETROVIC, V. — A new variant of chromosome 3 with unusual staining properties. J. Med. Genet, (in press). 64. ROGERS, J. G. and TAYLOR, A. M. — The Australian Experience. In Ethics and Human Gene tics: A cross-cultural Perspective, eds. D. C. Wertz and J. C. Fletcher, Springer-Verlag, Heidelberg, (in press). 65. ROUGH, D., CAMAKARIS, J. and LEE, B. T. O. — Copper transport in Escherichia coli. Molecular Biology and Chemistry. UCLA Symposia on Molecu lar and Cellular Biology, New Series, eds. D. Winge and D. Hamer, Alan R. Liss Inc., New York, NY (in press). 64

66. ROUGH, D., LEE, B. T. O. and CAMAKARIS, J. — Genetic and molecular basis of copper resistance in Escherichia coli. Molecular Biology and Chemistry. UCLA Symposia on Molecular and Cellular Biology, New Series, eds. D. Winge and D. Hamer, Alan R. Liss Inc., New York, NY (in press).

1988 INCOME Grants — Royal Children’s Hospital — NHMRC — Other Donations Interest Dividends Net Gain Sale of Investments Income — Other

67. SHERMAN, S. L., TURNER, G., SHEFFIELD, L., LAING, S. and ROBINSON, H. — Investigation of the twinning rate in families with the fragile X syndrome. Amer. J. Med. Genet. 30:625-631 (1988). 68. SRIVASTAVA, G., BORTHWICK, 1. A., MAGUIRE, D. J., ELFERINK, C. J., MERCER, J. F. B., MAY, B. K. and ELLIOTT, W. H. — Heme regulation of 5-aminolevulinate synthase mRNA in different rat tissues and during development. J. Biol. Chem. (in press).

EXPENDITURE Salaries and Wages Payroll On Costs Lab Consumables Equipment and Furnishings Equipment Maintenance Travel Library Audit Fee (no other benefits were received by the auditors) Central Service and Administration Costs

71. WAKE, S., HUTCHISON, W. and DAHL, H. -H. M. — Malignant hyperphenylalaninemia: New Ncol and Hinfl restriction fragment length polymorphisms detected with a cDNA probe. J. Med. Genet. 25:125-127 (1988).

74. WEBB, G. C., VOULLAIRE, L. E. and ROGERS, J. G. — Duplication of a small segment of 5p due to maternal recombination within a paracentric shift. Am. J. Med. Genet. 30:859-864 (1988). 75. WRAITH, J. E., BANKIER, A., CHOW, C. W., DANKS, D. M. and SARDHARWALLA, 1. B. — Geleophysic dysplasia. Amer. J. Med. Genet, (in press). 76. YEOH, G. C. T., EDKINS, E., McKENZIE, K., FULLER, S., MERCER, J. F. B. and DAHL, H. -H. M. — The development of PH in rat liver: in vivo and in vitro studies utilizing fetal hepatocyte cultures. Differentiation 38:42-48 (1988).14-04 1540/14-04 1540.

110,000 807,699 99,547 877,319 326,497 209,651 191,580 6,892

1987 $ 175,000 762,554 55,345 3,046,706 246,577 92,690 1,077,938 45,863

5,502,673

2,629,185

TOTAL INCOME

70. VOULLAIRE, L. E. and WEBB, G. C. — Complex chromosome rearrangements involving chromosomes 1:3 and 2:3 in two abnormal children. Clin. Genet, (in press).

73. WEBB, G. C., KRUMINS, E. J. M., EICHENBAUM, S. Z., VOULLAIRE, L. E., EARLE, E. and CHOO, K. H. — Non C-banding variants in some normal families might be homogeneously staining regions. Hum. Genet, (in press).

69. VISSEL, B. and CHOO, K. H. — Altered activity of restriction endonuclease Mnl-I cleavage of mouse satellite DNA. Nuc. Acids. Res. 16:4731 (1988).

72. WEBB, G. C., KEITH, C. G. and CAMPBELL, N. T. — Concurrent de novo interstitial deletion of band 2p22 and reciprocal translocation (3;7)(p21;q22). J. Med. Genet. 25:125-127 (1988).

