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The Murdoch Institute For Research Into Birth Defects Limited ANNUAL REPORT 1986

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The Murdoch Institute For Research Into Birth Defects

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Birth defects are abnormalities and diseases due to causes which are present at birth. They afflict two percent of babies - 5,000 a year in Australia placing great burdens on the families concerned. They kill more babies and children than any other cause.

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The Murdoch Institute is acknowledged as one of the world’s leading contributors to the understanding, prevention and treatment of birth defects.

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fi m » Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia.

The Institute is “working towards the ultimate goal that every child should be bom healthy and with normal abilities”.

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INTERNATIONALLY,

IN VICTORIA,

the Institute is known for - methods of quickly diagnosing and treating babies acutely ill with genetic diseases - discoveries about phenylketonuria, one of the most treatable causes of mental retardation - discoveries about genetic diseases which cause copper deficiency and about the importance of this element in nutrition - discovery of many individual genetic diseases - production of an outstanding computerised birth defects information system.

the Institute is at the centre of the system for provision of these genetic services and is involved in community education about genetic diseases and birth defects. The Institute was established in May 1986 and is located within the Royal Children’s Hospital. One hundred and twenty generous donors have contributed over $9 million to make this new Institute possible. The Murdoch family, the late Sir Jack Brockhoff and the Miller family have been especially generous. The Institute is recognised for its outstanding work by the National Health and Medical Research Council. It is one of only five Institutes in Australia to receive Block Grant funding. The work now encompassed by the Institute began in the 1960’s and has expanded rapidly since 1975. The present staff of 50 scientists, clinicians and research assistants has been recruited progressively over the last ten years. The establishment of the Institute marks the end of the beginning. Now we must get ahead with discovering more causes of birth defects, and inventing more treatments and methods of prevention. To achieve this our need for public support will go on and on.

WITHIN AUSTRALIA, the Institute has played a vital role in training clinical geneticists and in establishing standards of genetic diagnostic treatment and counselling services.

Chairman’s Report

Board of the Murdoch Institute

Finance Committee

Director’s Report

Possum

Clinical Genetic Services and Clinical Research

Phenylketonuria (PKU)

Genetic Research in the Royal Children’s Hospital 1962-86

Past Staff

Murdoch Family

The Late Sir Jack Brockhoff

The MiUer Family

Donations

Speech by the Prime Minister Launching of the Murdoch Institute

Murdoch Institute Opening Dinner

Staff Biographies

Murdoch Institute Lectures 1986

Visiting Scientists on Sabbatical Leave

Visitors to the Institute

Staff Involvement in Australian Scientific Community Activities

Overseas and Interstate Visits, Lectures and Seminars by Institute Staff

Postgraduate Degrees Awarded

Research Collaborations

Detailed Project Reports

Current Staff

List of Publications

Statement of Accounts

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It is particularly pleasing that we were able to thank Mr. Rupert Murdoch in person as well as his mother and sisters for the family’s great generosity. Mr. Murdoch responded on behalf of all donors and presented, as a gift to the Institute from himself and his sisters, a portrait of our Patron, Dame Elisabeth, painted recently by Mr. Wes Walters. We will have great pleasure in displaying this portrait in a place of honour in our new quarters when these become available. We are deeply grateful to The News Corporation Limited for sponsoring the function. While planning this report and the launching function we were thinking only of celebrating our formal establishment. In November 1986 we received news that gave a second and unexpected cause for celebration - the award of a Block Grant from the National Health and Medical Research Council. With the award of this gramt the Institute joins the Hall, Florey, Baker and Garvan Institutes as the only five medical research institutes in Australia funded by Block Grants.

tj Mr. Neil Walford 1 j

This is the first report of the Murdoch Institute for Research into Birth Defects Limited compiled a little over six months after the formal establishment of the Institute. The establishment of the Institute represents the successful conclusion of many years of striving towards a separate identity and reasonable financial security. It also signifies the end of the initial phase of fundraising and of the negotiation with our partner organisations - the Royal Children’s Hospital, the Royal Children’s Hospital Reseeurch Foundation, the University of Melbourne. The Institute was launched publically by the Prime Minister, the Honorable R. J. Hawke, A.C., M.H.R. at a dinner held in the Great Hall of the National Gallery of Victoria on Tuesday, February 17, 1987. On that occasion Professor Danks and I were able to express our gratitude to a large number of our generous donors who were present.

I want to express gratitude to the people who have played key roles in the establishment of the Institute. Two groups must be recognised - the generous private and corporate donors who have made possible our independent existence and the talented and devoted scientists on the staff of the Institute whose performance has earned the support of the National Health and Medical Research Council. These two aspects of the Institute and the two sources of financial support (private/corporate and government) will remain crucial and complimentary in our future. Elsewhere in this report we have listed ciU donations and promises committed to the Institute. Although many of these have been acknowledged previously in the Annual Reports of the Birth Defects Research Institute it seemed only appropriate that all of those who have contributed should be named in this our first Annual Report. Our three biggest supporters - the Murdoch family, the late Sir Jack Brockhoff and the Miller family - are acknowledged in special biographical notes. We intend to publish notes on other major supporters in future years. I wish also to thank the members of the Steering Committee who worked so hard during 1984 and 1985 to establish the Institute and the members of the Board who have directed the affairs of the Institute since its establishment. I want to express my own gratitude and that of the Board members to the staff of the Institute for their achievements in the past year and for the enthusiasm that they show for their future work.

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The story of the Murdoch Institute is in great measure the story of the career, for the last 25 years, of its Director, Professor David Danks. It all began in 1962, when he was the first member of staff - part time Research Fellow in what was then the Clinical Research Unit, a small new section within the Royal Children’s Hospital Research Foundation. Later called Genetics Research Unit, then Birth Defects Research Unit and now the Murdoch Institute. Professor Danks has been admirably supported along the way by a growing band of dedicated scientists, by some first class minds. From the very beginning he has been the leader and the main strategist, the builder of the team. The establishment of the Murdoch Institute marks an outstanding personal achievement by David Danks.

MR. N. WALFORD, B.Com., EC.A. Mr. Neil 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, stock broker and Company Director. He is a former Chairman of Repco Corporation, Costain Australia, Actrol Ltd., 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 companies.

MR. L.G. COX, B.Com., A.A.S.A., EA.I.M. Mr. Laurence Cox is the Vice-Chairman of the Board of the Institute and the Chairman of the Finance Committee. He is the Director of the Potter Partners’ Group of Companies and has been involved in the securities industry since graduating from Melbourne University in 1959. Mr. Cox has wide experience in both the domestic and international financial markets. As a member of the Potter Partners’ Management Committee he is responsible for the company’s joint venture with S.G. Warburg and Co. as well as its investment management activities. He has been on the Board of the Stock Exchange of

Melbourne Limited since 1982. His involvement with the Murdoch Institute began some five years ago when he helped form a small group to assist in the initial fundraising program.

DR. G.L. BARNES, M.D.,Ch.B.,ER.A.C.P. Dr. Graeme Barnes is the Board member nominated by the Royal Children’s Hospital. He is Director of Gastroenterology, a clinical department with strong research interests. Originally from New Zealand where he obtained his medical degree in 1965,. he was appointed to his present position at the Hospital in 1975. His major research interest is in the development of a rotavirus vaccine to prevent severe gastroenteritis in children.

MRS. J. CALVERT-JONES Mrs. Janet Calvert-Jones represents the Murdoch family on the Board of the Institute. She was a Foundation member of the Advisory Council for Children with Impaired Hearing (A.C.C.I.H.) and Honorary Secretary from 1969 - 1973. She is presently Chairman of the Council. Mrs. Calvert-Jones is also a Director of Cruden Investments Pty. Ltd. and has recently been appointed a Director of the Herald and Weekly Times Limited.

DR. B.R. CATCHLOVE, M.B., B.S., ER.A.C.P, ER.A.C.M.A., EH.A.

Mr. L.G. Cox

Dr. Barry Catchlove is the Chief Executive of the Royal Children’s Hospital. Dr. Catchlove graduated in medicine from Sydney University and is a Eellow of both the Royal Australasitm College of Physicians and the Royal Australian College of Medical Administrators. He came to the Royal Children’s Hospital in 1981 following a period as the Director of MedicaJ Services and Deputy Chief Executive Officer of the Royal North Shore Hospital, N.S.W. Dr. Catchlove is also Secretary of the Royal Children’s Hospital Research Foundation.

DR. R.G.H. COTTON, B.Ag.Sci., Ph.D., D.Sc. - Deputy Scientific Director

PROFESSOR D.M. DANKS, M.D., B.S., ER.A.C.P. - Scientific Director

MR. J.A. FITZGERALD

MRS.J. LEWISOHN,

PROFESSOR P.D. PHELAN,

Mr. John Fitzgerald has been Managing Director of International Public Relations Pty. Ltd., Australia’s largest public relations company, since 1982. This followed a period of 28 years in newspapers, the last 5 of which were spent as Editor of the Herald, Melbourne. As public relations consultant to Australia’s America’s Cup Defence of 1987 Ltd., he was central in the preparations to defend the America’s Cup off Fremantle. Mr. Fitzgerald is also Marketing Consultant to the Australian Olympic Federation which, for Los Angeles 1984, completed the most successful fund-raising in its history - $6.5 million.

B.A.

M.D., B.S., B.Sc., ER.A.C.P.

Mrs. Penny Lewisohn is a member of the Committee of Management of the Royal Children’s Hospit2d and represents the Hospital on the Board of the Institute. She has been a member of the Committee of Management since 1980 and is currendy a Vice-President. From 1970 1973 she worked with the Rural Finance Commission administering the Commonwealth Government Rural Reconstruction Scheme. She has been one of the three non-producer members of the Victorian Egg Marketing Bocird since 1981.

PROFESSOR G.J. FRAENKEL,

DAME PATRICIA MACKINNON

A.M., M.A., B.M., M.Ch., M.D.(Hon.)(Flinders), ER.C.S., ER.A.C.S., ER.A.C.M.A.

D.B.E.

Professor Peter Phelan is the Stevenson Professor of Paediatrics at the University of Melbourne and a distinguished thoracic physician. He graduated in medicine and science at the University of Queensland, and obtained his M.D. for studies on obstructive airway disease in infancy. He came to Melbourne in 1964 and spent some time as a Research Fellow in the Clinical Research Unit at the Royal Children’s Hospital before taking up a Postdoctoral Fellowship at Harvard University. 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 Gustav Fraenkel was educated at Perse School, Cambridge, and the University of Oxford where he graduated in Medicine in 1943. In 1958 he was appointed the Professor of Surgery, University of Otago, New Zealand. 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. W.H. HODGSON Mr. Bill Hodgson is the Deputy Managing Director of the National Austrtdia Bank Limited. He is also the Chairman of the Australian Resources Development Bank and of Carrington Confirmers Limited. Mr. Hodgson has had a long career with the Bank, joining the then National Bank of Australasia Limited in Perth, Western Australia, in 1945. He has held senior appointments since 1971 in London, Victoria and Queensland. In April 1981 Mr. Hodgson was appointed General Manager, Corporate and International Banking and after the restructuring associated with the National Australia Bank merger he became Genercd Manager, Corporate Banking. He was appointed Deputy Managing Director in January 1986. He is also a Director of the National Heart Foundation.

Dame Patricia has had a long and distinguished association with the Royal Children’s Hospital. She joined the Committee of Management in 1948 and served as President from 1965 - 1979. She was appointed a member of the Board of the Royal Children’s Hospital Research Foundation in 1965 becoming Chairman in 1967, a position she held until her retirement in 1985. Throughout her association with the Hospital and the Foundation she has been an untiring advocate for the support and growth of research. She is a Patron of the Royal Children’s Hospital Auxilitu'y, and the Royal Children’s Hospital Pied Pipers and an Honorary Life Member of the Royal Children’s Hospital Volunteer Service and the Uncle Bob’s Club.

MR. J.S. GUEST, A.M., O.B.E., M.B., B.S., B.Sc., ER.C.S., ER.A.C.S. Mr. James Guest is a distinguished Melbourne surgeon. He held the appointment of Honorary Surgeon Alfred Hospital 1952-76, and has been Consultant Surgeon there since 1976. He was a member of the Board of Management of the Alfred Hospital from 1970-76. He is a director of the Jack Brockhoff Foundation. Mr. Guest has served as a Member of the Board of the Cancer Institute, Peter MacCallum Hospital since 1967 and from 1983 has been its Chairman.

PROFESSOR G.B, RYAN, M.D., B.S., Ph.D., FR.C.P.A., ER.A.C.P. Professor Graeme Ryan is Detm of the Faculty of Medicine, University of Melbourne. A medical graduate of the University of Melbourne, he was appointed Professor of Anatomy in 1978. His major research interests, still based in the Department of Anatomy, are concerned with kidney structure, function and disease. He became Deputy Dean of the Faculty of Medicine in 1980 and was appointed Dean in 1986. He was appointed ViceChairman of the Academic Board tmd Pro ViceChancellor of the University of Melbourne in 1987. He is Chairman of the N.H.& M.R.C. Grants Committee and a member of the Medical Research Committee and Council of the N.H.& M.R.C. He is a member of Council of the University of Melbourne, and of the Boards of the Howard Florey, Baker, Walter and Eliza and Ludwig Institutes.

Finance Committee

CHAIRMAN: MR. L.G. COX

Director’s Report

MR. D.E. MEIKLEJOHN,

from research grants. I believe that our credibility with our peers who assess such grants will also influence the attitude of private and corporate donors in our favour.

B.Com., EA.S.A., C.P.A., A.A.U.Q.

MR. C.R ABBOTT, L.L.B.,C.P.A. Mr. Charles Abbott has been a partner in the Melbourne firm of solicitors, Blake and Riggall since 1970. Mr. Abbott’s expertise lies in banking and finance. He is a Director of Norwich Winterthur Insurance (Australia) Limited, Stronghold Assurance (Australia) Limited, Stirling Properties Limited, Group Property Services Limited, and is on the Australian Branch Board of the Norwich Union Life Assurance Society.

MR. D.T. CRAIG, A.C.A.(N.Z.) Mr. David Craig is the Director, Americas and Pacific Basin, ANZ Banking Group Limited. Mr. Craig joined the ANZ Bank in 1955 at Temuka, New Zealand. He has held a number of senior appointments in the ANZ Bank which have included Senior Manager, Customer Investments (1977); Controller AHQ(1979); General Manager, Management Services (1983); Executive Director, Grindlays Bank pic (1984); General Manager, Finance, AHQ(1985). Mr. Craig was a member of the Board of Grindlays during his stay in London. He is an associate chartered accountant (New Zealand).

MR. RJ. GRIFFIN, B.Com., A.S.I.A. Mr. Peter Griffin is the Chief Executive of Rothschild Australia Asset Management Limited. He is also a Director of N.M. Rothschild and Sons Australia Pty. Ltd. and N.M. Rothschild Asset Management Limited (London). Mr. Griffin was formerly a founding partner and Chairman of the Executive Committee of the prominent stockbroking firm, McIntosh Griffin Hamson, (now associated with Hoare Govett).

MR. G.E. HEELEY, B.Ec., EA.S.A. Mr. Geoff Heeley is the Executive General Manager Finance of BHP. He has been with the Financial area of the company since joining in 1956 as a cadet. He is a Director of Utah International Inc., Hematite Insurances Pty. Ltd., North West Shelf Development Pty. Ltd., Woodside Petroleum Ltd., Woodside Oil Ltd., Mid Eastern Oil Ltd., and Woodside Petroleum Development Pty. Ltd.. 10

Mr. David Meiklejohn is an Executive Director of Amcor Limited (previously APM) and is General Manager Commercial of the Group. In addition Mr. Meiklejohn is a Director of Kimberly-Clarke Australia Pty. Ltd., a Director of all Amcor’s major operating subsidiaries (including APM, Containers Packaging, Brown & Dureau and James Hardie Containers) and an alternate Director of Mayne Nickless Limited. Mr. Meiklejohn is also involved with a number of outside business associations. He has been a member of the executive committee of the Australian-New Zealand Business Council for some years and is currently Deputy Chairman. He is also a member of the Taxation and Government Expenditure Committee of the Business Council of Australia.

MR. F.D. RYAN, EC.S. Mr. Fergus Ryan is the Managing Partner of the Melbourne office of Arthur Andersen & Co. He is a Fellow of the Institute of Chartered Accountants in Australia and a member of the Australian Society of Accountants. Mr. Ryan is Chairman of the Graduate Careers Council of Australia, a Council member of the Royal Melbourne Institute of Technology, a Director of the Asthma Foundation of Victoria, and a Trustee of the Committee of Economic Development in Australia (CEDA). He has a specialist interest in international business which is reflected in his membership of the Pacific Basin Economic Council and the Australia-Japan Business Co-operation Committee.

I wish to thank a number of people who have played critical roles during the development phase of the Institute. The Steering Committee, which operated during 1984 and 1985 to develop a framework for the Institute and to raise the donations necessary to make it possible, played a most important role. We were fortunate to gather together twenty-five leading members of the Melbourne community who joined in this effort with great enthusiasm. Especially I want to thank NeU Walford for his leadership of this Committee and for his subsequent guidance, wisdom and friendship as Chairman of our Board. He has always been available to discuss our problems despite the pressures of his own business life. I also wish to express our thanks to Laurie Cox, John Fitzgerald and Bill Cowan for their different, but special, contributions to the Steering Committee. They were among the first to join the Committee and Laurie and John continue to play important roles on the Board and Finance Committee. •

I 1 Professor David Banks

Production of this first Report of the Murdoch Institute rates, along with the launching of the Institute on February 17th 1987, among the most exciting events of my life. It gives a tangible reality to the achievement of a dream of a separate and financially independent research institute in Melbourne studying the causes and prevention of birth defects. The award of the Block Grant from the N.H. & M.R.C. really capped it all to make 1986 a most remarkable year. I want to join our Chairman, Mr. Neil Walford, in thanking our generous donors who have made the new Institute possible and the scientists on our staff who have achieved the credibility necessary for the awtu'd of the N.H. & M.R.C. grant. In mentioning these two sources of financial support I must make it clear that we have reached the end of our beginning and that we must now move forward in both these facets of our existence - the achievement of top quality scientific results and the raising of private/corporate support. If the quality of our research is good enough we will maintain and expand our support

The origins of the Institute go back well before the existence of a Steering Committee and are described in some detail elsewhere in this Report. There are two people to whom I want to express a very special personal message of gratitude - Dame Patricia McKinnon and Dame Elisabeth Murdoch. Dame Patricia, as Chairman of the Research Foundation, had faith in our ability to establish an independent birth defects research institute from the moment that the idea was first mooted in 1980. She gave wonderful support at all phases of the process and I am only sad that she was not still Chairman of the Research Foundation when the Murdoch Institute achieved independence. It is pleasing that she agreed to be a Founding Member of the Board of the Institute. Dame Elisabeth has been far more than a most generous donor. She shared my dream of an independent institute researching the causes of birth defects from the time of the earliest discussions. I remember well the way she slipped a very generous cheque into my pocket at a Hospital function five years ago before we had even formally established an appeal, “just as a small encouragement”. I am very proud to direct an Institute which carries the name of Murdoch and which has this great Australian as its Patron.

I In thinking about the recent phase of establishment as an independent institute it is all too easy to forget the sources of our support in earlier years. I want to make very clear my gratitude to the Hospital and the Research Foundation for the encouragement and financial support received over the last 25 years, without which there would be no Murdoch Institute. I also want to thank my colleagues in other research groups who tolerated the preference given to our activities in the build up to our fundraising. Of course, the money which made it all possible came from successive Good Friday Appeals. Ultimately, then our existence depended upon the Herald and Weekly Times/3 DB/ HSV 7 and upon the generous people of Victoria. In striving to justify the faith of our supporters we will be thinking of them as well as of our more recent benefactors. In regard to the scientific achievements v/hich have allowed us to succeed in the eyes of our peers I owe a special debt to Dick Cotton with whom I have enjoyed a wonderful intellectual relationship over the last 18 years. During this time we have each raised, debated and discarded hundreds of scientific ideas, in an interchange which has enabled the few ideas that have become major projects to be well criticised before anything more costly than mental effort was spent on them! Over the later years it was a pleasure to bring John Rogers, Julian Mercer, Garry Brown, Graham Webb and Eric Haan into this intellectual process as well as good minds from outside our own group including Bill Cole, John Bateman and Nick Hoogenraad. With the growth of the Institute in the last two years the circle has widened considerably. I am delighted that the free wheeling interactions which have been so critical to our development are now occurring within this wider group. The balance between scientific independence and collaboration is a delicate one and preserving a proper balance is one of the real challenges we must face as we expand. This Report has some features which are peculiar to its status as our first Annual Report and other features which are intended to set a pattern for future Reports, at least for the next few years. A brief history of genetics research in the Royal Children’s Hospital is included as well as a list of all staff who have worked in the group. The list of present staff is annotated with the date when each individual joined the group and a few notes on the role of each individual. The list of Board Members and Finance Committee Members is annotated to give readers an idea of the background and talents brought to our management by these individuals.

I plan to choose two or three areas of the research within the Institute to highlight in each Annual Report. These will be presented at some length, but in non-teehnical language. I hope that they will prove interesting to our non-scientific readers. The work of other sections will be mentioned only very briefly with emphasis upon special achievements of the year. This year I have chosen to present in detail POSSUM, our computerised system for syndrome recognition, our work on phenylalanine hydroxylase, phenylketonuria (PKU) and related conditions and our interface with clinical services for genetics in Victoria. In future reports we will describe our work in enzymology, recombinant DNA research, trace element transport, embryology and clinical epidemiology. Short biographies of some staff members will be presented each year. This year we meet several people who play an important roles in the organisation of the Institute - Dick Cotton, the Deputy Scientific Director; Anne Ellis, our Business Manager; Barry Holt, our Laboratory Manager; and all the members of the Olive Miller Protein Chemistry group and the Clinical group. Other scientists will have their turn in future Reports. The front sections of this Report are directed to our non scientist readers. Other readers, scientists and staff of other research institutes, will want to see whether we are doing the sort of research they would expect of an Institute which bears a proud name and receives an NH & MRC Block Grant. For their benefit we include brief descriptions of aU the projeets in progress. While we have kept the descriptions of these projects relatively simple, our lay readers may find this section heavy going. It would be impossible for me to overemphasise the importance of collaboration between government, research institutes and private benefactors in our society. I would claim that our new Institute is remarkable among aU Australian Institutes for the balance that it has between government and private support from its establishment. We look forward to continuing this collaboration long into the future.

I am pleased to announce the involvement of the Institute in two teehnology joint ventures involving the Victorian Government. AMRAD, the Australian Medical Research and Development Corporation, brings the Victorian Government and eight research institutes into partnership with private investors. A very able Chief Executive, Dr. John Stocker, has been appointed. No specific collaborative projects have yet commenced. Our involvement in the second government joint venture has progressed much further. Computer Power Export was launched in December as a joint-venture between the Victorian Government (40%) and Computer Power Pty. Ltd. (60%). Our POSSUM system is one of the three projects in the initial portfolio of this international computer software publishing company. At the time of writing this report we are involved in negotiation with the Victorian Government to establish a formal collaboration between the Institute and the Departments of Health and Community Services for the provision of top quality genetic services for the State of Victoria. Good quality services have been established by the Department of Genetics of the Royal Children’s Hospital in collaboration with the obstetric hospitcils in the city. In recent times the financial support of these services has not been sufficient to allow them to keep up to date with the great advances that have occurred in the methods of preventing birth defects. We hope that a new collaboration will be established in which an adequately funded central clinical headquarters at the Royal Children’s Hospital will be an arm of the Murdoch Institute. We have been most grateful for the interest that the Department of Community Services has already shown in our research. They have provided a research grant to support our work on DNA methods of diagnosing Down’s syndrome.

We live in an era in which this type of collaboration is taking on a new meaning. Once it was considered almost improper for research workers in Institutes like this to take an interest in the commercial potential of their discoveries. Today, we are acutely aware that the future of our country depends upon the inventions of its scientists and the efficient commercial exploitation of these inventions.

Possum

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POSSUM is one of the most exciting developments of our new Institute. Its success illustrates the importance of long term persistence with something which was judged to be worthwhile, of keeping track of new technological developments and of developing effective partnerships. POSSUM stands for Pictures Of Standard Syndromes and Undiagnosed Malformations. POSSUM is also Latin for “I can” - T am able”. The POSSUM system can help doctors diagnose syndromes and is able (capable). We have good reason to believe that it is the best system of its type in the world, and that it is on its way to becoming accepted as the world’s standard system.

WHAT IS A SYNDROME? In this system our interest is confined to syndromes of birth defects. In this context, a syndrome is a recurring array of abnormalities, either anatomical or functional, occurring in different patients. Down syndrome could be regarded as a prototype which is familiar to all. Dr. Langdon Down first described patients with the features which were later named mongolism. His name is honoured in the title of this syndrome. We could till recognise the mental retardation, the characteristic facial appearance, body build, short stature and even the characteristic movements and mannerisms of patients with this condition. Recognition is instantaneous without need to analyse the individual components of the syndrome, just as recognition of a relative or friend met in the street occurs instantly, without analysis of the method of recognition. Equally it is possible to recognise any one of the 1000 syndromes which have now been identified, provided that one can remember each of the 1000 arrays of features and each of the 1000 characteristic appearances. The task of remembering all of these features is beyond most human minds and it is here that the computer has much to offer.

WHAT IS ACHIEVED BY DIAGNOSING A SYNDROME? Correct recognition of a syndrome has a great deal to offer in a practical way to the family concerned. In the longerterm it is the starting point of research into the basic cause of the syndrome. To be the parent of a child with a unique array of birth defects is a very lonely and anxious situation. Doctors can tell you little about what to expect in the future. You cannot talk to anyone else who has lived through the same

experience. Inevitably you feel that the doctors you are seeing must be incompetent because they cannot make a diagnosis and you wonder about other doctors in your own city or doctors in another country who might be able to resolve the matter or suggest some magic treatment. Doctors cannot tell whether a similar problem might recur in a future pregnancy. Contrast this with the situation when a baby is diagnosed as suffering from Down syndrome. Immediately the doctor is able to call upon his personal experience of a number of other patients with Down syndrome and upon the medical world’s experience of thousands of other cases. Although the exact development of that individual child with Down syndrome cannot be predicted precisely (any more than the details of the future development of a normal baby can be predicted precisely) at least the parents can be given a good estimate of the child’s future potential. The doctors will also know the complications or unusual illnesses to which children with Down syndrome are especially prone. Parents can be offered the opportunity to speak with other couples who have children with Down syndrome. Accurate predictions about the risk of recurrence of Down syndrome in future pregnancies can be offered and prenatal diagnosis can be made available for those few couples who have a high risk of producing another child with the condition. All of these helpful services become available to the parents because the syndrome was diagnosed correctly. Every experienced paediatrician can remember many couples whose anxieties about an abnormal baby have been diminished by correct diagnosis of the syndrome involved. Even though this diagnosis may not enable any specific treatment to be given, the parents stiU find contentment in knowing the nature of the condition and that other couples have dealt with this situation. They are no longer alone with their problem.. This knowledge regarding Down syndrome exists today because Dr. Langdon Down gave this particular condition a name 130 years ago. During the intervening years research workers have been able to collect together patients who have the same condition, to compare the backgrounds of those patients and the events preceding their birth, and to look at the characteristics of their parents. As early as 1932, studies of this type had already shown that children with Down syndrome were more likely to be born to older mothers, that it was very rare for a woman to have more than one child with Down syndrome and that nearly half of the few children born to women with Down syndrome also had the condition. These observations allowed two

very astute geneticists separately to hypothesise that Down syndrome might be caused by the inclusion in the fertilised egg of three doses of a chromosome rather than two. It was not until 1959 that scientists developed methods of examining human chromosomes and showed three chromosomes number 21 (trisomy 21). The investigation of the rare familial cases of Down syndrome revealed an hereditary chromosome rearrangement joining one chromosome 21 to another chromosome and predisposing to inclusion of an extra copy of this chromosome. The classification of groups of patients as having the same syndrome is the first step towards identification of the underlying defect. In another example, it was the recognition by Dr. John Menkes of a recurring pattern of abnormalities of hair and brain development accompanying poor growth and early death in male babies which led other doctors to attach his name to this X-linked inherited syndrome. Later, we recognised that a deficiency of copper could explain all of these features and a surge of understanding of this condition followed. Now prenatal diagnosis can be offered and experimental forms of treatment are being tested in the Institute.

