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The Murdoch Institute for Research into Birth Defects
The Murdoch Institute is devoted to advancing knowledge about genetic diseases and other types of birth defects so that prevention may become possible in the future. The basic research of the Institute finds practical expressions through its clinical arm, the Victorian Clinical Genetics Service, which provides a network of services throughout Victoria available to persons of all ages. The close integration of research and clinical service has provided a rich source of research ideas and prompt implementation of new knowledge.
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During its short history of eight years the Murdoch Institute has made important scientific discoveries, achieved recognition as one of Australia’s top medical research centres,
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the premier training centre of clinical geneticists, and established a network of clinical services which is serving as a model to other countries and states. The generous founding gifts of the Murdoch family, the late Sir Jack Brockhoff and other supporters set the Institute on a sound footing. Many of these generous friends still continue their support, but the Institute ■ ?■
desperately needs further supporters to complete the final stage of its establishment alteration of space purchased at the Royal Children’s Hospital to expand its laboratories and clinical facilities to the size required.
The Murdoch Institute for Research into Birth Defects Limited Royal Children’s Hospital Flemington Road PARKVILLE VICTORIA 3052 ACN 006 566 972 Postal address: Post Office Box 1100 PARKVILLE VIC 3052 Telephone: (03)345 5045 Facsimile: (03) 348 1391
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The Murdoch Institute
Scientific Director: Professor D.M. Danks Deputy Scientific Director: Dr. R.G.H Cotton Birth defects kill or maim i in 50 babies. The strain on families is enormous. The cost to the Australian community exceeds $2,000,000,000 a year. Research into the genetic causes of birth defects is starting to reduce these figures. The Murdoch Institute needs your support every year to maintain the momentum of
BOARD OF DIRECTORS Mr. L.G. Cox, Chairman Mr. W.FI. Flodgson, Vice Chairman
research into these genetic conditions. The Institute needs special support to complete its process of establishment by developing laboratories and other essential facilities, at a cost of over $5 million.
Professor |. Angus 1
Dr. G.L. Barnes Mrs. J. Calvert-Jones Dr. R.G.H. Cotton
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Professor D.M. Danks Mr. I. Davies Mr. ).A. Fitzgerald
Would you please give generously now. All donations are tax deductible. If you cannot give now, consider helping The Murdoch Institute through a bequest.
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Mr. P.|. Griffin Mr. J.S. Guest Mrs. A. McFarling Mr. A.S. Murdoch Professor P.D. Phelan Professor A.J. Pittard
The following is a suggested form for a bequest to the Murdoch Institute.
For further information contact the Business Manager, The Murdoch Institute for Research into Birth Defects. Royal Children’s Hospital Flemington Road, Parkville, Victoria 3052 AUSTRALIA Telephone; (03) 345 5045 Fax: (03) 348 1391
Mr. N. Clark, Chairman Sir Gordon Allard Mr. L.G. Cox Mr. D. Craig Mrs. A. Cronin Professor D.M. Danks Mr. J. Fitzgerald Mr. P. Griffin Mr. M. Handbury Mr. G.E. Heeley Mr. N. Miller Mr. D.E. Meikeljohn Dr. M. Robinson
Professor G.B. Ryan
I bequeath to The Murdoch Institute for Research into Birth Defects the sum of $ . (or part or all of residue of estate) to be applied for the purposes of the Institute.
BUILDING AND DEVELOPMENT APPEAL COMMITTEE
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FINANCE COMMITTEE Mr. P.J. Griffin (Chairman) Mr. LG. Cox Mr. C.P. Abbott Mr. D.T. Craig Mr. G.E. Heeley Mr. W.H. Hodgson Mr. D.E. Meikeljohn Mr. F.D. Ryan
VICTORIAN CLINICAL GENETICS SERVICES BOARD OF DIRECTORS Mr. L.G. Cox, Chairman Dr. G.L. Barnes Professor D.M. Danks Dr.). De Campo Mr. J.S. Guest Mr. G.E. Heeley Mr. W.H. Hodgson
Major Donors to the Murdoch Institute
FOUNDERS - Donors of $1 million or more. The Murdoch Family: Dame Elisabeth Murdoch Mr. Rupert Murdoch Mrs. Helen Handbury Mrs. Anne Kantor Mrs. Janet Calvert-Jones The late Sir Jack Brockhoff The Brockhoff Foundation The Scobie and Claire Mackinnon Trust
BENEFACTORS - Donors of $250,000 or more The Miller Foundation The Helen M. Schutt Trust
TRUSTEES - Donors of $25,000 or more Arthur Andersen Foundation Coles Myer Limited H. & L. Hecht Trust J.B. Were & Son Charitable Foundation Mrs. Joan Roxburgh Mrs. M.L. Griffin National Australia Bank Limited Qantas Repco Corporation Limited (Ariadne) The Broken Hill Pty Ltd The Danks Trust The Friends of the Murdoch Institute The Ian Potter Foundation The late Mr. Clive Roxburgh The late Mrs. L.B. Quayle The Morris Family Trust The News Corporation Limited The Percy Baxter Charitable Trust The Sidney Myer Fund
CORPORATE SPONSOR GROUP Corporations undertaking substantial future support The Broken Hill Pty Ltd National Australia Bank Limited
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Donations to the Murdoch Institute 1993
General The Jack Brockhoff Foundation Cruden Investments The Scobie & Claire Mackinnon Trust The Mijler Foundation Ltd
$ 350,000.00 250,000.00 100,000.00 50,000.00
M.H. Coins Patricia R. Shackell Dr. R.J. Collins Mr. & Mrs L. Barbieri Sir Gordon Allard Macleod Primary School Anthony Wells
200.00
100.00
200.00 200.00 150.00 150.00 150.00 100.00
The Broken Flill Pty Ltd Australian Fluntington’s Disease Association
50,000.00
National Australia Bank The Morris Family Trust
45,000.00
FI 81 L Hecht Trust The Securities Institute.
10,000.00 6,000.00
Bernard Dynes Brian Templeton Dr. Robin Small Mrs. G.A. Grimwade Grayson Andrew
Amcor Ltd Coca Cola Amatil
5,000.00
Lady Darvall
100.00 100.00
J.B. Were & Son Uncle Bob’s Club
5,000.00
Mr. & Mrs. K. Saville Mr. and Mrs. K. Troon T.M. Bryant Dr A.M. Penington
100.00
J.A. McLennan Pty Ltd M. & K. South
100.00
Jade Investments Dr. Geoffrey L. Gillam
100.00
W.H. Huffam P. Carpenter Jacaranda Wiley
100.00
B.J. Larwill H.D. Irish Dr. E.A. Lewis A.D. MacLean
100.00
Reliance Press Pty Ltd Anonymous Marfans Foundation
100.00
B.J. Darvell D.J. Lans Dame Patricia Mackinnon
50.00
G.L. Roseby Jason Leonidas Mrs. R.W. Mills Rosie Cunningham
50.00
Tanya Coleman Dr. Noel Cass
50.00
J.K. & J.E. Little Professor D.M. Danks Bonorge Pty Ltd (Training)
10,000.00
10,000.00
5,000.00
4,036.00 2,000.00 1,420.03 1,127.10
Anonymous Mr. G.E. Heeley Mr. Grant Stephenson
1,000.00
Dr. J.M. Gooch Mayne Nickless Ltd Mrs. N.B. Gantner McMullin Nominees Pty Ltd Mrs. B. Burzak-Stefanowski
1,125.00
1,000.00 1,000.00
1,000.00 1,000.00 900.00 800.00
Uncle Bob’s Club Mr. F.D. Ryan Mr. S.F. Gooley
800.00
Mr. E. Basarke S.F. Kimpton Kimberley Foundation Little Peoples Association of Australia
500.00
600.00 600.00
500.00 500.00 400.00
M.J. & A.J. Martin Becton Dickinson
370.00
Dr P Campbell
250.00
M.J. 8i D.l. Robinson Mr F Douglas Stephens Danielle & Craig Webber
250.00
300.00
250.00 200.00
s
100.00 100.00 100.00 100.00
100.00
100.00
100.00
100.00
100.00 100.00
100.00 100.00 100.00
100.00 85.00
50.00 50.00
50.00 50.00 50.00
50.00
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Dr E.K. Turner
50.00
Dr & Mrs G. Warne Gwenda A. Miller
50.00
N.H. Thies L. Markman J.E. Spong D.G. Bannister Dr. A. Weldon
50.00
P.F. Cosgriff J.G. Simpson Dr. N. McH Ramsey W.E. Swaney
50.00
G.E. Doery G. & F. Cran S.J. Crowden O.R. & T.L Greenwood J. Sampson Mr S.F. Wickramaslnghe A.C. a V.A. Walsh
50.00
50.00 50.00 50.00 50.00
50.00 50.00 50.00 50.00 40.00 40.00 30.00 25.00 25.00 25.00
Dr. M. Easton Dr. John Bartram Wendy Luscombe
25.00
Methodist Ladies College
20.00
25.00 20.00
The Institute thanks all those listed above for their generous support.
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Contents
Chairman's Report
Director's Report
Helping Families to Prevent Genetic Diseases
Improved DNA Testing for genetic disorders
Research in Progress
Victorian Clinical Genetics Service
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Chairman's Report
Neil Walford’s retirement as Chairman of the Institute was a great loss and I was most honoured to be asked to follow in his footsteps. Neil made an immense contribution in his i8 years of leadership and a more detailed recognition is set out on page 13 of this report. The responsibility of any Board is to ensure the continuity of first class leadership and management of the organisation. This will be our major task during 1994 as our founding Executive Director, David Danks, will retire in mid 1995 and it is important that we have his successor in place well before that time. David Danks was not only responsible for the establishment of the Institute, but has set extremely high standards in its conduct and will be very difficult to replace. We are conducting an International search and the Selection Committee, comprising representatives of the Board and the University of Melbourne, expects that a new Director can be appointed by the end of 1994. Whilst 1993 was very successful the challenges for 1994 and beyond are daunting. Our future is very dependent on the ability to fund growth in soundly based research activities and profound thanks are due to our donors past and present. The recession of the last four years has made it almost impossible to obtain major donations. Whilst we hope this position will change as the Australian economy recovers this will continue to be a major constraint on our activities. We must convince both State and Commonwealth Governments of the huge expansion of genetic services that will be necessary to deal with hereditary diseases of adult life. In this context we will continue to focus on the physical development of the Institute in order to accommodate the expanded activities of the Victorian Clinical Genetics Service, and to keep them in close contact with the Institute and the Royal Children’s Hospital. Close relations with the Hospital are very Important and during the year we appreciated the opportunity to consider the possible use of the renovated South-East Building. Unfortunately the costs are beyond our present resources and any move has been put on hold. During the year Bill Hodgson became Deputy Chairman and Peter Griffin was appointed as a'Director and also became Chairman of the Finance Committee. Their wide experience will be invaluable to the Institute. I would like to thank my Board colleagues for their valuable Input during 1993 and also to make special mention of the work of Mrs Ann McFarling and her “Friends of the Murdoch Institute”, Anne Cronin, Business Manager, and all members of the Institute staff.
Laurence G. Cox.
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App/eciation of Neil Walford, Chairman, Birth Defects Research Institute Fundraising Committee, 198^1-85 Chairman, Murdoch Institute Interim Board, 1985-86 Chairman, Murdoch Institute Board, 1986-93
I first met Neil Walford in his office in the elegant Victorian house in St Kilda Road which was then the Headquarters of Repco Ltd. The spacious office expressed the style and excellent taste with which I was to have the pleasure of becoming acquainted over the next 9 years. A mutual friend introduced me and it was my goal to persuade Neil to take on the role of chairing our Fundraising Committee. “Is this venture going to succeed? I expect things to succeed” opened our conversation. I assured him that I had no intention of wasting my time on something that didn’t succeed and we got on well from that moment. At that stage we had our eyes on raising $4.2 million. At the first meeting of the core members of the Fundraising Committee Laurie Cox, Bill Cowan and Neil persuaded me to list everything that we really needed for the sort of Institute we would like to develop and to work out what it would cost. This changed the figure to $10 million which became our target and was achieved.
Neil Walford
I believe we can all say to Neil • “with your leadership and guidance this thing has succeeded”. During his years in business Neil has had many careers and we are proud to have provided the setting for one of these. His style is to lead from the front and to assume what needs to be done can be done. I can only say that it is a great feeling for a Chief Executive to have a Chairman who so clearly expects you to get on and do what needs to be done, but is always happy to provide advice when requested. I am sure that all of those who worked with Neil will have their own personal memories of his contribution. Those who were intimately involved will know how much time and energy he put into ensuring that everything that needed to be done with the Institute was done well. Everyone who was fortunate enough to be present at the Launching Dinner in the Great Hall of the National Gallery in February 1987 will remember the sense of occasion which owed more to Neil’s style as Chairman than to any other single contribution.