1,423,138 102,832 403,948 178,814 18,207 59,693 7,706 1,752

1,256,516 116,220 288,965 222,456 26,780 31,493 26,768 2,750

144,007

148,149

TOTAL EXPENDITURE

2,340,097

2,120,097

SURPLUS FOR PERIOD

289,088

3,382,576

THE MURDOCH INSTITUTE FOR RESEARCH INTO BIRTH DEFECTS LIMITED

Balance Sheet

Statement of Sonrces and Applications of Funds

As at December 31, 1988

Year ended December 31, 1988

Notes CURRENT ASSETS Cash Accrued Income Investments

TOTAL CURRENT ASSETS TOTAL NON-CURRENT ASSETS TOTAL ASSETS CURRENT LIABILITIES Sundry Creditor Accrued Expenses

3 4

1988 $ 15,103 142 7,991,295

1987 $ 53,617 453 7,628,921

8,006,540

7,682,991

8,006,540

7,682,991

TOTAL CURRENT LIABILITIES

326,767 92,542 419,309

305,323 84,649 389,972

NON-CURRENT LIABILITIES Provision for Long Service Leave

99,588

94,464

TOTAL NON-CURRENT LIABILITIES

99,588

94,464

TOTAL LIABILITIES

518,897

484,436

NET ASSETS

7,487,643

7,198,555

MEMBERSHIP FUNDS

7,487,643

7,198,555

Notes SOURCES OF FUNDS Inflow of funds Outflow of funds

FUNDS FROM OPERATIONS

REDUCTION IN ASSETS Current assets Non-current assets INCREASE IN LIABILITIES Current liabilities Non-current liabilities

REDUCTION IN LIABILITIES Current liabilities Non-current liabilities TOTAL APPLICATIONS OF FUNDS

£ . . I'll*-S:.,

1987 $ 5,502,673 2,109,064

308,458

3,393,609

38,825

6 6

29,337

19,931 13,990

376,620

3,427,530

362,374

3,067,229

7 7

14,246

TOTAL SOURCES OF FUNDS APPLICATION OF FUNDS INCREASE IN ASSETS Current assets Non-current assets

1988 $ 2,629,185 2,320,727

360,301

376,620

3,427,530

THE MURDOCH INSTITUTE FOR RESEARCH INTO BIRTH DEFECTS UMITED

THE MURDOCH INSTITUTE FOR RESEARCH INTO BIRTH DEFECTS LIMITED 5. FUNDS FROM OPERATIONS

Notes to and forming part of the accounts

Grant — Royal Children’s Hospital Grant — Other Income — Other (Computer Power, bank interest) Donations Interest from Investments Dividends Net Gain Sale of Investment

•'i

Year ended December 31, 1988 1. STATEMENT OF ACCOUNTING POLICIES The significant accounting policies used in the preparation of these financial statements are as follows: 1.1

1.2

1.3

1.4

Basis of preparation of the Hnancial statements (a) The Murdoch Institute is a scientific institution and is exempt from income tax under Section 23(e) of the Income Tax Assessment Act. (b) The financial statements have been prepared in accordance with the historical cost accounting convention. Applicable approved accounting standards and Australian Accounting Standards have been adopted as the basis for preparing the financial statements. Accounting policies are consistent with those applied in the previous year. Investments Investments are stated at cost. Equipment The Murdoch Institute adopts the policy of writing off plant and equipment acquired for research and development activities in the year of purchase, against profit. This is a consistent policy with similar research institutions. As no future benefit will be derived from this plant and equipment, the prudent approach under AAS 13 is to write off the asset 100 per cent in the year of purchase. This accounting treatment is consistent with that of the prior year. The amount of the write-off was $178,814. Employee Benefits Provisions for employee benefits, which include long service leave, sick leave, holiday pay and other benefits are computed to cover expected entitlements at balance date. Contributions to employee superannuation funds are charged against operating profit.