WHY IS DIAGNOSIS OF SYNDROMES DIFFICULT? Recognition of a syndrome with which a clinician is really familiar is quite simple. In this sense “really familiar” means “have seen personally several times”, not just “have read about it and seen illustrations”. In other words, the clinician must be able to recall clearly aU the features of the condition including the visible features. Often the clinician has a vague feeling that he has “seen that face somewhere before”, but cannot remember where. Alternatively he may have no idea how he can connect together the features he sees in the patient. Ordinarily, his next recourse would be to thumb through books which describe and illustrate some of the known syndromes, searching for “that face”. The existence of over 1,000 syndromes makes this a mammoth task. Reduced to a simple statement the problem is to recall the features of known syndromes with which to match those present in the patient, especially the visible features.

HAVE OTHERS USED COMPUTERS FOR THIS PURPOSE? We were not the first to apply computers to this task. An American group and a German group started using computers back in the 1960s. We started our computerised system in 1972. Our first step was to decide which parts of the process should be retained by the human and which should be delegated to the computer. We decided that humans are much better than computers at recognising arrays of visible features. Each of us can recognise instandy hundreds of friends, relations and acquaintances. Yet we would have great difficulty in describing the features we use in this process of recognition sufficiendy accurately to teach them to a computer. Experienced doctors are also good at judging how much importtmce to place upon each symptom or sign. Weighting of features can be included in a computer program, but it is very difficult to include the subde variations in weighting which may be appropriate in different circumstances. All this comes naturally to the experienced clinician. These decisions dictated that our system of recording the features of known syndromes, and the features found in individual patients, should be kept very simple and that we would include photographs of known syndromes and undiagnosed patients. Further, it dictated that our system should produce for the clinician a list of diagnoses for him to consider, not just a single diagnosis chosen by the computer as the most likely. From its beginning the Melbourne system differed from the other systems. To this day no other system has photographs. Most of the other systems do use weighting of features and do try to make the diagnosis by computer.

HOW HAS POSSUM BEEN DEVELOPED OVER THE YEARS? The original system was designed by Professor Danks and Dr. John Btu-ry, a young New Zealand paediatrician who worked as a Research Fellow in 1972/73. Over the subsequent years a number of other trainees in clinical genetics working in the Department have contributed to the development of the system - Dr. John Rogers, Dr. Les Sheffield, Dr. David Sillence and Dr. Eric Haan. More recently. Dr. David Pitt worked on the system for several yetirs after his retirement from Kew Cottages. Over the last three years Dr. Agnes Bankier has played an outstcmding role in the development of POSSUM to its present state of sophistication.

The original system was quite primitive. The features of patients were punched on to 80-column cards and these were batch processed at a computing centre outside the Hospital. Searching the system involved punching a card about the patient and waiting for the opportunity to send a group of cards down to the computer centre and for a print out to return, a process which generally took several weeks.

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Then we heard of laser videodisc technology, which was just becoming available in 1984 and could be interfaced with a computer, so that the large number of individual frames stored on the disc could be accessed instandy. In fact, a single 12” videodisc can hold 50,000 frames, nearly 3 times the number in the system at present. Next, through the kind intervention of Mr. Rupert Murdoch, came the opportunity of collaboration from Computer Power Pty. Ltd., Australia’s leading computer software company, which is jointly owned by Mr. Roger Allen and by News Corporation. The last two years have been a very exciting time for all the clinical genetics group, especially for Agnes Bankier. Now we have a wonderfully sophisticated system.

HOW DOES POSSUM WORK? POSSUM operates on an IBM PC linked to a 12” laser videodisc player (Figure 1). To seek diagnostic assistance, the clinician types into the computer the code numbers corresponding to the five or six clinical features that he regards as particularly significant in his patient (Figure 2). He can instruct the computer to attempt to match all the features or perhaps to match any four of them, or he can demand that one of these four must be present and any

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Photographs were the strong feature of the system from the beginning. A standardised system of photography has been used without alteration since 1972. The photographs were mounted on cards and filed by sequential numbers to which the clinician was led by the computer. At a later stage the programs were upgraded for direct key entry and were moved on to the Hospital’s mainframe computer so that the system was available on-line within the building. However, the programme was still slow, taking 5-10 minutes to search the data bank and one stiU had to turn to the sets of mounted photographs kept on a bookshelf to get the photographic evidence. This arrangement worked quite well within the building, but it could not be shared with anybody else, except by the enormous labour of producing prints of the 1300 sets of 12 photographs of each patient.

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three of the remaining features may be matched. It takes the computer only two or three seconds to search the entire data base of over one thousand syndromes and to display a list of those which match to the required degree, starting with those that match most closely (Figure 3). Looking at this list, the clinician may be able to discard a large number of the suggestions. He may be left with four or five conditions about which he is uncertain, some of which are unfamiliar to him. He can ask the computer to tell him about any one of these conditions. A list of the features of the condition is then displayed, plus some references to books or journal articles and an offer of displaying photographs of patients with this condition (Figure 4). Photographs of affected individuals at different ages may be offered. The clinician selects the patients whose photographs he would like to see. Once again the delay is just three seconds before the first of these photographs flashes up on the screen (Figure 5). Further views of the same patient can be accessed instandy.

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The whole program is very simple to operate. There is an operating manual which can be read, or else the instructions can be displayed on the computer screen. The system can also be used to learn about a syndrome and has great potential as a teaching system for specialist paediatricians and geneticists. Although the method of presentation would be suitable for teaching medical students, most of the conditions included in POSSUM are too rare for them.

TO WHOM WILL POSSUM BE AVAILABLE?

Figure 3

Figure 1 - Dr. Agnes Bankier operates POSSUM

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POSSUM has been displayed at the International Congress of Paediatrics and at the International Congress of Human Genetics during 1986 and is now marketed world-wide by Computer Power Export, a newly established consortium of the Victorian Government and Computer Power Limited set up for the express purpose of publishing computer software internationally. Already we have firm orders or expressions of strong interest from over forty clinical geneticists around the world. We anticipate that most major paediatric hospitals in the western world will want a system like POSSUM. There are two alternatives available - one developed in London and another from the United States. Neither has pictures

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Ibi I bus, tl ekaoji tic NKiw il tin ease, • to tbni tte viw m SSss'n*to'Jwfi* th tnits f» tlds use, BC ti leto.________ Figure 4 and both are much slower to operate than POSSUM. We feel confident that POSSUM will be successful in the marketplace. Indeed, the London group has joined forces with POSSUM to some extent, allowing us to purchase their data so that we can add any information not already in POSSUM. Another high quality system has been developed in France, but it is available only in that country because it operates on the French telephone system. Dr. Segolene Ayme, the author of this system, has joined with us in the future development of POSSUM and we look forward to collaborating with her.

Clinical fenetic Services and Clinical Research

It would be good to have a single system of this type accepted by all geneticists around the world, just as the catalogue of human genetic diseases produced by Professor Victor McKusick ofJohns Hopkins Hospital is accepted without any argument or competition. Time alone will tell whether there is sufficient consensus to have just one system and whether POSSUM will be that system. We believe that it has a strong claim to this role because it is the only system with photographs and they are essential. Although other groups are talking of adding photographs to their systems, we know from experience the magnitude of that task.

From the start of work in genetics in 1962 research and the provision of services for diagnosis, treatment and counselling have developed together. Indeed, the special opportunities which exist in a very large paediatric hospital have influenced greatly the type of research progrcimme which has developed. Even compared to other large paediatric hospitals, around the world the Royal Children’s Hospital is an especially favourable place to conduct genetic research. It serves a large and well defined population drawing to it all cases of most genetic diseases which occur in Victoria, Tasmania and south-western N.S.W. As the service work and the research developed they took on separate names. A Department of Genetics developed in the Hospital and the research group had a series of names in the Research Foundation. Functionally, they have always been as one.

CLINICAL SERVICES Genetic counselling is the central service. The objective is to provide clients with the information that they need to reach important life decisions, generally decisions about future reproduction. Most often we are consulted by couples who have had an abnormal baby and wish to know the risk of repetition of the problem. On other occasions the client may have a disease and be worried about passing it on to offspring or may be a member of a family in which a particular disease has occurred without being affected personally. Some enquiries are about more general questions, such as the effect of marrying a cousin, or the risk involved when some drug has been taken during pregnancy or when the pregnant woman has been exposed to some industrial chemical or to X-rays.

Figure 5

In all of these circumstances the counsellor must first work out the statistical risk of the event about which the client is concerned and must consider whether any procedures exist which may modify this risk. Then comes the counselling that is, the presentation of this information in a form which is easily comprehended by the client and a discussion that helps the client to reach a decision. It is most important to listen to the client and to really understand the questions in her/his mind. The matter causing anxiety is not always the one which is most obvious to the counsellor. Sometimes the client has to develop confidence in the counsellor before revealing the question which is most concerning. Genetic counselling is a mixture of solid science and personal communication. The counsellor must be skilled in both aspects. The counsellor must be willing to spend plenty of time, sufficient to accomplish the purpose. Several discussions may be required.

In some medical centres all genetic counselling is undertaken by clinical geneticists. We do not believe that this is necessary or desirable. It is more satisfactory for genetic counselling about the more common genetic diseases to be undertaken by the medical specialists who diagnose and treat each disease. For instance, in Victoria, nearly all genetic counselling relating to cystic fibrosis, haemophilia and muscular dystrophy is undertaken by the clinics which care for these patients. The genetics group collaborates with these clinics whenever it becomes necessary (for instance, since the recent advent of DNA tests for these conditions) to ensure that the best possible services are made available. Our Genetics Clinic has always dealt mainly with birth defects whose cause is uncertain, and with the rarer genetic diseases in which no other clinician has developed a special interest. Other groups of patients have come to the Genetics Clinic because one or other of the geneticists has a particular interest or expertise in the condition. Correct diagnosis is the central issue in the scientific component of genetic counselling. Often the precision of diagnosis necessary for genetic counselling is greater than that required for clinical care. For instance, there are several muscle diseases which cause similar symptoms and need the same medical care, but are caused by different genes and follow different patterns of inheritance in families. The risk in future pregnancies may be 1 in 4 in one condition and nearly zero in another. Achieving the precise diagnosis needed for genetic counselling is not always easy. Geneticists are likely to know what subcategories of each disease exist, but may not have the technical skills necessary to distinguish one category from another. Medical specialists (e.g. neurologists, eye specialists, &c.) may have the technical skills, but may not be aware of the subtle differences which are important for genetic purposes. Close collaboration is needed. We have established joint clinics with eye specialists, skin specialists, orthopaedic surgeons, plastic surgeons and endocrinologists to deal with some of these difficulties. We hope soon to establish formal liason with relevant specialties in adult hospitals to cope with genetic diseases which develop symptoms first in adult life. The whole service has built up to a substantial size dealing with 20 to 25 couples each week in the various clinics. The first extensions of our genetic services into other hospitals occurred some years ago at the Royal Women’s Hospital and Queen Victoria Medical Centre. Each of these hospitals appointed a specific member of our team of clinical geneticists and gave aU other members of the team subsidiary appointments. This enables us to run an on-call roster for nights and weekends with one member on call for 19

genetic consultations throughout the city, thereby offering a much better service than could be offered by a solo geneticist appointed to any hospital. Early in the 1970s representations were made to the Health Department recommending the development of a “network of genetic services” serving all hospitals with its headquarters at the Royal Children’s Hospital. Eventually, in 1980, a modified system was introduced with a Department of Genetics at the Royal Children’s Hospital, and appointments of clinical geneticists at the two obstetric hospitals. An Expert Co-ordinating Committee on Genetic Services was established to oversee the development of the whole system. In practice this has worked reasonably well, except that it has been difficult to reconcile the need for genetics services to serve the whole Victorian community with the budgetary priorities of the individual hospitals, each with a prime interest in serving its own in-house requirements. The original system proposed would stiU be preferable and new submissions have been made to the Health Department to attach the central genetic service to the Murdoch Institute as its service arm, with a budget for the entire system administered through the Institute, rather than through the Royal Children’s Hospital. This approach has the full support of the executives and Committee of Management of the Royal Children’s Hospital and of the Expert Co-ordinating Committee. A good genetic service needs to integrate the requirements of paediatrics, obstetrics and adult medicine for genetic services. These requirements are changing rapidly with the development of new skills in prenatal and presymptomatic diagnosis by DNA technology. All of these procedures save serious emotional trauma to families and are highly cost-effective by reducing future expenditure for the long-term care of children with birth defects. As a Research Institute closely involved with practical matters relating to birth defects and genetic disease, we are determined to see that the results of our research, and of research overseas, are applied quickly and efficiently in Victoria. We are aware that a number of new discoveries turn out to be less valuable than they appear at first. We believe that our research workers represent a valuable resource of expertise to assist in the evaluation of new claims about diagnostic tests and methods of treatment before they are introduced into Victoria. These are strong reasons for maintaining a close link between research into genetic diseases and the provision of services for these diseases.

THE CLIVE AND JEAN ROXBURGH GENETICS CLINIC RECORDS SYSTEM Clive Roxburgh has been active in the Uncle Bob’s Club since its early days. This wonderful organisation began when a small group of men befriended some of the children at the Frankston Orthopaedic Section of the Royal Children’s Hospital. It was in the days when poliomyelitis, tuberculous infections of bone and joint and other chronic bone infections kept children in chronic ill health for many years, most of which were spent in hospital. These men formed a Club of friends prepared to be “Uncles” to these children and to subscribe a “bob” (shilling) a week - the Uncle Bob’s Club was launched. Over the subsequent 45 years the Club has raised well over $4 million for the Royal Children’s Hospital. Some years ago Clive and Jean Roxburgh established a special fund within the Uncle Bob’s Club. There it grew with accumulated interest. In 1984 they decided to give the accumulated interest and future interest to a specific project in this Institute. After discussion it was decided that conversion of the Genetics Clinic files to a computerised form and maintenance of these files would be an appropriate use of the funds. Over the last 25 years the Clinic has seen over 12000 families. These have been recorded on index file cards which lead us to the detailed files for each family. The index card system has been transferred to the Hospital mainframe computer and can be accessed by the Co ordinator at her desk. Each day she has 10 or 20 telephone calls about past or current patients. For most enquiries the information on the index card is sufficient, without needing to go to the detailed file. Previously she had to walk from her desk to a bank of 20 drawers which held the index cards. Now this information is literally at her finger tips as she sits at her desk. The new system gives her more information than the old cards. It tells her when the patient was seen last, what future appointments have been made and what test results or replies to requests for further information are still awaited. In addition to transferring the index cards to the computer, we have purchased a Lectrofile storage system to hold all our detailed files. This replaced 12 four-drawer filing cabinets.

scientists on a day by day basis, bringing to the research laboratories ideas that have arisen in the care of patients and seem to cast new light on existing research topics or to suggest new projects. A number of our major research projects have started in this way. However, their contribution goes far beyond the major projects, for many minor alterations to continuing projects have been suggested by clinical observations. It is this constant interplay between clinicians and laboratory workers which has characterised our research group.

A baby with multiple physical abnormalities was born to cousin parents. The defects did not fit any known syndrome. Two unknown chemicals were found in the urine. It took two yetirs to identify them, but eventually we discovered the first definite example of a genetic disturbance in chemical function which disrupts embryonic development. Several other examples of this process have been discovered since. Treatment was not possible, but we were able to offer the parents prenatal diagnosis in future pregnancies.

Many examples of clinical observations which led to laboratory projects could be given. Two will suffice to illustrate the point.

The observations which led us to discover malignant PKU comprise another example, discussed in detail in this Report, as does the discovery of copper deficiency in Menkes’ disease, a subject which will be discussed in detail in next year’s Report.

A baby severely iU with an acid-base imbalance and stiU at her birth weight at 5 months of age was found to have chemicals in her urine which suggested a fault in the breakdown of tyrosine, but the findings were different to all known diseases. Nonetheless a clinical decision was made to control strictly the dietary intake of tyrosine and she made a dramatic recovery. Now she is a beautiful, bright teenager and the disease is known as hawkinsinuria in her honour, the chemical present in her having proved to be entirely new to the world of chemistry as well as to medicine. In the process we had discovered a new step in the process involved in the breakdown of tyrosine in the normal body.

In the process of recording birth defect syndromes a number of conditions have been identified which had not previously been described and publications have arisen which have led to recognition of the same condition elsewhere in the world.

Mrs. Anne Glynn, Clinic Co-ordinator searches the computerisedfiles of the Genetics Clinic

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RESEARCH IN CLINICAL GENETICS Although our clinical geneticists have always carried a heavy service load they have also been very active in research. Their most important role in the research of the Institute is one which is hard to define or quantify. It comes through their interaction with the laboratory

The largest and most protracted research effort of the clinicians has been in the development of the system now known as POSSUM - a computerised system designed to assist clinicians in diagnosing birth defect syndromes. This system is discussed in detail in this report.

.—

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Phenylketonuria (PKU)

Phenylketonuria, phenylalanine hydroxylase and dihydropteridine reductase are big words likely to turn away anyone except a trained biochemist, so they will be reduced to PKU, PH, and DHPR respectively. PKU is a genetic disease. PH and DHPR are enzymes (biological catalysts) which can be at fault in PKU. The way they are related to one another is really very simple.

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The story of PKU is one of the great success stories of modern clinical genetics, but we believe that the story is still incomplete and that there are many improvements yet to be achieved in the treatment of patients with this condition. Untreated, PKU causes severe mental’retardation. Thanks to the efforts of a number of scientists working in different countries in the period 1934 to 1962 a very satisfactory method of early diagnosis and an effective treatment have been available for 25 years. Thousands of mentally normal children around the world owe a great deal to these scientists. The oldest of these patients are now having children themselves.

Mrs. Glynn uses the Lecirofile Storage System

A number of systematic family studies of birth defects have been conducted. In some instances the conditions concerned have been simple genetic ones - e.g. studies of cystic fibrosis to determine the incidence and to confirm recessive inheritance at a time (early 1960s) when rather foolish claims were being made by European groups about dominant inheritance rather than recessive. More often we have studied conditions in which there is known to be a genetic component to the cause, but not a single mutant gene causing till cases. The intensive and protracted studies of neonatal liver disease in the 1960s and 1970s fit into this'category. They identified several specific genetic diseases which can cause neonatal hepatitis, plus a less definable genetic contribution to susceptibility in the remaining cases, apparendy predisposing to infection by a virus or viruses. The same study showed that biliary atresia, also known as congenital absence of the bile ducts, was not due to failure of development of bile ducts as had originally been believed, but rather was the result of destruction of the bile ducts late in foetal life, apparently by a virus infection. No genetic contribution to this condition has been found.

...

A study of babies who died in the newborn period without kidneys or with severely malformed kidneys supported the view that there was a genetic contribution to this problem, and enabled us to separate two groups of patients for whom the risks in future pregnancies are quite different. Couples who have had a child with absent or severely abnormal kidneys, but no other abnormalities outside the urinary and genital systems, have a 5 % risk of producing a further

chUd with this problem. When a baby has died with malformations in other body systems, especially cardiac malformations, as well as the lethal renal defect, then the risk of recurrence of the abnormalities in the other body systems is quite high, but the risk of recurrence of the renal problem is low. A study of babies born with hydrocephalus revealed that the X-linked inherited form of this condition contributes over 10 % of cases and identified characteristics of the brain malformation in this condition which allow its recognition even when no previous cases have occurred in the family. In progress at present is a study of chondro dysplasia punctata (punctate epiphyseal dysplasia). These rather clumsy names describe abnormal patchy bone formation in the cartilaginous areas at the growing ends of the bones of a young baby. Previous classifications of these conditions have been unsatisfactory. We have been interested in these conditions for 15 years, and have accumulated data on over one hundred cases. As with all work on classification of birth defects, the separation of different classes of a condition is the first step towards discovering the precise cause of each class. This is already bearing fruit with these conditions, for workers overseas have found a specific biochemical defect in one of the rare subgroups of this condition. We will be applying the tests for this biochemical disorder to the other subgroups to determine whether less severe degrees of this defect or related defects are present.

Lisa Bullock was bom with PKU - a treatable genetic disease. Thanks to treatment she now has a child which will be normal.

TYRosine

PHEnylaianine

Body proteins

Figure 1

The disease was discovered by a Norwegian paediatrician in 1934. The basic biochemical fault was demonstrated by an American biochemist in 1948. A German paediatriciam devised the dietary treatment in 1956 and an American microbiologist invented the cheap and efficient newborn screening test in 1962. Today every baby in Australia has a Guthrie test performed on the fourth day of life. A drop of blood is soaked into a filter paper, aJlpwed to dry and posted to a central laboratory. Abnormal results are known before the age of 10 days and dietary treatment can generally be started before 2 weeks. The end result is a level of intelligence comparable to that of the normal brothers and sisters.

BASIC BIOCHEMISTRY OF PKU To understand the disease we need to look at a few facts about the particular biochemical reactions that are involved. This is very simple if the awkwtu-d names of the chemicals are ignored. The human body makes the thousands of proteins which determine its structure and function from simple units called amino acids. Twenty amino acids are used. Most of these amino acids come from the proteins in our food which are broken down to amino acids in the intestine before they are absorbed. To understand PKU we need to consider just two of the twenty amino acids - phenylalanine (PHE) and tyrosine (TYR). Both are derived from food protein and both are necessary to form body proteins (Figfure 1). Ideally, the body needs to be able to make individual amino acids if insufficient is available, and to dispose of excess. PHE is one of six amino acids which cannot be made in the body and are termed “essential”. Excess can be disposed of only by conversion to TYR, which, in turn, can be broken down to COj and water (Figure 2). 23

Normal

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PHE + O2 PHEnylalanine

TYR

TYRosine-------- CO2+H2O

Synthesis

Figure 2

The conversion of PHE to TYR is catalysed by the enzyme PH (Figure 2) which requires for its activity a cofactor which we will call BH^. It is made in the body by a series of reactions which need not concern us for the moment. In the process of converting of PHE to TYR, BH, is changed to an inactive form (qBH^) which is reactivated to BH^ by the other enzyme we wish to consider, DHPR (Figure 3). We see the whole process if we combine Figures 2 and 3. PKU occurs when PHE cannot be converted to TYR. This happens when PH fails to function adequately. This is usually caused by a genetic defect in PH itself, but can also be caused by a genetic defect in DHPR (or in one of the enzymes involved in making BH J. It was the form due to a defect in PH which was discovered in 1934, is known as classical PKU, is responsible for 98% of all cases of PKU and responds so well to dietary treatment. The form due to a defect in DHPR was discovered only in 1974, has more serious effects (“malignant PKU”) and is responsible for about 1 % of cases and causes progressive brain damage even with dietary treatment.

CLASSICAL PKU When PHE is not converted to TYR adequately, PHE accumulates (Figure 4) in the bloodstream and has a toxic effect upon the brain. The accumulation of PHE begins only after birth and takes several weeks to affect the brain. This is why the dietary treatment can be effective provided it is started within a few weeks after birth. Before birth any excess in PHE passes across the placenta into the mother’s blood and is broken down in her body. The principle of the diet is quite simple - each day’s food contains just the amount of PHE which is required for making new body proteins and no more (Figure 5). In

BH4

qBH, DHPR

proteins

• PH

abilities. Some doctors argue that a more subtle effect upon the brain may be occurring in all patients who are taken off the diet and that the ill-effects may become apparent in middle age. A few clinics continue treatment throughout life. In Melbourne we follow the more generally accepted practice, believing that continuation of the diet causes emotional stress in all patients and that alleviation of this stress is more important than trying to prevent hypothetical long-term effects. Only in 30 years time will we find out which opinion is correct.

Figure 3

practice, this is not very difficult to achieve. Most of the protein required is given as a powdered mixture of the other 19 amino acids which are needed to make body proteins. The child then eats a diet which is quite similar to that consumed by vegetarians, containing no meat, fish, eggs, cheese or mUk, but including measured amounts of a number of other foods with a low content of protein, and liberal quantities of non-protein foods (carbohydrates and most fruits and vegetables). In young babies a special formula is used which provides the right balance of PHE and other amino acids. The treatment is monitored by measuring the rate of growth and by measuring the level of PHE in the blood at regular intervals. This sounds very restrictive, but, in fact, the parents soon come to take the whole process in their stride. They attend a special clinic every three or four months and perform blood tests each month between these visits, learning to use the same blotting paper method of dispatching the sample to the laboratory as was used in the newborn testing. Most families learn to cope with this diet very satisfactorily, achieving accurate dietary control and accepting fully that they have a “normal child who happens to eat different foods”. Unfortunately, in a proportion of families, the diagnosis and the diet remain a focus of smouldering discontent and the child comes to use the diet as a means of manipulating the family and the rest of the world, becoming unhappy and risking brain damage because of inadequate control of the PHE levels in the body. Because it is clear that the main harmful effect of the untreated disease is upon the growing brain of the younger child, the diet is generally discontinued at 7 to 10 years of age. Hundreds of children have been observed after ceasing the diet and nearly all of them have continued to progress well, but a small number have shown some loss of

In PKU

Proteins in food

TYR-------- CO2+H2O

Body proteins

Figure 4

When one describes the complex process involved in diagnosing and treating PKU - testing 60,000 babies each year in Victoria to diagnose six cases and treating each of these six with a complex diet which uses expensive synthetic protein substitutes - some might query the whole process on economic grounds. In fact, the costs are greatly outweighed by the savings. It costs $30,000-40,000 to diagnose and treat one case of PKU. This sum would provide special education or institutional care for a retarded child for just one or two years out of the 40 or 50 year lifespan of an untreated PKU patient. Although PKU is one of the success stories of modern genetics, there is still quite a long way to go before the success is complete. Still hanging over us are the concern about subtle late effects upon the brain and the problem of coping with female patients during pregnancy. Very careful control of the blood phenylalanine level from before conception is essential if the baby is to be born normal. Otherwise, severe brain damage occurs, plus physical malformations in some cases. The baby does not inherit PKU, but does suffer brain damage because the mother’s high levels of PHE pass across the placenta and damage the developing foetad brain. The disease harms a growing

brain and the foetal brain is growing very rapidly. Keeping in contact with all female patients and persuading all of them to start the diet before becoming pregnant will not be easy.