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David Danks
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Genetics and the Community
Director's Report
It is sad to have to record Mr Neil Walford’s resignation, but numerous happy memories of his contributions will remain. It was characteristic of him to recognise that the process of appointing my successor and helping him/her to settle into the role should not be interrupted by a change of Chairman and to step aside at an early stage in the process. We are all very pleased that Mr Laurie Cox has accepted election as Chairman. He was the
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Laurie G. Cox Member, Birth Defects Research Institute Fundraising Committee,1983-85 Member, Interim Board, Murdoch Institute, 1985-86 Deputy Chairman, Murdoch Institute Board, 1986-93 Chairman, Finance & Investment Committee, 1986-93 Chairman, Murdoch Institute Board, 1993-
Peter Griffin Member, Finance 8i Investment Committee. 1986-93 Chairman, Finance & Investment Committee, 1993Member of Board, Murdoch Institute. 1993-
first businessman to agree to help us to raise the funds that we required to establish the Institute and helped us in many ways, especially as Chairman of our Finance & Investment Committee. We welcome the appointment of Mr Bill Hodgson as Deputy Chairman of the Board and Mr Peter Griffin as Chairman of the Finance & Investment Committee and a member of the Board. In 1993, we have had to consider several important general issues which will have a major influence on our future research and clinical activities - the rapid expansion of genetic knowledge and its application to community health, the role and control of gene therapy and the government funding of medical research. I have judged it timely to discuss the issues, especially the first, in this year’s Report. All our major projects have moved along well this year. The conversion of Dick Cotton’s chemical cleavage of mismatch (CCM) technique for detection of mutations to an enzyme cleavage method (ECM) has been a special achievement. We plan to expand work on this method, and other possible related
Bid Hodgson Member, Birth Defects Research Institute Fundraising Committee, 1983-85 Member, Interim Board, Murdoch Institute, 1985-86 Member, Murdoch Institute Board 1986Deputy Chairman, Murdoch Institute Board, 1993-
methods, in 1994 for reasons which are made clear in other sections of the Report. Our scientists in the Trace Element Research Group have made good use of their discovery of the gene at fault in Menkes disease and we expect to see rapid progress towards a full understanding of cellular copper transport in the next few years. Progress continues in the work on the way in which the centromere
controls chromosomal function. A bold new technique is starting a surge of exciting results in the analysis of the factors controlling cell migration in the developing embryo. More about these and other projects can be found in the section “Research in Progress”. This year we are experimenting with a new format for our Report and we hope you will tell us whether you think it is a step forwards or backwards. This section is written for our lay friends and supporters and as an introduction to new friends. Two separate sections describe our research to our scientific colleagues in Australia and overseas and present our formal Company Report and Financial Statements for the year of 1993. The problem of achieving adequate laboratory and office accommodation has been mentioned
During 1993 there has been a considerable increase in the amount of publicity given to advances in genetic knowledge and the applications that the media imagine may follow. Unfortunately a great deal of the reporting has been inaccurate and sensational. It is important for us to ensure that new genetic knowledge is used effectively and appropriately. We also need to find ways of educating the general public about the good uses to which the knowledge can be put and reassuring them that inappropriate use can be prevented. Attention to these matters has come to occupy a large part of my time and I have decided that this would be an appropriate year in which to focus on the applications of genetic knowledge in this annual report.
repeatedly in past Reports and has occupied our thoughts for much of 1993. Our Cytogenetics Laboratory was almost strangled by the combination of a rapid growth in work
that we set great importance upon coordination of basic research with the application of knowledge in the diagnosis, treatment and counselling of patients with genetic diseases. It was this attitude which determined that the
referred and a lack of space for expansion. At year’s end a temporary solution was found through the cooperation of the RCH Play Therapists who kindly moved from their offices
Victorian Clinical Genetics Service should be established as a subsidiary of the Murdoch Institute and function as a single service throughout the whole State, providing clinical
on the 4th Floor, adjacent to the Cytogenetics Laboratory, to temporary quarters in our vacant space in 10 West, as a step towards a permanent new home in the North-West Building. We are
services in all the hospitals that need them. It also makes it logical for the Institute and the clinical service to be located side by side in the Royal Children’s Hospital. Overall we are very pleased with the integrated arrangement that we
grateful to them and pleased that we are able to offer them Improved facilities. Our next move is to draw up detailed plans for conversion of 10 West to new laboratories for Cytogenetics, DNA Diagnostic Testing and Newborn Metabolic Screening and a vacant laboratory ready for the use of the new Director. A preliminary estimate of $1 million and the good return on our investments in 1993 has encouraged the Board to approve this step. Before proceeding we considered very carefully an opportunity offered by the Hospital of moving into 4 floors of the South-East Building. Unfortunately, our investigations showed that the costs of conversion were far beyond our means.
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All of our regular supporters will be well aware
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have achieved. All the overseas geneticists who have spent periods working with us as researchers or clinicians have commented favourably upon this arrangement. On the other
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hand, we have been much less successful than our colleagues in New South Wales in convincing the
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State Government to provide adequate resources for genetic services. They have achieved a staff
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establishment which is two to three times greater than the Victorian level, and are negotiating for a
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further doubling by 1997. We must increase our efforts to change this situation.
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Gene Therapy
The centre section of this report, devoted to a lay person’s explanation of some of our activities, attempts to provide simple descriptions of the advances that are occurring in our ability to analyse the detailed structure of genes (DNA Diagnostic Laboratory) and to apply this knowledge to the analysis of chromosomes (Cytogenetics Laboratory), following a general description of the progress that is occurring in genetic knowledge and the new clinical services which this knowledge will make possible. This leaves
patients always seems more urgent than the prevention of diseases. Modern genetics offers a particularly powerful approach to the prevention of many serious diseases of childhood and of adult life. A reaiiy efficient network of diagnostic services coupled with counselling and the availability of prenatal
rather little to be said in the formal report on
and presymptomatic diagnosis is required, not mass screening of the whole adult population or of all babies as the media keeps
the activities of the VCGS which is therefore
on suggesting. Preventive services should be
quite brief this year.
available without personal cost to encourage maximal use. The dramatic recent discoveries regarding hereditary forms of common cancers
For several years we have been talking about establishing genetic clinics in adult hospitals, but have not had the staff to pursue this idea vigorously. There was also some reluctance on the part of the adult hospitals. Dramatic changes in diagnostic tests for the important adult diseases like Huntington disease and myotonic dystrophy, and the impending vast role of these tests in the control of cancer, has changed these attitudes at the same time as Dr Mac Gardner’s arrival has made it possible for us to offer a service. It Is very pleasing that, by the time this report is published weekly clinics will have started at the Royal E'
it has always been difficult to persuade governments to finance preventive approaches in medicine. The treatment of individual sick
Melbourne Hospital and St Vincent’s Hospital. We would like to be able to provide full-time genetic counsellors to these two hospitals, in addition to the weekly visit by Dr Gardner, but we can only afford to send Ms Mary-Anne Young along with him to the weekly clinics.
may prove the factor which finally convinces governments of the importance of this approach.
Back in 1985 it was apparent that gene therapy of genetic diseases would start within the next decade and we were able to persuade the NHSiMRC to set up guidelines before there were any proposals for the use of this new form of treatment. As things have turned out the use of gene therapy in cancer has moved ahead much more quickly than its application to hereditary diseases. The guidelines have needed considerable revision and it has become clear that voluntary guidelines will be inadequate. Although this form of treatment is not really so radically different from conventional treatment, this is certainly not the perception of the media and some minority groups, it is very important to see that the introduction of these treatments proceeds in a manner which is above criticism.
Staff and visitors
We were pleased to have Dr Mac Gardner join us on a long term basis as an additional experienced clinical geneticist. He had demonstrated his skills as a clinician and a researcher over the years in which he was the only geneticist in Dunedin.
Dr Philip Welch of Halifax, Nova Scotia worked in our clinics for the first half of the year. Dr Moshe Chemke joined us for study leave in August, coming from israel. He has a particular interest in syndromes and in the POSSUM and OSSUM systems, to which he is contributing greatly. Dr Alasdair Hunter of Ottawa was with us just three months, studying the social adjustment of dwarf patients with bone dysplasias. We have also enjoyed several other enthusiastic young visitors. Dr Shamima Rahman from London has carried out a clinicai/laboratory study in the mitochondrial research group. Dr jolanda Flipsen from Rotterdam has played an important role in starting up a survey of disabled people in Victoria to identify those with fragiie-X
Eileen Treacy
syndrome, the most frequent hereditary cause of mental retardation. Dr Stefan Mundlos came from Mainz and has studied the biochemical basis of inherited bone diseases, in our clinics and in Dr John Bateman’s Orthopaedic Research Laboratory. His wife Dr Christine Mundlos has helped out in our overburdened cytogenetics laboratory. Dr Connie Sham, a qualified clinical geneticist from Hong Kong, spent six months with us gaining further experience in molecular cytogenetics and DNA diagnostic techniques.
Our clinical team was further strengthened by the temporary addition of Dr Geoff Woods, a young clinical geneticist from the UK, and Dr Eileen Treacy, a young Irish doctor, who trained in paediatrics, genetics and metabolic diseases in Canada. Geoff will stay until late 1994, but Eileen left in
My own efforts have focussed upon the promotion of the importance of modern
January 1994. Dr Geoff Thompson moved to Dublin in April after providing good care of our metabolic disease patients for several years and doing interesting research
genetic services to governments. Commonwealth and State, and the establishment of satisfactory systems of controlling the introduction of gene therapy in Australia, it is interesting that both of these activities have been dominated by new discoveries about the genetics of cancer.
on their diseases. Moshe Chemke
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Possum / Ossum
We have decided to combine POSSUM and OSSUM into a single system, particularly because the demand for OSSUM by clinical geneticists and paediatricians interested in birth defects has been much greater than anticipated. Unfortunately it has not proved as popular as we had hoped with paediatric radiologists. We announced the combined system at the meeting of the American Society of Human Genetics in New Orleans in October and have been pleased by the response. A new marketing arrangement is allowing those who would like to order the combined system to have the two separate systems immediately while awaiting delivery of the combined system in mid 1994. All this has required renegotiation of our business arrangements with Computer Power Ltd and I am grateful to Mr Ron Baxter and Mr ]ohn Marquet for the fine spirit of these negotiations. The agreement reached will make it financially feasible for us to continue the ongoing development of the information content of the systems without further drain on our research resources which had previously carried a heavy burden.
Scientists' News
We were pleased when the NH&MRC agreed to promote Dr Dick Cotton to Senior Principal Research Fellow, the NH&MRC name for professorial level in the research world. This is a further indication of the high regard in which
We have valued their advice of commercial prospects of several projects and now they have agreed to fund one aspect of Dr Andy Choo’s work on the centromeres of human
of invitations as guest speaker at congresses. Dr Andy Choo’s invitation to edit a book on In Situ Hybridisation Protocols for Humana Press indicates his international recognition.
chromosomes. If the centromere can be isolated it could be used to develop a human artificial chromosome. This would be of great
Other good news from NH&MRC came when the 1994 Projects Grants were announced and we learned that Dr jim Camakaris’ application had scored very highly in its peer review. For many years his work on copper transport has been supported through our NHMRC Program Grant or Block Grant. The copper work has been my
potential interest for gene therapy.
NH&MRC Grants and Infrastructure Costs
own particular interest and this arrangement may not prove satisfactory after my retirement,
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report is the support of infrastructure costs. When the NH&MRC was established to provide research grants to universities the level of Commonwealth Government support
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per academic staff member was relatively generous and it was reasonable to expect the overhead costs of the few extra research staff to be carried out of general revenue. Over the
so it is good that he will now have Independent support. Of course, the close collaboration between Julian Mercer and Jim
Following Senator Richardson’s proposal to increase the funding of medical research to
Camakaris will continue.
taken a strong interest in the Australian Medical Research and Development Corporation
2% of the health budget there has been intensive scrutiny of the organisation and achievements of the NH&MRC including an independent review by Professor Bienenstock from Canada. Many discussion papers have
(AMRAD) since its establishment. Their principle focus is upon becoming the first ever Australian
been released from the NH&MRC, and from other organisations, regarding the various
based and Australian owned major international pharmaceutical company. They have a secondary interest in developing and marketing Australian discoveries in the fields of
ways in which the NH&MRC distributes its funds for research. Block Grant funding of research institutes has been one of the topics to attract attention and some critics have
diagnostic tests and scientific equipment.
described the scientists in the institutes with Block Grants as “privileged” because they do
As a shareholding Member Institute, we have
not have major teaching commitments. They conveniently ignore the substantial involvement of our scientists in clinical activities, and the restrictions that are placed upon our scientists in seeking to expand
POSSUM Team: Clockwise : Cathy Rose, Sofia Mercer, Moshe Chemke, David Danks, David Silience, Anne Cronin, Agnes Bankier.
their activities into new fields. None of our scientists can apply to the NH&MRC for a new Project grant and only very modest increments
his research is held and we all congratulate him. His journal. Human Mutation, is going from strength to strength and he is in demand as a speaker on mutation detection techniques and on phenylketonuria at international congresses. He and a British colleague organised a special workshop of the experts in this field in Oxford in 1991 and a second workshop of the same type was held in 1993 in Northern Italy and proved a great success. Dr Julian Mercer’s discoveries about the Menkes disease gene have also brought him a number
funded institutes. It is appropriate the institutes should have to recruit both private and public financial support, but it is sad to see the old interstate rivalries given formal support. An important issue raised in his
are available at the 5 yearly reviews. All new developments have to be encompassed within our existing Grant by curtailment of some other activity.
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years, the proportion of university staff supported by research grants has risen greatly, infrastructure funding of universities has been reduced and independent research institutes which receive no other Commonwealth support have developed. The prohibition against using NH&MRC funds for infrastructure costs hurts them very badly. The Victorian Government recognises the problem and provides some help, but our current support of about 7% of total expenditure is far below the real extent of the problem. Of course, many private and corporate donors also wish to see their money spent on specific research work and not on administrative and housekeeping costs. Somehow, we must convince governments that if they want effective collaboration with private sources of funding they must be prepared to pay most of the unglamorous, but essential, infrastructure costs.
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We are very fortunate that many of our private benefactors do understand that money is needed for all aspects of running this Institute, from the occasional exciting “breakthrough” down to the work books in
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which the scientists record the laboratory results and the cleaning of their laboratories.
Professor Bienenstock’s report released recently, advocates setting limits on the proportion of an institute’s funding which can be provided by a Block Grant and taking account of the geographic distribution of
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Thanks to all who help us
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The Friends of the Murdoch Institute (President, Mrs Anne McFarling) continue to provide valuable financial support and to help us to become better known in the community. Our faithful major supporters have continued to stand by us despite the pressures of the recession. As always, I have had special support from many people - Mr Walford, Mr Cox, the Board and Finance Committee, the senior scientists who form my Executive Committee i and the wider range of scientists and clinicians who join them in the Executive Committee 2, Dr Agnes Bankier (Coordinator of Clinics), Mrs Anne Cronin (Business Manager), Mr Barry Flolt (Laboratory Manager) and Mrs Lee Jackson (my Personal Secretary). I want to thank all these people, the remainder of the staff and all our friends and supporters for making 1993 another successful year.