2. INVESTMENTS At Cost — Shares — Listed on a prescribed stock exchange — Unlisted Government Bonds — listed on a prescribed stock exchange Other investments

Total Market Value of Investments Shares Government Bonds Other Investments

1988

1987

2,245,508 16,000

3,152,787 16,000

2,261,508

3,168,787

5,729,787

1,537,076 2,923,058

7,991,295

7,628,921

2,374,627

2,552,701 1,605,479 3,251,477

5,683,714

t i

3. SUNDRY CREDITOR Royal Children’s Hospital (This is a suspense account which is used for payments to creditors. The hospital pays the creditors on behalf of the Institute.) 4. ACCRUED EXPENSES Salaries and Wages Holiday Pay

7,409,657

326,767

305,323

37,600 54,942

33,453 51,196

92,542

84,649

1987 $

110,000 907,246 6,892 877,319 326,497 209,651 191,580

175,000 817,899 45,863 3,046,706 246,577 92,690 1,077,938

2,629,185

5,502,673

38,514 311 38,825

2) Non-current assets

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INCREASE IN LIABILITIES 3) Current liabilities — Creditor, Royal Children’s Hospital — Accrued Expenses

21,444 7,893

19,931

29,337

19,931

4) Non-current liabilities — Long Service Leave Transfer 7. APPLICATION OF FUNDS INCREASE IN ASSETS 1) Current Assets — Cash at Bank — Investments — Accrued Income

13,990

362,374

25,750 3,041,026 453

362,374

3,067,229

2) Non-current Assets REDUCTION IN LIABILITIES 3) Current Liabilities — Sundry Creditors — Grants in Advance

319,132 41,169 360,301

♦

8,058,341

6. SOURCES OF FUNDS REDUCTION IN ASSETS 1) Current Assets — Cash at Bank — Accrued Income

1988 $

4) Non-current Liabilities — Payments from Long Service Leave Provision 8. REMUNERATION OF DIRECTORS REMUNERATION Amounts received or due and receivable from the company by directors of the company Number of directors whose remuneration was within the following bands. $85,000 — $90,000 $80,000 — $85,000 SUPERANNUATION BENEFITS Superannuation contributions paid in respect of directors. The directors believe that the provision of full particulars would be unreasonable.

14,246

86,251

81,019

No.

1 1

9,658

7,486

THE MURDOCH INSTITUTE FOR RESEARCH INTO BIRTH DEFECTS LIMITED

THE MURDOCH INSTITUTE FOR RESEARCH INTO BIRTH DEFECTS UMITED Directors’ Report

The Directors have pleasure in submitting their report for the year ended December 31, 1988. 1. DIRECTORS The names and relevant details of the Directors of the company in office at the date of this report are set out below: Names and QualiHcations Dr G. L. Barnes, M.D., Ch.B., F.R.A.C.P. Dr Barnes is the Director of the Department of Gastroenterology, Royal Children’s Hospital. He rep resents the Hospital on the Institute’s Board. Mrs J. Calvert-Jones Mrs Janet Calvert-Jones was a Foundation member of the advisory council for children with Impaired Hearing (Vic.) and has been its Chairman since 1973. Mrs Calvert-Jones is also a Director of Cruden Investments Pty. Ltd. and member of the Council of the University of Melbourne. She has recently been appointed Chairman of The Herald and Weekly Times Limited. Dr B. R, Catchlove, M.B., B.S., F.R.A.C.P., F.R.A.C.M.A., F.H.A. Dr Catchlove is the Chief Executive of the Royal Children’s Hospital. Dr Catchlove came to the Royal Children’s Hospital in 1981 following a period as the Director of Medical Services and Deputy Chief Executive of the Royal North Shore Hospital, N.S.W. Dr Catchlove is also Secretary of the Royal Children’s Hospital Research Foundation. Professor M. G. Clark, B.Sc., Ph.D., F.I.Biol. Professor Michael Clark is Professor of Biochemistry, University of Tasmania. A science graduate of the University of New South Wales he was appointed to the Chair in 1985. His major research interests are concerned with the molecular mechanisms of hormone action, particularly as these relate to development of obesity and insulin-resistant diabetes. He is Chairman of the Training Awards Committee, and member of the Medical Research Committee of the National Health and Medical Research Council of Australia. Dr R. G. H. Cotton, B.Ag.Sci., Ph.D., D.Sc. Dr Cotton is Deputy Scientific Director of the Institute. An agricultural science graduate from Melbourne University, he developed a special interest in bioche mistry and has since followed a career in medical research. Mr L. G. Cox, B.Com., A.A.S.A., F.S.I.A. Mr Laurence Cox is Vice-Chairman of the Board of the