OUR RESEARCH ON PH When we began our research on PKU our objective was to determine precisely the various biochemical defects which could cause PKU. Experience with other genetic diseases which were better understood at that time had taught us to expect that a number of different alterations within the gene coding for PH might be able to cause PKU. To achieve our objectives we started by purifying PH and determining in detail the structure of the normal protein. In the late 1960s and early 1970s one had to study the protein itself and it was quite unthinkable to suggest studying the gene direcdy. The new techniques for isolating and analysing genes did not come on the scene until the mid 1970s. When they did come they revolutionised the whole approach to this type of research and even changed the long term objective of the work. Originally, we wanted to determine the different alterations which we expected in PH because we thought that some of these might be treated by direct manipulation of the enzyme, without requiring the long-term diet. While this still remains a possibility , the goal has now changed to developing methods of correcting the gene itself. It may prove best to replace the whole gene without needing to know the precise location of the fault within the gene or it may turn out that correction of the fault within the gene is a better approach. This approach would depend upon precise knowledge of the alteration present in each patient. These goals would have seemed sheer fantasy in 1968. They are not near achievement in 1987, but are near enough to become an objective, albeit a distant objective.

Figure 5

CO2+H2O

Purification of PH had eluded a number of other research groups around the world, partly because it was difficult to get access to enough liver tissue to purify the enzyme and partly because the human enzyme is very unstable, breaking down into fragments very soon after death. Dick Cotton developed a very elegant method of purification which relied upon the complexity of the PH reaction with involvement of both PHE and BH^. This method was so specific that it could purify PH in a single step. Not only did this save a lot of time and work, but it also achieved the purification quickly enough to prevent breakdown of the enzyme. Although this method was eventually applied successfully to the human enzyme, it was originally developed using monkey liver PH. This choice, dictated initially by access to monkey livers from Commonwealth Serum Laboratories, proved fortunate because monkey PH turned out to be much more stable than human PH. This was a great advantage during the development of the technique. Next, antibodies were made against the purified PH and these antibodies have been used extensively in analysing the structure and function of the enzyme. A few words must be said about antibodies. They are molecules made by the body’s immune system in response to foreign chemical molecules, generally proteins, which may enter the body, often as proteins on the surface of bacteria or viruses. When a foreign protein is injected into an animal (or when it enters a human body) the immune system makes a number of different antibodies, each of which recognises a different part of the protein molecule. The serum of an immunised animal contains a mixture of these different antibodies. This mixture is very useful for some experimental purposes. For other purposes one would like to separate each of the different antibodies and to make use of the specific ability of each of these antibodies to recognise a different region of the molecule. Until the mid 1970s this was not possible, but in 1975 Kohler and Milstein working in Cambridge developed a method of making each of these antibodies separately - an achievement which won the Nobel Prize. They are called monoclonal antibodies. The postdoctoral fellow in Cesar Milstein’s laboratory who did the experiments which opened the way to the development of this technique was a young Australian called Dick Cotton! It was natural that he should use monoclonal antibodies extensively in his studies of PH. The various antibodies against PH which have been developed in our laboratory over the years are very useful for purifying PH and for recognising different functional regions of the molecule. Enzymes catalyse chemical 26

about the structure of the protein which was needed to confirm that it was the correct gene. By mid 1984 it became clear that we should clone the gene ourselves so that we could work with it as we wished. Techniques had changed so much in the intervening 4 years that the isolation was achieved in five weeks by Henrik Dahl.

Figure 6

reactions in the body by binding on their surface the chemicals that have to interact and holding them together so that the interaction occurs. Then they must be able to release the newly formed chemicals and start the process over again. Eventually we need to be able to describe the parts of the molecule to which PHE and the cofactor BH, bind and explain how these regions of the molecule change after the PHE and BH, have interacted. Presumably a change in the shape of the molecule allows TYR to be released along with qBH^, the oxidised form of BH^. (Figure 6). Good progress has been made and Dick Cotton and his collaborators in Melbourne and overseas are closer to achieving this ultimate goal than any other group in the world. With the advent of techniques for analysing genes (DNA) it became apparent that progress in understanding PH would be more rapid if we could isolate the gene than if we were to continue studying the protein alone. Julian Mercer and Dick Cotton worked out a strategy for isolating the PH gene and began this work in 1979. At that time the isolation of genes coding for very abundant proteins had become fairly routine, but isolation of a gene for less abundant proteins like PH was on the edge of technical feasibility. The strategy devised was expected to take about 3 years to isolate the gene. About 12 months later we learned that an American group had isolated a gene which they believed to be the one coding for PH. They had used exacdy the same strategy that we had chosen and had taken 3 years! We spoke with them immediately, felt almost convinced that they had isolated the correct gene and suggested collaboration in proving the validity of the result and in future research. This negotiation was protracted and never satisfactory, even though Dick Cotton and Robert James eventuaOy provided the information

By this time the American scientists had developed an indirect method of prenatal diagnosis, but were not making the gene probe available to laboratories wanting to use the test. We issued our probe to all laboratories around the world who wanted it and set up the prenatal diagnostic test for use within Australia. In practice, most Australian couples who have one child with PKU are so pleased with the outcome of the dietary treatment that they do not feel concerned about producing a second affected child. Of course, couples differ in their attitudes on these matters and some couples will want to use prenatal diagnosis. We believe that the test should be available for them. Studying both the PH gene and the protein, we are moving closer to a complete understanding of the normal gene and its function. Molecular geneticists have still not developed simple and satisfactory ways of identifying the mutation in a gene in each family with the disease. The process is still very laborious. Only a small number of mutations have been defined. Our long-term goal is to understanci in detail the PH gene and the way it is controlled so that we can develop a method of gene replacement as a one-shot cure of PKU. Ethically, it will be appropriate to offer gene therapy in PKU only after there has been considerable experience with gene therapy in other life threatening or disabling diseases. After all, it is a marginal improvement in outcome that we are seeking - certainty that delayed subtle effects upon the brain will not be a problem, freedom from risk to the offspring of affected females and avoidance of a cumbersome diet. Nonetheless, we do believe that gene replacement will later become so precise and sophisticated that it will replace dietary treatment in PKU. We plan to be part of this achievement by contributing to the development of methods of inserting genes and by understanding the structure and function of the PH gene in great detail. This is a very long-term goal and we do not expect to be working continually on the PH gene during the intervening years. We expect to continue our detailed analysis of the PH gene and protein for several more years until we have reached a point of knowledge sufficient for the purpose. Then we can put the knowledge aside until experience of gene therapy has accumulated.

MALIGNANT PKU We and others interested in the details of the PH enzyme reaction (Figure 3) had been looking out for cases of PKU caused by defects in other components. In 1974, we and groups in London, Heidelberg and Bethesda observed cases which failed to respond clinically to the dietary treatment. These patients had insufficient BH^ to keep the PH reaction working. This cofactor is produced in the body by a series of chemical reactions, each of which is catalysed by a specific enzyme, and is recycled by DHPR (Figure 3). The series of chemical processes involved in producing BH^ includes at least five steps. Defects in each of these steps has now been shown to cause PKU. The first patients we described with a form of PKU due to deficiency of BH^ turned out to have a defect in DHPR. We therefore began an intensive study of this enzyme. First, something needs to be said about the disease that occurs in patients with a deficiency of DHPR, or indeed, a deficiency of any of the other enzymes involved in the production of BH^. These diseases have a more devastating effect than ordinary PKU (generally called classical PKU). This is because the BH^ cofactor is also required for at least two other enzymes in the body, enzymes which are involved in the production of chemical messenger molecules in the brain (neurotransmitters) which are very important in brain development and function. Consequently a patient with a deficiency of BH^ (whether due to DHPR deficiency or to other enzyme defects) has aU of the problems of PKU plus an underproduction of important neurotransmitters. Treatment with the PKU diet relieves only part of the problem, leaving unrelieved the deficiency of neurotransmitters causing progressive brain damage and death. This disease warrants the term malignant PKU. It was this peculiar behaviour of some patients with PKU which first attracted our attention of ourselves and that of scientists in London, Heidelberg and Bethesda in 1974. It is possible to administer substances which the brain can convert into the missing neurotransmitters and this does improve the progress of these patients with malignant PKU, but does not always produce a normal child. This is a more serious disease than classical PKU and most couples choose to use prenatal diagnosis in subsequent pregnancies.

OUR RESEARCH ON DHPR For this reason and because we wanted to understand the role of BH^ in the body more fully, we decided to purify DHPR from human tissues and to analyse it. It proved 27

Genetic Research in the Royal Children’s Hospital 1962 to 1986

easier to purify than PH. It is a very stable enzyme which remains fuUy active in liver tissue even twenty-four hours after death. It is present in aU tissues of the body and can be studied in patients with the disease using white blood cells or cells cultivated from a fragment of skin. The enzyme was purified, antibodies against it were made, the structure of part of the enzyme was determined and this information has been used to isolate the gene. Isolation of the gene proved a little more troublesome than with PH but was achieved. Then we were able to develop a method of prenatal diagnosis using the isolated gene. Now we are able to diagnose DHPR deficiency prenataUy by either of two methods - the measurement of the activity of the enzyme in the cells obtained from a chorion villus sample or by amniocentesis, or using DNA analysis. We plan to undertake further studies of the mutations in DHPR in some of the patients we have studied and we are considering whether to embark upon more extensive studies of the role of BH^ in the human body. There are reasons to suspect that it is quite important in other neurological diseases and to suspect that it has functions other than as cofactor to the three enzymes which have already been mentioned. Decisions about the further work for this project will await Dick Cotton’s return from sabbatical leave in September 1987.

David Danks In this first report of the Murdoch Institute it seems appropriate to describe its origins. Inevitably this involves some rather personal comments on the factors which influenced my choice of a career in medical genetics and gathered together those colleagues who have made possible the establishment of this Institute. To trace the origins, I must go back forty years to a phase in the development of the Royal Children’s Hospital (then the Children’s Hospital), which I did not witness personally. During the latter half of the 1940s Lady Latham, President of the Committee of Management, set out to persuade talented paediatricians to devote themselves exclusively to their specialty, to accept full-time salaried positions in the Hospital and to regard research as important. Her successor. Lady Murdoch, sustained and expanded this approach. This established an attitude which made the Children’s Hospital of the 1950s a very exciting place to attend as a medical student and to work as a Resident Medical Officer. This I can say from personal experience. Outstanding among those who created this atmosphere, especially in regard to research, was Dr. Howard Williams. It was the fresh and inquisitive approach to medicine that I encountered in Howard’s ward as a student that influenced me to become a paediatrician. Later, he drew my attention to the need for new developments in three fields in paediatrics - neurology, psychiatry and genetics. The opportunity of training in genetics arose in Britain in 1960. Dr. John Fraser Roberts and Dr. Cedric Carter were prepared to take me on as a trainee if a salary could be found. At that time, Howard Williams and Dame Elisabeth Murdoch, were busy setting up the Royal Children’s Hospital Research Foundation, but they found time to persuade the Trustees of the Felton Bequest to provide a grant for one year. After this followed a National Institutes of Health Training Fellowship, to work with Professor Victor McKusick at Johns Hopkins, completing my good fortune in working under under the two men who are today acknowledged as most important in the establishment of clinical genetics - Carter and McKusick. Returning to Melbourne in 1962,1 slipped with no noticeable effort on my part, into positions created by my fairy godparents - Howard WUliams and Dame Elisabeth - as half-time Associate Paediatrician and half-time Research Fellow in the Clinical Genetics Unit.

Before shifting to the more matter-of-fact chronicles of genetics research in the Royal Children’s Hospital Research Foundation, I must acknowledge several individuals and organisations who gave grants and scholarships in the crucial etirly years of our work. The sums concerned were smtill by today’s standards, but without them, the Institute would not exist today. The Uncle Bob’s Club Travelling Scholarship helped me to go to Britain in 1959; the Felton Bequest supported me in London in 1960-61, and gave further research grants in 1962-65; the Apex Foundation for Research into Mental Retardation and the 3Rs Fund gave grants in the early years. The Percy Bttxter Charitable Trust helped with some of the equipment we needed to start our work. Just recently I found the item we purchased gathering dust on a shelf - a Madas electric calculator which cost $500 in 1963 and was considered remarkable because it could add up the squares of numbers. Today’s equivalent of that sum would buy a very sophisticated microcomputer. My training project in London was on neonatal hepatitis and in the process, I learnt about infantUe liver disease. This remained my field of research for a number of years after my return and it proved necessary to take on diagnosis and treatment of all liver disease in order to identify the diseases I wished to study. For fifteen years I was the consultant in paediatric liver disease for Melbourne and we developed quite an international reputation for treatment of diseases like bile duct atresia, as well as for our research. It was strange that this research led to the conclusion that the apparent congenital abnormality of the bile ducts (biliary atresia) was in fact the result of destruction of the ducts in the later part of pregnancy by a virus infection, whereas the condition that had always been assumed to be purely the result of virus infection (neonatal hepatitis), was at least partly caused by genetic factors. Although the interaction of infections and genetic susceptibility had a considerable interest, the study of the biochemical basis of genetic disease became a greater interest, and it was good to be able to hand over the responsibility for the management of liver diseases to Dr. Arnold Smith in 1976. The early research on liver diseases involved close collaboration with Dr. Charlotte Anderson and Dr. Valerie Burke, in the Gastroenterology Research Unit, in studies of the effects of liver disease upon fat absorption, and also in genetic family studies of cystic fibrosis.

My first research grant from the NH & MRC was given to employ Dr. Kathleen Hayes, a paediatrician/virologist, to study the consequences of cytomegalovirus infection during pregnancy. Kath Hayes did some fine research here and subsequently at the Queen Victoria Medical Centre and at Fairfield Hospital. In 1967 I was appointed half-time Reader in Human Genetics in the Department of Genetics of Melbourne University, while maintaining a half-time position as Medical Geneticist in the Clinical Research Unit of the Research Foundation. Postgraduate students then joined in the research and in 1968 Dr. Richard Cotton started work on inborn errors of metabolism, supported by the Elizabeth Sweet Fellowship of Melbourne University. Our partnership has continued ever since, except for the three years that Dick spent furthering his training overseas in 1970-73. It has been a good and enjoyable partnership because we have different, but complimentary, talents and because each respects the other’s skills and point of view. We each like to hypothesise rather wildly, and we each respect the other’s criticism of these hypotheses. Since 1968 the emphasis of our research has been increasingly upon the biochemical basis of genetic diseases and this was the title of the Program Grant we eventutJly won from the NH & MRC in 1982. Indeed, very similar broad titles were used for all our NH & MRC applications from 1969 onwards, and it is fortunate that they tolerated this rather unusual style of “mini-program” proposals for all those years (except for a notable episode when our applications were rejected in 1979). It was fortunate because it allowed us to develop a style of research which particularly suited our situation, working in a very large and busy children’s hospital. We learnt to recognise new metabolic diseases when they came along and to recognise unusual features of known diseases which might give a new lead to the understanding of the biochemical basis of these diseases. The dialogue between basic science and clinical skills has been very important. The search for new inborn errors of metabolism began in earnest in 1970. An assistant (Miss Patricia Tippett) set up simple and rapid methods of identifying abnormal levels of amino acids in urine and more sophisticated methods of analysing organic acids in urine and we provided a close clinical liaison between the wards and the laboratory. This lifted the diagnostic rate far above that achieved with the methods that had been used previously in the Hospital’s Department of Pathology and in 1973 we combined forces with the Department of Clinical Biochemistry in a collaboration which has continued ever since.

lid

Another important event occurred in 1973 when we begtm a collaboration with Professor Berthold Halpern of the Department of Chemistry of Wollongong University, a world expert in mass spectrometry. This proved an exciting, enjoyable and highly successful collaboration. Over the next 8 years, we were among the most successful groups in the world in discovering new inborn errors of metabolism and it was tragic that this collaboration ended abruptly two years before Bert Halpern’s intended retirement when he died of a heart attack. We owe a very special debt to him and we still miss his friendship, his infectious enthusiasm and his expertise in chemistry.

These notes have identified the way in which our three biggest areas of research - phenylalanine hydroxylase, copper transport and inborn errors of metabolism in general have developed. Omitted from the story is the discovery in 1974 of unusual variants of PKU in which a different group of enzymes is at fault and most recendy, the isolation of the gene responsible for one of these variant conditions. This work has also been led by Dick Cotton. Dr. Frank Firgaira, as a PhD student and Postdoctoral Fellow, played an important part in purifying and studying the enzyme and Henrik Dahl has recendy isolated the gene.

Dick Cotton began to study phenylalanine hydroxylase, the enzyme at fault in phenylketonuria (PKU) soon after he arrived in 1968 and he is stUl discovering new and interesting things about its structure and function. Although it was one of the first inborn errors of metabolism to be recognised, it has been one of the most difficult to understand completely and Dick Cotton is acknowledged as one of the world leaders in this subject. Since 1975, he has been very ably assisted by Ian Jennings and more recently has collaborated with Dr. Choo, Dr. Mercer and Dr. Dahl. This work is described in detail elsewhere in this report.

There have been many other smaller biochemical projects which cannot be mentioned in this brief outline, but all have contributed to the development of the Institute to the present level. Work in cell culture, in the production of monoclonal antibodies and their application to many different projects, clinical studies of individual rare genetic diseases or family studies of somewhat more frequent birth defects and genetic diseases, cytogenetic studies of unusual patients with chromosomal abnormalities have all contributed to the progress. Another very notable piece of work in the Institute has been the development of POSSUM, a computer assisted system for the diagnosis of birth defect syndromes, but this is the subject of a special section in this report.

The next important event was the discovery, in 1971, that copper deficiency could explain all the abnormadities observed in Menkes’ disease, a fatal X-linked inherited disease, described first in 1962. Research on this disease and related conditions has increased our knowledge about the way copper is made available to carry out its functions in the cells of the body, but a great deal remains to be learned. The initial work was carried out with Elizabeth Cartwright (a Ph.D. student). Dr. Peter Campbell and Mr. Brian Stephens of the Department of Pathology. Then Dr. Jim Camakaris joined the team as a Research Fellow. Later he became Lecturer and then Senior Lecturer in Human Genetics in the Department of Genetics at Melbourne University, but he has continued to collaborate in the copper research . Most of the research has been carried out by postgraduate students, some at the University and some in the Institute. Dr. Julian Mercer joined in this work after he isolated the gene coding for metallothionein in the rat, and he went on to study the function of this gene in mutant mice and in sheep, which utilise copper in an unusual way. Recently, Dr. Harry McArdle has joined the copper group, coming from a group which studies the transport of iron in the body.

Professor Berthold Halpern

For nearly ten years, the search for new inborn errors of metabolism and the study of the known ones was carried out by research assistants working with Dick Cotton and myself, but since 1980 this work has escalated to a new level in the hands of Dr. Garry Brown, a medical graduate trained subsequently in biochemistry, who has a great understanding of human metabolism and great skill in studying it. As well as maintaining our record in the recognition of new inborn errors of metabolism Garry has made an intense study of a particularly interesting and important enzyme called pyruvate dehydrogenase which is at the focal point of the body’s energy metabolism. Many other groups in the world have studied this enzyme and all have had difficulty in measuring its activity. Garry has purified the protein and has collaborated with Henrik Dahl in isolating the gene coding for the most critical subunit of the enzyme. They are poised ready to reap great benefits from the analysis of the patients we have seen over the years in Melbourne and the patients that have baffled research workers in clinics around the world.

The group has known several homes. The first was a fibroplaster extension on the roof of the Ward 12/14 building of the old Hospital in Pelham Street, in which Dr. Charlotte Anderson, Dr. Rudge Townley, a secretary and I shared a total area of approximately 10 square metres. In the new Hospital in 1963 to 1973 we occupied several different small areas on the 8th floor for offices amd laboratory. Then, while the new 9th and 10th floors were being added we moved to the new Genetics Building at the University, returning to larger laboratories on the 10th floor in 1977. The space available on the 10th floor has increased over the intervening years and will be increased further in the future.

Past Staff I

The group has also had several changes of name. Throughout its development it was within the Royal Children’s Hospital Research Foundation. Originally an anonymous section of the Clinical Research Unit it later became the Genetics Research Unit. In 1981 an International Peer Review was conducted by Professor Cedric Carter of London, Professor Leon Rosenberg of Yale and Professor Chev Kidson of Brisbane. The report was favourable and the Board of the Research Foundation decided to assist the group to expand and, eventually, to achieve independent standing. The steps towards this goal were taken at surprising speed - renamed the Birth Defects Research Institute in March 1981, short-listed for a Commonwealth Research Centre award late in 1981, awarded an NH & MRC Program Grant in 1982, successful fundraising drive in 1984-5, formally established as the Murdoch Institute for Research into Birth Defects in May 1986, awarded an NH & MRC Block Grant in November 1986.

I 32

j.—

Elizabeth Cartwright 1969-74 K.H. Choo 1976-78 Frank Firgaira 1977-80 Judy Finger 1979-83 Sue Herd 1979-85 Marie Phillips 1979-83 Trevor Stevenson 1981-85 Peter Koopman 1982-85 Samantha Wake 1982-86 Greg Peterson 1983-86

Postdoctoral Fellows:

Students: Ph.D.:

John Connellan 1970-73 Stan Im 1973-74 Kym Faull 1975-76 Roger Truscott 1976-79 Eric McCairns 1979 Peter Royce 1979-80 Choon Wong 1980-81 leva Lazdins 1980-81 Frank Firgaira 1981-83 Stuart Smith 1981-83 Chris Cobbett 1982-83 Robert James 1982-84 Isabel Roberts 1985-86

First home of Genetics Research

Scientists (Senior): Kath Hayes 1969-73 Eric Haan 1982-85 Graham Webb 1980-86

)

Students: M.Sc.: Meredith Patrick 1970-71 Clarence Yap 1975-77 Jeff Mann 1976-80 Carol Wicking 1982-84 Stewart Fabb 1984-86 Anna Longworth 1984-86

Scientists (Junior):

Clinical Genetics (Staff):

Pat Tippett 1970-73 Judy Hammond 1974 Peter Schlesinger 1975-78 Roslyn Kushinsky 1984-85

Eric Haan 1983-85 David Pitt 1980-83 Ed Wraith 1986

Research Assistants:

John Barry 1973 Les Sheffield 1974-76 Arnold Smith 1974 David SiUence 1975-77 Eric Haan 1978 Agnes Bankier 1981-82

Paul Clarke 1968-69 Don Kiritsopoulos 1970 Ann Opray 1970-71 HalinaGibas 1970-71 Linda Dimech 1971-73 Vaughan Buchanan 1971-74 Ann Nankivell 1972 Jennifer Stirling 1972 Ian McNiece 1973-74 Ian Wilmot 1973 Paul Grattan 1974-75 Judy Clark 1974 Karen Radok 1974-75 Susan Smout 1975-78 Jeff Mann 1976-86 Owen Gwynne 1979-81 Ursula Klug 1979-80 Diane Beck 1982-85 Julie de Blaquire 1982-83 Jenny Thompson 1983 1985-86 Ann Maloney 1981-82 Joanna Wedgewood 1984 Pina Puglielli 1984-86 Dawn Hobson 1985-86 Margaret Kerr 1985-86 Hildegard Reiter 1985-86

Clinical Genetics (Trainees):

Research Assistants (Clinical): Lucille Hutchinson 1972-75 Sue Rosshandler 1982-83

Co-ordinator, Genetics: Imasjurk 1977 Sue Downes 1978 Robin Forbes 1978-84

Secretaries: Jan Burnett 1962-64 Rosalie Brown 1965 Margaret Ryder 1966 Jill Cullum 1967-71 Louise Wilson 1981-86 Deborah Davis 1984-85

Fundraising Consultant David Hall 1984-85

Murdoch Family

The support of this Institute is very much a family affair for the Murdochs. All who know them recognise each member of the family as a remarkable person, but would agree that the Murdoch family is an entity which is even greater than the sum of its individutil members. The family seems to have an existence of its own. To be a guest at any family function held at Cruden Farm, small or large, is like being accorded temporary membership of this wonderful family. Being greeted by the various family members, as they move through the gathering company, is a cumulative experience of welcome, not a series of separate encounters. At the Institute’s launching in the Great Hall, it was this feeling of entering the Murdoch family which made the event so successful. Like all really successful organisations, the Murdoch family has an undisputed leader who leads firmly but lovingly - described so appropriately and so affectionately by her famous son as “Mum” and as “The Matriarch”. All who work in the Institute are proud of our great Australian name, Murdoch, and delighted that Dame Elisabeth is our Patron. Dame Elisabeth is one of the really special women of our times - special for her ability, for her energy, determination and gentle forcefulness, for her recognition and encouragement of excellence, for her wide range of interests, for her generosity and selflessness and for showing how a woman can achieve so much while still retaining those important characteristics which are properly regarded as those of a lady. She has never needed women’s liberation - can one imagine a social structure which could hold her captive? Many community activities have benefited from Dame Elisabeth’s generosity, especially those in which she has a special interest, but so many less well-known charities. Her love of gardening is seen in the beauty of the garden she has created with her own hands at Cruden Farm, in the way she uses it to encourage the lovers of gardening in the community and in the establishment of a Chair of Landscape Architecture at the University of Melbourne. When her grandson was born deaf she threw her energy and her financijd support into the development of services for the deaf throughout Victoria. The love, and deep knowledge, of art which she and Sir Keith shared, has channelled energy and money towards the development of the National Gallery of Victoria. She helped to establish tapestry weaving in Victoria, revels in the success of the Victoria Tapestry Workshop and is immensely proud to see one of its finest works of art as the sole decoration of the

Great Hall. How appropriate it was that the Institute should be launched there and launched just three days before the opening of the Keith and Elisabeth Murdoch Courtyard at the Gallery. For Dame Elisabeth the most absorbing community activity of all was her involvement with the Royal Children’s Hospital. Joining the Committee of Management of the Children’s Hospital (not yet Royal) in 1933, she remained a member until 1966. She was President from 1954 to 1966, taking over the reins from the redoubtable Lady Latham and carrying the Hospital through all the battles of establishing a new building on a new site in the corner of Royal Park. The official opening by Her Majesty The Queen on February 25, 1963 was a very proud moment for her. On that occasion the Queen invested her Dame of the British Empire in recognition of her services to the community, especially to children. Although much less public events, the establishment in 1960 of the Stevenson Chair of Paediatrics and of the Royal Children’s Hospital Research Foundation were seen by Dame Elisabeth as matters of very great importance. Like Lady Latham, she was acutely aware of the importance of academic standards to the Hospital and of the vital role of research in establishing and maintaining these standards. The Research Foundation was her very special interest and she stayed on as Chairman until 1968 and as a Director until 1979 contributing to many important decisions and giving great encouragement to the research workers, especially the younger ones.

Helen Hamdbury is the eldest of the four children of Keith and Elisabeth Murdoch. In her one senses a blend of the homeliness, reserve and resourcefulness of an Australian farmer’s wife, which she is, and the forceful logic and determination appropriate in a Director of Southdown Press and Progress Press, other roles which she tackles with enthusiasm. Immediately warm and friendly, her conversation is economical and probing, always keen for new information of interest, not just an exchange of words. She and her husband Geoff Handbury, a warm enthusiast for any subject raised in conversation, play very active roles in the community in which they live near Horsham. Before moving to Western Victoria twelve years ago they were equally busy in Melbourne in Legacy (Geoff was Melbourne President in 1973), in Red Cross and in many other community activities. Rupert Murdoch is so well known that it is quite unnecessary to comment on his business achievements or his public personality. It is obvious that the means of

endowing the Institute are of his making and that he played a key, and ultimate, role in the decision which created the Murdoch Institute. It was pleasing to know that the proposals for the future Institute were worked over by a business mind of this calibre before a decision was made. The Institute expects to be judged by its performance, not just by the emotional pull of its objectives. Personal contact with him has been limited because of his residence overseas, but has revealed a personal warmth and enthusiasm for the objectives of the Institute, rapid comprehension of technical information, restless energy (maternally inherited) and a fund of probing and intelligent questions about the feasibility of planned strategies. Those who heard him speak at the Institute’s launching would recognise these characteristics, many of which are shared by his wife, Anna. He was quick to recognise the potential of the one Institute project which actually overlapped his own activities - POSSUM as described in this Report owes much to his visit and to the

The Murdoch Family (missing - Prudence and Lachlan Murdoch)

In all her community activities. Dame Elisabeth’s involvement has been characterised by her passionate enthusiasm for each objective and her injection of personal effort as well as money. How fortunate we are that birth defects and this Institute are among her speciail interests. Before passing on to the second generation of our supporting family some mention must be made of the late Sir Keith Murdoch, whose influence is always quietly apparent behind the actions of the family. Their feeling of togetherness with his memory was never more apparent than when they gathered in 1986 to celebrate the day that would have been his one hundredth birthday. As can be seen from the photograph taken on that day it was not a morbid occasion. Rather it was a time to contemplate his enjoyment, had he been able to see the success of his widow, his children and his grandchildren. If present he would have marvelled, without any surprise, at her role in it all. Of course, he would have taken particular pride in the business and publishing achievements of his son, Rupert. 35

The late Sir Jack Brockhoff

The Miller Family

collaboration with Computer Power Pty. Ltd. which he set up. We look forward to showing that our achievements measure up to his demanding standards.