Helping Families to Prevent Genetic Diseases
Background
Genetic counselling first began in the 1930s, but it only became widely practiced in the late 1950s and 1960s. Clinical and diagnostic methods plus recording of a family pedigree allowed geneticists to advise family members about the risk of having children with the disease or developing the disease themselves. Most serious early childhood diseases have a 1 in 4 risk of recurrence and the genetic diseases of adult life are generally passed on to half of the offspring. Couples had to decide between accepting the risk or refraining from having further children. Fortunately, adoption
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3.0 143 2.8 93 140
3.0 14.5 2.8 59 140
r 7 1.2 23 3.7 9.1 2.8 93 140
7 1.2 23 3.7 9.1 2.8 93 140
1.5 0.7 1.7 3.7 9.1 2.8 93 140
7 ij 2J 3.7 9.1 2.8 93 140
1.5 1.2 1.7 3.0 14.1 23 93 140
was often available as an alternative. Some geneticists had a background in diseases of adult life and focused their attention on these, but the majority came from paediatrics because genetic disease was so prominent in this age group. The bias toward paediatricians has been particularly strong in Australia where there are still very few clinical geneticists with a background in adult medicine. During the 1950s the chemical structure of genes (DNA) was discovered and the biochemical consequences of mutations became understood. Techniques were developed for examining chromosomes in patients. All of this caused a surge of interest in genetics, particularly in paediatrics, ■ because the new knowledge made some childhood genetic diseases treatable. In the 1970S prenatal diagnosis by amniocentesis provided a dramatic step forward, giving geneticists a method of helping couples to avoid the risk of having a
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child with genetic disease by terminating a pregnancy which had been proved to be affected. The development of chorion villus sampling (CVS) in the 1980s moved this technique forward to 10 to 12 weeks’ gestation instead of 16 to 20 weeks. Initially chromosome analysis and biochemical studies allowed diagnosis of several hundred genetic
diseases. Most of these conditions were rare, but the families concerned were very grateful. In the 1980s DNA tests made it possible to
7 0.7 23 3.7 9.1 23 {93 140
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This pedigree of a family with Huntington disease displays the work involved in deciding whether we could offer the male consultand (C) a presymptomatic test. Each of the pairs of numbers shown beside the vertical lines is the result of a separate test, each of which took a scientist 5 days. (Circle = female; pink = affected)
identify the faulty gene in foetal cells, especially in CVS samples, and made prenatal diagnosis available for several of the most common genetic diseases of childhood - cystic fibrosis, Duchenne muscular dystrophy, thalassaemias, haemophilias. These advances further focused the attention of both paediatricians and obstetricians upon genetic services.
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The new DNA tests also started to have some impact upon the difficult counselling problems
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of adult onset diseases, like Huntington disease (dementia associated with abnormal body movements) and myotonic dystrophy (progressive muscle weakness) in which patients were generally diagnosed after completing their families. The first tests could only track a defective gene from generation to generation. They are cumbersome and the results are never really precise. ■I
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However, they still have a place in some diseases. More recently direct detection of the fault within the gene has speeded up testing and provided very precise answers the isolation of the genes causing Huntington disease and myotonic dystrophy and demonstration of a single type of fault in each disease has converted these from the most laborious and imprecise tests in our laboratory to the least laborious and most sensitive. A scientist who previously spent a whole year testing 25 families with Huntington disease can now do this work in a week. With the rapid progress in identifying genes through the Human Genome Project, we are likely to have prenatal or presymptomatic diagnosis (as appropriate in individual diseases) for all genetic diseases within 10 years. The problem will be to persuade governments to allocate sufficient resources to provide the laboratory tests and especially This is another Huntington disease pedigree tested for the expanded triplet repeat which causes the disease. Two of the chiidren of the affected mother (pink symbols) were shown to have the expanded repeat sequences (upper red band). The lower bands show the variability in the two normal genes in the unaffected family members and in the single normal gene in those with the disease. All of this work was completed within 2 days, along with testing another family.
to provide the counselling that is absolutely essential. This will remain very laborious despite all of the technical advances. One cannot change the time it takes to help people understand new, and sometimes devastating, information. Extra work with the conditions which we have always known to be inherited will probably treble the requirement for genetic counselling and double the DNA diagnostic laboratory
Even if we improve the technology loo fold, testing the 20,000 new cases of cancer in Victoria each year, will need a vast increase in the number of laboratory scientists doing the
work in our community. Other types of conditions which most people would not regard as inherited will cause a much greater increase in the need for counselling and DNA testing. I refer to the common forms of cancer.
tests and counsellors available to explain it all to the family members concerned. The costs concerned will be covered many times over by the reduction in numbers of patients needing
It has recently become apparent that cancer is an inherited disease in 5 to 20% of patients with each of the common cancers of adult life
treatment of advanced and incurable cancers, and the society will benefit financially and in human terms.
- eg., colon, breast, ovary, prostate, lung cancer and melanoma. Family members who inherit the gene concerned have close to a 100% risk of developing cancer. A major
If we are to believe the media, all of this is leading towards a horrendous future with every baby tested at birth for susceptibility
task ahead is to find an efficient method of identifying the families with inherited forms of cancer, in order to achieve early diagnosis
to everything that will ever happen during its life and all this information readily available to insurance companies and employers, with citizens ranked in various orders of value according to their genetic make-up!
and care in other family members. The strategy is clear enough - we need to identify the four or five different genes which can cause hereditary cancer of a particular organ, develop quick and cheap tests for
It should not be difficult to restrict testing to providing answers to serious questions that
finding the mutations (faults) which can occur in these genes, and apply these tests to every patient who develops that type of cancer. Those with no evidence of an inherited cause can be reassured that the risk to other
pose a major threat to the health or happiness of an individual or family or to restrict access results. We are not proposing community-wide screening for cancer susceptibility, but orderly
family members is low and the relatives of those with the hereditary forms of cancer can be counselled and offered testing. Half can be reassured and the others can be offered careful surveillance, early diagnosis and curative surgery. Great technical improvements in mutation detection will be required, and it may be that Dick Cotton’s enzyme cleavage of mismatch will provide
I;
the basis for achieving this.
Insurance companies and employers may expect people to divulge the results of predictive tests, it should prove possible to reach a sensible bargain with insurance companies because they will be among the big winners if we find a way of anticipating lo to 20% of ail forms of cancers and curing these patients, most of whom now die prematurely of their disease. These individuals will live
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longer as a result of the testing and the insurance company will benefit. The question of employers is a little more difficult. Obviously neither they nor insurers should be able to just look up the test results of any potential employee. However, there may be situations when it is beneficial to an individual or to society for genetic susceptibility to be taken into account in choosing employees. Some Canadian wheat silo operators introduced testing of all employees for ai-antitrypsin deficiency, a genetic defect which causes greatly increased susceptibility to dust-induced lung disease. The employers argued that they were doing this to protect the
testing of cancer patients to identify those with hereditary cancers. This is just an extension of
individuals concerned, and the unions argued that they were discriminating against them. Would you like to be a passenger on a jumbo
the approach that has been so successful in helping families with inherited genetic diseases which afflict young children. We also know
jet flown by an individual who has a biochemical disturbance which makes him almost certain to have a coronary artery
similar, but less frequent, genetic diseases which confer 100% risk of very early onset of coronary artery disease, and are taking a similar approach in these families. We will find similar sub-groups within each of the common diseases of adult life, like Alzheimers disease
attack before the age of 40? Careful thought and reasonable discussion is going to be needed to resolve these questions.
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and multiple sclerosis. In all of these situations, the strategy is fairly simple and
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not very invasive of privacy. The existing privacy of medical records can ensure the privacy of the results.
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Improved DNA testing for genetic disorders
This proved to be a very exciting year for those scientists and doctors dealing with families with Huntington disease. For 10 years scientists had been trying to identify the gene that is at fault in this disease and at last this was achieved by an international collaboration between a number of laboratories. To the great delight of those of us who have been trying to give young adult members of the affected families the information that they want before they start a family it turned out that all patients with Huntington disease have the same type of fault in the gene. This changed gene testing for this disease from one of the most laborious procedures carried out in our laboratory to one of the simplest. The scientists discovered that the basic fault in Huntington disease is an increase in the number of times a three letter DNA word, CAG, is repeated in one region of the gene. This change is known as an expanded triplet repeat The actual appearance of an analysis of the Huntington disease triplet repeat. The arrow points to expanded repeat sequences present in individuals
1 J
Similar progress had been made a year earlier in a disease called myotonic dystrophy which is also caused by expansion of a triplet repeat sequence in all patients. Here the triplet is CTG and the degree of expansion of the triplet repeat sequence is much greater than in HD. Some patients have as many as 2,000 CTG triplets. As in Huntington disease, the symptoms of myotonic dystrophy generally develop in mid adult life after a family has been started or sometimes after the family has been completed. Diagnosis of one of these diseases in a person in the 40’s immediately made them very worried about the possibility that they had passed the faulty gene onto some of their children and made it very difficult for the young adult children to know what to do about having a family. Now that we can prove which children have inherited the faulty gene and which have not we can remove some of this anxiety.
sequence. Normal individuals have between 15 and 30 repeats, whereas individuals with Huntington disease have more than 37 repeats in their gene. Although the scientists have still not worked out what this gene actually does in the brain and how the expansion of the CAG
find it very difficult to cope with knowing in advance that they will later develop progressive muscle weakness (myotonic dystrophy) or dementia (HD). However, the
as the polymerase chain reaction (PCR) we can accurately measure the number of triplet repeats in any individual’s HD gene. This test can be performed in one day and a scientist can test 10 or more individuals at a time. The previous procedure required to determine whether a family member had inherited Huntington disease involved a complicated series of tests each taking 2 weeks to perform. Extensive family studies were needed and often key individuals were unavailable or had died. One scientist in our laboratory was previously able to obtain useful results for only about 20-
opposing ways. Every few weeks scientists somewhere in the world describe a method of testing for another genetic disease and we have to work out how to provide these new tests for Victorians. This would lead to a huge increase in our workload if it were not for the counteracting effect of improved technology upon the speed of carrying out tests. The scientist who was previously spending his whole year testing patients with Huntington disease is now able to take on testing for a number of other diseases. Indeed the 4 scientists who have been working in our DNA laboratories for the last 6 years are today performing more than ten times as many tests as they were performing 6 years ago, thanks to technical advances.
Of course it is still difficult for a person who has inherited the altered gene to decide what to do about having a family and some people
triplet repeat causes degenerative changes which lead to loss of control of movements and to dementia, this new knowledge has allowed us to offer a much more efficient method of testing for the disease. Using a process known
The story of testing for these two diseases has been described in some detail to illustrate the way in which the rapid advances in genetic knowledge are affecting the workload of our DNA Diagnostic Laboratory in two
majority of people find that uncertainty about their future is even more difficult to handle than certainty. It is very important that the options available to family members should be carefully discussed before anybody undergoes testing of this type. Our clinical geneticists and genetic counsellors spend a great deal of time with these families before any testing is undertaken. This approach is especially important in Huntington disease in which the possibility of future dementia is so hard to accept. Ms Sue Mansie and Dr Edmond Chiu, of the Department of Psychiatry at the Royal Melbourne Hospital, have worked together in this important area
30 patients a year. Now he can achieve this
of counselling, along with our Dr Les Sheffield, for a number of years. Recently Sue Mansie formally joined the
in a week or two.
establishment of the VCGS.
Unfortunately Huntington disease and myotonic dystrophy are exceptional in that all patients have the same fault in the gene and one method of testing can be used in all families. The more usual situation, especially in the rarer genetic diseases, is to find a different fault in the gene in each family. One of the great challenges of the next 5 years is to develop a single relatively simple test that can be applied to every gene to recognise all possible faults. More than 15 years ago we recognised this as one of the great technical challenges of modern clinical genetics and we have spent very many hours debating possible techniques over this time. Out of this came Dick Cotton’s chemical cleavage of mismatch method and now his new enzyme cleavage of mismatch technique (see Research in Progress). However, even this test is currently a hundred times more laborious than the test we need. We plan to put even more effort into
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17
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Molecular methods in cytogenetics
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many DNA techniques over the last 3 years. The first reason has been to replace rather unreliable or very time consuming cytogenetic tests by DNA
their relatives who have inherited the gene can be recognised and treated before their cancers have spread.lt is most probable that several different types of test will be needed because ATG
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Each vertical line represents a different mutation in a colon cancer gene. The height of the line indicates how many different families have been found with the same mutation. Each mutation needs to be slightly different.
tests like those used in the DNA Diagnostic Laboratory. The second application has been to use DNA methods as an adjunct to theexamination
some will suit one gene and others will suit another. The tests that we have already for expanded triplet repeats are rapid and cheap and enough to test hundreds or thousands of patients. At the moment we know only 7 diseases in this category, but more will certainly be found. Recently scientists in Holland and America have noticed that almost all of the 200 different gene faults that cause a hereditary colon cancer reduce
to identify all mutations. We have faith that the ingenuity of the thousands of scientists working on gene technology will provide us with the tests which will allow us to handle the huge workload of hereditary cancers even though we will need to be able to test every one of the 20,000 Victorians who develop cancer each year for possible faults in 4 or 5 different genes. About $100 is currently spent on ordinary pathology tests on the tumour In each of these patients and we feel sure that society could afford to spend a further $100-200 evaluating all new cancer patients for the possibility that their cancer is hereditary. Although 40 extra scientists would be needed and a similar number of counsellors, the cost of detecting each relative at risk would be approximately $1,000.
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of chromosomes under a microscope to allow identification of specific regions of chromosomes. Cytogenetics is the science of examining chromosomes under a high quality microscope
type of test that the DNA Laboratory uses in these other conditions. The change in technology has speeded up testing and has, at last, given us reliable tests for the mildly affected females and for the female carriers.
to recognise changes which are large enough to be visible. Even with best equipment it is very hard to recognise changes affecting only a small part of a chromosome. Each chromosome contains
In Prader-Willi syndrome and the Angelman syndrome a small proportion of patients showed visible changes in one particular part
been limited to recognising changes that involve 50 to 100 genes, or more. These new approaches are helping them to refine the
What we really need is a single, simple test able
T1 T2 T3 T4 T5 T6 T7 T8
which this type of fault was recognised and this discovery spurred the discoveries in myotonic dystrophy and Huntington disease.) Now we are able to test family members for this disease for carrier status by the same
about 5,000 genes, and cytogeneticists have
the length of the protein formed. This made it possible for them to devise a simple test to use in all patients. This test is likely to be applicable to some other genes.