Institute and the Chairman of the Finance Committee. He is a Director of the Potter Partners’ Group of Companies, Chairman of the Australian Stock Ex change (Melbourne) Limited and a Director of the Australian Stock Exchange Limited. Professor D. M. Banks, M.D., B.S., F.R.A.C.P. Professor Danks has been the Scientific Director of the Institute from its incorporation, having been head of the Genetics Research Unit from which it evolved since 1973. In 1975 he was appointed the Stevenson Professor of Paediatrics of the University of Mel bourne, transfering to the Chair of Paediatric Research in 1983. He is also Executive Director of the Victorian Clinical Genetics Services. Mr J. A. Fitzgerald Mr Fitzgerald has been Managing Director of In ternational Public Relations Pty. Ltd., Australia’s largest public relations company, since 1982. This followed a period of 28 years in newspapers, the last five of which were spent as Editor of The Herald, Melbourne. Mr Fitzgerald is corporate affairs advisor to some of Australia’s largest corporations. Professor G. J. Fraenkel, A.M., M.A., B.M., M.Ch., Hon. M.D., F.R.C.S., F.R.A.C.S., F.R.A.C.M.A., Hon. F.F.A.R.A.C.S. Professor Fraenkel was the Foundation Dean of the School of Medicine at Flinders University South Australia, 1970-1984. He is the Co-ordinator of Research and Chief Executive, Royal Children’s Hospital Research Foundation (appointed 1985), and represents the Foundation on the Board of the Institute. Mr J. S. Guest, A.M., O.B.E., V.R.D., B.Sc., M.B., B.S., F.R.C.S., F.R.A.C.S. Mr Guest is a distinguished Melbourne surgeon. He held the appointment of Honorary Surgeon, Alfred Hospital 1952-1976, and has been Consultant Surgeon there since 1976. He was a member of the Board of Management of the Alfred Hospital from 1970-1976. He is a Director of the Jack Brockhoff Foundation. Mr Guest has served as a Member of the Board of the Peter MacCallum Cancer Institute since 1967 and from 1983 has been its Chairman. Mr W. H. Hodgson Mr Hodgson has been Deputy Managing Director of the National Australia Bank Limited since 1986. He is the Chairman of the Australian Resources Deve lopment Bank and of Carrington Confirmers Limited, and a Director of the National Heart Foundation.

Mrs P. M. Lewisohn, B.A. Mrs Lewisohn is a member of the Committee of Management of the Royal Children’s Hospital and represents the Hospital on the Board of the Institute. She has been one of the three non-producer members of the Victorian Egg Marketing Board since 1981. Mrs I. McFarling Mrs Ann McFarling has had a long interest in the Royal Children’s Hospital through three generations of her family serving on the Board of Management, two of whom became President. In the early 1960’s she formed a junior auxiliary, Kateena, and was President of this for a number of years. She worked in public relations for over 15 years which included successfully running her own company. Professor P. D. Phelan, B.Sc., M.D., B.S., F.R.A.C.P. Professor Phelan is the Stevenson Professor of Paediat rics at the University of Melbourne and a distinguished thoracic physician. He was the Director of the Department of Thoracic Medicine at the Royal Children’s Hospital from 1974-1983 when he was appointed to the Chair of Paediatrics. Professor G. B. Ryan, M.D., B.S., Ph.D., F.R.C.P.A., F.R.A.C.P. Professor Ryan is Dean of the Faculty of Medicine, Vice-Chairman of the Academic Board and Pro Vice-Chancellor of the University of Melbourne. He is Chairman of the NH & MRC Grants Committee and a member of the Medical Research Committee and Council of the NH & MRC. Professor Ryan is also a member of Council of the University of Melbourne, and of the Boards of the Howard Florey, Baker, Walter and Eliza and Ludwig Institutes. Mr N. Walford, B.Com., F.C.A. Mr Walford was elected the first Chairman of the Board in June 1986 following the incorporation of the Institute. He has been a chartered accountant, footwear manufacturer, stockbroker and Company Director. He is a former Chairman of Repco Corpora tion, Costain Australia, Actrol, a former Commissioner of the State Bank and a former partner in Ord Minnett. He is also currently Chairman of Electrolux Pty. Ltd. and a Director of various public and private com panies. 2. At the date of this report, and since the date of the previous report, no Director has declared any interest in any contract or proposed contract with the company.