In the 1960s he negotiated the merger with William Arnott Pty. Ltd. to form The Australian Biscuit Co. Pty. Ltd. and conducted a takeover battle with Nabisco, U.S. A. for Swallow and Ariel Ltd. For the last years before his retirement in 1973 he was Managing Director of ArnottBrockhoff-Guest Pty. Ltd.,

Anne Kantor is very familiar with the consequences of birth defects in society, through her personal work as a social worker with the Citizen’s Welfare Service. Although she does not work specifically with handicapped persons, inborn handicaps crop up disproportionately in all disadvantaged groups in the community. On meeting Anne the conversation will soon turn in some direction which will reveal the concern for other people that is an important part of her personality. Even when she was too busy rearing her own children to take on full-time work she was busy organising kindergartens and working for the families of prisoners at Pentridge through the Prisoners Aid Society (now VACRO). Her husband Milan, a Czechborn lawyer, has brought a special European dimension to the family. Janet Calvert-Jones is the Murdoch known best to the Institute staff, because she represents the family on the Board. One might have expected the family representative of the principal donor to see this as a watching brief, but Janet has thrown her full energy into the development and promotion of the Institute. (The odds against the genes for energy passing from mother to all four children are substantial - obviously their effects are dominant and probably those who did not know him are forgetting the contribution of similar genes from the father). Anyway, Janet has energy in plenty and uses it to the great benefit of the Institute. How she found time to play a big part in organising the launching dinner in a month which included the celebration of her daughter’s wedding and of her own 25th Wedding Anniversary is hard to understand. The effort she puts into the Institute is the more amazing when one remembers her heavy commitment to the development of services for deaf children through her work for the Advisory Council for Children with Impaired Hearing at ‘Taralye’. The achievements and good personal adjustment of her deaf son, James, are a tribute to her well-balanced parenting and that of her husband John An officer in the British Army (13/18th Royal Hussars) at the time of their marriage, John Calvert-Jones has made a very successful second career as a stockbroker and has contributed his own individual style to the Murdoch family.

Soon after his great public gift in 1979 Jack Brockhoff was knighted and in 1980 he married for a second time. Lady Brockhoffjoined him in his full-time occupation of giving away the income of the Foundation in a wise and humane manner. Together, they took great delight in researching worthy causes, especially those concerned with sick, handicapped or neglected children. (Although married briefly in the 1930s, Sir Jack had no children.) Apart from Sir Jack the initial Directors of the Foundation were Sir Henry Bolte, Sir Ernest Coates, Mr. G.H.O’D. Crowther, Mr. James Guest, and Mr. Robert Symons. Mr. Les Sutherland, his long-term associate in the business, became Secretary of the Foundation. By astute management the corpus of the Foundation increased substantially despite grants in excess of $300,000 annually. By June 30, 1985, after Sir Jack’s further legacy, the Directors reported assets in excess of $18.5 million, after distributing $803,000 in 1984/5. Today Brockhoff Foundation ranks as one of Australia’s most notable charitable benefactions.

SirJack Brockhoff

Sir Jack’s grandfather began A.F Brockhoff & Co., biscuit manufacturers, in a bakery in West Melbourne in 1881. Ninety-eight years later his grandson established the Brockhoff Foundation with a gift of $5 million and an apologetic comment that “it’s all I can afford at the moment”. When he died in 1985 his will added a further $5 million. Jack Brockhoff was born in 1908 and was educated at Wesley College where he described his scholastic record as “only average” and he “played all sports, but only made the seconds”. Despite these self-effacing comments he refused his father’s offer of a clerical position at £3 a week when he left school in 1926, claiming his services were worth more than this. After working with a firm of chartered accountants for two years he did join the family company in 1928, at £4 per week. After some overseas travel observing biscuit manufacturing with a special interest in costing and budget control he returned to manage these aspects of the company through the depression years. During the years that he and his two brothers directed the company he tried his hand at all aspects of management and, as Managing Director, he moved the factory to a new garden setting in Burwood in the early 1950s, becoming Chairman of Directors soon after the move.

)

It was very fortunate indeed for the Institute that our work on the prevention of birth defects turned out to combine Sir Jack’s two special interests in helping disabled children and in medical research, and to satisfy his long-standing desire to find some particular cause to which he could make a major contribution. He decided to donate $1 million towards the construction and equipping of our laboratories and to commit his Foundation to future annual support in perpetuity. Sadly, Sir Jack was already terminally ill at the time of our discussions and his illness progressed rapidly. We were able to arrange a media conference about the Murdoch, Brockhoff and Miller benefactions just a few days before his death. Sir Jack was too ill to attend, but insisted that the statement released to the media should describe an even more generous annual contribution than he and the Directors of the Foundation had previously envisaged. Scientists and visitors who enter “The Jack Brockhoff Laboratories” at the Institute will be reminded of this gentle and generous man who felt so strongly that children should not have to endure disabilities.

Mr. Noel Miller

Noel Miller was born in 1905 and trained in accountancy with Flack & Flack, Chartered Accountants. In 1931 he joined J.B. Were & Sons, Sharebrokers, and later became General Manager of their Investment Trusts. During World War II he was Personal Assistant to the Finance Member of the RAAF Air Board, a position he took up soon after his marriage to Olive Whiteside. In 1947 he joined Ian Potter & Co. becoming a partner in 1952. He made a great success of his career as a stockbroker and to this day he remains active in Potter Partners as an Investment Consultant. In their personal life Noel and Olive Miller showed a strong religious conviction and were active in a wide range of activities of the Presbyterian church. Mrs. Miller invested a great deal of personal energy in the community service activities of the congregations to which they belonged at different stages of their lives - the Ewing Memorial Church, and those at Ashburton and Mornington. He was Treasurer of St. Hilda’s College for 12 years from its foundation, a member of the Council of the Christian Television Association for 10 years. He has served for 30 years as a member of the Central Finance Committee of the Presbyterian Church, and subsequendy of the Uniting Church.

FUNDS DONATED

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In 1964 he established the Miller Foundation. Amongst its beneficiaries, the Foundation has established the Olive Miller Trust for Scholarships at St. Hilda’s College and has supported generously several important new Church activities. Following Olive Miller’s death 1984, Noel Miller, his sons Andrew and Richard, their wives Jean and Pamela and their respective children were doubly determined to see that the Miller Foundation continued to use its funds to alleviate the problems of our society which concerned her so greatly. The Murdoch Institute is fortunate that its objectives matched well with those of the Miller family. We are proud that our protein chemistry group is now named in honour of Olive Miller and we enjoy our continuing association with the family over the work of this group. Andrew, Jean and Pamela have all had their own special associations with the Royal Children’s Hospital. Sadly, Sam Miller, second son ofJean and Andrew, died of a malignant tumour late in 1985 after spending long periods in the Hospital undergoing medical and surgical treatment. It is typical ofJean and Andrew that they are now among those who volunteer to help other couples who are battling to cope with a child with cancer. Pam is a biochemist and worked for several years in the Gastroenterology Research Unit.

Mrs. Olive Miller «

$5,000,000*

Murdoch Family Sir Jack Brockhoff & Brockhoff Foundation -t- $250,000 p.a. Miller Family Trust Helen M. Schutt Trust The Scobie and Claire Mackinnon Trust Dame Elisabeth Murdoch Anonymous Marie Louise Trust (Mrs. M.L. Griffin) Percy Baxter Charitable Trust J.B. Were Charitable Foundation The Ian Potter Foundation Repco Corporation Limited H. & L. Hecht Trust Mrs. L.B. Quayle The Dcmks Trust Clive and Joan Roxburgh (Uncle Bob’s) -($4,000 p.a. Estate of K. Minnett G.J. Coles & Co. Limited National Australia Bank Arthur Anderson & Co. Foundation The George Alexander Foundation Smorgon Family Charitable Foundation Sportscraft Foundation (Mr. D. Bardas) Mr. & Mrs. N. Walford Commonwealth Bank Corporation Amcor Ltd. Morris Family Trust Pierce Armstrong Foundation (Mrs. Marjorie Pierce) Pacific Dunlop Limited Willi2un Angliss (Victoria) Charitable Foundation The Amelia Eliza Holland Trust Linfox Transport Group W.R. Grace Australia Limited Potter Partners Associated Broadcasters Services Limited Mayne Nickless Limited Union Fidelity Trust Co. (MKA Bell Memorial Fund) Melbourne Securities Holdings Pty. Ltd.

1,500,000* 500,000* 300,000* 155,000 125,000 90,000* 40,000 30,000* 26,000 25,000 25,000* 25,000* 25,000 20,000 20,000* 17,774 15,000* 15,000 10,000 10,000 10,000 10,000* 10,000 10,000 8,500 6,000 5,000 5,000 5,000 4,892 3,000* 3,000 2,000 2,000 2,000

2,000 2,000

OR PROMISED AS AT 31 DECEMBER 1986

Dame Hilda Stevenson Australian Guarantee Corporation Budget Rent-a-Car (Mr. B. Ansett) Oakes family and friends (in memory of Christopher) Mr. L.G. Cox The George Hicks Foundation Mr.& Mrs. B. Redpath Roche Bros. Pty. Ltd. Monies Beatrice Australia Limited Taxation Services of Australia (Victoria) Pty. Ltd. Moonie Oil Co. Limited Mcllwraith Davey Industries Limited Mr. K. Veall Mr. David Haines Brambles Industries Limited Comalco Limited Visyboard Pty. Ltd. (Mr. R. Pratt) Dame Patricia Mackinnon Boral N.& S. Rockman Charitable Fund Amatil Limited Blue Circle Southern Cement Limited Anonymous Lloyds of London Little Peoples Association Tarrant family and friends (in memory of Claire) Carlton and United Breweries Limited FAI Insurance Group (Mr. L.J. Adler) Chase NBA Group Limited Entrad Limited International Combustion Australia Limited Kraft Foods Limited Moore Business Systems Australia Limited ACI Qintex Ltd. Gordon & Gotch Limited Leighton Holdings Limited Mr. & Mrs. PE Morris Mr. A.B. McMuUin Mr. C. Macek Mrs. G. Grimwade Mr. & Mrs. Bryant Henderson’s Industries Limited Costain Australia Limited Cadbury Schweppes Pty. Ltd.

1,250 1,500 1,140

1,049 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 804 741 600 539 500 500 500 500 500 500 500 500

National Mutual Royal Bank Limited Sir John Holland Dame Beryl Beaurepaire Austen & Butta Limited Lanes Motors Pty. Ltd. Yakka Pty. Ltd. Qantas Airway Limited Capel Court Corporation Limited Macquarie Charitable Foundation Phillips Fox & Masel Arthur Robinson & Hederwicks Mr. & Mrs. G.J. Lather Purves and Purves Billy Guyatt Discount City Mr. R. Green Mrs. J. Dowdy Pepsi Cola Co. of Aust. Ltd. Mr. & Mrs. L. Barbieri Cooper Charitable Fund Sir Robert Southey Aldus Limited Mr. E. Marriott Mr. R.E. Dowland Mr. M. Joss Cussons Pty. Ltd. Tenrow Australia Pty. Ltd. Crusader Oil Macdonald Hamilton Hoescht Australia Limited Mr. PC. Trumble Mr. A.C. Goode Mr. Krslovic Thompson Douglas Mr. H. Sahin Mr. & Mrs. Pedretti Foster High School

200

200 200 200 200 200 200 200 200 200 160 150 150 100 100 100 100 100 100 100 100 65 60 50 50 50 50 50 50 50 25 20 25 20 20 10

* Spread over periods varying from 3-10 years.

500 500 500 350 350 350 300 300 250 250 250 39

Speech by the Prime Minister THE HON. R.J.L. HAWKE, A.C., M.R

LAUNCHING OF THE MURDOCH INSTITUTE FOR RESEARCH INTO BIRTH DEFECTS MELBOURNE, 17 FEBRUARY 1987

About 15 months ago, I had the honour, with the Premier, John Cain, of opening the new building for the Walter and Eliza Hall Institute of Medical Research. In its 72 years, that Institute has proven itself a world class medical research institute.

The Murdoch family’s place is already firmly fixed in Australian history thanks to the outstanding contributions made to the Australian community over the decades by Murdochs including Sir Keith Murdoch, Sir Walter Murdoch and Rupert Murdoch.

Alongside the growth of the Walter and Eliza Hall Institute, Melbourne residents have witnessed the growth of two other major institutes of medical research, the Howard Florey Institute of Experimental Physiology and Medicine and the Baker Medical Research Institute.

Western Australia’s second University bears the Murdoch name. And now this Institute bears witness to the Murdoch family’s generous contribution to the community. It must be unique in Australia for the one family to have given its name to two major institutions - a record of which the family can be proud.

Tonight we are celebrating the rise of Victoria’s fourth medical research institute of national and international significance: the Murdoch Institute for Research into Birth Defects. The creation of the Murdoch Institute - indeed the flourishing of medical research in Melbourne in general is the result of the vision, generosity and skills of many people: donors, administrators, and scientists alike. The goal of the Murdoch Institute is described in the words: “to work towards the day when every chUd is born healthy with normal abUities”. There could hardly be a higher or more worthy ambition. At this gathering tonight we see some of the people whose generosity has given the Institute the resources to strive towards that goal, and some of the people whose research skills win contribute towards achieving it. Tonight we formally launch an Institute which in fact has a history of high quality research stretching back over 20 years. Its genesis and growth within the Research Eoundation of the Royal Children’s Hospital was made possible through the generosity of the Victorian public through the Good Friday Appeal, which raised millions of dollars for the research work. The achievements of that research group are known internationally, especially for discoveries about phenylketonuria; about genetic defects in the utilisation of copper in the body; for the discovery of a number of metabolic diseases; and for the development of methods of diagnosing and treating metabolic diseases. Its computerised syndrome diagnostic system is highly regarded and will shortly be released internationally, possibly to become the world’s standard system. But the transformation of this research group into the Murdoch Institute opens a vital new chapter in its growth.

I noticed that some questions have been raised, usually by those not entirely disinterested, about Rupert’s “Australianness”. I had cause to say at a private function recendy, in the presence of many Americans, that the crucial test of his loyalties was provided by the America’s Cup challenge off Fremande - Rupert barracked for an Australian victory! I observed parenthetically that this was one of the very few occasions when Rupert was on the losing end of any contest.

But our responsibilities as a community do not end there. For we must also take the responsibility of endeavouring to protect future generations of Australian children from suffering debilitating birth defects.

It is clear Australia has lagged behind Britain, Canada and the U.S.A. in private sector support of medical research. But if sport and the arts can become the object of corporate promotion, so surely should medical research.

This is where Murdoch Institute will make its mark, for research into the causes of birth defects offers the best hope of early detection and prevention of birth defects.

The private sector donors to the Murdoch Institute are leading the way, I hope, towards significant improvements in level of private support for medical research.

The truism that today’s research becomes tomorrow’s medical practice has already been borne out in numerous examples such as immunisation against rubella.

Their support will amount to $9.5 million, which is a very firm foundation indeed for an Australian research institute. It represents great faith in the ability of Australian researchers to compete successfully in the difficult but fundamentally important task the Institute has set itself.

New discoveries of molecular genetics are making prenatal diagnosis available for more and more genetic diseases, including, in very recent years, thalassaemia, haemophilia, muscular dystrophy and cystic fibrosis. The skills and facilities needed for these diagnoses are specialised, and the tests are not cheap. However, in every one of these diseases the cost of preventing a single case is less than one tenth the cost of caring for a single patient.

By their encouragement and generosity, the Murdoch family, along with a number of other donors, are targeting a very complex medical problem which has serious implications for many Australian families.

Twenty years ago it would have seemed unbelievable that by 1987 scientists would have isolated the defective genes responsible for dozens of genetic diseases and would be using this knowledge for prenatal diagnosis.

In today’s Australia, birth defects are the most important paediatric health problem. Each year 5,000 Australian babies are born with birth defects which will kill or cause long-term disability. Each of these births brings anguish to the family concerned - tmger, self-accusation, sadness and the long-term burden of coping with a child with an intellectual or physical disability.

For it is to government that families look for help in coping with problems of this type. Families look to the health services to tell them what is wrong and why it went wrong. Families need help in assisting the child to live with the disability and to maximise its abilities. Government services provide hospitals and special education centres and the transport to travel to and from them. They provide direct financial aid when additional costs are involved. In extreme situations, government agencies are called upon

These research skills cannot however be acquired or applied by a community without support, including financial support, from many sources.

Now even genetic diseases are becoming preventable. Early prenatal tests are now available for over a hundred different genetic diseases.

Nothing could better demonstrate his commitment to this country than this extraordinarily generous $5 million contribution by Rupert Murdoch and his family.

The total cost of birth defects to the Australian community cannot be calculated, because one cannot put a price on the heartache and emotional cost suffered by disabled children and their families. However, spending by Federal and State Governments on special services to Australian children suffering intellectual or physical disabilities is in the order of hundreds of millions of dollars a year.

to provide total care and support. Governments are also involved in rehabilitating those many people with disabilities who can learn to take their places in society.

Today, the correction of defective genes in the cells of a patient seems hard to believe, yet some scientists anticipate such progress in the next 20 years. The Murdoch Institute may well play an important role in the advancement of science towards the goals of prevention and curative treatment of birth defects. Located in Australia’s largest paediatric hospital, some of the Murdoch Institute’s staff are involved in diagnosing genetic diseases and counselling families, and in planning the rational development of services. Others are studying fundamental aspects of biology like the control of gene action, the biochemical processes involved in embryonic development and the role of an essential element like copper in maintaining normal health. So, in short, to have the Murdoch Institute working in this field can only foster further advances, better techniques, improved health care, and new hope for many fcunilies.

For its part, the Government is pleased to have played a role in backing the venture. It was once popular to criticise low Government allocation of money to medical research in Australia relative to other Western nations. My Government has increased this allocation in real terms each year since it was elected, during difficult years which have seen reductions in research expenditure in many other countries. Over the years. Government funds have been made available to medical research through the National Health and Medical Research Council. Block grants of large sums for repeated 5-year periods have been awarded by the N.H. & M.R.C. to the Walter and Eliza Hall Institute, to the Howard Florey Institute and more recently to the Btiker Institute. Now the N.H. & M.R.C. has decided to award Block Grants to the Murdoch Institute as well as to the Garvan Institute in Sydney. The Government’s support for the Murdoch Institute amounts to $3.7 million in real terms over five years. So the Institute we launch tonight is a product of a true spirit of co-operation and collaboration between the private and public sectors. In formally launching the Murdoch Institute for Research into Birth Defects, I convey the Government’s best wishes to the Institute and its future; I again express my thanks to the Murdoch family and the other private sector donors for their magnificent gesture; and, beyond the Institute itself, I commend to other research areas in Australia this fine and fruitful example of collaborative effort between the public and private sectors.

It is always remarkable to observe the quality of Australia’s research base, which has produced and is producing so many figures of world renown. 41

The Murdoch Institute Opening Dinner

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The Murdoch Institute

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Professor David Banks, Scientific Director

Mr Neil Walford, Chairman of The Murdoch Institute Board

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The Murdoch Institute

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Cl The Hon. R.J. L. Hawke, A.C., M.P., Prime Minister ofAustralia

Mr Rupert Murdoch, A.C.

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Staff Biographies

ADMINISTRATION Mrs. Anne Ellis, our Business Manager, Joined the Research Foundation in 1975 as Administrative Assistant to the Co-ordinator of Research. Her background training in science and her experience as a virologist at Fairfield Hospital plus a recently developed interest in business management provided a good background for this administrative role. Anne played an important role in encouraging research in Hospital departments, gaining the confidence of the staff and helping with their applications for internal and external research grants. She soon moved from assisting the Co-ordinator of Research to organise the annual allocation of the Research Grants to managing this process on her own with minimal assistance from the Co-ordinator. She was also able take over the production of the annual report of Research within the Royal Children’s Hospital. For several years now she has effectively administered the Research Foundation, making the role of the Co-ordinator of Research very simple. This made it possible for Professor Danks to run the Birth Defects Research Institute and to conduct a fundraising campaign whUe also responsible for the Research Foundation.

Mrs. Anne Ellis

When Professor Fraenkel succeeded Professor Danks as Co-ordinator of Research, she tackled the difficult task managing both the Foundation and the Institute. With tact and hard work she succeeded. However, the workload of managing the two organisations will soon become too great even for her. Then she will become full-time Business Manager of the Murdoch Institute. All research workers in the Hospital have reason to thank Anne Ellis.

Despite her workload she continued to do a small amount of laboratory work and found time to undertake an accountancy course, obtaining honours in every subject. Barry Holt, Laboratory Manager, began his career in this Hospital as a trainee technician in the Pathology Department in 1957. When the Royal Children’s Hospital Research Foundation was set up in 1960 he was working as a Research Assistant to Dr. Donald Cheek. After a period at the Adelaide Children’s Hospital from 1962 he rejoined Dr. Cheek in the United States in 1968, managing his laboratory at Johns Hopkins Medical School. In 1973 he returned to Melbourne ahead of Professor Cheek to set up the laboratories that Professor Cheek would use as Director of the Research Foundation and was responsible for the design of the 10th Floor laboratories which were built between 1974 and 1976. His skill in design and his foresight in installing a number of unusual features, such as piped deionized water, has been recognised with gratitude by all those who have worked in the laboratories in the succeeding decade. When Professor Cheek resigned in 1980 Barry stayed on as Laboratory Manager in the Research Foundation and now manages laboratories for both the Research Foundation and the Murdoch Institute. Later Barry will become full time Laboratory Manager of the Murdoch Institute. The extra pressure that will trigger this change is likely to arise when detailed planning of our new laboratories commences. Barry has a number of valuable attributes. His skills in laboratory planning have been very valuable. Laboratory safety is another important responsibility. He is energetic in seeking the best source of supply for consumables and for major equipment. Supplier’s representatives quake when Barry Holt sits down to bargain with them about contracts. Most important of aU is Barry’s loyalty and devotion to his duties regardless of the personal effort involved.

After nearly three years in the Research Foundation Dick went to the United States and then to the U.K. for further experience, returning in 1973 to become the senior laboratory scientist in the group. The latter two years abroad were spent with Dr. Cesar Milstein in the M.R.C. Molecular Genetics Laboratory at Cambridge University. There he introduced cell culture and cell fusion methods into a laboratory which was studying the chemistry of immunity. He was able to show that fusion of two myeloma (tumour) cells making different antibodies produced a hybrid cell which retained the specificity of both parent cells. This discovery led immediately to the experiments which produced the first monoclonal antibodies, initiating one of the two branches of modern biotechnology. These latter discoveries won the Nobel Prize for Dr. Kohler (Dr. Cotton’s successor as a Postgraduate Fellow) and Dr. Milstein.

Mr. Barry Holt

OLIVE MILLER PROTEIN CHEMISTRY GROUP

♦

Dick Cotton, the Head of the Group, was one of a number of agricultural science graduates from Melbourne University to develop a special interest in biochemistry as the result of the enthusiastic teaching of Professor Frank Hird. This interest was stimulated further during his Ph.D. studies with Dr. Frank Gibson at the Department of Microbiology and subsequently as a Postdoctoral Fellow in Canberra where Professor Gibson took up the Chair of Biochemistry at the John Curtin School of Medical Research. His thesis and postdoctoral work involved studies of the metabolism of two related amino acids, phenylalanine and tyrosine, in the bacterium E. coli. This background made it logical for him to study the fundamental aspects of the disease phenylketonuria (PKU) when he joined the Research Foundation in 1968. He threw himself into the study of the enzyme at fault, phenylalanine hydroxylase (PH), with characteristic energy. The method of purification of PH developed is used in many laboratories. Over the subsequent years Dick Cotton has become a leading authority on PH, on PKU and upon other enzyme defects which can cause PKU. This work is described in some detail elsewhere in this Report.

Dr. Dick Cotton

In 1980, during a period of sabbatical leave at the Dunn School of Pathology (famous as the place where Professor Howard Florey worked on penicillin), Dick became interested in the study of embryonal carcinoma cell lines as a model of embryonic development. Embryonal carcinomas are tumours in which a number of different tissues develop in a manner which resembles embryonic development. He developed a method of growing these tumours from a single cell which allowed genetic studies to be undertaken after he returned to Melbourne. This led on to a number of projects in Melbourne.

I’

At present Dick is on sabbatical leave in the Department of Biochemistry in Oxford, working with recombinant DNA techniques. We look forward to the new ideas and projects with which he will return in September 1987. Dick Cotton’s most important characteristics are his enthusiasm, his extremely broad knowledge of, and interest in, biology, his capacity for innovation and his good humour. He reads very widely and has a keen eye for innovations developed in other disciplines which might help his work. His interaction with Professor Danks over many years has been important in the development of the Institute. Ian Jennings joined Dick Cotton in his work on phenylalanine hydroxylase immediately after graduation. He soon demonstrated his competence and has established a permanent place for himself in the research team. Through the years he has shown more and more independence in his work and his relationship with Dick Cotton has become that of a collaborator rather than an assistant. His advice is sought by the other members of the staff, especially about the production of monoclonal antibodies and their application to the evaluation of enzymes. A man of few words, Ian can be moved to emotion about cricket. Wendy McAdam joined the laboratory while still completing her Medical Technology course and has grown in skills and self-confidence to become an important member of the Protein Chemistry Group and of the Institute as a whole. Recruited initially to assist with work on precision two-dimensional gel electrophoresis, she remains the resource person for this and related techniques and has become an authority on peptide purification by HPLC and FPLC of peptides. She has worked extensively with dihydropteridine reductase. In her quiet way, she has become one of the important personalities of the Institute, always quick to volunteer when extra help is required with any task, whether collation of our Institute grant application or organisation of a staff party.

CLINICAL GENETICS GROUP

couple, the introduction of a part-time Social Worker (Mrs. Margaret Sahhar) to the group, and the establishment of genetic clinics in Tasmania, among others. In recent years he has taken particular interest in helping couples to cope with the grief associated with the birth of an abnormal baby or with the death of a baby.

John Rogers trained in paediatrics at the Royal Children’s Hospital, in Sheffield and in London, at the Hospital for Sick Children, Great Ormond Street. During his second spell on the resident staff of the Royal Children’s Hospital he decided to choose a career in medical genetics. An opportunity arose for him to undertake formal training in the Department of Medical Genetics at Johns Hopkins Hospital under Professor Victor McKusick. During his three years in Baltimore John amassed a great experience in clinical genetics, especially in the management of patients with hereditary bone and connective tissue disorders. He also undertook laboratory research on nerve growth factor in association with Dr. S.H. Boyer. During this phase of his training his skills in clinical diagnostic work became obvious.