The new method of causing the colon cancer gene to make its protein product in the laboratory and measuring its length gives a diagnostic result in almost all patients. In each vertical track we see an upper band which is the protein made by the normal gene and a lower band at a different position in each patient which is the shortened protein made by the mutant gene. A normal person would have only the upper band.
About 3 years ago the gene at fault was isolated and the type of unstable triplet repeat which has been discussed in relation to Huntington disease and myotonic dystrophy was recognised as the cause of the problem, (indeed fragile-X was the first condition in
The VCGS Cytogenetics Laboratory has introduced
Looming ahead is the huge challenge of identifying all those patients with cancer who have the hereditary forms of the disease so that
kd
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analysis beyond this limitation.
of chromosome 15. Only a minority of patients showed this change and very similar changes
Three quite important conditions associated with mental retardation have been particularly
were sometimes seen in quite normal individuals. Both conditions cause mental retardation, but the associated features are
challenging to cytogeneticists. These are the fragile-X syndrome, the Prader-Willi syndrome and the Angelman syndrome.
quite different. Babies with Prader-Willi syndrome have very poor appetite and fail to
The fragile-X syndrome is the most common hereditary cause of mental retardation and second
gain weight, but later they develop voracious appetites and become very obese. Children with Angelman syndrome have strange wobbly
only to Down syndrome in frequency among all causes of this problem. It used to be recognised by seeing a stretched and narrowed region near
head and hand movements leading to a descriptive name of “the happy puppet syndrome”. We now know the Prader-Willi syndrome is caused by the lack of a paternal
the lower end of an X chromosome. It looked as though the tip of a chromosome might break off; hence the name. The problem was that cells had
copy of a particular small part of chromosome. The father’s chromosome may have this piece missing, or the child may have inherited 2 copies
to be treated in a special way to see this change and it was present in only a small percentage of X chromosomes, even in boys with the severe effects of the condition. Girls may have a milder
of the chromosome 15 from the mother
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form of the condition or may be mentally normal carriers who are at risk of having sons with mental retardation. Many of these women do not show
and none from the father. The situation is just the reverse in Angelman syndrome which is caused by loss of
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Fragile X Chromosome
Fragile X DNA
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an adjacent part of chromosome 15 from the maternal chromosome or inheritance of 2 copies of the paternal chromosome 15. Quite simple DNA tests can now be performed to diagnose those conditions with much greater confidence. The ultimate importance of these developments is that all the family members at risk of producing children with the fragile-X syndrome can be identified and counselled about their risks, with prenatal diagnosis available to those
Fragile X syndrome: Father and son on left do not have Fragile X syndrome, the three sons on the right have the Fragile X syndrome.
who wish to use it. In Prader-Willi and Angelman syndrome we can explain the cause of the child’s problem and predict the particular problems like excessive appetite which will develop in the future so that steps can be taken to prevent (for example) gross obesity. We can also reassure most parents of children with these 2 conditions that they are unlikely to have a second affected child because they are not often inherited. The other general approach has been to use purified genes to “light up” or “paint” segments of chromosomes which are known to be the location of the corresponding chromosomal gene. The acronym FISH (Fluorescent In Situ Hybridisation) describes the ability of a purified gene to attach itself to the corresponding gene in the chromosome, and to give a fluorescent signal by appropriate labelling of the gene probe. This can be used to identify tiny marker chromosomes which may be found in addition to a normal set of chromosomes in the cells of some patients or to determine the components of a chromosome rearrangement produced by exchange of fragments between
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chromosomes. In both these applications the
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probes used may be mixtures of several different genes all of which are located in a particular region of a chromosome.
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Unusual patient with Down syndrome in which the third chromosome 21 is attached to a chromosome 4. The yellow dots show two small chromosomes 21 and also light up the end of the larger chromosome (arrows).
These mixtures are known as chromosome paints because they light up the whole of a chromosome or a large part of it. Painting of a marker chromosome with genes from chromosome 21 may reveal that it is a fragment of a chromosome 21 and that the patient has a partial Down syndrome. Quite often it turns out that the small fragment of chromosome present as a marker is derived from either the X chromosome or the Y chromosome. Babies who are born with only 45 chromosomes (instead of the usual 46) and only one X chromosome, without a second X or a Y chromosome, have a condition known as Turner syndrome which comprises short stature and failure of development of the ovaries. If a marker chromosome derived from a Y chromosome is present in addition then there is a high risk of developing cancer in the abnormal ovaries and surgical removal of the ovaries is important. Detailed description of more complicated chromosome rearrangements may also be achieved allowing the doctors to give the parents a much more accurate prediction of a child’s future development. A very complicated example which could not have been sorted out without chromosome painting is worth describing. Present applications of DNA techniques in the Cytogenetics Laboratory have done a great deal to help us sort out changes which were far too subtle to see under the microscope without these special aids, but we can only apply these improvements when we have some clue about the identity of the chromosome which is altered. There are reasons to believe that there are hundreds of babies born with abnormalities caused by the loss or doubling up of a fragment of chromosome containing several genes. We need to develop a technique which can allow us to recognise this type of change wherever it might occur in one of the 23 chromosomes. It would be boring tohave no future challenges!
I'
Research in Progress
The aim of this section is to provide our lay readers with a brief overview of the work going on in the institute. For our scientist colleagues it may be more satisfactory to look at the detailed project descriptions in the latter part of the report.
The Olive Miller Protein Research Group
Head: Dick Cotton.
.
Scientific Officer: Ian Jennings. Postdoctoral Fellows: Enzo Palombo (to May), Peter Smooker (to May), Rima Youil. Research Assistants: Marita Black (to May), Rania Horaitis (from July), George Makris, Tamara Gough, Michelle O’Brien (from May). PhD Scholar: Susan Ramus.
The work of this group focuses upon the enzymes (biological catalysts) which are at fault in patients with phenylketonuria (PKU)
PKU Family Trees (Pedigrees).
and the development of methods of detecting mutations (faults) in genes in genetic diseases.
Family A
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retardation and our Newborn Screening Laboratory tests every baby in Victoria for both of these conditions. Although the
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PKU and hypothyroidism are the two most common treatable causes of mental
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dietary treatment of PKU is effective, it is Pottontl dtlA400:l65T
complex and costly and we would like to develop simpler methods to make lifelong
Family B
treatment affordable. Administration of a modified form of the enzyme that is missing or insertion of a normally functioning gene
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are possible approaches. Very detailed knowledge of the structure of the enzyme and the gene is necessary to develop either of these treatments.
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23
Enzymes are very large protein molecules and in some cases only a small part of the enzyme is really important. A synthetic replica of this critical part may be effective as a treatment. Although Ian Jennings’ work on the PAH enzyme which is at fauit in most cases of PKU has identified several regions of a molecule that are particularly critical it seems very likely that the pieces in between are also important to hold the critical pieces close enough to one another to function properly. The analysis required is very time consuming and Rania Horaitis has joined Ian to move this work along more quickly. It has long been known that the severity of the brain damage in different patients with untreated PKU varies quite widely and also that the amount of protein that treated PKU patients can tolerate in their diet varies quite extensively. Studies conducted by Susan Ramus, Eileen Treacy and Sue Forrest have
Centromeres/transgenic mice supported by the Brockhoff Foundation
The chemical cleavage of mismatch method developed by Dick Cotton in 1987 has been used widely, but this is fairiy slow to use, difficult to learn and involves the use of toxic chemicals. Recent experiments conducted by
Head: Andy Choo. Scientific Officers: Elizabeth Earle, Anna
Rima Youil and Michelle O’Brien have shown that the chemicals can be replaced by an enzyme which can be extracted from certain
Michalska. Postdoctoral Fellows: Clara Gaff, Helen Trowell, John Martyn (from October). PhD Scholars: Desiree Dusart, Camille McQuillan, Paul Kalitsis (from February).
viruses. The test procedure is simpler and there are no toxic chemicals involved. More work is needed to find a way of extracting enough enzyme for widespread use and to be sure that this method can identify all of the possible mutations that can occur in genes. The ultimate aim is a method which can allow scientists to find the mutations in each patient
The main project of this group, the detailed analysis of the structure of the centromeres of human chromosomes, is such a team effort that identification of individual contributions
with just a few hours work. This capability
is difficult in such a brief description. A second line of work involves production of
will be essential for the methods of cancer detection which we envisage in the future (see Helping Families to Prevent Genetic Diseases).
transgenic mice with defects engineered in particular genes as models of human diseases by Anna Michalska.
shown that while differences in the actual mutation (fault in the gene) are mainly responsible for this variation there must be
1;
Cell multiplication in the body depends upon replication of each gene, doubling of chromosomes and separation of the two
.thUassaeinla: ECM (genomic)
some other factors involved because considerable variation in severity has been found even within small groups of patients with the same gene mutations. As an adjunct
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daughter chromosomes, one going into each of the two daughter cells. This is achieved by attachment of spindle fibres to specific regions of each chromosomes called centromeres to
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to this study the origins of the gene mutations causing PKU in Victoria have been traced. As expected from studies in other countries,
pull the chromosomes into the daughter cells. The centromeres also allow the members of
these mutations have come particularly from the Celtic populations of Ireland and
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Western Scotland. More and more of the genes which are at fault in hereditary diseases are being isolated, allowing prenatal diagnosis of severe childhood diseases or presymptomatic diagnosis of diseases with onset in adult life. Efficient application of these methods requires the ability to rapidly determine the mutation in a gene in a particular family. This is quite !il|
likely to be different from the mutations observed in other families.
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chromosomes pairs to recognise one another in the special type of cell division that is needed to form egg and sperm cells. Sexual reproduction requires each parent to contribute one of each gene pair to the offspring. Understanding the way in which the centromere works is one of
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the big fundamental questions remaining Enzyme cleavage mismatch for a fl-globin mutation.
unanswered in the science of genetics. The group has made very good progress in working out the arrangement of the large blocks of repetitive DNA (sequences of code
letters repeated hundreds of times) which exist in the centromeres. Their decision to study chromosomes 13, 14 and 21, those chromosomes most often involved in errors of cell division, some of which cause birth defects, has proved fruitful. The focus is now upon small unique regions of DNA buried within this repetitive DNA and upon specific proteins which bind to these unique regions and also to spindle fibres. These are likely to be particularly important.
Back L to R: Andy Choo, John Martyn, Anna Michalska, Paul Ka Iits is, Helen Trowell. Front L to R: Heather Davidson, Clara Gaff, Camille McQuillan, Elizabeth Earle.
If the team can recognise the critical region of the centromere and insert it into a piece of DNA containing all of the elements of a chromosome except a centromere they should produce a tiny artificial chromosome which can replicate and divide, persisting in a stable condition through many cell divisions. This would be very useful for gene therapy to carry the relevant gene into body cells and maintain it in a functional condition. In addition, we anticipate that a full understanding of the way in which a centromere functions will provide new understanding why some egg cells contain two copies of chromosome 21 leading to the birth of a baby with Down syndrome. This and other errors of chromosome separation in the formation of egg and
1,'
sperm cells account for nearly a quarter of the serious birth defects in our community and for about half of all miscarriages.
J'The project which led to the production of mice which are unable to make a metal binding protein called metallothionein was
I
discussed at length in last year’s report. Many scientists believed that this protein was of critical Importance in detoxifying toxic metals like mercury and cadmium, in controlling the toxicity of essential metals like copper and in maintaining the supply of zinc. To our surprise the mice proved to be sensitive only to cadmium toxicity, and not to acute copper overdose. II
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They showed no signs of zinc deficiency. Long term experiments analysing the details of utilisation of copper and zinc in these animals are in progress. We have also embarked on a breeding program to cross these mice with strains of mice with inherited defects in the utilisation of copper. In all of these mice metallothionein levels are increased in the affected organs and we anticipate that the disease may behave quite differently if this protein cannot be produced. Early results are showing that lack of metallothionein does make these conditions more severe.
Defects of mitochondrial function
Head: Henrik Dahl. Senior scientist: David Thorburn. Clinician: Geoff Thompson (to April), David Danks (from April). Postdoctoral Fellow: Rozanne Blok, (Helen Schutt Fellow), Fumie Takabuko (to August). Trainee Research Fellow: Shamima Rahman. Scientific Officers: Denise Kirby, Wendy Hutchison. Research Assistants: Effie Tsostis, Kay Sellers. PhD Scholar: Jamie Fitzgerald (to July),
This year the group focused particularly on a
Mitochondria are tiny organelles inside our cells which generate the energy required for the cell to carry out its functions. There are hundreds or thousands of mitochondria in each cell and each of them contains hundreds of different proteins which collaborate together to produce energy. The system is very compiex.
condition called Leigh syndrome, a degenerative brain disease of young babies. Shamima Rahman has sifted the clinical information on 109 patients from all States to classify 36 as definite cases and 31 as probable cases. A specific defect in energy production has been found in 40% of patients, but we believe it is likely that defects in energy production not detectable by
It is made even more complex because some of these proteins are controlled by genes carried within the mitochondria themselves, not by the genes located in the chromosomes
present methods were probably present in the remaining individuals. Some of the defects that were found were in the genes carried
in the nucleus, which are responsible for controlling all our other functions. The way in which these two groups of genes interact in controlling the function of mitochondria is poorly understood. However it is apparent
within the mitochondrion and others were in genes carried in the nucleus. This difference is of great importance in counselling families because mitochondrial gene defects are inherited from the mother only and are passed on to all of her children, although the severity of the effects may vary greatly from one child to another. The pattern of inheritance is more
that a very wide range of disease in both adults and children can be caused by failure of energy production in mitochondria. In general, diseases due to deficient energy production affect multiple organs, especially brain, muscle and heart. However, there are
predictable in cases caused by defects in the nuclear genes. Laraine Peters and Wendy Hutchinson are making detailed analyses of
also other diseases in which just one organ (for instance, the light sensitive retina of the eye) is damaged. It is particularly difficult to understand why patients who have very similar
the nuclear gene defects.
symptoms may turn out to have defects in quite different components of the energy production system and patients with defects in the same component of the mitochondrion
The enzyme pyruvate dehydrogenase is involved in energy production in mitochondria and was studied in detail and successfully for several years by Henrik Dahl and his colleagues. This area of research has been scaled down during 1993 with diversion of effort towards the study of mitochondrial energy defects. However several interesting findings have still emerged in relation to mutations of the pyruvate dehydrogenase (PDH) Eia gene. One patient studied by Fumie Takabuko and Wendy Hutchison was
Back L to ft: Wendy Hutchison, Shamima Rahman, Laraine Peters, Georgia Polidoros, Moira Graham. Seated L to ft; Henrik Dahl, Rozanne Blok.
shown to have a defect in the part of the gene which allows the PDH enzyme to enter the mitochondria. This process is not completely understood and the change in this patient’s enzyme is providing new knowledge. The evolution of the PDH Eia gene proved very interesting, especially when James Fitzgerald extended the study to include marsupials. This gene, and a few others, are found on the X chromosome in all placental mammals, but on one of the chromosomes not involved in sex determination in marsupials. Analysis of these genes is giving new understanding of the evolution of sex determining mechanisms in mammals.
may develop quite different symptoms.