3. The principal activities of the Institute during the course of the financial year were to promote and undertake medical research into the understanding, prevention and treatment of birth defects. 4. The net surplus of the Institute for the financial year was $289,088. 5. In the opinion of the Directors the results of the Institute’s operations during the financial year were not substantially affected by any item, transaction, or event of a material and unusual nature. 6. At the date of this report: (a) The Directors are not aware of any circumstances that would render the values attributed to current assets in the accounts misleading. (b) No charge on the assets of the Institute exists that has arisen since the end of the financial year and secures the liability of any other person. (c) No contingent liability has arisen since the end of the financial year. (d) The Directors are not aware of any circumstances not otherwise dealt with in the report or accounts that would render any amount stated in the accounts misleading. 7. No contingent or other liability has become enforceable or is likely to become enforceable, within the period of twelve months after the end of the financial year, that, in the opinion of the Directors will or may substantially affect the ability of the Institute to meet its obligations when they fall due. 8. Since the end of the previous financial year, no Director of the company has received or become entitled to receive a benefit other than a benefit included in the aggregate amount of Directors’ remuneration shown in the accounts by reason of a contract made by the company or a related corporation with a Director or with a firm of which he is a member, or with a company in which he has a substantial financial interest. By Order of the Board

R. NEIL WALFORD (Director)

LAURENCE G. COX (Director) Melbourne, 12th April, 1989

THE MURDOCH INSTITUTE FOR RESEARCH INTO BIRTH DEFECTS LIMITED Auditor’s Report To the Members of the Murdoch Institute for Research into Birth Defects Limited We have audited the accompanying accounts being the Balance Sheet, Statement of Income and Expenditure, Statement of Sources and Applications of Funds, Notes I to 8, and Directors’ Statement thereon in accordance with Australian Auditing Standards. As an audit procedure it was not practicable to extend our examination of donations beyond the accounting for amounts received as shown in the books and records of the Institute. Subject to this, in our opinion, the accounts are properly drawn up in accordance with the provisions of the Companies (Victoria) Code so as to give a true and fair view of: (i)the state of affairs of the Institute at December 31, 1988 and of the surplus of the Institute for the year ended on that date; (ii)the other matters required by Section 269 of that Code to be dealt with in the accounts; and are in accordance with applicable approved accounting standards and Australian Accounting Standards.

TOUCHE ROSS & CO.

Acknowledgments The Murdoch Institute for Research into Birth Defects Limited acknowledges the following donations'. Printing by Brownhall Printing Pty Ltd.

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Color reproduction by Wilke Color.

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B. JAMIESON — PARTNER Chartered Accountants

PRINTERS

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Melbourne, 12th April, 1989

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Paper by Dalton Fine Paper.

DAilPTfP

Statement by Directors 1. In the opinion of the Directors of the Murdoch Institute for Research into Birth Defects Limited: (a) The accompanying Statement of Income and Expenditure is drawn up so as to give a true and fair view of the surplus of the Institute for the year ended December 31, 1988. (b) The accompanying Balance Sheet is drawn up so as to give a true and fair view of the state of affairs of the Institute as at December 31, 1988. (c) As at the date of this statement, there are reasonable grounds to believe that the Institute will be able to pay its debts as and when they fall due. 2. The Institute’s accounts have been made out in accordance with applicable approved accounting standards and Australian Accounting Standards.

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A.C.P. POLYCHROME LIMITED

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Typesetting by The Herald and Weekly Times Limited.

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By Order of the Board R. NEIL WALFORD (Director)

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LAURENCE G. COX (Director) Melbourne, 12th April, 1989

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