Over the years Dr. Rogers has proved astute in recognising unusual variants of clinical conditions and in extracting from the features of patients clues to the likely underlying defect. These skills have been important in the development of the good liason between the clinical tmd research work of the Institute. He has also developed a strong interest in teratology, particularly in the teratogenic effects of drugs. A sabbaticcJ spent with Dr. Thomas Shepard in Seattle encouraged these interests which he now puts to good use as a member of the Congenital Malformations Subcommittee of the Australian Drug Evaluation Committee.

John Rogers returned to Melbourne 1976 and was soon “thrown in at the deep end”, being asked to cope with the clinical genetics work single handed while Professor Danks was on sabbatical leave during 1977. John Rogers did not sink - in fact he swam very strongly and emerged from this difficult year with a reputation as a clinical geneticist which has been further enhanced in the intervening years.

I 46

Les has been appointed Medical Geneticist in charge of the Genetics Clinic at the Royal Women’s Hospital, and has become the clinician involved with DNA diagnostic tests. He is also dealing with visually handicapped individuals especially those with retinitis pigmentosa (in collaboration with the Victorian Eye and Ear Hospital). His work in epidemiology will be discussed in a subsequeht Annual Report. Agnes Bankier was the first to undertake the formal training programme in clinical genetics established in the Birth Defects Research Institute in the early 1980s. She had previously completed a brilliant undergraduate course at Monash University, suffered a disappointing exposure to laboratory research early after graduation and then set her heart upon a clinical career in paediatrics. She proceeded through her paediatric training with ease and soon came to realise that she would like to work in some field which gave her an opportunity to mix research with clinical work. Fortunately she chose genetics.

John has been responsible for a number of valuable innovations in our clinical genetics service - the practice of sending a written summary of the consultation to each

Dr. John Rogers

1 Dr. LesShJfield

Isabel Roberts’ stay in the Protein Chemistry group as a postdoctoral fellow was too short, but she contributed considerably to the progress of the group during her stay. She came in 1985 to purify and characterise a protein factor which had been shown to retard the differentiation of embryonal carcinoma cells in culture. Her previous experience in protein chemistry suited her to this task and she made good progress, handing over to Wendy McAdam in October 1986 a process which produces the quantities of protein required for the characterisation.

We had hoped that he would then return to set up an epidemiology group here in Melbourne, but the opportunity of heading his own genetics clinic at the Adelaide Children’s Hospital proved too tempting. Over the next nine years he established a very good genetic service in Adelaide, but he discovered that there was not sufficient time to undertake the research in epidemiology which interested him. Consequendy, he moved back to Melbourne to join the Institute in 1985.

Les Sheffield is one of our team of clinical geneticists as well as heading our research activities in epidemiology. His research interests'began when he undertook a B.Med.Sci. year as a medical student. He trained in paediatrics at the Royal Children’s Hospital and in genetics in the Genetics Research Unit. Then he went to McMaster University in Canada for formal training in epidemiology.

During the latter part of her training she became interested in our computerised syndrome identification system which was then known as CAMSIS and was in the hands of Dr. David Pitt. When David retired, Agnes took over responsibility for this system. Soon afterwards came the opportunity to improve the system by using interactive video disc technology, in partnership with Computer Power Pty. Ltd. Agnes has thrown her great energy into the development of POSSUM and has every justification in feeling immensely proud of her achievement and of the recent launching of POSSUM on the international market. At the same time she has gained the respect of all with whom she deals for her skills as a clinical geneticist. She is now an established member of our Genetics Clinic team and is becoming known internationally for her knowledge of syndromes.

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Ed Wraith was born on Tyneside and trained in paediatrics and metabolic disease in Manchester. He came to Melbourne in 1985 on a registrar interchange, mainly to obtain additional experience with metabolic diseases. The roster allowed him only 6 months as our Registrar in 1985, but Dr. Haan’s departure left a vacancy for a consultant caring for metabolic patients in 1986. Ed filled this role with distinction, proving expert in clinical care and very energetic in clinical projects on metabolic diseases. His lively personality and delightful sense of humour made him one of the most popular people ever to work in the group.

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dealing with the many organisations concerned with the care of the deaf. Jim has handled all of these problems with great skill and we are confident that he will make a first class clinical geneticist. John Christodoulou has completed the first year of his training towards a career in the management of metabolic diseases in children. We view this as a special area within genetics and have encouraged him to undertake a full programme of training in genetics as described for Jim McGill. However, John’s project is in the laboratory and occupies a large proportion of his time. He is purifying and studying the enzyme malonyl CoA decarboxylase and expects to use this work in a thesis for a PhD. John came to us from the Children’s Hospital, Camperdown, in Sydney, where he undertook his basic training in paediatrics.

Sue Tomkins and Sharon Grosvenor are the two secretaries attached to the clinical group. Sue has been with us since 1977 and has made a very important place for herself in the work of the unit and in the hearts of all of the members of the unit. We all admire the professional standard of her work and her determination in coping with a busy schedule despite her physical frailty. Sharon has joined the group more recently, but has quickly established herself as someone upon whom we can rely.

Dr. Ed Wraith

Jim McGill has just completed the second year of his formal training in clinical genetics within the Institute. His basic training in paediatrics was in Brisbane and he came to Melbourne for part of his advanced training. He worked in general paediatrics, first as a Registrar and then as a Fellow, but then chose a career in clinical genetics. As a Trainee Fellow his time is split between formal lectures in genetics at the University, involvement in discussions of genetic topics in the seminars and other meetings of the Institute, clinical experience and a major research project on genetic causes of deafness. This survey of selected groups of deaf individuals in the community is a major undertaking requiring careful planning and great tact in

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As Genetics Clinic Co-ordinator, Anne Glynn plays a pivotal role in the operation of our genetics service. The Co-ordinator is responsible for maintenance of records, for organisation of clinics, for chasing up the information that is required before patients are seen and the results of tests which become available after their visits. Most important of all, she is the first contact person for a new patient coming to the clinic, speaking to the patient on the telephone when arranging the initial appointment, and also dealing with referring doctors. She is assisted, very ably, by Mrs. Jo Wells, who has played a major role in moving our genetics files onto computer.

When Michael White retired and shifted to the Research School of Biological Sciences in Canberra, Graham moved with him and worked there until at the end of the 1970s, when he Joined us as Head of the Cytogenetics Laboratory and as a research worker in this field. Over the next few years the Cytogenetics Laboratory at the R.C.H. became the reference laboratory in Victoria for difficult cytogenetics problems. There is no doubt that Graham Webb’s was the best opinion available about an unusual chromosome rearrangement which was difficult to interpret. His knowledge of the rearrangements of chromosomes that have occurred during insect evolution gave him a great facility in the interpretation of these unusual human karyotypes.

Jane Halliday studied genetics to an M.Sc. level at Monash University before starting a family. Fortunately she contacted us when she wanted to return to the workforce and she has proved a valued member of our clinical research team ever since. Working part-time, she has been responsible for our ongoing surveillance of the results of prenatal diagnosis in Victoria and for several family studies including one on infantile hydrocephalus and an ongoing interest in the fragile X syndrome. Her adaptability and resourcefulness are important attributes. Judy Dodge also studied genetics to the M.Sc. level at Melbourne University and Monash University and then retired from the workforce for some years, raising a family. We were very pleased when Jane Halliday encouraged her to think ofjoining us. Judy has become a second resourceful, adaptable part-time member of our clinical research group. She has taken a particular interest in the establishment of DNA diagnostic tests, in cost-benefit analysis of some of these procedures and in the computerization of our clinic records.

Professor Michael J.D. White. He took an interest in several unusual cytogenetic cases recognised in Melbourne and he worked with Dr. David Danks in organising courses in human genetics for medical and science students.

Unfortunately, Graham decided that the strain of combining research and the supervision of a large laboratory was proving too great. We were sorry to see him leave. Jeff Mann joined the research group on a part-time basis to supervise the breeding of mice when we began to study mutant strains. He chose to undertake a part-time M.Sc. in the other half of his time, working on the mottled

Dr. Graham Webb

Mann

DEPARTURES FROM THE INSTITUTE Graham Webb came to the Genetics Research Unit and the Department of Genetics from a career in evolutionary cytogenetics. This broad background allowed him to contribute in many important ways to the development of the Murdoch Institute. In his own research he made good progress in the establishment of the technique of in situ hybridisation of DNA probes to human chromosomes, a line of work which grew out of a B.Sc.Hons. student project. After completing his Science degree Graham Webb worked as a science teacher, but soon returned to the University for postgraduate studies in genetics. He worked in the Department of Genetics as a Demonstrator and part-time Ph.D. student of that famous evolutionary cytogeneticist.

Visiting Scientists on Sabbatical Leave

Murdoch Institute Lectures 1986 mutants. After two years, this research had become so successful that he was employed full-time. The thesis which resulted was of a very high standard and is still an important resource for our ongoing work on copper transport. Later, it seemed of advantage to his career and to the Institute for him to work overseas to learn some of the modern techniques of mouse embryo manipulation. Working with Dr. Anne McLaren in the M.R.C. Mammalian Genetics LFnit in London for 18 months, he contributed significantly to their research, completed the major part of a Ph.D. at the University of London, and he became highly skilled in techniques of interest to us. During his time in London Jeff developed an intense interest in the earliest events of embryonic development occurring soon after fertilisation, especially in the curious differential effects of maternally-derived and paternallyderived X-chromosomes. He taught us about these matters after his return and convinced us of the relevance of this line of research to the Institute. However, it was also clear that this line of research was more closely aligned with that of the Centre for Early Human Development at Monash University. When an attractive offer came he decided to move to the Centre.

DR. C. BELL, Department of Physiology, University ofMelbourne. Histochemical and biochemical analyses of the peripheral nervous system.

In recent years we have enjoyed having a number of well known scientists work in our laboratories during period of sabbatical leave. They bring fresh ideas, attitudes and techniques and contribute greatly to the morale of the Institute as well as to its work. We are proud that scientists of this calibre want to spend their leave periods with us. This year brought some notable visitors.

DR. G. CAMPBELL, Department ofAnatomy, University ofMelbourne. Smooth muscle cells and their role in atherogenesis. DR. D. KEMP, Walter and Eliza Hall Institute. Update on the malaria vaccine. DR. N. ISAACS, St. Vincent’s Hospital Medical Research Institute. Progress in the structure of platelet factor 4: binding to heparin and DNA. DR. G. FINCHER, Department ofBiochemistry, Latrobe University. Characterisation of a cDNA encoding barley /3-glucanase: potential commercial applications. DR. K. MURRAY, Department of Chemistry, Monash University. Bioinorganic model complexes. DR. J. FUNDER, Prince Henry’s Medical Centre. Steroids, neuropeptides - a potpourri.

Dr. Ian Craig, Dr. Sally Craig and a PhD student Neil Fraser, came from the Department of Genetics, University of Oxford and spent a hectic 3 months studying the intriguing paradox of XX males. The human Y chromosome contains male determining genes and the development of male gender characteristics without any visible Y chromosome has puzzled doctors of over 20 years. Now that gene probes are available it is becoming clear that the essential male determining genes have been shifted to the X chromosome in XX males. The Craigs analysed the X chromosomes of the cases available in Melbourne with some findings which will bring nearer the eventual goal of this work, the isolation of the male determining genes.

Professor Jim Haworth spent the first 4 months of the year in our clinical group with some forays into the laboratory activities. He had recently relinquished the chairmanship of the Department of Paediatrics, at the University of Manitoba in Winnipeg, Canada. This very demanding role has kept him away from his personal interest in inborn errors of metabolism. He was looking for an opportunity to catch up on the progress which had occurred while he was otherwise occupied. It was a pleasure to give him the exposure to clinical cases and research which he desired. In return we learned much from him as a wise and very experienced clinician with an interest in biochemistry.

Ian, Sally and Neil fitted into our group very smoothly and close contact has been maintained since they returned to the U.K. We hope that Neil may return for post-doctoral experience in 1988.

DR. G. TREGEAR, Howard Florqi Institute. New developments and applications of peptide synthesis. DR. J. BALDWIN, Department ofZoology, Monash University. Energy metabolism in animals - a comparative approach.

Dr. Ian Craig

DR. I. KOLA, I.V.F Centre, Queen Victoria Medical Centre. Teratogen effects on mouse preimplantation embryos. Professor George Brownlee

Professor George Brownlee of the Dunn School of Pathology of the University of Oxford, is with us at the time of writing. He arrived in Melbourne in September and will stay here for a whole year. His research time will be split between our Institute and the Hall Institute. He developed an outstanding reputation as a molecular geneticist in the MRC Laboratory in Cambridge, before moving to the chair he now holds in Oxford. His deep interest in the molecular detail of the immune response to influenza virus provides an interface with the work at the Hall Institute. His other special interest is in the application of molecular techniques to the resolution of

significant problems in human genetic diseases - hence his decision to spend part of this leave with us. He is pursuing two very demanding projects. One involves evaluation of a technique which may allow relatively rapid identification of the minute changes in genes which underly most genetic diseases. In the other project he and Ruth Brown are trying to isolate the part of the human X-chromosome which looks fragUe in the fragile-X form of mental retardation. His enthusiasm for science and his critical analytical mind are having a very good influence in our laboratories and we are all learning a great deal from him.

!■

Mrs. Janet Guthrie lectures in nutrition and physiology at the Lincoln Institute. During an 8 month sabbatical leave she joined Harry McArdle in studying the uptake of copper into cultured human fibroblastic cells. She made a valuable contribution to the research in the trace element laboratory and also to the interpersonal relationships there.

Visitors to the Institute

Staff Involvement in Australian Scientific Community Activities

Professor Rudi Jaenisch, Whitehead Institute, Boston, U.S.A. - to discuss collaborative projects with Dr. Marie Dziadek and Dr. John Bateman (Orthopaedic Research Unit).

PROFESSOR BANKS

DR. SHEFFIELD

Member, Recombinant DNA Monitoring Committee and Scientific Sub-committee, Department of Industry, Technology and Commerce.

Member, Australian Ionising Radiation Advisory Council.

Member, Scientific Council and Research Grants Committee, Adelaide Children’s Hospital. Co-opted Member, Medical Research Ethics Committee, National Health and Medical Research Council.

Member, Congenital Malformations Subcommittee, Consultative Council on Obstetric and Paediatric Mortality and Morbidity, Health Department, Victoria.

Professor Peter Steinert, National Institutes of Health, Bethesda, U.S.A. - lecture and discussion on genetic control of keratin production.

Chairman, Expert Co-ordinating Committee on Genetic Services, Health Department, Victoria.

Chairperson, Prenatal Diagnosis Committee, Human Genetics Society of Australasia.

Member, Congenital Malformations Subcommittee, Consultative Council on Obstetric and Paediatric Mortality and Morbidity, Health Department, Victoria.

Convenor, Working Party on Genetic Counselling, Human Genetics Society of Australasia.

Professor Kohei Shiota, Congenital Anomaly Research Centre, Kyoto, Japan - lecture and discussion on abnormalities identified by careful examination of aborted foetuses. Dr. Geoff Sherwood, Hospital for Sick Children, Toronto, Canada - discussion of common interests in inborn errors of metabolism. Dr. Frances Smith, Department of Microbiology, Mt. Sinai Hospital, New York, U.S.A. - lecture and discussion on new methods of identifying mutations in cloned genes.

Member, Expert Co-ordinating Committee on Genetic Services, Health Department, Victoria.

Professor Arno Motulsky, Department of Human Genetics, University of Washington, Seattle, U.S.A. lecture and discussion on genetic predisposition to atherosclerosis.

Chairman, Neonatal Metabolic Screening JointCommittee, Human Genetics Society of Australasia and Australian College of Paediatrics.

Member of Council, Human Genetics Society of Australasia.

Member, National Health and Medical Research Council Regional Grants Interviewing Committee.

DR. COTTON Member, National Health and Medical Research Council Regional Grants Interviewing Committee.

DR. ROGERS Registrar, Australian College of Paediatrics (until May 1986). Chairman, Victorian Branch, Human Genetics Society of Australasia. Member, Paediatric Examination Committee, Royal Australasian College of Physicians. Member, Congenital Malformations Subcommittee, Australian Drug Evaluation Committee. Convenor, Clinical Genetics Subcommittee, Human Genetics Society of Australasia. Member, Expert Co-ordinating Committee on Genetics Services, Health Department, Victoria. Mrs. Janet Guthrie

Member, Committee of National Association of Loss and Grief, Victoria.

Overseas and Interstate Visits, Lectures and Seminars by Institute Staff PROFESSOR BANKS

DR. DZIADEK

Internationcil Congress of Human Genetics, Berlin, ER.G.

Gordon Conference on Basement Membranes, New Hampshire, U.S.A.

- Training of clinical geneticists in Australia. - Use of computers in the genetic clinic. - Menkes disease and related conditions. Society for the Study of Inborn Errors of Metabolism, Annual Meeting, Amersfoort, The Netherlands. American Society for Human Genetics, Annual Meeting, Philadelphia, U.S.A. - Expression of caeruloplasmin gene in rat tissues, Metals, Trace Elements and Mammalian Development, Sydney - Copper deficiency in man and animals. Australian Biotechnology Conference, Melbourne Medical applications of DNA techniques.

Connective Tissue Society of Australia and New Zealand, Annual Meeting, Melbourne - Basement membranes: heterogeneity in structure and function. Symposium on Mammalian Embryogenesis, Melbourne The extracellular matrix in mouse development.

DR. MERCER

Mount Sinai Medical Centre, New York, U.S.A. - Use of a cDNA with complete mRNA sequence in the expression of active human factor IX in transgenic mice. Department of Medical Research, Veterans General Hospital, Taipei, Taiwan, Republic of China. Regulation and expression of cloned genes in ceU culture and transgenic animals.

DR. BROWN Society for the Study of Inborn Errors of Metabolism, Annual Meeting, Amersfoort, The Netherlands. Structural and genetic studies of normal and mutant forms of the human pyruvate dehydrogenase complex. International Congress of Human Genetics, Berlin.

Australian Institute of Medictil Laboratory Scientists, Sydney - The role of DNA diagnostic tests.

Molecular Approaches to Developmental Biology, U.C.L.A. Meeting, Keystone, Colorado, U.S.A. Metallothionein gene expression in rat liver during development.

DR. COTTON

Texas A & M University, College Station, Texas. Seminar on metallothionein genes.

International Society for Study of Trace Elements in Humans, Palm Springs, U.S.A. - The role of albumin in copper uptake by fibroblasts.

8th International Symposium on Pteridines and Folic Acid Derivatives, Montreal, Canada

Mt. Sinai Hospital, New York - Seminar on phenylalanine hydroxylase.

MRS. GUTHRIE

- A model for hyperphenylalanaemia due to tetrahydrobiopterin deficiency.

Metals, Trace Elements and Mammalian Development, Sydney - Zinc, copper and metallothionein in RNA in sheep liver during development.

- A monoclonal antibody reacting with all three aromatic amino acid hydroxylases. - Correlation of dihydropteridine reductase cross reacting material with non-responsiveness to a tetrahydrobiopterin load.

DR. ROGERS 1st International Achondroplasia Conference, Rome Sponsored representative of the Little People’s Association of Australia.

DR. SHEFFIELD American Society for Human Genetics, Annual Meeting, Philadelpia, U.S.A. - A genetic study of 64 patients with the Robin malformation complex.

DR. BANKIER International Pediatric Congress, Hawaii - Presentation and demonstration of POSSUM. International Congress of Human Genetics, Berlin, ER.G. - demonstration of POSSUM.

Australian and New Zealand Society for Cell Biology, Annual Meeting, Sydney - Characterisation of a new, small basement membrane protein, BM-40.

Postgraduate degrees awarded

MSc Stewart Fabb “An investigation of first meiosis using molecular methods” Anna Longworth “Localisation of gene copies to chromosomes by in situ hybridisation”

PhD Peter Koopman “Characterisation of in vitro differentiation of embryonal carcinoma cells” Greg Petersen “The sheep metallothionein gene family”.

DR. McARDLE

Metals, Trace Elements and Maunmalian Development, Sydney - Role of albumin in copper uptake into cells.

Australian Biochemical Society, Annual Meeting, Melbourne - Structure and regulation of sheep metallothionein genes. Organisation and Expression of the Genome, Lome Isolation of cDNA clones for the copper binding protein, caemloplasmin.

DR. CHOO American Society for Human Genetics, Annual Meeting, Philadelphia - Expression of active human blood clotting factor IX in transgenic mice. Australian and New Zetiland Society of Cell Biology, Annual Meeting, Sydney - The human factor IX gene: transfection and expression in cultured hepatoma cells and production of transgenic mice. Organisation and Expression of the Genome, Lome Amplified expression of cloned rat phenylalanine hydroxylase in cultured mouse LTK-ceUs. Australian Biochemical Society, Annual Meeting, Melbourne. - Vectors for the expression and amplification of cDNA in mammalian cells: expression of rat phenylalanine hydroxylase. 55

Research Collaborations

Detailed Project Reports

PKU RELATED PROJECTS

analysis of biological samples from patients with Menkes’ disease and mottled mouse mutants.

Metallothionein mRNA levels in Menkes’ and normal cell cultures

Dr. Barry Lee, Department of Genetics, University of Melbourne - Copper transport in E.coli.

R. Farrell, J. Camakaris, J. Mercer

Dr. Ian Walker, Centre for Cancer Studies, University of Melbourne - Analysis of allelic forms of phenylalanine hydroxylase. Dr. Frank Morgan, Medical Research Institute, St. Vincent’s Hospital - Sequencing of dihydropteridine reductase peptides. Dr. NeU Isaacs, Medical Research Institute, St. Vincent’s Hospital - Crystal structure of dihydropteridine reductase and of PH7 monoclonal antibody.

Dr. George Yeoh, Department of Physiology, University of Western Australia - Regulation of phenylalanine hydroxylase during development.

Dr. Brian May and Professor Bill Elliott, Department of Biochemistry, University of Adelaide - Regulation of 6-amino levulinic acid synthetase during development in the rat.

Dr. E. Edkins, Department of Physiology, University of Western Australia - Analysis of rat phenylalanine hydroxylase promotor.

Dr. S. de Souza, Department of Immunology, Scripps Clinic, La Jolla, California - Regulation of antithrombin III during development in the rat.

Dr. Wilf Armarego, Department of Biochemistry, John Curtin School of Medical Research, Canberra - Synthesis of pterins.

Dr. Ian Walker, Centre for Cancer Immunology, University of Melbourne - Sequencing of pyruvate dehydrogenase peptides.

Dr. Istvan Tork, Department of Anatomy, University of New South Wales - Use of PH8 monoclonal antibody in immunohistochemistry of the brain stem.

Prof. Steve Goodman, University of Colorado, Denver Molecular analysis of electron transport flavoproteins.

Dr. M. Parniak, Lady Davis Institute for Medical Research, McGill University, Quebec, Canada Interaction of PHI monoclonal antibody with rat liver phenylalanine hydroxylase. Dr. G. Bracco, Regina Margherita Children’s Hospital, Turin, Italy - Dihydropteridine reductase deficiency. i

Dr. Bruce Kemp, Department of Medicine, Repatriation Hospital - Peptide synthesis. Prof. Charles Coutelle, Academy of Science, East Berlin Prenatal diagnosis of PKU by gene probes. Dr. E. Edkins, Department of Physiology, University of Western Australia - Analysis of rat phenylalanine hydroxylase promoter.

COPPER PROJECTS Prof. John Howell and research students. Department of Veterinary Pathology, Murdoch University, WA. Studies of copper toxicosis in sheep. Prof. Gerhardt Schreiber, Department of Biochemistry, University of Melbourne - Studies of rat caeruloplasmin. Dr. Tony Widd, Department of Chemistry, LaTrobe University - Electron spin resonance studies of albumincopper complexes. Dr. George Legge and research students. Department of Physics, University of Melbourne - Proton microprobe 56

MOLECULAR GENETIC PROJECTS Dr. Kathie Raphael, CSIRO, Division of Animal Production, Blacktown, N.S.W. - Production of transgenic mice which express human clotting factor IX.

Metallothionein (MT-I and MT-II) mRNA levels have been measured at a range of intracellular copper (Cu) concentrations in continuous lymphoid cell cultures from normal individuals and from patients with Menkes’ disease. The data obtained does not support the hypothesis that Menkes’ cells synthesise MT mRNA in a partially constitutive fashion, but suggest that copper is present in a form which induces MT mRNA more strongly in the mutant cells. At low extracellular Cu concentrations Menkes’ cells have increased levels of MT mRNA, but this can be attributed to increased intracellular Cu levels. At high intracellular Cu concentrations normal cells have higher MT mRNA. We believe that the Menkes’ cells may have a larger pool of MT protein which can handle the excess Cu without so much synthesis of new protein.

EMBRYOLOGY PROJECTS

♦fie*

Dr. Rudi Jaenisch and Dr. Angelika Schnieke, Whitehead Institute, Boston (and Dr. John Bateman, Orthopaedic Research Unit, R.C.H.R.E) - Changes in collagens in mice with disrupted collagen aJI) gene. Dr. Rupert Timpl and Dr. Mats Paulsson, Max-PlanckInstitut fiir Biochemie, Munich - Assessment of role of basement membrane proteins using specific antibodies.

jfi

f , ‘“7...8"”9 dW fia|' MENKES,

normals

CLINICAL/EPIDEMIOLOGICAL PROJECTS Mr. Ron Batagol, Pharmacy Department, Royal Women’s Hospital - Risks of drugs taken during pregnancy. Dr. John Mulley, Department of Histopathology, Adelaide Children’s Hospital - Statistical interpretation of results of DNA diagnostic tests. Dr. Segolene Ayme, Institut National de la Sante et de la Recherche Medicale, Marseille - Further development of POSSUM syndrome data system. Dr. Michael Baraitser and Dr. Robin Winter, London Dysmorphology Data Base, London - Data for POSSUM.

“Northern blot’of metallothionein mRNA isolatedfrom Menkes and normal continuous lymphoid cell lines, incubated at various copper concentrations, showing excess mRNA in the mutant cells at low copper concentrations, but not at high concentrations. Copper concentration in medium 0.7 ng/ml 1 and 7 2 and 8 2.6 ixg/ml 3 and 9 3.7 ng/ml 13.7 fig/ml 4 and 10 5 and 11 18.6 fcg/ml 6 and 12 22.5gLg/ml Tracks are paired and copper concentrations span a range of 0.7 fig/ml (1 & 7) to 22.5 gcg/ml (6 & 12). Tracks

Copper transport in cells derived from Wilson’s disease patients J. Camakaris, L. Paul Previous studies have shown that Wilson’s disease fibroblasts have increased levels of copper (Cu), and that the excess Cu is bound to a metallothionein-like protein. Similar findings have now been made when using Wilson’s disease continuous lymphoid cell lines (CLCs) with elevated concentrations of total Cu in the culture medium. Wilson’s disease CLCs have elevated levels of a •’’‘Culabelled metallothionein-like protein. RNA has been extracted from one of these lines and increased levels of MT-II mRNA were detected. Further studies will be directed towards analysing the relationship between intracellular Cu concentrations and MT mRNA levels in CLCs derived from a number of different Wilson’s disease patients. Caution is needed in interpretation of these data as the increase in MT mRNA in Wilson’s disease cells is small and furthermore, levels of MT mRNA in CLCs appear to vtu-y with growth rate.