Laraine Peters (from March). The first step in tackling this problem has been to establish a number of techniques for examining the components of the energy production system in muscle and liver samples and in cells cultured from blood or
The genes in a mitochondrion are arranged in a circle. The extent of the novel deletion observed in our patient is shown by the dark line inside the circle. It deletes almost half of the genes.
skin (Denise Kirby and David Thorburn), and methods of analysing the mitochondrial genes (Rozanne Blok, Henrik Dahl and Shamima Rahman). These techniques are
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all working nicely and have been applied to the analysis of samples sent to us from paediatricians, and paediatric neurologists, in all cities of Australia. We serve as a reference laboratory for these tests. We have also examined samples stored from patients referred in earlier years.
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*# Head; David Thorburn. Clinicians: Geoff Thompson (to April), Eileen Treacy, David Danks (from April).
9
Scientific Officer: Denise Kirby. Research Assistants: Kay Seller, Effie Tsostis.
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Southern blot ofCHO genomic DNA probed with Hamster Menkes (Mnk) PCR product.
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This group is involved in both diagnostic work and research projects on metabolic disorders. They collaborate closely with the staff of the Royal Children’s Hospital Department of Clinical Biochemistry, especially with Mr James Pitt.
The Scobie and Claire Mackinnon Trace Element Research Group
Head: Julian Mercer. Senior Associate: Jim Camakaris (University of Melbourne). Scientific Officer: Andrew Grimes. Postdoctoral Fellows: Suzanne Rogers, James Spencer (from May). Research Assistants: Sharon Gross, Jenny Paynter, Paul Lockhart, Cathy Economou. Technical Assistant: Leanne Bailey. PhD Scholars: Leigh Ackland (to April),Rohan Farrell (to August), Loreta Ambrosini (from
One line of work involves using stable isotopes (chemically altered forms of elements) to trace the metabolism (chemical inter-conversion) in several different genetic and metabolic diseases. In one condition called methylmalonic acidaemia there has been great interest in the importance of production of toxic chemicals by bacteria in
L to R: Kay Seller, Effie Tsostis, David Thorburn, Denise Kirby.
February). BSc Hons Scholar: Michael Petris. Last year’s research report gave a detailed description of the recent isolation of the gene which is at fault in Menkes disease, a rare
the large intestine and an international trial of antibiotic treatment has been conducted. The samples collected for this trial are now being analysed. Another study, mentioned
condition in which babies die of copper deficiency because the process of copper absorption from the food and distribution of copper within cells is defective. In the last few months groups in St Louis, Toronto and Boston used the knowledge that we developed
earlier, involved using stable isotopes to measure the function of the enzyme which is deficient for PKU in the whole patient rather
about this gene to isolate a very similar gene which is at fault in Wilson disease, another defect of copper transport which results in
than in a liver sample.
toxic damage to liver and brain.
Trimethylaminuria is a distressing condition
Intensive collaborative work by the whole group has focussed on understanding the full detail of the Menkes disease gene, which is very large and complex, and studying the
in which individuals have a body odour like rotting fish. This occurs because an enzyme in the liver is unable to break down a compound which comes from fish or eggs in the diet. Elimination of these foods does not completely control the problem because bacteria in the large Intestine can make the offending chemical from other foods. A trial using antibiotics to control the bacteria in the intestine, conducted by Eileen Treacy, is giving very encouraging results, and she also started studies of the genetic fault, which are continuing.
faults in the related gene in the various strains of mice which we have used in our research on Menkes disease. We were pleased to confirm that the faults in these mice are indeed in the equivalent mouse gene. Our analysis of the protein produced by this gene suggests that it carries copper across cell membranes, probably from the inside of the cell to the outside. This belief was
confirmed dramatically by results obtained in some cultured cells that have been studied extensively in Jim Camakaris’ group. These cell lines are able to grow well in high levels of copper which are lethal to normal cells because they are unusually efficient in excreting copper back into the culture medium. They have very high levels of the Menkes disease protein and ten or twenty copies of the gene instead of the normal two copies. This cell culture system will be very valuable in allowing us to understand exactly how this protein carries copper out of the cells. Good progress has also been made in studying a new copper binding protein discovered in cultured white blood cells and work is proceeding towards isolation of the gene that encodes this protein so that we can analyse exactly what role it plays in copper transport. Other experiments using the metallothionein deficient mice produced by genetic engineering methods in Andy Choo’s
Embryology Group
Head; Don Newgreen. Postdoctoral Fellow: Susan Bevan. Research Assistants; Richard Kerr, Joseph Minichiello. BSc Honours Student: Adrian Westwood (from February to November). With the addition of a postdoctoral fellow and a BSc honours student, and the evolution of Don Newgreen’s plans for the group, there has been a considerable surge in their activities and a pleasing cohesion between the different projects that they pursuing. In collaboration with Dr Dion Venter of the Department of Anatomical Pathology particularly good progress has been made with a very ambitious approach to isolating
cells in the chick embryo. Neural crest cells are derived from the developing nervous system and disseminate throughout the
Studies of a child with Menkes disease who was treated with copper injections from the first day show that delivery of copper to the
embryo to many sites where they organise the development of structures as separate as
produced by the Menkes disease gene is needed to deliver copper to these sites.
Mel NC Mei NC
[CAZjCAZjCAb jcTS]
Differential display of embryonic cell-type specific cDNAs.
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genes which play important functions at the various stages of migration of the neural crest
group were described in that section.
places inside cells where it is needed has been only partially successful, leaving effects tike one of the milder variants of the disease. This supports the idea that the protein
Standing L to R: Richard Kerr, Don Newgreen, Joe Minichiello. Seated: Susan Bevan.
facial bones, components of the heart, nerves of the intestine and pigment cells in the skin. By painstaking dissection of many hundreds of chick embryos it has been possible to accumulate 5-10,000 cells from each of 8-10 different stages of neural crest cell migration. Different genes are likely to be functional in these cells at the different stages of their migration. The proteins that these genes produce during their brief period of activity are likely to play key roles in the process of cell migration. There are elegant molecular techniques which can match the genes that are being expressed in cells at one stage with those being expressed at another stage and select out only those which are functioning at only one of the stages, ignoring those which are functional at both stages.
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Two other important projects involve analysis of the signals which tell the cell nucleus about changes that are occurring on the surface of a cell so that specific genes can be turned on or off. Another project involves analysis of a complex sugar molecule called proteoglycan which has been shown to stop the movement of neural crest cells. Getting a cell to the right place involves signals which tell it when and where to move and other signals that tell it to stop moving.
Developmental research in the diagnostic laboratories.
We have always arranged for
the techniques that they use. This especially applies to laboratories using DMA techniques which are evolving at such a very rapid pace. In this particular Annual Report we have chosen to present a special discussion of the application of DNA diagnostic tests. This incorporates the research and development work going on in the DNA Diagnostic Laboratory and the Cytogenetics Laboratory. Our diagnostic work of mitochondrial diseases and enzymology and metabolism is really an offshoot of research in these areas which has Cytogenetics Laboratory
presented briefly. In addition to the matters described here each physician is involved in reporting in the medical literature
list, but are not discussed here.
Dr Agnes Bankier continued development of POSSUM/OSSUM:
The team working with Agnes comprises John Marquet (Computer Power), Catherine Rose, Sophia Mercer, Max Robinson, Moshe Chemke (visiting geneticist since August), Anne Cronin,
Back L to R: Margaret Olsen. Eileen Treacy, Mac Gardner, Geoff Woods. Seated L to R: Agnes Bankier, Jo Wells, Mary-Ann Young.
photographs, presented more clearly and with better colour balance and accompanied by more useful commentaries. We were recently very pleased to welcome an imminent visitor of particular relevance to POSSUM/OSSUM - Dr Jennifer Howse who is President of the March of Dimes in the United States. This huge charitable organisation,
products, some of which are intended for use in diseases that occur during pregnancy, poses a problem, it is aiways possibie that some drugs may have harmfui effects upon the fetus. Of course, aii drugs are evaiuated in pregnant iaboratory animais before being released on the market, but there is always the risk that some teratogenic effect may be peculiar to humans. It is desirable to identify
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Women’s Hospital and is devoting a little more of his time to this work.
initiated by Franklin Roosevelt to raise funds to conquer poliomyelitis, has devoted its major attention to birth defects for many years and has had a strong interest in information systems about birth defects. We were pleased to hear from Dr Howse that POSSUM is highly regarded in the USA.
collected some interesting data on the patterns of utilisation of prenatal diagnostic tests in Victoria, examining factors which seem to play a part in determining which
Dr Les Sheffield - studies of drugs in pregnancy and of prenatal diagnosis:
L to R: Ron Batagol, Helen McNeil, Les Sheffield, Jane Halliday.
women taking these potent drugs and to make sure there is no unusual frequency of birth defects in their babies. Traditional methods of undertaking these studies are extremely labour intensive and costly. Mr Batagol has recently retired from the Royal
Mrs Jane Halliday, who has worked with Dr Sheffield for a number of years, has been undertaking studies towards a PhD with Dr Judith Lumsiey of the Centre for the Study of Mothers’ and Children’s Health. She has
women will use these procedures and documenting the frequency of any complications of the procedures.
Dr Mac Gardner
Susan Nash, David Danks.
been reported in the sections above. This was always going to be a busy year because updates of the video discs containing illustrations were due for both systems. It was this coincidence which provided the opportunity to consider combining the systems together. For various reasons mentioned elsewhere in the report we decided to produce a single system which will be marketed by the
1
has added his incisive knowledge of the bone conditions which are covered by OSSUM. We are confident that the users of the new system will find that they have access to a considerably enhanced collection of
our full-time clinicians to have joint appointments between the VCGS and the Murdoch Institute in order to encourage an active involvement in research. These various research activities are
each year a number of unusual clinical observations or new birth defect syndromes. These reports are included in the publication Each of our laboratories is involved in continual development and improvement of
If the joint system is well received we will later merge the two systems more completely. Cathy Rose and Moshe Chemke have shown remarkable persistence and attention to detail in revising all of the commentaries and Professor David Sillence from Sydney
Research by clinical geneticists
Institute. Time was too short to completely integrate the two search systems and for the time being users will have to choose which database they wish to search, but moving from one to the other is quite simple.
Although Dr Les Sheffield has moved to spend a greater proportion of his time in clinical work he is retaining his interest in birth defects caused by mechanisms other than defective genes. For some years he has worked with Mr Ron Batagol, Head Pharmacist of the Royal Women’s Hospital, to devise methods of linking computerised records of drug prescriptions at the major obstetric hospitals with the computerised records of the Victorian Congenital Malformations Register. The continued development of potent and very effective new pharmaceutical
Before Mac Gardner joined us he and Grant Sutherland, of Adelaide, produced a very useful book published by Oxford University and entitled Chromosome Abnormalities and Genetic Counselling. This was an immediate success and there is already a demand for a new edition, so Mac is busy with this work. Nonetheless, he is still finding plenty of time to take a lively interest in many other projects.
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Victorian Clinical Genetics Service
His major project is a survey of individuals with mental retardation, or lesser degrees of intellectual disability, to identify all families in Victoria with the fragile-X syndrome. This is primarily a service activity of the VCGS and is discussed in that section of the report. Mac has been particularly successful in recognising families which may contribute to basic research being conducted by various colleagues in New Zealand and elsewhere in the world and he is continuing to find such families
The increase in demand for genetic services continues, especially in relation to inherited diseases which have their onset in adult life. An even more rapid increase in this demand is anticipated in future years. We see this as such an important problem that we have chosen to make it the feature article of this Annual Report. It will also form the spearhead of our approach to the Victorian Government for urgent and major expansion of genetic service in the 1994/95 budget.
in Melbourne, maintaining old New Zealand collaborations and striking up some new Australian collaborations. He has been undertaking research on chromosome and DNA instability syndromes before he joined us and he has continued to extend this
It is pleasing to announce that before this
interest on families that we know with these conditions in Melbourne.
because there are large numbers of inherited diseases of the nervous system and muscle that start in mid-adult life and great progress has occurred in the understanding the genetics of these conditions in recent years. Diagnostic
report is published we will actually have started our clinics in the Royal Melbourne Hospital and St Vincent’s Hospital, following successful negotiations during 1993. In both hospitals the closest collaboration will be with neurologists
gene tests are now available for the most of them. We have been able to introduce tests for a number of these conditions into our DNA Laboratory without adding any staff because new technical developments have allowed each staff member to handle more tests. However, there is a limit to the increase in work capacity that can be achieved in this way. The other close collaboration expected at the Royal Melbourne Hospital Is with gastroenterologists Interested In colon cancer and the surgeons who operate on these conditions. Particularly rapid progress is occurring in the understanding of genes which can cause colon cancer.
mi,
We have felt able to start these two adult hospital clinics because of the arrival of Dr Mac Gardner, a very experienced New Zealand clinical geneticist, who joined us in june. His predecessors in this position (Dr Ron Davidson and Dr Jack Insley) were very able, but were here for limited periods and we felt uncomfortable about starting a new service when we were not certain who could carry it on in the following years. Unfortunately, we are
able to offer only one clinic per week staffed by Mac Gardner and Mary-Anne Young, one of our genetic counsellors. We feel that each large adult hospital needs a full-time genetic counsellor and a half-time clinical geneticist, but at least we are making a start. In addition, we were fortunate to have major clinical contributions throughout 1993 from Dr Geoff Woods from Birmingham/Oxford and Dr Eileen Treacy from Montreal, both already fully qualified and working as clinical geneticist and metabolic geneticist, respectively. The clinical load at the Royal Women’s Hospital Is also increasing rapidly and we are needing to provide two, and sometimes three, clinical geneticists at our weekly clinics. Ann Robertson is in constant demand by the obstetric staff of the Hospital for clinical genetic referrals and the time has come when we should really have a second genetic counsellor there, especially because we are now being asked to play a bigger role in the counselling about thalassaemia. The need for help with this disease is increasing rapidly following migration from South-East Asia where it is particularly common. The demand for our services is also increasing rather rapidly in Tasmania. Other country centres are providing a steady flow of work for our outreach clinics which are being conducted by John Rogers and Geoff Woods (Tasmania), Agnes Bankier (Geelong), Les Sheffield (Albury / Wodonga) and Mac Gardner (Sale / Traralgon and Warrnambool). The cytogenetics laboratory has experienced a difficult year, coping with a greatly increased workload in cramped conditions which made an increase in staff physically impossible. At year’s end relief is in sight through the cooperation of the Play Therapists who have agreed to move to temporary accommodation in our empty loW
We really enjoyed the large group of young clinicians who spent 1993 in the VCGS and/or Institute. Dr Connie Sham from Singapore spent 3 months in cytogenetics and a similar time in the DNA diagnostic laboratory. Dr Jolanda Flipsen from Rotterdam divided her time between laboratory and clinical aspects of the fragileX syndrome. Dr Stefan Mundlos from Mainz split his time between the Orthopaedic Research Laboratory and our bone dysplasia clinic, while his wife Dr Christine Mundlos worked part-time in the cytogenetics laboratory. Dr Shamima
Dr. John Rogers.
Rahman from London studied both molecular and clinical aspects of Leigh syndrome, a devastating neurological disease of young children. The new position of Nurse Coordinator for metabolic diseases has proved successful and Pauline McGrath was well suited to the role. Her assistance was particularly crucial after Dr Geoff Thompson stopped clinical work at the beginning of the year in order to tidy up his research before departing in April. Dr Eileen Treacy provided very valuable help to Professor Danks who resumed primary responsibility for a large number of metabolic disease patients. Pauline McGrath has indicated her intention of moving to Sydney for personal reasons early in 1994. Fortunately we have been able to appoint a very able successor in Ms Sue Casanelia, a very experienced paediatric nurse who has dealt with our patients in Ward 5 East for many years and has completed successfully our Genetic Counselling Course. The team looking after
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metabolic diseases was completed when Ms Dorothy Francis joined the VCGS as our dietitian dealing with metabolic diseases. Dorothy has had a long and illustrious career in the dietary management of metabolic diseases, starting at the RCH and followed by a 25 year period as dietitian in charge of this work at the Hospital for Sick Children in London.
wing. A third successful international staff exchange brought Arthur Ma to our laboratory from Vancouver and Louise Hills to replace him there. V
She achieved an international reputation for her personal work and for the books on the dietetic management of metabolic diseases which she produced, it was natural for the very important work on genetic counselling and presymptomatic testing in Huntington
The term genetic counsellor describes a person from a background other than a medical degree who has acquired the range of knowledge and experience necessary to counsel people about genetic diseases, it is a discipline which was first established
disease families to begin in the Department of Psychiatry at the Royal Melbourne Hospital where there has been a strong interest in this disease for many years, led by Dr Edmond Chiu. Ms Sue Mansie had been the social worker to the Huntington Disease Association
in the United States over 20 years ago and is offered as a formal tertiary course in
for several years and had developed a particular interest in this aspect of the disease. Her dual qualification in medical social work and nursing prepared her well for a career as a genetic counsellor. When new knowledge of the location of the gene on chromosome 4 made gene tracking possible the Victorian
training in counselling.
Health Promotion Foundation agreed to support Sue’s work in offering this test to the families and we took on the laboratory work. Now that there is a much better test the demand from family members has increased considerably and it became clear that the time had come for Sue’s work on Huntington disease to be incorporated with work on all other genetic diseases within the VCGS. The Health Department kindly agreed to add her position to our establishment and Ed Chiu welcomed this formalisation of arrangements. We are pleased to welcome Sue as a valued senior member of our team
several Graduate Schools in the USA. These courses offer formal teaching about various aspects of genetics, physiology, biochemistry, embryology andmedicine, plus formal
About 10 years ago several universities and CAE’s in Australia discussed the idea of establishing a course of this type, but it was
More recently, with the great developments in prenatal diagnostic testing for early onset diseases and presymptomatic testing for adult onset genetic diseases, a substantial demand for genetic counsellors has developed. With considerable encouragement from Professor Ron Davidson, who had been responsible for establishing an in service training course for
The Human Genetics Society of Australasia has established formal criteria for qualification as
genetic counsellors at McMaster University in Canada, and financial support from the Sidney Myer Fund, we embarked upon our first training course at the beginning of 1992. At that time we had appointed 4 genetic coordinators to help us in various aspects of our work, 3 with backgrounds in nursing and
in thinking about the way we should integrate genetic counsellors into our service we have decided that it was important to consider what is involved in genetic counselling and what background is needed for each part of
one with a background in teaching biology. The course was intended to involve learning
concluded that the minimum number of students needed for a viable course (at least 10 a year) would very quickly satiate the demand in Australia. It seemed better to recruit individuals with a background in one
by personal inquiry in a problem solving situation, assisted by a staff member as facilitator. It quickly became apparent that this method of interactive learning required a group larger than 4, and so 4 additional trainees were recruited following advertisement of the course. These were all
of the relevant disciplines - nursing, a science degree in genetics, medical social work or perhaps psychology - and to offer in service training.Our own involvement began back in
people with a desire to move into a career as a genetic counsellor. Each was employed in a manner which allowed attendance of one half day a week to attend the course.
the early i97o’s when we encouraged two young women who had completed a BSc in genetics to undertake a Medical Social Work Course before joining us. They both proved very able, but moved away for different
a genetic counsellors and this course has been designed to comply with these criteria. Indeed Les Sheffield and Margaret Sahhar have been two of the principal authors of the training requirements of the HGSA.
the task. The first step in genetic counselling is to be certain of the diagnosis of the disease present in the family. Then one needs to be able to understand the way it is inherited and to convey this information in a simple way to the family members. Equally important is a knowledge of the medical effects of the condition and an ability to explain these. Finally, one must be able to help people to reach their own decisions about the risks involved. People with a medical background clearly have an advantage when the diagnosis is uncertain and in knowing about the medical implications of a large number of rare diseases. Genetic counsellors are often particularly skilled in communicating with people.
Thanks are due to the efforts of Ron Davidson, Les Sheffield (who took over the leadership after Ron returned to Canada),
personal reasons.
Margaret Sahhar (our social worker, who played a very important role in the counselling aspects) and other staff members who acted as facilitators for the various sections of this course. The course wound up in the latter half
of genetic counsellors. A special comment on the initiative that we have taken in training genetic counsellors seems appropriate now that the first two year course has been completed and 8
of 1993 with a quite demanding problem solving test which most candidates
trainees have qualified.
handled well.
We try to use our clinical geneticists to deal with rare diseases and when the diagnosis is uncertain, and to focus the work of genetic counsellors upon the conditions which are more common and those in which people need special help to adjust to the information they receive. Huntington disease is an excellent example and Ms Sue Mansie has made a great success of her role with that condition. Ann Robertson and Mary-Anne Young with their extensive experience in midwifery are particularly well suited to working at the interface with obstetricians in relation to prenatal diagnostic work in our obstetric hospital clinics. Margaret Olsen has concentrated upon cystic fibrosis and the
If
fragile-X syndrome. ■
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The leadership that Mrs Margaret Sahhar has provided in the formalisation of the training requirements for genetic counsellors and in establishing their role within the genetics
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services has focussed appropriate attention on this important member of our team. Starting as a member of the RCH Social Work Department and allocated to assist the Genetics Clinic in the late 1970’s, Margaret’s career moved progressively towards specialisation in our field and she has been on our staff since the VCGS was established in 1987. Her influence upon all clinical staff, trained and trainee, has been great. She can be relied upon for a special blend of knowledge, common sense, compassion and wisdom. Her special interest in parent and patient support groups has been greatly appreciated by all of the Margaret Sahhar.
15 groups that she has helped to start and/or sustain. i,
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37
staff Murdoch Institute
Victorian Clinical Genetics Services
Scientific Director:
Clinical Fellows
Technical Assistants
Executive Director:
David Danks, A.O., M.D., B.S., F.R.A.C.P.
Catherine Rose, M.B., B.S.
Evelyn Boyer
David Danks, A.O., M.D., B.S., F.R.A.C.P.
Deputy Scientific Director: Richard Cotton, B.Ag.Sci., Ph.D., D.Sc.
Business Manager: Anne Cronin, B.Sc., B.Bus.(Acc.), A.S.A., C.P.A.
Laboratory Manager: Barry Holt, B.App.Scl.(M.T.), A.A.I.M.L.S.
Scientists (Senior) Richard Cotton, B.Ag.Sci., Ph.D., D.Sc. Jim Camakaris, B.Sc.(Hons.), Ph.D. K.H.A. Choo, B.Sc.(Hons.), Ph.D. Henrik Dahl, Ph.D. Julian Mercer, B.Sc.(Hons.), Ph.D. Donald Newgreen, B.Sc.(Hons.), Ph.D.
Scientific Officers and Research Assistants Leanne Bailey Tina Colgan, S.R.N. Marjorie Crawford, A.R.M.l.T. Elizabeth Earle, A.A.I.M.L.S. Cathy Economou, B.Sc.(Hons.) Andrew Grimes, B.App.Sci. Sharon Gross, B.Sc., Grad.Dip.Diet Ian Jennings, B.Sc. Richard Kerr, B.Sc. (Hons.) Denise Kirby, B.Sc.(Hons.) Paul Lockhart, B.Sc.(Hons.) Ourania Horaitis, B.Sc. (Hons) Wendy Hutchison, B.App.Sci.(App.Biol.) Helen McNeil, M.I.Biol. George Makris, B.Sc.(Hons.) Sofia Mercer, S.R.N.
Les Sheffield, B.Med.Sci., M.B., B.S., M.Sc., D.C.H., F.R.A.C.P.
Joseph Minichiello, M.Sc. Tamara Gough, B.App.Sci. Michelle O’Brien, B.Sc. (Hons.)
Research Fellows
Jenny Paynter, B.Sc.(Hons.) Kaye Seller, B.App.Sci. Janet Shaw, B.Sc. (Hons.)
Susan Forrest, B.Sc.(Hons.), D.Phll.(Oxon.) David Thorburn, B.Sc.(Hons.), Ph.D.
Kim Spence, B.Sc.(Hons.) Effie Tsotsis, B.Sc.
Clinical Scientists Dietitian:
Agnes Bankier, M.B., B.S., F.R.A.C.P. John Rogers, M.B., B.S„ D.C.H., F.R.A.C.P.
Dorothy Francis
Postdoctoral Fellows
Ph.D. Scholars
Susan G. Bevan, Ph.D. Rozanne Blok, B.Sc.(Hons.), Ph.D.(Helen
Loreta Ambrosini, B. App. Sci.
M.Schutt Fellow) Clara Gaff, B.Sc.(Hons.), Ph.D. John Martyn, B.Sc.(Hons.), Ph.D. Anna Michalska, M.Sc., Ph.D.(Adelaide) James Spencer, B.Sc.(Hons.), Ph,D, Helen Trowell, B.Sc.(Hons.), Ph.D. Rima Youil, M.Sc., Ph.D.
Desiree Dusart, B.App.Sci. Jane Halliday, B.Sc.(Hons.) Paul Kalitsis, B.Sc. Laraine Peters, B.Sc.(Hons.) Camille McQuillan, M.Sc. Susan Ramus, B.Sc. (Hons.) Rohan Farrell, B.Sc. (Hons.)
Mandy Baxter Sophie Gazeas Moira Graham
Clinical Geneticists:
Michelle Guneratne
Agnes Bankier, M.B., B.S., F.R.A.C.P. John Rogers, M.B., B.S., D.C.H., F.R.A.C.P. Les Sheffield, B.Med.Sci., M.B., B.S., M.Sc.,
Administration Assistant Accountant: Sue Nash, B.Bus.(Acc.) Personnel Assistant: Debbie Zombolas Personal Assistant to the Director: Lee Jackson Secretaries: Debbie Davis Kristine Yeomans Receptionists: Kay Buckle, Fiona Keltle
D.C.H., F.R.A.C.P. Mac Gardner, M.B., Ch.B., M.Sc., F.C.C.M.G., H.G.S.A.C.C.G. Geoff Woods, M.D., Ch.B., M.R.C.P.
Metabolic Physician Geoffrey Thompson, M.B., B.S., F.R.A.C.P., M.D., Ph.D. Eileen Treacy, M.B., B.Ch., B.A.O., F.R.A.C.P.
Fundraising Executive/OSSUM Marketing Manager
Scientists - DNA Diagnosis
Maxwell Robinson, M.B., B.S., M.R.A.C.P.,
Michaela Balnaves, B.Sc.(Hons.) Janice Brasch, B.Sc.(Hons.), M.Sc.
F.R.A.C.P.
Steven Nasioulas, B.Sc.(Hons.) Andrea Twomey, B.Sc.(Hons.)
Accounts Clerk: Cathy McMillan
Photography/Graphic Design: Michele Winsor
Scientists - Cytogenetics Howard Slater, B.Sc., Ph.D., Dip.R.C.Path., H.G.S.A.C.C. ■ Scientist -in-charge Melissa Curtis, B.Sc. Sue Dale, B.Sc.(Hons.) Julie Davies, B.Sc., H.G.S.A.A.C. Dean Foster, B.App.Sc. David Francis, B.Sc.(Hons.) Yvonne Harney, B.Sc.(Hons.), Ph.D. Louise Hills, B.Sc. Ralph Oertel, B.Sc., H.G.S.A.A.C. Vida Petrovic, B.Sc., H.G.S.A.A.C. Anne Robertson, B.Sc., H.G.S.A.C.C.