Copper resistant variants of Chinese hamster ovary cells J. Patton, J. Camakaris

Prof. Gerhardt Schreiber and research students. Department of Biochemistry, University of Melbourne Tissue specific expression of acute phase proteins in rat embryos.

Cu causes a similar differential induction of MT-1 and MT-II mRNA synthesis in both normal and mutant cells. MT mRNA levels in continuous lymphoid cells appear to vary with growth rate. Extensive gel filtration analysis of extracts from normal cells has revealed a novel ''‘‘Cubinding protein which will be analysed further.

Two copper (Cu) resistant Chinese hamster ovary (CHO) cell lines have been isolated in previous years. Studies utilising somatic cell hybrids have revealed that the resistance phenotype in one of the Cu-resistant variants is dominant whilst in another variant (isolated using a different selection protocol) the resistance phenotype is recessive. Uptake of Cu and metallothionein levels are previously shown to be normal. A major effort has gone into studying the kinetics of efflux of the ‘’^Cu from these variants. The studies suggest that there is an enhanced rate of efflux of “’^Cu from both variants. However, truly quantitative analysis of efflux has not been possible as the nature and size of the respective intracellular efflux pools is not known. Although homogeneously staining regions and double minute chromosomes have not been detected in the Curesistant variants, the variants do however have some

i Recendy a computer program has been developed to present the elemental content of fibroblasts in a threedimensional manner. The program uses a contour representation to indicate elemental intensities at particular points within the scanned cell. The program allows better display of elemental distribution than was achieved with the previous dot-map representation.

The copper-resistance plasmid, PRT1004, allows E.coli to tolerate extracellular Cu levels up to 3-fold higher than those which parental (non-plasmid) strains can tolerate. At least 4 genes are responsible for the copper-resistance encoded by the major resistance determinant on the plasmid. One of these genes was found to encode a 26Kda protein. Furthermore a chromosomal encoded 11 kda protein was found to be inducible by copper in parental strains - investigations are in progress to determine whether this is a metaUothionein-like protein

6R0WTH CURVES OF NORHAL (CNO) AND COPPER-RESISTAITT VARIANT (CuR)

Further analyses have been done on Menkes’ and normal fibroblasts, and these have enabled normal ranges to be established. Studies on intestinal mucosa from normal and brindled mice have commenced tmd studies on kidney are being planned.

Elemental analysis in cultured fibroblasts and mouse tissues using the proton microprobe G. Allan, B.Kirby and G. Legge (School of Physics, University of Melbourne); J. Camakaris, L. Paul, H. McArdle, D. Danks. The proton microprobe has been used as a highly sensitive analytical tool for determining concentrations and distributions of trace elements in individual cultured fibroblasts growing on nylon foils. The occasional ’spot’ contaminations with heavy metals have been eliminated by replacing the stainless steel supports with polyethylene supports. The collection of back-scattered protons has enabled determination of the elements C, N, O, Na and Mg. Normalisation can now be made to a greater range of elements and elemental sweeps of Na and Mg can be performed.

Growth curves of normal Chinese Hamster Ovary cells (CHO) and a copper-resistant variant derivedfrom these (Cu R), at a range of extracellular copper concentrations.

chromosomal rearrangements. Further studies are required to determine whether there is a direct association between the chromosomal rearrangements and the Curesistant phenotype.

H. McArdle, J. Guthrie, L. Ackland, S. Gross, J. Camakaris, L. Paul, D.M. Danks

Genetic and biochemical analysis of copper transport in Escherichia coli

Escherichia coli is an organism in which it should be relatively easy to use mutants to identify all the steps of copper transport and mechanisms of copper tolerance. Although some steps may be different in mammalian systems, the findings in E.coli will provide important leads for research in mammalian cells.

A number of copper-sensitive mutants have been isolated, some of which are also copper-dependent. The coppersensitive phenotype facilitates cloning of the “wild-type” genes which code for components of copper transport. Plasmid constructs containing fragments of‘Svild-type” DNA were isolated which restored normal copper tolerance to a copper-sensitive/copper-dependent mutant. Restriction analysis and sub-cloning are in progress.

Contour maps and three-dimensional maps showing distribution ofphosphorus and copper in (a) a normalfibroblast and (b) a fibroblastfrom a Menkes’patient. C

Preparation of high specific activity copper-64 P.J. Sorby and E.L.R. Hetherington (Australian Atomic Energy Commission, Research Establishment, Lucas Heights, N.S.W.) andj. Camakaris

Contour maps showing the distribution of copper (a, b) and iron (c,d) in mutant mouse ileum. Dense areas are located around the tip of the villus showing local higher concentrations of metal.

In an attempt to increase the specific activity of ®'‘Cu, the Szilard-Chalmers reaction was investigated; copper phthalocyanine was used as target material. The ^’Cu produced was purified on BioRad Chelex 100 chelating resin. Consistently high yields were obtained with specific activities as high as 40 TBq g'. This material has been used in a wide range of studies involving cultured cells and mice - initial rates of Cu uptake into cultured cells, Cu accumulation experiments over 48 hr, mouse tissue Cu distributions, labelling of Cu-binding proteins. The high specific activity ®*Cu has behaved reproducibly in these studies and has enabled experiments to be carried out at much lower Cu concentrations than was previously possible.

Mechanism of copper transport in plasma and uptake from plasma into the various classes of body cells

J.Camakaris; B.T.O.Lee, and G. Adcock (Genetics Department, University of Melbourne)

concentrations is required (e.g. kinetics of Cu uptake across cell membranes) or when high specific activities of labelled proteins are desired.

Copper-64 (®*Cu) produced at the Australian Atomic Energy Commission by the neutron irradiation of Cu produces the radioisotope, ®*Cu with the relatively low specific activity of ITBq g '. This, together with the short half-life of ®^Cu (t)^ = 12.8 hr) has placed limitations on in vitro studies where labelling with low total Cu

Copper (Cu) is known to be present in plasma in a number of forms - as a component of caeruloplasmin, attached to albumin, and to a lesser extent, amino acids, especially histidine. Recently, Cu metallothionein has been identified in rat plasma and there has been a claim of an additional Cu transport protein called transcuprein. Kinetic studies in whole animals or humans have shown clearly that when Cu is absorbed from the intestine it appears first attached to albumin in the plasma and 24-48 hrs later a considerable amount is present incorporated into caeruloplasmin. It has therefore been natural to suggest that albumin acts to transport Cu from the intestine to the liver where the Cu is incorporated into caeruloplasmin and transported to other tissues. However, the experimental proof of these various steps varies from moderately strong to flimsy. In recent years many studies have measured accumulation of Cu in, and release of Cu from, cells in culture or in suspension, but many of these experiments have paid litde attention to the form in which the Cu may have been present in the culture medium. Most media have contained amino acids and/or albumin. Differences have been noted between cells from normal individuals and cells from humans or mice with mutations affecting Cu transport. To fully interpret these results, it seems important to have a much clearer idea of the physiological mechanisms of uptake of Cu into various cells in the body from the carriers present in plasma. 59

ORIGIN

28S

Plexus 18S

Polyadmylated RNA from choroid plexus (2 /xg), and other tissues (7 fig) was analysed by electrophoresis in a 1% agarose gel containingformaldehyde and transferred to nitrocellulose. Thefilter-bound RNA was hybridised with labelled ceruloplasmin cDNA. After washing, thefilter was anlaysed by autoradiography. Some degradation is evident and the slightly greater mobility of the mRNA fromyolk sac, placenta and testis was due to uneven migration in the gel. No ceruloplasmin mRNA was detected in stomach, adrenal gland, kidney, spleen and small intestine.

A number of studies have been performed with continuous lymphoid cell cultures in this laboratory in recent years. These have now been augmented by systematic studies of the delivery of Cu to mouse hepatocytes in suspension and to human fibroblasts from culture.

Copper uptake by fibroblasts H. McArdle, J. Guthrie, L. Ackland, S. Gross Fibroblastic cells are of interest because they produce large amounts of a copper (Cu) enzyme, lysyl oxidase. When labelled Cu complexed to albumin is added to medium the label is taken up by fibroblasts. No specific receptor for albumin or for Cu-albumin could be demonstrated on these cells. Experiments which varied the concentration of labelled and unlabelled Cu and albumin suggested that it is the component of labelled Cu which dissociates from albumin which is available for uptake. This suggested that histidine or another amino acid might act as an intermediary between Cu-albumin and the fibroblast, but addition of these amino acids inhibited uptake and a range of experiments showed that the uptake of labelled Cu was dependent upon the pool of free labelled Cu in all these experiments. 60

These studies are considered to rule out albumin, histidine and other amino acids as the specific mediators of Cu uptake into fibroblasts. The alternate conclusion that Cu is taken up from a pool of free ionic Cu is the correct interpretation of the experimental results, but is not necessarily an appropriate interpretation of physiological happenings in the body.

Copper accumulation in continuous lymphoid cells J. Camakaris, L. Paul; G. Hanson, J. Pilbrow (Department of Physics, Monash University, Clayton, Victoria). Continuous lymphoid cell lines were incubated in basal culture medium (RPMl) under various conditions in the presence of 3 fiM copper-64. Copper-64 accumulation in the cells was measured at time intervals between 30 minutes and 26 hours. Supplementation of the basal medium with L-histidine or foetal calf serum caused a marked inhibition in accumulation of copper-64.

Ceruloplasmin (CP) is a candidate favoured by many authorities for the role of transport of Cu to cells like fibroblasts. Experimentally, Cu uptake from Cu-CP could be shown, but no specific receptor could be demonstrated for CP or for Cu-CP. CP is a complex protein which is known to be quite labile in experimental conditions and it is possible that the methods of labelling the protein might alter its binding capacities. Several different methods of labelling have been used and none revealed any specific binding. Rat CP labelled in vivo was also tested against human cells without showing any evidence of a receptor. A number of metabolic inhibitors failed to influence the uptake of Cu from Cu-CP.

The only significant primary copper complex detected by EPR spectroscopy, in unsupplemented medium, was the Cu-histidine complex (RPMl contains approx. 100 /tM histidine). EPR studies also revealed that an unidentified Cu-complex (apparently not Cu-histidine) gradually accumulates in the cells following incubation periods of up to 30 minutes.

At present we have not been able to demonstrate a specific role of albumin, histidine or CP in the uptake of copper into fibroblasts in culture. We have yet to evaluate the metallothionein, transcuprein and the histidyl tripeptide which is found in plasma.

Further studies will be aimed at examining the distribution of copper-64 amongst Cu-binding proteins following incubation of cells in the various media, and to identify the EPR positive copper complex associated with the cells.

Uptake of copper by hepatocytes

Nature of copper binding to albumin

H. McArdle, S. Gross

H. McArdle; Dr. A. G. Wedd (Latrobe University).

Mouse hepatocytes will survive in culture for a number hours and show normal metabolic activities, but they need a medium more complex than that necessary for experiments with fibroblasts. Minimum satisfactory medium was found to be Hank’s balanced stJt solution with 10% foetal calf serum. In this medium hepatocytes accumulate to Cu from mass Cu-albumin complex but that no specific receptor for either albumin or Cu-albumin could be demonstrated. Experiments varying in concentration of Cu and albumin indicated that it was the pool of Cu which dissociates from albumin which is taken up. The addition of histidine and other amino acids caused a stimulation of uptake by hepatocytes (in sharp contrast to the results with fibroblasts) which was most marked with histidine. Further experiments showed that the cells do recognise histidine-Cu differentially from histidine alone or Cu alone. These results confirm earlier claims that Cu dissociates from albumin in the plasma and forms a transient complex with histidine which is the form which is taken up by hepatocytes.

During the experiments with hepatocytes and fibroblasts a compound called Chelex was used to remove loosely bound Cu from the Cu-albumin complex. Chelex is a gel which has iodoacetamide groups capable of chelating heavy metals. In the literature it has been suggested that Chelex can remove copper from its specific binding site on albumin but the published evidence was only circumstantial. We have used electron spin resonance to examine this question and have found that Chelex could remove only non-specifically bound Cu from 2ilbumin and could not remove the metal bound to the specific binding

site. This is as one might expect considering that the association consonant of albumin for Cu at a specific site is 10"M.

Analysis of tbe metallotbionein gene family in sbeep M.G. Peterson, J.EB. Mercer An alteration in structure or regulation of the metal binding proteins, metallothioneins (MT) could explain the unusual transport and distribution of copper in the sheep. In this species, biliary excretion of copper is much less efficient than in rats or healthy humans and resembles the situation found in Wilson’s disease in man. We have continued our analysis of the sheep MT gene family reported in 1985. Two overlapping cosmid clones have been isolated and they contain four complete MT genes (la, Ib, Ic and II) and one truncated pseudogene. The complete sequence of these genes has been determined and their expression in sheep fibroblasts has been studied. The MT-Ia, -II and -Ic genes produce substantial amounts of mRNA in response to various heavy metals, the levels found being in the order stated from highest to lowest; levels of MT-Ib mRNA were just detectable. Only MT-II produced any mRNA in response to dexamethasone. MTIc responded very weakly to copper compared with MT-Ia and -II. Each gene shows a characteristic pattern of response to various inducers, but the functional significance of this is unclear.

Metallotbionein mRNA levels in sbeep livers during development J. Smith, A. Grimes and J.EB. Mercer; J. McC. Howell (Murdoch University, Perth). To further explore the possibility of differential regulation of individual members of the sheep metallothionein (MT) gene family and to investigate the mechanisms of MT gene control during development, we determined the levels of MT-Ia, -lb, -Ic and -II mRNA in livers of sheep of from 30 days of gestation through to one year after birth. The most interesting finding was that levels of MT mRNA were very high early in gestation and fell steadily to reach

Sheep metallothionein gene cluster.

cMT9 CMT11 Ikb

cosmid

MT-n

MT-Ic

MT-Ib

MT-Ia

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adult levels soon after birth. The MT mRNA levels in adults were still higher than in rats. Hepatic zinc levels were found to be closely correlated with the MT mRNA and zinc concentrations were also surprisingly high (3,000 Hg/g dry wt) early in gestation. There was no evidence of differential regulation of the individual MT genes, the same relative levels being maintained throughout fetal life. The high MT mRNA levels and close correlation with zinc levels suggests that perhaps sheep have an unusual mode of MT gene regulation.

Analysis of liver metallothionein mRNA levels in adult sheep J.EB. Mercer, A. Grimes; R Gill andJ.McC. Howell (Murdoch University, Perth). We undertook the analysis of MT-Ia, -Ic and -II mRNA levels in sheep liver samples provided by our collaborators at the Murdoch University. There were three groups of animals - control, Cu-supplemented and Cusupplemented/thiomolybdate treated. Liver Cu levels were very high in most of the Cu-supplemented animals, as expected. MT mRNA levels were quite variable, but surprisingly high (compared with rat liver) in many animals. As in sheep fibroblasts in culture, the relative mRNA levels were Ia>II>Ic>Ib. To our surprise, there was no correlation between MT mRNA levels and the Cu concentration in the livers. However, we did find a significant correlation of MT mRNA levels and hepatic zinc concentrations, perhaps suggesting that the differences in MT mRNA between individuals might be related to dietary zinc intake. Taken in conjunction with results obtained in fetal and newborn sheep we feel that there must be something unusual about MT gene regulation by zinc in sheep liver and that copper accumulation may be a secondary consequence.

HEPATIC MT mRtJA VERSUS ZINC IN DEVELOPING SHEEP 25000O

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Caeruloplasmin mRNA distribution in rat tissues and in response to inflammation

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J.EB. Mercer and A. Grimes; A. Aldred and G. Schreiber, (Biochemistry Department, University of Melbourne).

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800

1600

2400

3200

Zinc Concentration (pg/g dry wt.)

Surprisingly high levels ofzinc and metallothionein mRNA are found in livers of foetal sheep and there is a good correlation between the two.

HEPATIC MTIA mRNA VERSUS ZINC IN ADULT SHEEP

Zinc Concentration (pg/g dry wtj

CP gene expression was observed in the choroid plexus of the brain, yolk sac, placenta and testis in addition to the liver, the well-studied site of production. All of these tissues are at the interface between adjacent extracellular body compartments and may be involved in maintaining homostasis in these compartments. Synthesis of CP in these tissues may be important in the transfer of Cu across the barriers between compartments or CP may have another role in a specific compartment, e.g. oxidation of serotonin and catecholamines in the cerebrospinal fluid. No CP mRNA was detected in RNA from stomach, adrenal glands, kidney, spleen and small intestine.

J.EB. Mercer, A. Grimes

A. Grimes and J.EB. Mercer

We have isolated a cDNA clone for human CP. In order to analyse the regulation of the CP gene by various physiological factors we need to use experimental animals. Rats and mice are the most suitable. Accordingly, we isolated a number of cDNA clones for rat CP by screening a rat liver cDNA library with our human CP cDNA. The

Using the rat caeruloplasmin (CP) cDNA we analysed the levels of CP mRNA in rat liver following turpentine injection, the standard method of inducing an acute phase response to inflammation. Levels of CP mRNA reached a maximum of 350% of normal 36 hours after injection and returned to normal within 60 hours. This demonstrates that the increase in plasma CP known to occur during the acute phase response is caused by an increase in mRNA production.

Ceruloplasmin mRNA and copper treatment in rats

Isolation of a cDNA clone for rat caeruloplasmin Ceruloplasmin (CP) is a blue glycoprotein which contains 6 copper (Cu) atoms and accounts for about 90 % of the Cu present in blood plasma. The various functions of this protein are not well understood, although there is some evidence that it can function as a Cu transport molecule. Levels of CP are elevated in response to inflammation and during pregnancy.

obtained a complete predicted amino acid sequence for rat CP. Rat liver contains a single CP mRNA of 3,800 nucleotides which falls in between the sizes of the two CP mRNA species in human liver, both of which code for the same protein, differing only in the lengths of their prime 3’ untranslated regions.

There is a correlation between zinc and metallothionein mRNA in adult sheep.

largest clone contained a 2kb insert that includes most of the CP coding region. We obtained the DNA sequence of the ends of the clone and the predicted amino acid sequence of this region of rat CP is highly homologous to the human sequence. Unfortunately, we have yet to isolate a clone covering the whole sequence, so we have not yet

Ceruloplasmin (CP) mRNA levels were determined in RNA from livers of rats that had been treated with Cu salts by injection. There was no clear response of the mRNA to this large does of Cu. The animals had also been starved for various times during the initial experiments and when starvation time was plotted against CP mRNA levels, a decrease of the mRNA was noted. This decrease was prevented by a prior injection of Cu chloride. This result suggests that normal CP mRNA production may require a constant intake of Cu in the diet and that Cu already present in the liver may not be available for CP production. This interesting suggestion is being investigated.

Olive Miller protein group - overview R.G.H.Cotton The work of this group focuses on two topics - enzyme defects related to phenylketonuria (PKU) and a factor found to retard differentiation of mouse embryonal carcinoma cells in culture. Within the Institute there is close collaboration with members of the DNA group, with the cell culture laboratory and with the embryology group. All the projects related to PKU, whether by protein chemistry or DNA techniques, are gathered together in this section of the Report.

Immunochemical analysis of phenylalanine hydroxylase I. G. Jennings, R.G.H. Cotton. The enzyme deficient in PKU, phenylalanine hydroxylase (PH) is complex and we are assessing the role of monoclonal antibodies in determining which amino acids in the sequence are responsible for the function of the enzyme. We have now completed an analysis of an initial series of 11 antibodies. These antibodies exhibited a range of effects on enzyme activity, but none were directed to the active site. However, three were found particularly useful cmd are now being studied in collaboration with other workers. The antibody PH8 appears particularly valuable. Dr. Istvan Tork, Department of Anatomy, University of New South Wales, spent much of 1986 in Lausanne using the antibody to produce the first atlas of the serotonergic neurons of the human brain stem. It should be useful in analysis of pathological changes in the brain stem. Antibody PHI is currently being used to analyse the mechanism of activation of phenylalanine hydroxylase and PH7, an antibody to the phosphopeptide of PH, is being used by Dr. Fisher in Liverpool to study regulation of PH in response to hormones. Most recently we have isolated a new series of antibodies which appear to bind at the pterin binding site as they also react with dihydropteridine reductase and dihydrofolate reductase.

Analysis of normal and mutant dihydropteridine reductase W. McAdam, I.G. Jennings, R.G.H. Cotton Dihydropteridine reductase deficiency leads to a rare variant form of PKU and we have studied this enzyme fof many years since we identified one of the first patients known with this defect. This year we isolated a series of monoclonal antibodies to this enzyme and most recently we cloned the gene.

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Southern blots ofgenomic DNA probed with DHPR clones. Showing restrictionfragment length polymorphisms.

We had known for some time that the defect in patients was heterogeneous as some patients had normal levels of inactive protein in their cells whereas others had no protein. Further, we knew that some patients responded to the BH^ load test developed in our Institute, whereas others did not. Analysis of patients produced strong support for the hypothesis that non-responding patients have normal levels of an inactive protein. There are indications that the symptoms occur earlier or are more severe in these latter patients. The mechanism of this effect is of great interest.

Purification of a factor retarding the differentiation of embryonal carcinoma cells I. Roberts, W. McAdam, R.G.H. Cotton A factor affecting the rate of differentiation of embryonal carcinoma (EC) cells was recently described from this laboratory. As the reactivity at least appears novel we have decided to characterise the factor to decide if it is a new factor involved in normal differentiation. An improved assay was developed which facilitated the analysis of purified fractions and large quantities of conditioned medium were carried through the purification process. We are confident that we will obtain information about the amino acid sequence of the protein in the near future.

Molecular analysis of human dihydropteridine reductase H.-H.M. Dahl, W. Hutchison, W. McAdam, EJ. Morgan (St. Vincent’s Hospital Medical Research Institute), R.G.H. Cotton.

and the expression of the protein. It was shown that DHPR consists of a 26,000 dalton protein chain present as a dimer in the active form of the enzyme. It appears to be expressed in most tissues, but is present at a higher level in kidney and liver. DHPR has been purified to apparent homogeneity, and monoclonal and polyclonal antibodies to the enzyme have been produced. The overall amino acid composition is known and the amino acid sequence has been determined for several peptides generated by trypsin and cyanogen bromide cleavage. To extend the study of DHPR, we have isolated cDNA clones from a human fetal liver cDNA library (constructed in the expression vector Xgtll), by screening this library with affinity purified polyclonal antibodies to DHPR. This led to the isolation of two independent clones, each approximately 1400 bp long. The cDNAs were sequenced and the predicted amino acid sequence was shown to include several of the known peptide sequences. These initial clones are hybridised to synthetic oligonucleotide probes corresponding to the known peptides based on the determined amino acid sequence. The clones did not contain the full coding sequence, but, after screening many more clones one containing the full coding sequence and a short part of the 5’ untranslated region was obtained.

Analysis of the rat phenylalanine hydroxylase gene promoter

Phenylalanine hydroxylase in developing rat liver

H.-H. M. Dahl, D. Hobson, J. E B. Mercer, R. G. H. Cotton.

J.EB. Mercer and A. Grimes; G. Yeoh and E. Edkins, (University of Western Australia).

Last year we reported the isolation of full-length cDNA clones for both rat and human PH. We reported that the human and rat PH enzymes are highly homologous and that PH mRNA in rat hepatoma cells is inducible up to 20 fold over the basal level by treating the cells with hydrocortisone.

Many enzymes in the liver of the rat appear at characteristic times during development, such as just before birth (late foettil). Just after birth (neonatal) and after weaning. It is generally considered that the appearance of these enzymes is related to dietary and physiological changes that accompany birth and weaning, but little is known about the nature of the inducers (possibly hormones) controlling the enzyme activities or about the level at which control is exerted (e.g. gene activation or activation of a preformed protein).

In order to study the regulation and expression of this enzyme we have isolated a genomic DNA fragment containing the 5’ end of the cDNA sequence. The 16 kb fragment was identified by screening a rat liver ng and non-coding sequence. We are currendy trying to locate the exact starting point for the initiation of transcription of this gene by primer extension experiments and direct mRNA sequencing. With this completed we can start the investigation of specific promoter and operator elements.

Cloning of phenylalanine hydroxylase cDNA from a PKU liver J.EB. Mercer, A. Grimes, H. Dahl and R.G.H. Cotton.

We have used our DHPR cDNA clones to look at the mRNA level in cell lines established from patients with hyperphenylalaninaemia by RNA blotting techniques. The cDNA probes detect a 1700 bp mRNA. Of the 13 patients that we have analysed, one has no DHPR mRNA, but the others have levels that do not differ significantly from control samples. Southern gel analysis of genomic DNA revealed no major deletion or rearrangement in any of these cell lines. These results, combined with previous analyses of DHPR protein, allow definition of at least three types of mutation - mRNA“, CRM-, mRNAL CRM", CRM*.

We have samples of liver from a PKU patient who died of unrelated abnormalities in the newborn period. RNA has been isolated and Northern blot analysis has demonstrated the presence of a normal sized phenylalanine hydroxylase mRNA. The amount of this mRNA was low (about 20%) compared with a normal adult, but was the same as in age matched controls. This result shows that the mutation in this patient is not a complete gene deletion or gross RNA splicing defect. The mutation could be a single base change that either alters the amino acid sequence or disrupts mRNA translation during protein synthesis. To identify the mutation we have constructed a cDNA library from this patient. After screening about 500,000 clones from the library we isolated 5 different phenylalanine hydroxylase clones. Unfortunately none of these included the full coding sequence for the protein, and we concluded that the library is unlikely to contain such a clone. To be sure that the mutation has been correctiy identified a complete sequence is required and so we have not further analysed these clones. We are considering alternative cloning strategies that may improve our chances of identifying this mutation.

Three restriction fragment length polymorphisms (RFLPs) have been identified using two restriction enzymes (MspI, Ava II). These RFLP show some linkage disequilibrium and only 60% of individuals are informative (expectation 80% if no dis-equilibrium). Nonetheless, they do provide a second method of prenatal diagnosis additional to our previous method using enzyme assay. The RFLP method should be applicable in a wide range of laboratories.

The study of DHPR has been one of the major interests of the Protein Chemistry group for several years. This has led to considerable knowledge about the molecular properties

We have determined the time of appearance of phenylalanine hydroxylase protein and mRNA levels in rat liver during development and our results show that the mRNA first appears on day 18 of gestation, closely followed by the protein and enzyme activity. Thus the enzyme belongs to the late foetal cluster of enzymes. Using foetal hepatocytes in culture the synthesis of the enzyme appears to be regulated by both cAMP and dexamethasone. The exact nature of the regulation is stUl being investigated.

Pyruvate dehydrogenase S. Hunt, R. Scholem, W. Hutchison, H. Dahl and G. Brown. The pyruvate dehydrogenase (PDH) complex, which converts pyruvate to acetyl CoA, is one of the major enzyme systems involved in the regulation of energy metabolism. The PDH complex is of additional importance in man because deficiency of its activity is the most common enzyme defect encountered in patients with primary lactic acidosis. We have been studying the structure, function and genetic organisation of the PDH complex in normal human tissues and in patients with PDH deficiency. In previous years, we purified the enzyme from bovine human heart and obtained a series of antibodies which we have used in an immunochemical analysis of normal and mutant forms of human PDH. In the past year, we have used these antibodies to isolate cDNA clones corresponding to the El subunit. This subunit is of particular importance as it contains the enzyme active site of the El or pyruvate decarboxylase component as well as the phosphorylation sites involved in regulating the activity of the entire complex. The El subunit is the site of the genetic defect in the majority of patients with PDH deficiency. 65

64 ■.Jf-

3 4

Using these cDNA probes, we have already obtained preliminary results at gene structure and mRNA levels in a number of patients with PDH deficiency.