I
Marie Thorpe, B.Sc.(Hons.) Cathryn Vaux, B.Sc. Lucille Voullaire, M.Sc., H.S.S.A.C.C. Arthur Ma, B.Sc., R.T.(C.G.), C.L.S.P.(C.G.) Susan White, B.Sc, Dip. Ed.
4
International Visitors
Technical Assistant
Dr. Moshe Juan Chemke, Rehovot, Israel.
Lynda Phillips Dr. Jolanda Ftipsen, Utrecht, The Netherlands.
Laboratory Assistant
Dr. Alasdair Hunter, Ontario, Canada.
Bozena Jezierski Ian Brooks
Dr. Stefan Mundlos, Mainz, Germany.
Neonatal Screening Laboratory Ivan Francis, B.Sc., Dip.Comp.Sci. - Scientist-inCharge Leonard Bonacquisto, B.Sc.(Hons.), Dip.Comp.Sci. Karina Forshaw, B.App.Sci. Maureen Ryan Nick Tzanakos, B.App.Chem.
Dr. Connie Sham, Hong Kong. Dr. Eileen Treacey, Toronto, Canada. Dr. Philip Welch, Nova Scotia, Canada.
Genetic Clinic Co-ordinators Pauline McGrath, S.R.N. Margaret Olsen, Dip.App.Biol. Ann Robertson, S.R.N. jo Wells Mary-AnneYoung, S.R.N.
Social Workers Sue Mansie, S.R.N., B.S.W. Margaret Sahhar, B.A., Dip.Soc. Studies
Business Manager Anne Cronin, B.Sc., B.Bus.(Acc.), A.S.A., C.P.A.
Laboratory Manager Barry Holt, B.App.Sci.(M.T.), A.A.I.M.L.S.
Secretary Joanne Hogg
Administrative Assistant Michelle Halden Sharon Vandersluis
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Research Studies
r Olive Miller Protein Laboratory
R.G.H. COTTON This has been a. busy and successful year for the laboratory during which we farewelled Peter Smooker and Phil Dickson. Peter, who is now at the Animal Health Laboratories at Attwood made an excellent contribution towards the DHPR program. He documented and expressed many mutations and, with Tamara Gough, nearly completed the gene structure. Susan Ramus transferred from a research assistant to a PhD student this year to study illegitimate transcription at the phenylalanine hydroxylase locus. Tamara Gough transferred to a part-time MSc from being a full-time assistant in the Tissue Culture Laboratory and is completing the description of the DHPR gene locus. Michelle O’Brien joined us as a research assistant to assist Rima Youil in the use of enzymes to detect mutations. Rania Horaitis also joined us during the year to assist Ian Jennings in the characterisation of our interesting substrate mimicking antiidiotype antibodies.
Back L to R: Dick Cotton, Ian Jennings, Rima Youil, Melanie-Jane Gibbs, George Makris. Front L to R: Susan Ramus, Orania Horaitis, Michelle O'Brien.
Structure function studies of phenylalanine hydroxylase (PAH). I.G. Jennings, P. Dickson (University of Melbourne), B. Kemp (St Vincent's Hospital, Research Institute). During the year our studies on truncation mutations of phenylalanine hydroxylase to find the minimum length necessary for enzyme
bound this substrate and by the identification
Methods of mutation detection.
in this region of sequence conservation between other pterin binding enzymes.
R. Youil, M. O'Brien, B. Kemper (Cologne).
Studies directed at expression of PAH in Baculovirus have begun and enzyme obtained by this method is to be crystallized and the 3-dimensional structure determined by x-ray crystallography.
Structure function studies of a pterin mimicking antiidiotype antibody. /. G. Jennings, M. Black (to May), O Horaitis, B. Kemp (St Vincent's Hospital, Research Institute). Progress towards our objective of defining the regions of the pterin mimicking antibody which actually bind to the pterin site on numerous pterin binding proteins has been frustratingly slow. However in the last months we have sequenced 6 of 12 complementarily determining regions of our two well studied antibodies and have ordered synthesis of these peptides for activity studies. We hope to soon understand how amino acids can mimic the pterin ring structure.
Dihydropteridine reductase (DHPR) mutations and gene structure. P. Smooker (to May), T: Gough.
activity were completed. The fact that a reasonably large piece was needed for activity disappointed us a little, however it appears this length is necessary for the proper functioning of the active site. The other main activity, the systematic search by in vitro mutagenesis of amino acids essential for pterin substrate binding is nearing completion. This was based ori previous work which had identified a 27 amino acid peptide which
We have characterised the mutations on 30 alleles in the collection of cells from 28 patients with DHPR deficiency. Those characterised most recently were valuable as they were able to indicate intron position due to the mutation affecting splice sites. This has made definition of the gene structure much easier and currently the final experiments including definition of the promoter are being performed to complete this task.
Our work on the initial assessment of T4 Endonuclease 7 is almost complete. We have found that examples of all classes of single base pair differences between two DNA strands up to i.4kb can be detected by this enzyme. Examples of deletional differences were also detected. Only two mutational events out of 20-30 so far studied have not been recognised. Further work will focus on these problems. Because of the potential utility of the enzyme and the interest generated at the American Society of Human Genetics Meeting at New Orleans we are attempting to generate larger quantities of the enzyme for others to use.
Phenylalanine hydroxylase mutations in phenylketonuria (PKU). S. Ramus, D. Pitt, S. Eorrest (DNA Diagnostic Laboratory). This project embodies our wish to characterise all the phenylalanine hydroxylase mutations in a cohort of 55 untreated patients. At the same time we are looking to develop a method which will enable rapid detection of any unknown mutation in a newly diagnosed patient. Standard methods have been used to identify 73 mutations in 84 alleles in our study group of 42 families. The characteristics of illegitimate transcripts, which enable mRNA from peripheral tissues to be studied are currently being defined. It is hoped that a rapid protocol will be found which can identify the remaining unknown mutations and may be suitable for clinical use in the direct detection of causative mutations.
Retinitis pigmentosa mutations in rhodopsin. G. Makris, S. Forrest (DNA Diagnostic Laboratory), R. Youil, L. Sullivan (Eye and Ear Hospital), M. Loughnan (Eye and Ear Hospital). The illegitimate transcription approach is being studied in this disease, but some difficulties are being encountered in the amplification of the DNA for study. However we have identified three new mutations causing this disease.
Disorders of Mitochondrial Energy Generation
H-H.M. DAHL Molecular analysis of mitochon drial DNA defects. R. Blok, E. Tsotsis, S. Rahman, D.R. Thorburn, H.-H.M. Dahl. Patients with suspected OXPHOS defects have been screened for the presence of mtDNA deletions and common point mutations. We have evaluated various ways of making this as streamlined as possible. Deletions are detected by Southern blot analysis, preferably of muscle or liver mtDNA. Southern blot analysis also enable us to search for insertions and to some extent to quantitate the level of mitochondrial DNA. This is important in mtDNA depletion syndromes and for interpretation of enzyme measurements in some syndromes with mitochondrial proliferation. We are also screening for the common point mutations in MELAS, MERRF and NARP using PCR amplification/restriction
and respiratory failure, and pathologically by the finding of multifocal symmetric lesions in the basal ganglia, thalamus, brainstem or spinal cord. Until recently, Leigh syndrome had been regarded as an autosomal recessive disorder. Defects of pyruvate dehydrogenase (PDH) or the mitochondrial respiratory chain have been found in some patients, and in the last two years there have been several case reports of patients with mitochondrial DNA (mtDNA) point mutations. Since we receive tissue samples or cell lines from most Australian cases of Leigh syndrome, we have attempted to determine the incidence of mtDNA mutations in Leigh syndrome. Ten of 56 suspected Leigh syndrome families were found to have mtDNA point mutations. The mutations (and number of affected families) were 8344A->G (1), 8993T->G (6) and 8993T->C (3). The other molecular causes of Leigh syndrome that we have found were six families with PDH deficiency (of which at least three have mutations in the X-linked PDH Eia gene), six with complex IV deficiency and three with
enzyme cleavage analysis.
complex I deficiency.
We have found deletions and point mutations in a number of patients. DNA sequence
Our results confirm the biochemical and
analysis has also revealed several novel polymorphisms. In addition, nuclear and mitochondrial DNA is being sequenced in selected patients.
Mitochondrial DNA mutations in Leigh syndrome. 5. Rahman, H.H.M. Dahl, R. Blok, D.M. Danks, D.R. Thorburn. Leigh syndrome (subacute necrotising encephalopathy) is a progressive neurodegenerative disease that usually presents during infancy or early childhood. It is characterised clinically by psychomotor regression, hypotonia, lactic acidosis and brainstem abnormalities such as nystagmus
genetic heterogeneity of Leigh syndrome. Ten of 25 families with a molecular diagnosis had mtDNA point mutations and would be expected to show maternal.inheritance, though with variable severity. The other families could show X-linked pseudodominant inheritance (PDH Eia) or either autosomal recessive or maternal inheritance (complex IV and I deficiency). Accurate genetic counselling of these families is only possible if the molecular defect can be identified.
Molecular basis of cytochrome c oxidase deficiency. L. Peters, D.R. Thorburn, H.-H.M. Dahl.
repair systems does not function effectively to repair damaged nuclear DNA. To investigate the role, if any, that nuclear-encoded repair enzymes might play in the repair of mitochondrial DNA (mtDNA), we have analysed
Of the 13 COX subunits 3 are coded for by the mtDNA (subunit I, II and III) and 10 in the
a group of 5 patients with various nuclearencoded repair defects (Fanconi anaemia,
nucleus (IV, Va, Vb, Via, VIb, Vic, Vila, Vllb, Vllc, VIII). We have -8 families who appear to have isolated Complex IV (cytochrome c oxidase or COX) deficiency. The affected offspring in four of the families were born to
Cockayne syndrome. Xeroderma pigmentosum). The age of the patients ranged from 3 to 50 years. Using PCR amplification we searched for a skb deletion, common in the mtDNA of elderly people. This deletion
consanguineous parents (first cousins) and the defect is therefore likely to be in a nuclear encoded gene. No mutation has yet been described in a nuclear encoded COX gene and we have started to investigate these families by Western blot. Northern blot. Southern blot and DNA sequence analysis. Western blots were done with crude human muscle mitochondrial preparations and purified
was not detectable in young control individuals or in any of the patients with the nuclear-encoded DNA repair defects, but the deletion was clearly detectable in DNA from 80 and 88 year old control individuals. It would appear from this study, that the skb deletion is no more prolific in our 5 patients with nuclear-encoded repair defects than in
bovine cytochrome c oxidase. However, not all COX subunits are detectable. The 10 nuclear encoded proteins are all relatively small, and the genes have been cloned (cDNA clones). We have designed oligonucleotide primers to the 5’ and 3’ untranslated regions of the human nuclear encoded COX genes and are using these to PCR amplify the genes in unaffected controls and in affected patients. So far we have investigated the COX subunit VIb in our patients, but no mutation has been found.
DNA repair defects. L. Peters, G. Woods, H.'-H.M. Dahl. At least three different nuclear-encoded DNA repair systems protect the nuclear genome from lasting damage due to UV light and chemical carcinogens. Photo reactivation, excision repair, and post-replication repair mechanisms are present and active in most cells. In patients with nuclear-encoded repair enzyme defects one or more of the DNA
normal subjects.
Pyruvate dehydrogenase Eia mutations. H.-H.M. Dahl, F. Takakubo, L. Hansen (Aarhus University, Denmark), D.R. Thorburn. Mutations were characterised in two Danish patients with pyruvate dehydrogenase (PDH) Eia deficiency. One has a 33 bp duplication (l307ins) affecting the coding region. The duplicated DNA contains 13 bp of the 3’ end of intron 9 and 20 bp of the 5’ end of exon 10. Another patient has a 7 bp deletion (S3i2fs) in exon 10. This mutation has been described in 2 other unrelated patients and is present in a region of the chromosome that appears relatively unstable. This is also the case in a girl with a 5 base pair duplication (R302fs) at the junction of intron 9 and exon 10. A possible reason for the instability of this region could be that the sequence has high homology to the topoisomerase II consensus cleavage site.
Human Centromere and Down Syndrome
Finally, we have detected a novel mutation and a polymorphism in a girl with PDH Eia deficiency. The mutation changes a highly
Thus, it seems that the testis-specific isoforms of PDHA were created at least twice since eutherian/ marsupial divergence. Both genes
conserved alanine to a proline residue (A175P). The mutation is an unusual 6 to C substitution in a CpG dinucleotide.
arose independently via retroposition to different autosomes suggesting that retroposition as a mechanism for gene duplication may be common.
Evolution of the pyruvate dehydrogenase Eia genes. J. Fitzgerald, J. Graves, 5, Easteal, H.-H.M. Dahl. In eutherian mammals, two isoforms of the gene coding for the pyruvate dehydrogenase (PDH) Eia subunit are present. PDHAi maps to the X chromosome and is expressed in somatic tissues. PDHA2 is autosomal (mapping to chromosome 4 in humans and chromosome 19 in mice), intronless and testis-specific. PDHA2 is likely to have evolved in response to the absence or functional inactivity of the X chromosome in male spermatogenic cells. We have cloned the marsupial PDH Eia gene and shown that only a single PDHA gene is present in marsupials. It maps to an autosomal region in marsupials that translocated to the eutherian X chromosome following eutherian/marsupial divergence. Furthermore, since it contains introns and is expressed in somatic tissues, marsupial PDHA is homologous to the eutherian PDHAi and not to the testis-specific PDHA2. We have studied the evolution of these genes. The results suggest that there are no differences in substitution rate between the two PDHA isoforms or between humans and rodents. Furthermore, the data indicates that human PDHA2 arose later than rat Pdha-2. It has also been hypothesized that the mutation rate is much higher in the human male germ line than in the female germ line. We find that there is no significant difference between the two isoforms for both humans and rodents suggesting that chromosomal location has no effect on substitution rate.