Malonyl CoA decarboxylase

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J. Christodoulou and G. Brown.

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Analysis of mRNA for the Ela. subunit ofpyruvate dehydrogenase in culturedfibroblasts. Total RNA extracts from culturedfibroblasts were analysed by northern blotting using a pyruvate dehydrogenase Ela subunit cDNA probe. The results show twoforms of mRNA detected by this probe which differ in size (1.65 and 3.3 kb). Track 1 is RNA from a normal control, track 2from a patient with severe pyruvate dehydrogenase d^iciency and tracks 3 and 4 from two patients with a lesser degree of enzyme dficiency. In track 5 are size markers. Thefibroblastsfrom the patient with severe pyruvate dehydrogenase dficiency have a marked reduction in the level of mRNA for the Ela subunit.

We have obtained cDNA clones which correspond to the entire mRNA of the El subunit and their nucleotide sequences have been determined.

Malonyl CoA decarboxylase is a mitochondrial enzyme which converts malonyl CoA to acetyl CoA. However, the role of this enzyme in intermediary metabolism is unclear as there is no known mechanism for generating malonyl CoA within the mitochondrion under normal circumstances. We are in a unique position to determine the biological role of malonyl CoA decarboxylase following the identification of two patients with genetic defects involving this enzyme. The patients have a variety of clinical and biochemical abnormalities which, when fully understood, should clearly define the function of the enzyme and its interaction with other metabolic pathways. Our approach to this enzyme defect is first to establish the structure and biochemical properties of the normal enzyme. As the protein appears to be well conserved, we are purifying the enzyme from bovine liver and using this preparation to generate antibodies. These will be used to analyse the structure of the human enzyme. At the same time, we are studying the kinetic properties of the human enzyme and the effects of malonyl CoA on other ultramitochondrial metabolic pathways. It is expected that once the basic properties of the enzyme have been established and suitable immunological reagents are available, we shall proceed to investigate the structure of the gene for malonyl CoA decarboxylase and its expression in different tissues by isolating cDNA clones corresponding to the mRNA. We should then be in a position to define the nature of the genetic defect in the two patients.

Cytochrome oxidase K. Hayasaka, D. Kirby and G. Brown.

The predicted amino acid sequence has a leader sequence which is typical for proteins destined for uptake into the mitochondrion and also contains all three phosphorylation sites. (The sequence of these sites in bovine and porcine PDH were available from other workers).

Cytochrome oxidase deficiency is the most commonly recognised defect of the electron transport chain in man. However, the clinical spectrum of cytochrome oxidase deficiency is still poorly understood and specific genetic defects of individual subunits of the complex are rarely defined.

Two classes of cDNA clones were obtained which differ in length, but share the same open reading frame. These correspond to two distinct mRNA molecules, both of which have been found in a variety of human tissues. The relationship between these two mRNA species is at present unclear.

It is now clear that a number of patients with cytochrome oxidase deficiency have a generalised defect although clinically only the central nervous system is involved (often manifesting as Leigh’s syndrome). Other patients apparently have tissue-specific deficiencies and, as a result, have isolated organ disease such as myopathy.

To define the genetic basis for the various manifestations of cytochrome oxidase deficiency and to improve the accuracy of diagnosis, we are developing a number of immunochemical methods to analyse cytochrome oxidase structure in man.

pathways which have not been considered before and which cannot be fitted into current biochemical knowledge. It is extremely unlikely, therefore, that these interactions would ever be studied without the stimulus of the clinical problems of this patient.

The enzyme complex has been purified from bovine heart and antibodies to this preparation are being obtained. From our own previous studies, we know that these antibodies will cross-react with human cytochrome oxidase and will enable us to characterise specific genetic defects in this electron transport chain complex.

Two further patients were studied in collaboration with Dr. Alf Poulos at the Adelaide Children’s Hospital. In fact, four patients with very similar clinical abnormalities have been observed. These comprise an unusual and specific facial appearance, poor growth and neurological function, muscle weakness, joint contractures and early death. Two of the patients were siblings - one of these was studied biochemically. Another similar patient from another family was also studied. The fourth patient, from a third family, was classed with the others on clinical grounds alone. The patients who were studied showed an array of abnormalities of peroxisomal functions comparable to those seen in Zellweger’s syndrome. However, the clinical features did not match this diagnosis, nor did the finding of high levels of threonine in cerebrospinal fluid and plasma. A different disorder of peroxisomal function seems to be present.

New inborn errors of metabolism D.M. Kirby, R.D. Scholem, S.M. Hunt, G.K. Brown; H. CroU, D. Mitchell and J. Pitt (Department of Clinical Biochemistry, Royal Children’s Hospital). Each year we investigate a number of patients with new or unusual metabolic diseases. Usually, the first clues to the nature of the biochemical abnormality in these patients come from urine metabolic screening tests performed by the Department of Clinical Biochemistry. We then follow up these studies in an attempt to define the basic enzyme defect and determine its biochemical consequences. In the past year, we have studied two patients in considerable detail. The first is a young boy who has a complex array of metabolic abnormalities including lactic acidosis, hypertriglyceridaemia, severe neutropenia'and a gross deficiency of urea cycle amino acids. Many of these metabolic abnormalities have responded to the administration of large doses of the urea cycle amino acid, citruUine. There is at present no precedent for this condition, nor has it been possible to explain all the biochemical abnormalities in terms of known metabolic pathways. The second patient is a young girl with an extremely rare, but well recognised, enzyme defect, sulphite oxidase deficiency. This condition results in multiple abnormalities in the metabolism of sulphur-containing amino acids, leading to severe central nervous system damage. Only two other patients with this condition have been described and both were diagnosed many years ago before many of the sophisticated techniques we now use were developed. We have therefore studied this enzyme defect and its consequences in considerable detail to provide firm data for future studies of this disease and the related metabolic pathways. The first of these two patients illustrates particularly clearly the reason for pursuing this type of investigation. His novel combination of metabolic problems serves to direct attention to interactions between metabolic

Cerebral lactic acidosis E. Wraith, D. Kirby, R. McCaskill, J. Rogers, David Danks. Starting about 10 years ago we have debated the existence of a group of patients with lactic acidosis which has a profound effect on the brain without causing significant bodily acidosis. Three further patients seen recendy with severe anatomical brain abnormalities and profound neurological disturbance, but no acidosis, convinced us. Analysis of lactate levels in cerebrospinal fluid (c.s.f) showed that the c.s.f. to plasma lactate ratio was much higher in the six patients with these clinical features than in other cases. Five of the six patients had defects in pyruvate dehydrogenase. It is clear that c.s.f. lactate measurement is an essential test in young babies with profound brain disease including those with hydrocephalus and hydranencephaly. We are interested in looking for some specific metabolic reason for this distinct category of cases.

Expression of human blood clotting factor IX in transgenic mice K. H. Choo, M. G. Peterson and W. McAdam in collaboration with K. Raphael (C.S.I.R.O., Sydney). Factor IX is an essential component of the blood clotting system. A defect in this factor leads to the severe bleeding disorder of haemophilia B or Christmas disease which affects 1 in 30,000 males. Patients are currendy treated by 67

injection with factor IX concentrates prepared from pooled plasma. The treatment carries the risk of infection by blood-borne viruses such as those responsible for hepatitis and AIDS. Some human proteins (e.g. growth hormone) are now produced in genetically engineered bacteria, but factor IX is a very complex protein requiring various modifications after initial synthesis. We have explored the feasibility of synthesis in transgenic animals, in which all these processes should be functional. Transgenic mice were generated by microinjection of a cloned full-length human factor IX cDNA into the pronucleus of fertilised eggs, followed by reimplantation back into pseudopregnant foster mothers. Out of 7 transgenic animals produced, 5 were shown to express the factor IX protein in their plasma. Four of these expressed only low levels of protein. The highest expressor was characterised in detail and was shown to produce mRNA and protein which were identical to those of the human factor IX. The protein was functionally active as measured in a one-stage blood clotting assay. The level of activity was approx. 150% that of normal human plasma factor IX activity. Having demonstrated that we can achieve high levels of gene expression in a small animal, and that the transgenic trait is heritable in a Mendelian fashion, our next step is to try to generate larger transgenic animals (e.g. in pigs or sheep) which may produce large quantities of active human factor IX. It is of considerable basic interest that our particular DNA construct achieved in vivo expression while those of others (R. Palmiter and R. Brinster, personal communication) have failed. We plan to collaborate with these workers to determine which features of the DNA construct are important for expression.

Isolation and analysis of chromosomespecific repetitive DNA enriched in the centromeres of human chromosomes K.H. Choo, R. Brown, B. Vissel, G. Filby, G. Webb and M.E. Earle. We and a number of other workers have recently identified a family of repetitive sequences which are localised predominantly to the centromeric region of all the human chromosomes. These sequences are called the a-sateUite family of repetitive DNA, and these are a few thousand copies on each chromosomes. No specific role has been assigned to these sequences, although their chromosomal position and the observed conservation of the sequences in different chromosomes suggest a role related to the structure and/or function of the centromere. To study the 68

role, and to understand the evolution, of these repetitive DNAs, it is necessary to analyse and compare sequences from the different chromosomes. We have already isolated recombinant DNA clones containing a-satellite sequences specific for the centromeres of chromosomes X (TRX) and 17 (TR17). Using the TR17 probe, we have shown that, in the humtin genome, homologous sequences are organised principally as five major polymorphic (PstI) forms of tandem repeats which have molecular weights between 2.0 - 2.7 kb. We have studied the higher-order organisation of these major forms by isolating and analysing a series of cosmid clones. The results indicated that the different polymorphic forms of the a-satellite DNA are closely linked, and that most of the repeat arrays are greater than 20 kb in size. In addition, we showed that arrays of DNA are relatively frequently interrupted by non-homologous genomic DNA. Our data suggest a complex and irregular pattern of organisation of the repetitive DNA in the human genome.

Studies of the fragile-X (Fra Xq27) syndrome of mental retardation R. Brown, G. Brownlee, H.-H. M. Dahl, J. Halliday, J. Dodge; D. Loesch and D. Hay (LaTrobe University). Collection of family data, analysis of observations of the physical abnormalities and psychometric tests on family members have continued through 1986. In the latter months planning commenced for a survey of all retarded children and adolescents in Victoria. In the laboratory the hypothesis that the X-chromosome may have an unusual tendency to break at the fragile-site was put to the test by forming human-mouse cell hybrids. One cell line contained a fragment of the X-chromosome which appeared to correspond to the region proximal to the fragile-site fused with a mouse chromosome. Two human genes (HPRT and factor IX) known to be located in this region were retained, but not the genes for factor VIII and the fragments ST14 and DX13 known to be distal to the fragile site. To truly evaluate the hypothesis many more hybrids should be made and examined to determine whether the break point is always, or frequendy, at the same position. This would take a long time and instead it seemed better to try to clone and analyse the junction point of the one hybrid already obtained. A cosmid library has been constructed and is being screened for inserts which contain both human and mouse sequences. Such fragments should contain the junction site and may include the fragile site.

Detection of chromosomal trisomies by DNA hybridisation H.-H. M. Dahl, K. H. Choo, R. Brown, E. Earle, G. Filby. Down’s syndrome (chromosome 21 trisomy, mongolism) is the best known and most frequent of the human chromosome abnormalities. It is the most common definable cause of mental handicap. Unfortunately there is no cure in sight. Prevention by prenatal diagnosis is used extensively. The present method of prenatal detection is by cytogenetic analysis of cells from the amniotic fluid or chorion villi sampling. This is a slow, and labour intensive procedure and its application is therefore limitec) by cost, to older women (37 years and over in Victoria) who have a greater risk of having a child with Down’s syndrome. We have investigated the possibility of applying recombinant DNA techniques to simplify the analytical procedure, so that more women can be offered the test. We developed an assay in which the number of copies of chromosome 21 can be estimated by hybridisation of a DNA probe specific for chromosome 21 to total cellular DNA, using a DNA probe specific to another autosome as a reference. We have now improved the hybridisation conditions and probe labelling procedures, and are endeavouring to increase the sensitivity by using several chromosome 21 specific probes. A number of unique probes have been isolated from a chromosome 21 library and are being characterised and tested in the Down’s syndrome DNA assay Several other trisomies (chromosomes 13 and 18 especially) are also clinically important though less frequent than

Four patches of signal detected in the interphase nucleus of an XXXX cell using a probefor the X-chromosome specific a-satellite repetitive DNA sequences.

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trisomy 21. If a DNA test were to replace cytogenetic procedures it would need to detect all three of these trisomies. DNA probes which hybridise to the repeated a-satellite sequences around the centromeres of human X-chromosomes give a signal strong enough to allow the X-chromosomes to be counted in interphase cell nuclei. We have shown this using isotope labelling of the probe and others have used fluorescent labels. It should be possible to use 3 different fluorescent labels for the 3 chromosomes of interest and to identify trisomy of any of them. Unfortunately, chromosomes 13, 18 and 21 have a-satellite sequences which are very similar to those on several other chromosomes and no probe of this type has been found with the specificity required. Another possible approach involves using a mixture of 20 to 50 chromosome-specific single-copy probes to “paint” a chromosome and make it visible during interphase. Some progress has been made towards this goal for chromosome 21.

Cell culture collection K. J. Fowler, P. I. 'Wajngarten, P. B. Gatehouse. A large number of cell lines were received from overseas and interstate laboratories during 1986. These included transgenic mouse embryo variant cell lines for characterisation by John Bateman and Marie Dziadek, various hamster-human and mouse-human hybrid cell lines which include human chromosome 21 for use by Bryce Vissel, and lactic acidosis and metabolic disease cell lines for enzyme studies by Garry Brown. Work in the tissue culture laboratory has been speeded up since we demonstrated that a domestic microwave oven can be used to thaw rapidly and warm cell culture media prior to use on cells. We are grateful to Philips Consumer Products, who kindly donated the oven.

Monoclonal antibodies K. J. Fowler, P. I. Wajngarten. A monoclonal antibody which detects rotavirus common group antigen was developed some years ago in conjunction with the Department of Gastroenterology. This has now been incorporated into a kit which doctors and clinicians can use for rapid diagnosis of rotavirus infection. The kit is known as KadaiCha and has been developed by ICI Australia Operations Pty Ltd. Diagnostics Division. It is due to be released in May 1987 at the Australian Society for Microbiology in New Zealand. Antibodies specific to serotypes 1, 2 and 4 have been produced.

Classification of chondrodysplasia punctata L.J. Sheffield, J. Halliday, E Jensen (on leave from Queen Victoria Medical Centre) and V. Mayne (Department of Radiology.

Anti-idiotypic monoclonal antibodies to phenylalanine hydroxylase have been produced and a new panel of monoclonal antibodies to DHPR. Monoclonal antibodies to the N-terminal sequence of a factor involved in causing hypercalcaemia in malignancy have been produced with Professor T. J. Martin’s group (Department of Medicine, Melbourne University at the Repatriation General Hospital).

Mild forms of the Sanfilippo syndrome

Teratogenic risks of drugs taken during pregnancy

Monoclonal antibodies to synthesized peptides for a glucose transporter sequence which corresponds to the outer and inner membrane have been produced with M. Dunlop and J. Proietto (Department of Medicine, Melbourne University at Royal Melbourne Hospital). Production of mouse myeloma and hybridoma IgA continues with Colin Jones (Department of Immunology, Royal Children’s Hospital). 70

In addition, a handbook on drugs used in pregnancy, published previously by Mr. Batagol, is being reviewed with the intention of publishing a second edition. This information is useful to obstetricians, paediatricians and general practitioners in the community as well as within the Royal Women’s Hospital. The Congenital Abnormalities Subcommittee of the Australian Drug Evaluation Committee has developed proposals for a grading system to classify the degree of risk

J.E. Wraith, A. Bankier, D.M. Danks

J.E. Wraith, J.G. Rogers, D.M. Danks

In the intervening years the total series on our files has risen to 110 cases. We expect to make real improvements in the classification during the analysis of these cases. Over half of the radiological data has been reviewed and the clinical and genetic review will be undertaken during 1987.

The aim of this study is to evaluate the computerised prescription system now in use at the Royal Women’s Hospital as a research resource. All drugs prescribed to antenatal patients are recorded on computer as are observations regarding the delivery and findings in the baby. Each component of the system will have to be assessed before the data can be interpreted. For instance one cannot be sure that prescribed drugs are actually taken and other drugs prescribed elsewhere will not be recorded. Evaluation of aiU aspects is likely to take three years.

Computer calculation of genetic risk The advent of DNA linkage tests for use in presymptomatic and prenatal diagnosis has increased the need for computer programs to combine together the information from pedigree analysis, from conventional tests and from the new tests. Several different computer programs which are available have been evaluated and those bests suited to our purposes in the genetic clinic and in our DNA laboratory have been established. The need for careful evaluation was apparent when one program gave erroneous results in some pedigrees. The error in the program has not been traced, but we are satisfied that those we have chosen are reliable.

L.J.Sheffield and R. Batagol (Pharmacy Department, Royal Women’s Hospital). Attempts at producing anti-idiotypic serotype 3 specific monoclonals continue with Barbara Coulson (Department of Gastroenterology), Norman Mermelstein and Don Roberton (Department of Immunology).

two or three patients previously described around the world that this mild phenotype was peculiar to the Sanfilippo B syndrome.

L.J. Sheffield, J. Halliday, J. Dodge, D. M. Danks.

Chondrodysplasia punctata is a naime given to a group of disorders of bone growth in which patches of abnormal calcification are seen in the epiphyses (growth areas at the ends of bone). The existing classification has been quite unsatisfactory partly because it was developed by comparing published cases with one another rather than by analysing extensive personal experience. In 1976 we described 26 cases of a mild form of the condition which had passed unnoticed by others. Later we realised that the end results of this condition in adults had been called Binder’s syndrome by plastic surgeons who were called upon to correct the residual nasal deformity. We know that there were two or three other groups of cases, distinct from the named categories, among the 41 cases we reviewed in 1976.

Dr. Barbara Coulson, Department of Gastroenterology, using the Kadaicha Rotavirus diagnostic kit.

associated with various drugs used in pregnancy. After careful review of this system some modifications have been suggested. This grading system will be used in the new edition of the handbook.

The clinical manifestations of the Sanfilippo syndrome (mucopolysaccharidosis III) have been remarkably consistent in most cases, despite the fact that deficiency of any one of four different enzymes can produce this syndrome. Of all of the mucopolysaccharidoses this is the one with the most profound effect on brain function and the least marked effects on the remainder of the body. Consequently, the diagnosis was often missed, even in children with the very characteristic clinical progression arrest of developmental progress at the age of five or six years, followed by loss of previously acquired skills and the development of an aggressive hyperactive pattern of behaviour, leading eventually to total loss of neurological functions, with death at mid to late adolescence. Sanfilippo syndrome was diagnosed 15 years ago in one boy who was first seen with mild intellectual impairment at the age of five years. There was a phase of mild hyperactivity and slight aggression, but this passed and he has shown no significant loss of skills over subsequent years. In the last 12 months four further patients with mild to moderate mental retardation have been diagnosed in their teens or early twenties. In all five patients there were minimal signs of soft tissue involvement by the disease, with slightly coarse facial appearance and bone structure. Clearly, it is important to consider the Sanfilippo syndrome as a possible diagnosis in patients with moderate mental retardation and minimal somatic features. Enzyme assays showed that 3 patients fitted into Sanfilippo B, and 2 into Sanfilippo C categories, contradicting a claim based upon

Geleophysic dysplasia Ten years ago, Jurgen Spranger from Mainz in West Germany and John Opitz from the United States described patients with a very unusual “happy-looking” facial appearance, flexion deformity of the fingers, severe mucoid chcmges in multiple heart valves and named the condition geleophysic (happy countenance) dysplasia. They noted one apparently similar patient described in the cardiology journals some years previously. In 1984 they updated their experience with one or two additional cases and published extensive analysis of liver tissue in which patchy storage of an excess glycoprotein material was described. About that time we observed a similar patient in Melbourne, but did not find the changes in the liver described by Spranger. Recently a further case was encountered in Melbourne and Dr. Wraith remembered a similar patient seen in Manchester. Careful comparison of these patients confirmed the close similarity of all four of them with the condition described by Spranger and his colleagues. Liver tissue available from two of the four patients failed to show the type of storage described by Spranger, but changes in fibroblastic cells were quite remarkable in one of the patients and warrant further investigation.

In situ hybridization of cDNA probes to chromosomes G. C. Webb, E. Earle, R. Brown. Techniques for hybridizing DNA probes to human chromosomes have improved gradually over the last two or three years, but most groups still find that the results with any new probe are unpredictable. Two different techniques have been in use during the year - one developed over several years by Graham Webb; the other introduced by Ian Craig during his sabbatical leave and carried on by Ruth Brown. Notable successes during the year include localization of genes for glutathione-S-transferase 2 and 1 acid glycoprotein, both in collaboration with Dr. Philip Board of the John Curtin School of Medical Research.

Unusual chromosomal abnormalities encountered in patients S.M. Dale, D. DuSart, R. Hutchinson, M.A. Leversha, V. Petrovic, J. Roberts, P. Stoddart, L.E. Voullaire, G.C. Webb. In the course of routine diagnostic cytogenetic analyses interesting cases are found. The preparation of these cases for publication involves extra work for cytogeneticists already carrying a heavy routine work load. The appointment of L.E.VouUaire, M.Sc. in a part-time capacity, to co-ordinate the publication of papers dealing with work form the routine laboratory has facilitated this process. This year three such cases were published. The first of these dealt with a child with a partial deletion of chromosome 11 with the break occurring at the site of a familial fragile site. A second paper dealt with a case where coloboma of the right eye and Hirschprungs disease in a child were associated with multiple chromosomal rearrangements including a small deletion in chromosome 2. The third paper presented a child who had minor dysmorphic features, mental retardation and congenital isotropia which was found to be caused by absence of the right, and hypoplasia of the left lateral rectus muscles, while chromosomally the child had partial trisomy of chromosome 7 due to an unbalanced insertional rearrangement which was maternally inherited. The chromosomal diagnosis of fragile X associated mental retardation is a significant component of the work of the laboratory. In 1986 the laboratory collaborated in a project investigating the phenotypic variation in male transmitted fragile X undertaken by Dr. D. Loesch and Dr. D. Hay of Latrobe University.

Embryology group - Overview Marie Dziadek Two broad areas of research are planned - assessment of the roles of extracellular matrix in embryonic development and development of methods of producing mutant mice with characteristics with embryological interest by insertion mutagenesis.

Specific roles of extracellular matrix seem likely during embryonic development in relation to controlling cell proliferation, cell migration, cell shape and cell differentiation. The first step is to analyse variations in the structure of embryonic and adult extracellular matrices (especially basement membranes) to get some indication of the relationship between structure and function. Factors controlling synthesis and degradation of basement membranes are likely to be important in the assembly and remodelling which goes on during embryonic development.

Mutations are very powerful tools in the study of normal processes and this is especially true in embryology. Much has been learnt from the careful analysis of spontaneous and induced mutations in mice over the last 50 years. Recent experience with the mouse mutant produced by Dr. Rudi Jaenisch by insertional disruption of the collagen I gene has illustrated the power of the new molecular methods of inducing mutations which are more easily identified than those occurring spontaneously or produced by the older mutagens. We believe that this method of inducing mutations can be made even more specific and are actually recruiting personnel to explore this approach. We believe that embryo derived stem cell lines will be the raw material for these studies and Kerry Fowler is currently working at establishing such cell lines of normal karyotype as a part-time M.Sc. project - learning a number of the key techniques from Jeff Mann before he moved to Monash University, where he will still collaborate in this work.

Monoclonal antibodies to basement membrane components M. Dziadek, R. Clements, K. Mitrangas, H. Reiter, K. Fowler Monoclonal antibodies to the basement membrane glycoproteins, laminin and nidogen, are needed for use in studies of the structural heterogeneity of basement membranes, and the changes in basement membranes during remodelling. We have immunised rats with mouse antigens and although we could detect a strong immune response using specific radioimmunoassays we have managed to generate very few monoclonal antibodies. The hybridisation procedures are being modified. We have isolated and characterised one monoclonal antibody against nidogen, which also reacts with a stable proteolytic fragment of this protein. This monoclonal antibody appears to recognise all embryonic and adult basement membranes which we have studied by immunofluorescence using unfixed frozen sections. Brief fixation in 70% ethanol abolishes reactivity in cJl embryonic basement membranes apart from Reichert’s membrane, and several, but not all, adult basement membranes (e.g. tubular, but not glomerular, basement membranes in adult kidney). This monoclonal antibody has been very useful in our studies on the susceptibility of basement membranes to proteolytic degradation We have not been able to produce good monoclonal antibodies to laminin. This is surprising because laminin is an extremely antigenic glycoprotein. However, we have

isolated 6 monoclonals which show partial binding to radiolabelled laminin (5-20%), but do not react with basement membranes by immunofluorescence. These antibodies have different reactivities on tissue sections, some recognising epithelial structures, and others binding to muscle layers. It would be interesting to determine whether they recognise variant laminin molecules present in other tissues, or are merely cross-reacting with other molecules which may have similar epitopes, but this question does not have a high priority at present.

We have used polyclonal antibodies which recognise two different domains of laminin in immunofluorescent reactions on tissue sections. While antibodies against the short arms recognise aU basement membranes, antibodies against the long arm show restricted reactivity. Most spectacular was the heterogeneous staining of kidney tubules. Our interpretation is a differential staining of proximal and distal tubular basement membranes, and we are using additional histological techniques to verify this. We still hope to isolate monoclonal antibodies to these domains of laminin which will allow us to study the structural and functional significance of these staining patterns.

Analysis of basement membrane structure

molecules. We hope to use these antibodies to demonstrate the complex in sections of embryonic tissues which contain too little basement membrane to attempt purification of the complex.

Proteolytic degradation of basement membranes M. Dziadek, K. Mitrangas Remodelling of basement membranes is associated with various developmental events, including branching morphogenesis of epithelial glandular organs and epithelial-mesenchymal trEmsformations. We wish to investigate the factors which control very localised remodelling of matrices. Site specificity could be controlled by a localised milieu of proteases, an altered structure of basement membranes at certain sites which renders it more susceptible to degradation, or a combination of both. Nidogen appears to be the basement membrane component most susceptible to proteolytic degradation. Fragments produced after degradation do not interact with other basement membrane components, and would be readily solubilised from the matrix. We have incubated sections of various embryonic and adult tissues with enzymes known to degrade nidogen in different ways, and

M. Dziadek, R. Clements

The organisation of basement membranes depends on the interactions between component molecules. Heterogeneity in basement membrtme structure in different tissues may arise by differences in such interactions, which may alter the interactions between cell surface receptors and the matrix. Intermolecular interactions have been elucidated using components isolated from the FHS tumor as a model basement membrane. However, it is not at all clear whether these interactions are responsible for maintaining the integrity of the tumor basement membrane in situ, and whether similar interactions operate in other basement membranes. Laminin and nidogen interact with each other particular strongly, and intact laminin-nidogen complexes can be isolated from the FHS tumor matrix, but it is not clear whether a laminin-nidogen complex ctm be purified from normal tissues. Using procedures established for the FHS tumor, we have recently isolated this complex from mouse placenta. We are currently trying to increase the yields, to compare the complexes isolated from both tissues with respect to affinity and protein domains involved. We have also immunised rats with the laminin-nidogen complex to produce monoclonal antibodies which recognise the complex, but neither of the individucJ

Embryonic basement membranes appear to be structurally different to adult basement membranes, shown here by a difference in the susceptibility of nidogen to proteolytic degradation. Immunofluorescent staining of control sections of embryonic mouse lung (a) and adult mouse kidn^ (c) shows nidogen localized in basement membranes of both tissues. After treatment of sections with trypsin, nidogen immurwstaining disappears in embryonic lung (b) but persists in adult kidn^ (d).

Current Staff

used immunofluorescence with our monoclonal antibody to monitor the disappearance of this fragment of nidogen from the matrix. Nidogen in many embryonic basement membranes was more readily degraded by trypsin and thermolysin than that in sections of the EHS tumor, while in the majority of adult basement membranes (e.g. blood vessels) it is relatively resistant to degradation. However, nidogen in Reichert’s membrane appeared particularly stable against degradation by these enzymes. Nidogen in most embryonic and adult basement membranes could be almost completely solubilised by 2.0M guanidine hydrochloride, whereas nidogen in Reichert’s membrane was little affected by this treatment. Thus, basement membranes do differ in their susceptibility to degradation or solubilisation.

Analysis of basement membrane assembly

Administration:

Basement membrane components are localized at the interface between epithelial (e) and mesenchymal (m) compartments of embryonic tissues sueh as stomach (a) and lung (b), but are also associated with mesenchymal celb. In regions of the lung undergoing branching morphogenesis, these mesenchymal cells may be contributing to new assembly of basement membranes.

M. Dziadek Various cell types are known to synthesise all or some of the basement membrane components which have been characterised so far. In general, it is assumed that epithelial and endothelial cells which are in close contact with basement membranes are mainly responsible for the synthesis and deposition of the matrix on their basal surfaces. Our studies on the synthetic repertoire of isolated epithelial and mesenchymal components of embryonic salivary gland, lung and visceral yolk sac show that mesenchymal cells synthesise both nidogen and laminin. We are currendy determining whether both the epithelial and the mesenchymal cells contribute components to the basement membrane at their interface, and whether variations in the respective contributions may account for heterogeneity in assembly. The mesenchymal extracellular distribution of laminin and nidogen indicates that these components may also interact with interstitial matrix collagens and fibronectin. Our studies show that nidogen within the mesenchymal matrix is much more soluble and more readily degraded by proteases than nidogen present within the actual basement membrane of embryonic tissues.

Production of embryo-derived cell lines K. Fowler, J. Mann, R. Cotton, M. Dziadek We plan to study mouse mutations which cause malformations or embryonic lethality. Insertion mutagenesis of embryo-derived stem cell lines will be the first step. These cells could then he introduced into normal embryos by injection or aggregation procedures. Colonisation of the germ line of the host embryo would result in appearance of offspring having the niutation. Unlike embryological mutations which have arisen spontaneously or have been established after chemical or radiation mutagenesis, we will be able to identify the molecular defect by using the inserted DNA as a genetic probe. We are attempting to generate pluripotential stem cell lines from preimplantation mouse embryos following procedures established in other laboratories. Although the initiators of these procedures have described a 25 % success rate, we have only one possible cell line to date. Better results are anticipated with experience.

David Banks, M.D., B.S., ER.A.C.P. - Scientific Director, 1962Dick Cotton, B.Ag.Sci., Ph.D., D.Sc. - Deputy Director, 1968-70, 1973Anne Ellis, B.Sc., B.Bus.(Acc.) - Administrative Officer, 1975Barry Holt, B.App.Sci.(M.T.), A.A.I.M.L.S. Laboratory Mamager, 1972Sue Hirst - Fundraising Assistant (P/T), 1985Anneke Veenstra, Cert.App.Sci.(M.Lab.), Ass.Dip. App.Sci.(Media Production) - Graphic Designer/ Photographer, 1985-

Scientists (Senior): Dick Cotton, B.Ag.Sci., Ph.D., D.Sc. - 1968-70; 1973 Protein Chemistry Jim Camakaris, B.Sc., Ph.D., 1975 - Trace Element K.H. Choo, B.Sc.(Hons.), Ph.D., 1979-80; 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 Marie Dziadek, B.Sc.(Hons.), D.Phil.(Oxon.), 1985 Embryology Harry McArdle, B.Sc.(Hons.), Ph.D., 1985 - Trace Element

Research Assistants: Ian Jennings, B.Sc., 1975 - Protein Chemistry Andrew Grimes, B.App.Sci., 1975 - DNA Denise Kirby, B.Sc.(Hons.), 1976 - Enzymology Kerry Fowler, B.App.Sci.(M.L.T.), 1976 - CeU Culture Leigh Ackland, M.Sc., 1978 - Trace Element Ruth Brown, M.Sc., 1980 - Cell Culture NeU Francis, B.App.Sci.(M.L.S.), 1979 - Trace Element Wendy McAdam, 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 McCaskiU, B.App.Sci.(M.L.T.), 1981 Enzymology Wendy Hutchison, B. App.Sci.(Applied Biology), 1984 DNA Judy Dodge, B.Sc.(Hons.), M.Sc., 1985 - Clinical Peter Gatehouse, 1985 - Cell Culture Richard Clements, B.Sc., 1986 - Embryology Sharon Gross, B.Sc., Grad.Dip.Diet., 1986 - Trace Element

Kathy Mitrangas, B.Sc.(Hons.), 1986 - Embryology Haley Vogel 1986 - Trace Element/DNA Sue Hunt, B.Sc.(Hons.), 1978 - Enymology Peter Wajngarten, B.App.Sci.(M.L.S.), 1984 - Cell Culture

Postdoctoral Fellows: Samantha Wake, B.Sc.(Hons.), 1986 - DNA/Clinical Kiyoshi Hayasaka, M.D., Ph.D., 1986 - Enzymology

Students: Ph.D.: Janet Patten, B.Sc.(Hons.), 1982Les jones, B.Sc.(Hons.), 1980Bryce Vissel, B.Pharm.(Hons.), 1986John Christodoulou, M.B., B.S., 1986-

Clinical Genetics (Staff): David Banks, M.D., B.S., ER.A.C.P, 1962John Rogers, M.B., B.S., D.C.H., ER.A.C.P, 1976Agnes Bankier, M.B., B.S., FR.A.C.P., 1983Les Sheffield, B.Med.Sci., M.B., B.S., M.Sci., D.C.H., ER.A.C.P, 1985-

Clinical Fellows: Jenny McGUl, M.B., B.S., 1986Jim McGill, M.B., B.S., 1985John Christodoulou, M.B., B.S., 1986-

C y togeneticist s: Margaret Leversha, B.Sc.(Hons.), 1978Sue Dale, B.Sc.(Hons.), 1978-83, 1985Desiree Dusart, B.App.Sci., 1983Paula Stoddart, 1986Rhonda Hutchinson, M.Sc., 1986VidaPetrovic, B.Sc., 1986LucUle VouUaire, M.Sc., 1985Julie Roberts, B.Sc., 1986-

Co-ordinator, Genetics: Ann Glynn, B.S.W., 1985-

Secretaries: Lorraine Thompson (White) 1971-77; 198- (P/T) Sue Tomkins 1977Margaret Turnbull 1981Sharon Grosvenor 1985Julie Taylor 1986Jo Wells 1983- (P/T) AUsa Whitley 1986- (P/T)

List of Publications, 1986

IN PRESS PREVIOUS REPORTS, NOW PUBLISHED

MERCER, J.EB. AND WAKE, S. An analysis of the rate of metallothionein mRNA poly(A) shortening using RNA blot hybridization. Nucleic Acids Research 13:7929-7943 (1985)

BYRNE, E., DENNETT, X., GROTTY, B., TROUNCE, L, SANDS, J.M., HAWKINS, R., HAMMOND, J., ANDERSON, S., HAAN, E.A. AND POLLARD, A. Dominantly inherited cardioskeletal myopathy with lysosomal glycogen storage and normal acid maltase levels. Brain 109:523-536(1986).

ROGERS, J.G., GREENAWAY, J.C., MIRKES, RE. AND SHEPARD, T.H. Methacrylic acid as a teratogen in rat embryo culture. Teratology 33:113-117 (1986). WEBB, G.G. AND FABB, S.A. Probing murine male meiosis using unique DNA flanking the immunoglobulin heavy chain genes. Cytobios 43:159-165 (1985).

COLE, WG. AND KIRBY, D.M. Urinary free amino acids in osteogenesis imperfecta. Bone 7:13-16 (1986). COTTON, R.G.H. A model for hyperphenylalaninaemia due to tetrahydrobiopterin deficiency. J. Inher. Metab. Dis. 9:4-14 (1986). DAHL, H.H.M. AND MERGER, J.EB. Isolation and sequence of a cDNA clone that contains the complete coding region of rat phenylalanine hydroxylase. Structural homology with tyrosine hydroxylase, glucocorticoid regulation and use of alternate polyadenylation sites. J. Biol. Chem. 261:4148-4153 (1986). DANKS, D.M. AND BROWN, G.K. Inborn errors of metabolism in the neonate, in Textbook of Neonatology (ed) N.R.C.Robertson, Ghurchill Livingstone, Edinburgh, 1986. FINCHER, G.B., LOCK, P.A., MORGAN, M.M., LINGELBACH, K., WETTENHALL, R.E.H., MERCER, J.EB., BRANDT, A. AND THOMSEN, K.K. Primary structure of the (1 —3,1 ->4)-/3-D-glucan 4glucanohydrolase from barley aleurone. Proc. Natl. Acad. Sci. USA 83:2081-2085 (1986). GUDDAT, L.W., COWAN, S.W, MACHIN, K.J., ISSAACS M.W AND COTTON, R.G.H. Crystallization and preliminary carystallographic data of the FAB fragment on an anti-phenylalanine hydroxylase monoclonal antibody. J. Mol. Biol. 186:479-480 (1985). HAAN, E.A., SCHOLEM, R.D., CROLL, H.B. AND BROWN, G.K. Malonyl coenzyme A decarboxylase deficiency. Clinical and biochemical findings in a second child with a more severe enzyme defect. Eur. J. Paed. 144:567(1986).

ALDRED, A., GRIMES, A., SCHREIBER, G. AND MERCER, J.EB. Rat caeruloplasmin: Molecular cloning and gene expression in liver, choroid plexus, placenta, yolk sac and testis. J. Biol. Chem. (in press).

COTTON, R.G.H. Inborn errors of pterin metabolism, in Folates and Pterins: Vol. 3 - Nutritional, Pharmacological and Physiological Aspects, (ed) R.L.Blakley, J. Wiley & Sons, Inc., 1986. COTTON, R.G.H. A model for hyperphenylalaninaemia due to tetrahydrobiopterin deficiency. Chemistry and Biology of Pteridines 1986.

COTTON, R.G.H., DAHL, H.H.M., MERCER,

BATEMAN, J.E, CHAN, D., WALKER, L, ROGERS, J.G. AND COLE, WG. Lethal perinatal osteogenesis imperfecta due to the substitution of arginine for glycine at residue 547 of the pro 1(1) chain of type 1 procollagen. J. Biol. Ghem. (in press). BROWN, G.K., HUNT, S.M., MITCHELL, D.K. AND DANKS, D.M. Profound neurological illness, relieved by protein restriction, in a baby with a transient disturbance in the metabolism of ingested isoleucine. Eur. J. Pediatr. (in press).

COULSON, B.S., TURSI, J.M., McADAM, WJ. AND BISHOP, R.E Derivation of neutralizing monoclonal antibodies to human rotaviruses and evidence than an immunodominant neuralization site is shared between serotypes 1 and 3. Virology 154:302-312 (1986).

BROWN, G.K. AND HAAN, E.A. Therapeutic mutation in inherited metabolic disorders in Recent Advances in Clinical Mutation, Vol. II, (ed. M.L. Wahlquist and A.S. Tinswell, John Libby, London, 1986.

DAHL, H-H.M., HUNT, S.M., HUTCHISON, WM. AND BROWN, G.K. The human pyruvate dehydrogenase complex: isolation of cDNA clones for the El subunit, sequence analysis and characterisation of the mRNA. J. Biol. Chem. (in press).

BANKIER, A. AND HAAN, E. Autosomal dominant Brachmann de Lange syndrome. Am. J. Med. Genet. 25: 163-165, 1986. BATEMAN, J.E, CHAN, D., MASCARA, T, ROGERS, J.G. AND COLE, WG. Collagen defects in lethal perinatal osteogenesis imperfecta. Biochem. J. 240: 699-708, 1986.

BOARD, P.G. AND WEBB, G.C. Isolation of a cDNA clone and localisation of human glutathione S-transferase 2 genes to chromosome band 6pl2. Proc. Nat. Acad. Sci. (USA) (in press).

HUNT D.M., WAKE, S., MERCER, J.EB. AND DANKS, D.M. A study of the role of metallothionein in the inherited copper toxicosis of dogs. Biochem. J. 236:409-415 (1986).

CHOO, K.H., BROWN, R., WEBB, G., CRAIG, I.W, AND FILBY R.G. Genomic organisation of human centromeric alpha satellite DNA: Characterisation of a chromosome 17 alpha satellite sequence. DNA - J. Molec. Biol, (in press).

MANN, J.R. DDK egg-foreign sperm incompatibility in mice is not between the pronuclei. J. Reprod. Fert. 76:779-781 (1986).

CHOO, K.H., RAPHAEL, K., McADAM, W. AND PETERSON, M.G. Expression of active human blood clotting factor IX in transgenic mice. Nucleic Acids Research 15: 871-884, 1987.

J.EB., JENNINGS, I., HAAN, E.A., CHOW, C.W., DANKS, D.M. AND MORGAN, E Molecular biology of phenylalanine hydroxylase. J. Inher. Met. Dis. 9: Suppl.2, 206-208, 1986. COTTON, R.G.H., JENNINGS, I., BRACCO, G., PONZONE, A. AND GUARDAMAGNA, O. Tetrahydrobiopterin non-responsiveness in dihydropteridine reductase deficiency is associated with the presence of muttmt protein. J. Inher. Metab. Dis. 9: 239-243 (1986). GOULSON, B.S., FOWLER, K.J., WHITE, J.R. AND COTTON, R.G.H. Non-neutralizing monoclonal antibodies to a trypsin-sensitive site on the major glycoprotein of rotavirus which discriminates between virus serotypes. Arch. Virol, (in press).

HALLIDAY, J., CHOW, C.W, WALLACE, D. AND DANKS, D.M. X-linked hydrocephalus: a survey of a 20 year period - Victoria, Australia. J. Med. Genet. 23:23-31 (1986).

JENNINGS, LG., RUSSEL, R.G.McR., ARMAREGO, W.L.E AND COTTON, R.G.H. Functional analysis of the effect of monoclonal antibodies on phenylalanine hydroxylase. Biochem. J. 235:133-138 (1986).

PUBLISHED AND ACCEPTED FOR PUBLICATION SINCE 1985 REPORT

CHOO, K.H., FILBY, R.G., JENNINGS, I.G., PETERSON, G. AND FOWLER, K. Vectors for expression and amplification of cDNA in mammalian cells: expression of rat phenylalanine hydroxylase. DNA 5: 529-537 (1986).

CHOO, K.H., FILBY, G., GRECO, S., LAU, YF AND KAN, YW. Cosmid vectors for high efficiency DNAmediated transformation and gene amplification in mammalian cells: studies with the human growth hormone gene. Gene 46: 277-286, 1986.

DAHL, H-H.M., HUTCHINSON, W, McADAM, W., WAKE, S., MORGAN, EJ., COTTON, R.G.H. Human dihydropteridine reductase: characterisation of a cDNA clone and its use in analysis of patients with dihydropteridine reductase deficiency. Nucl. Acids Res. (in press). DANKS, D.M. Mathison lecture - Double helix: double joy. Chiron l:(No.4)13-18 (1986). DANKS, D.M. Molecular genetics in medicine. Chiron l:(No.3)26-30 (1985). DANKS, D.M. Of mice and men, metals and mutation. J. Med. Genet. 23:99-106 (1986).

DANKS, D.M. AND WRAITH, J.E. Prenatal Diagnosis, in Fetal and Neonatal Neurology and Neurosurgery, (ed) M.I.Levene, M.J.Bennett andj. Punt, Churchill Livingston, Edinburgh. DANKS, D.M., WRAITH, J.E. AND BROWN, G.K. Inborn Errors of Metabolism, in Fetal and Neonatal Neurology and Neurosurgery, (ed) M.I.Levene, M.J.Bennett and J. Punt, Churchill Livingston, Edinburgh. DANKS, D.M. Acute neonatal illness in inborn errors of metabolism, in Paediatric Emergencies, (ed) J.A.Black, Butterworths, London (2nd Edn). DeAREE, M.A., HECHT, E, SUTHERLAND, G.R. AND WEBB, G.C. Guidelines for the diagnosis of fragile X. Clin. Genet. 29:95 (1986). D’SOUZA, S.E. AND MERCER, J.EB. Antithrombin III mRNA in adult rat liver and kidney and in rat liver during development. Biochem. Biophys Res. Comm, (in press). FONG, L.V., WRAITH, J.E., CHOW, C.W, MENEHAM, S. Endocardial fibroelastosis occurring in the Maroteaux-Lamy syndrome. Clin. Cardiol, (in press). GIBBS, R., CAMAKARIS, J., HODGSON, G. AND MARTIN, R. Molecular characterisation of 1251 decay and X-ray induced CHO HPRT mutants. International J. Radiation Biology (in press). GIBBS, R., CAMAKARIS, J., STEVENSON, T. AND MARTIN, R. Structural antJysis of 1251 decay and X-ray induced HPRT-mutants. Proc. Australian Biochemical Society 18: 4 (1986). HAAN, E.A., BROWN, G.K., MITCHELL, D., DANKS, D.M. Succinic semialdehyde dehydrogenase deficiency - a further case. J. Inher. Metab. Dis. 8: 99 (1985). HAAN, E.A., JENNINGS, LG., CUELLO, A.C., NAKATA, H., CHOW, C.W, KUSHINGSKY, R., BRITTINGHAM, J. AND COTTON, R.G.H. A monoclonal antibody recognizing all three aromatic amino acid hydroxylases allows identification of serotonergic neurons in human brain. Brain Research (in press). HAAN, E.A., SCHOLEM, R.D., PITT, J.J., WRAITH, J.E. AND BROWN, G.K. Episodes of severe metabolic acidosis in a patient with 3-methyl glutaconic aciduria. Eur. J. Pediatr. (in press). HETHERINGTON E.L.R., SORKY, PJ. AND CAMAKARIS, J. The preparation of high specific activity copper-64 for medical diagnosis. Int. J. Applied Rad. and Isotopes (in press). JIEQING, Z., ALLAN, G.L., LEGGE, G.J.E, McCALLUM, J.C., McKENZIE, C.D. AND CAMAKARIS, J. Recent advances with the scanning microprobe. Nuclear Techniques 4: 5-14 (1986). KEITH, C.G., WEBB, G.C. AND ROGERS, J.G. Absence of a lateral rectus muscle associated with duplication of 7q34 to 7q36. J. Med. Genet, (in press). 77

KOOPMAN, P. AND COTTON, R.G.H. The response of embryonal carcinoma cells to retinoic acid depends on colony size. Differentiation 31: 55-60 (1986). KOOPMAN, P. AND COTTON R.G.H. Pluripotent differentiation of single F9 embryonal carcinoma cells. Exp. Cell Res. 168: 567-571 (1987). KOOPMAN, P. AND FOWLER, K.J. The rapid heating of culture media with microwaves does not impair cell growth. Aust. J. Med. Lab. Sci. 7:55-56 (1986). LIM, B.C., McARDLE, H.J., MORGAN, E.H. Transferrin-receptor interaction and iron uptake by reticulocytes of vertebrate animals - a comparative study. J. Comp. Physiol, (in press). LOESCH, D.Z., HAY, D.A., SUTHERLAND, G.R., HALLIDAY,J., JUDGE, C. AND WEBB, G.C. Phenotypic variation in male transmitted fragile X: genetic inferences. Amen J. Med. Genet, (in press). LOVELLBADGE, R.H. AND MANN, J.R. Whole genome transfer in mammals. In Exploiting New Technologies in Animal Breeding (eds. Smith, King and McKay) Oxford University Press (1985). McGILL, J., MADDISON, T, COLLINS, K., POWELL, H. AND HAAN, E. I^igh’s disease. Med. J. Aust. 144:111 (1986). McGILL, J.J. AND ROBERTON, D.M. A new type of transient diabetes mellitus of infancy? Arch. Dis. Child. 61:334-336(1986). MANN, J.R. AND LOVELLBADGE, R.H. The development of XO gynogenetic mouse embryos. Development (in press).

SCHNIEKE, A., DZIADEK, M., BATEMAN, J., MASCARA, T, HARBERS, K., GELINAS, R. AND JAENISCH, R. Introduction of-the human proal mouse:human hybrid type 1 collagen. Proc. Nad. Acad. Sci. U.S.A. (in press). SMITH, S.C., McADAM, W.J., KEMP, B.E., MORGAN, EJ. AND COTTON, R.G.H. A monoclonal antibody to the phosphorylated form of phenylalanine hydroxylase: definition of the phosphopeptide epitope. Biochem. J. (in press). SPEER, A., REISS, O., COBET, G., HANKE, R., DAHL, H., COTTON, R.G.H. AND COUTELLE, C. Typing of families with classical phenylketonuria using three alleles of the Hindlll linked restriction fragment polymorphism, detectable with a phenylalanine hydroxylase cDNA probe. Clin. Genet, (in press).

VOULLAIRE L.E., WEBB, G.C. AND LEVERSHA, M.A. Chromosome deletion at llq23 in a child with fragility at the same site in a parent and a sibling. Hum. Genet, (in press). WEBB, G.C., KEITH, C.G. AND CAMPBELL, N. Concurrent de novo deletion of band 2p22 and reciprocal translocation (3;7)(p21;q22). J. Med. Genet, (in press).

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WICKING, C.A., SCHOLEM, R.D., HUNT, S.M. AND BROWN, G.K. Immunochemical analyses of normal and mutant forms of human pyruvate dehydrogenase. Biochem. J. 239: 89-96 (1986). WONG, C.T, McARDLE, H.J., MORGAN, E.H. The effect of iron chelator on placenta uptake and transfer of iron in the rat. Am. J. Physiol, (in press).

MERCER, J.EB. AND GRIMES, A. Isolation of a cDNA clone for human caeruloplasmin that includes the complete N-terminus. FEBS letters 203:185-190 (1986). MERCER, J.FB. AND GRIMES, A. Variation in the levels of hepatic metallothionein mRNA during rat development. Assessment of the role of hepatic zinc as a primary inducer. Biochem. J. 238: 23-27 (1986).

MERCER, J.EB., McADAM, W, CHAMBERS, G.W AND WALKER, I.D. The W and L allelic forms of phenylalanine hydroxylase in the rat differ by a threonine to isoleucine substitution. Biochem. J. 236:679-683 (1986). PETERSON, M.G. AND MERCER, J.EB. Sequence and regulation of the sheep metallothionein-la gene. Eur. J. Biochem. 160: 579-585 (1986). PHILLIPS, M., CAMAKARIS, J. ANDDANKS, D.M. Comparisons of copper deficiency states in the murine mutants blotchy and brindled. 2. Changes in copper dependent enzyme activity in 13 day old mice. Biochem. J. 238: 177-183 (1986). ROGERS, J.G. AND DANKS, D.M. Cutis laxa with delayed development. Aust. Paediatr. J. 21:281-283 (1985).

79-

A.:,

The Murdoch Institute for Research into Birth Defects Limited

STATEMENT OF INCOME AND EXPENDITURE FOR PERIOD ENDED 31 DECEMBER, 1986 1986 INCOME Grants - Royal Children’s Hospital - Other Donations Interest

$ 230,000 394,127 1,596,023 643,341

TOTAL INCOME

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

2,863,491

1,128,835 37,504 263,612 288,808 18,526 53,048 13,400 2,350 81,429

TOTAL EXPENDITURE

1,887,512

SURPLUS FOR PERIOD

975,979

BALANCE SHEET AS AT 31 DECEMBER, 1986 1986 MEMBERSHIP FUNDS Accumulated Funds

$ 3,815,979 3,815,979

Represented By: CURRENT ASSETS Cash at Bank Investments (Market Value $5,509,733)

27,867 4,587,895 4,615,762

Less CURRENT LIABILITIES Sundry Creditor - Royal Children’s Hospital Accrued Expenses Provision for Long Service Leave Grants in Advance

624,455 64,718 69,441 41,169 799,783 3,815,979

The Murdoch Institute for Research into Birth Defects Limited

NOTES TO AND FORMING PART OF THE FINANCIAL STATEMENTS:

AUDITORS’ REPORT To The Members Of The Murdoch Institute For Research Into Birth Defects Limited

1. Statement of Accounting Policies: a) The Financial statements have been prepared: i) on the basis of the historical cost convention ii) in accordance with the prescribed requirements set out in schedule 7 as in force immediately before 1 October, 1986. b) Fixed Assets Generally, grants received for the purpose of acquiring equipment are specifically designed to meet the cost of individual items of equipment. All items of equipment are written off in the year of purchase. The amount of the write off in 1986 was $288,808. 2. The Institute was incorporated on 20 May, 1986. 3. This being the first Financial Report of the Institute no comparative figures are shown.

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 period ended 31 December, 1986. 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 31 December, 1986. 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. Melbourne by Order of the Board 8 April, 1987.

The Murdoch Institute for Research into Birth Defects Limited

We have audited the accompanying accounts being the Balance Sheet, Statement of Income and Expenditure, Notes 1 to 3 and Directors’ Statement thereon in accordance with Australian Auditing Standards. In our opinion the accounts are properly drawn up in accordance with the provision of the Companies (Victoria) Code so as to give a true and fair view of: (i) the state of affairs of the Institute at 31 December, 1986 and of the surplus of the Institute for the period ended on that date; (ii) the other matters required by Section 269 of that Code to be dealt with in the accounts; Emd are in accordance with Australian Accounting Standards and applicable approved accounting standards.

- R. Neil Walford (Director) Melbourne 8 April, 1987.

TOUCHE ROSS & CO.

- Laurence G. Cox (Director)

B. Jamieson - Partner CHARTERED ACCOUNTANTS

DIRECTORS’ REPORT The Murdoch Institute For Research Into Birth Defects Limited The Directors submit their report made in accordance with a resolution of the Directors with respect to the surplus of the Institute for the financial year ended 31 December, 1986 and the state of the Institute’s affairs at that date. (1) The name of the Directors in office at the date of this report are: Graeme L. Barnes James S. Guest William H. Hodgson Elisabeth J. Calvert-Jones Penelope M. Lewisohn Barry R. Catchlove Una P. Mackinnon Richard G. Cotton Peter D. Phelan Laurence G. Cox Graeme B. Ryan David M. Danks R. Neil Walford John A. Fitzgerald Gustav J. Fraenkel (2) The financial year commenced on the date of incorporation. May 20, 1986 and ended on December 31, 1986. At the date of incorporation the Institute was transferred from the Royal Children’s Hospital Reseau'ch Foundation. (3) The Principal activities of the Institute during the course of the financial year were to promote and undertake medical research related to birth defects. (4) The net surplus of the Institute for the financial year was $975,979. (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) The Institute continued its research into the incidence and causes of birth defects. (7) No matter or circumstance has arisen since the end of the year that has significantly affected or may significantly affect the operations of the Institute in subsequent financial years. (8) There are no likely developments which will significandy affect the operations at the Institute. by Order of the Board Melbourne 8 AprU, 1987. 4

- R. Neil Walford (Director)

Cox (Director)

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