Methylation and regulation of PDH Eia gene expression. W. Hutchison, J. Fitzgerald, R. lanello, H.H.M. Dahl. We have previously reported on the presence of somatic and sperm-specific forms of the human and mouse pyruvate dehydrogenase (PDH) Eia genes. To further understand how these genes are turned on and off, we have analysed the methylation pattern of the regulatory regions of the mouse PDH Eia genes. We have focused on 300 bp from the promoter region of the testis specific form. Rocco lannello has shown by transfection studies and generation of transgenic mice that less than 200 bp of the promoter region is required for testis-specific expression of this gene. The gene is expressed mainly in pachytene spermatocytes. The methylation pattern has been analysed in DNA from pachytene spermatocytes, round spermatids and heart. This showed that nearly all CpG dinucleotides in the heart DNA are methylated. An exception from the hypermethylation pattern is two CpG dinucleotides that are part of an SP-i binding site. The same promoter region in pachytene spermatocytes and round spermatids is hypomethylated. We have also investigated the methylation pattern in a 200 bp segment of the somatic mouse PDH Eia gene promoter from a male mouse. The results have shown that all the CpG dinucleotides in this region are unmethylated. Combined with analysis of transcription factor binding these results may add to our understanding of gene regulation.
K.H.A. CHOO Long-range analyses of the centromeric regions of human chromosomes 13, 14 and 21. H. Trowell, /t. Nagy, B. Vissel, K.H.A. Choo. Alpha-satellite, satellite III and satellite I DNA have been proposed as candidate components of a functional human centromere. Over the years, we have isolated a relatively large number of subsets of these three DNA from the pericentric regions of the human acrocentric chromosomes. Using pulsed field gel electrophoresis, we have constructed longrange maps of the various centromeric markers for chromosomes 13, 14 and 21. These maps cover -2.3 megabases of sequence for each chromosome, and the results demonstrate that within this centromeric region, chromosomes 13 and 21 have a similar organisation that is partially shared by chromosome 14. A discrete satellite III domain was identified on each chromosome within the boundaries of the alpha-satellite DNA. No satellite I was detected within the defined centromeric regions. These maps therefore exclude the role of satellite I in centromere function and provide the first localisation of a discrete satellite III domain within the human centromere. In a separate study (see below), we have obtained evidence for the binding of a previously unidentified nuclear protein to the DNA at the junction of the satellite III and alphoid DNA domains. Our long-range maps also revealed a region within the centromeres of all three chromosomes which contains DNA that is neither alpha satellite nor satellite III. Work is now in progress to isolate this DNA for detailed characterisation. The maps we have constructed should provide the necessary skeleton for the further dissection of the functional roles of existing and new centromeric sequences, and should facilitate completion of the Human Genome Mapping Project by bridging across the centromeres of these three chromosomes.
Cloning of a functional marker centromere with no detectable alpha satellite, satellite III or CENP-B protein. D. Dusart, E. Earle, J. Martyn, C. Gaff, L.E. Voullaire, H. Slater, V. Petrovic, Jen-i Mao, K.H.A. Choo. We have initiated a programme to investigate an unusual human supernumerary marker chromosome 10 designated mar del(io) with the aim of employing the special characteristics of this chromosome to help us identify the functional component(s) of the human centromere. This marker chromosome has an active centromere at a primary constriction as indicated by its full mitotic stability in cultured fibroblasts and lymphocytes. Fluorescent in situ hybridisation (FISH) using alpha satellite and satellite III DNA as probes failed to detect any signal at the site of primary constriction whereas the sensitivity of our method would have enabled us to detect less than 2okb of these sequences. This result indicated a reduction by at least two orders of magnitude in the amount of these two types of DNA in the centromere of the mar del(io) chromosome. No CENP-B centromere-binding protein could be demonstrated, although the presence of at least some other centromeric proteins was confirmed using a CREST antiserum. In collaboration with Dr. J. Mao of Collaborative Research Inc. who supplies us with chromosome 10-specific probes, we have begun the construction of a detailed map of the marker chromosome. Purification of this chromosome by Flow-sorting and microdissection, and its isolation in a somatic cell hybrid are currently underway.
Extensive meiotic instability of acrocentric p-arm in Robertsonian translocation Down families, and identification of a potential recombination hot spot. C.l. McQuillan, H. Trowell, H. Davidson, E. Earle, P. Kalitsis, D. Dusart, R.H. Martin, L. Shaffer, K.H.A. Choo. Five percent of Down patients carry a t(i4q2iq) Robertsonian translocation chromosome in addition to two normal chromosomes 21. Our goal is to understand the molecular aetiology of such transloctions and we aim initially to identify the DNA that is found at the translocation breakpoint. Pulsed field gel electrophoretic analysis of 10 different t(i4q2iq) families using a satellite III probe for the proximal p-arm of chromosome 14 reveals extensive alterations in the probands that are significantly above the background level seen in control families. Complete stability of the centromeric and pericentric p-arm domains is observed for both patient and control families. These results point to a recombination “hot spot” on the acrocentric p-arm that is responsible for the observed meiotic instability and chromosomal translocation. Further studies will aim at defining more closely the molecular nature of the “hot spot”. As a part of these studies, we have already isolated a host of new short-arm satellite III sequences that are expected to be useful position markers in assisting us to derive a long-range DNA map spanning the “hot spot” and the Robertsonian translocation breakpoint.
A novel nuclear protein that binds specifically to centromeric alpha satellite DNA. C. Gaff, D. Dusart, A. Nagy, P. Kalitsis, R lannello, K.H.A. Choo.
relatively common occurrence. Characterisation of these marker chromosomes should provide useful information on the relative importance of the various sequences found within the human centromere.
Alpha satellite and satellite III are the two key DNA components of the human centromere. The exact arrangement of these two components within the centromere is presently unclear and is currently being studied in this laboratory (see earlier section). We have recently identified the first junction sequence which contains both these DNA. Detailed sequencing of such a structure isolated from each of chromosomes 13, 14 and 21 indicated a high degree of conservation of both types of DNA surrounding 200 bp of the junction. Further away from the junction, polymorphic length variations, by multiples of 5 bp of the GGAAT type was observed for the satellite III DNA. Using an 18-bp DNA found at the junction of this sequence, we have demonstrated its binding to a new protein in Hela nuclear extract. Detailed examination of this protein by competitive binding with a host of mutant oligonucleotides has confirmed the specificity of the protein for the 18-bp DNA.
Testing candidate DNA sequences for centromeric function.
Characterisation of the centromeres of supernumerary human marker chromosomes'. L.E. Voullaire, H. Slater, V. Petrovic, Y. Harney, L. Hills, J. Davies, P. Kalitsis, E. Earle, K.H.A. Choo. In addition to the mar del (10) marker chromosome described above, we have employed cytogenetics staining and fluorescent in situ hybridisation techniques to characterise four new human supernumerary chromosomes. These supernumerary marker chromosomes are each present in a karyotype with a deleted chromosome and are shown to be derived from the same chromosome as the deleted chromosome. The marker chromosomes are for the most part stable during mitotic division both in vitro and in vivo. In all cases the deleted chromosomes contain the alpha satellite DNA and pericentric euchromatin
P. Kalitsis, G. Woods, A. Michalska, E. Earle, K.H.A. Choo.
that are normally present on the cognate chromosomes. In two cases the supernumerary chromosome also contain the same chromosome-specific alpha satellite as the deleted chromosome partner. In the
To date, a number of DNA sequences have
other two cases, the supernumerary chromosomes are deficient in alpha satellite
been postulated to play a major part in centromere function. These include alpha satellite and satellite III DNA in man, and minor satellite DNA in mouse. Such postulations have been based principally on the observed physical location of these DNA on, or in the vicinity of, the centromere, and the ability of these DNA to bind nuclear protein. No convincing direct functional
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analysis has so far been reported. In our study, we aim to establish an assay to allow us to determine if a particular DNA sequence can function as a centromere in mammalian cells. A number of DNA constructs have already been prepared and are currently being tested in different mammalian cells.
as determined by in situ hybridisation at low stringency. The origin and molecular nature of the centromeres in these latter cases are presently unknown. This and the above study involving the mar del (10) chromosome indicate that stable marker chromosomes with centromeres that are devoid of most or all of the alpha satellite DNA that normally resides abundantly within a centromere may be a
Study of proteins of unknown function in mouse mutants generated by targeted gene disruption. A.E. Michalska, H. Trowell, P. Kalitsis, K.H.A. Choo. We report the successful generation of transgenic mice deficient in the metallothionein MT-I and MT-II genes. The mutations were introduced into embryonic stem (ES) cells by homologous recombination. Chimeric mice resulting from the targeted ES cells transmitted the disrupted alleles through their germline. Flomozygous animals were born alive, and appeared phenotypically normal and fertile. Absence of MT proteins was confirmed by direct measurement in liver extracts. Challenging the mutant animals with moderate levels of CdS04 indicated their greater susceptibility to cadmium toxicity than wild type animals. We hypothesise that most functions of MT-I and MT-II can also be carried out by other metal binding proteins, and that the essential functions of these two MT proteins may become apparent only when the animal is placed under chronic stress. We are currently examining some of these functions in collaboration with the Trace Elements Laboratory and a number of external collaborators. Experiments are also underway to employ the homologous recombination technique to disrupt several other genes in which the functions of their corresponding proteins are unclear.
The Scobie and Claire Mackinnon Trace Element Group
J.F.B. MERCER, J. CAMAKARIS During this year the activities of the group have been concentrated on the extension of our work with the Menkes gene. In particular we have been working on the mouse homologue, and the analysis of the mottled mouse mutants which are thought to be mutations of the Menkes gene in mice. We have made good progress in this area. We have isolated and sequenced the mouse cDNA, and showed a high degree of identity with the human Sequence which confirms the predicted sequence of the human protein. We have identified the molecular basis of one mouse mutant and found abnormalities in another. Another area we are concentrating on is the structure of the Menkes gene and although this is a complex task, since the gene is very large, we hope to have this completed in the first part of 1994. We have commenced a more detailed analysis of our Menkes patients. We find the majority of our patients do not have the Menkes mRNA yet only one patient so far has a deletion of the gene. Given the complex structure of the gene it is predicted that many cases of Menkes disease wilt involve errors of RNA processing.
Top L to R: Paul Lockhart, Julian Mercer. James Spencer. Centre: Andrew Grimes. Sharon Horton, Tania Voon. Front L to R: Jenny Paynter, Loreta Ambrosini.
Isolation of the clones covering the entire coding sequence of the Menkes gene. A. Grimes, P. Lockart, J. Paynter, J. Mercer. The first clones of the Menkes gene that we isolated only contained the metal binding motifs (Nature Genetics paper). In order to complete our analysis of the gene we required all the mRNA sequence to be represented in our clones. We achieved this by a combination of cDNA library screening and by using the PCR reaction from cDNA prepared from RNA. Since the mRNA is so large (8.5 kb) we have not found a cDNA which contains the entire sequence. This is not important when using the pieces as gene probes, but we will be faced with piecing together the bits to form a continuous coding sequence. We hope to have this in the next year. Having the entire coding sequence in clones is allowing us to determine the genomic organization (next section).
Structure of the Menkes gene (MNK) in humans.
An exciting finding which was based on the earlier work of Jim Camakaris with copper
j. Mercer, L. Ambrosini, J. Spencer.
resistant Chinese Hamster Ovary Cells, has been the discovery that copper resistance in the cells is associated with amplification of the
During the isolation of the gene we established an overlapping series of lambda clones covering about 300 kb around the Menkes gene locus. By using the cDNAs we have mapped the exons of the MNK gene to
Menkes gene. This is the first real biological data to support the proposed copper efflux role,of the protein. Rohan Farrell passed his Ph-D examination and is currently a postdoctoral fellow in the laboratory of Dr Denis Winge in the School of Medicine, University of Utah, USA. The coming year promises to yield much more information about the function of this protein and the nature of the mutations which cause Menkes disease.
about 150 kb of this. The most surprising result came when we used a PCR product which extended more 5’ than any of the cDNA clones used previously. This clearly showed that the 5’ end was quite some distance away from the breakpoint, and that there may well be a large intron (about 50 kb) between the start of the coding sequence and the 5’ untranslated region/promoter which may be characterized by a marked cluster of rare cutting enzymes, suggesting a CpG island . We are constructing a detailed restriction
map of the region, using the restriction enzymes Eco Rl, Hind ill and Bam HI. We have sequenced one exon from lambda 3.18, and it contains the first metal binding site. Perhaps there will be one exon for each metal binding site.
Isolation of the mouse homologues of the Menkes gene. A. Grimes, P.Lockart, J. Paynter, Herman Dierick (University of Michigan), J. Mercer. We have put much of the group’s energies into this project. By using a combination of library screening and PCR methods we obtained cDNA clones which covered most of the coding sequence of the mRNA. We were unable to find the 5’ end since the libraries did not contain it, and the sequence between mouse and human was too different in this region to permit PCR. The predicted protein sequence indicated a high degree of identity with the human protein. All the functional domains identified in human were present: 6 metal binding sites, transmembrane regions, phosphorylation regions and ATP binding
The dappled mouse has a DNA rearrangement of the MNK gene. L. Ambrosini, J. Spencer, J. Mercer. The dappled mouse is one of the most severe of the mottled mutants in mice, the affected pups die at about 15 days of gestation. Southern blots of DNA from normal, heterozygous and mutant embryos showed that restriction fragment changes are associated with the mutation. The complexity of the changes suggested that this was not a simple polymorphism, but probably involved a deletion and possible rearrangement in the gene. By using probes from various regions of the gene, we could localize the change to a relatively small (zokb) region in the centre of the gene. Thus a major deletion involving another locus is not involved. The embryos with this change do not express the RNA. It is interesting that in humans failure to express the RNA results in Menkes disease, lethal in childhood, but in mice it is foetal lethal. Perhaps developing mice require more copper?
sites. The differences between mouse and human indicated that the structural constraints on the molecule were more severe
An RNA change is found in the blotchy mouse.
in the transmembrane regions compared with the metal binding regions, (see Figure). This may reflect the fact that the metal binding regions are in the cytoplasm and can adopt a number of conformations which are still compatible with activity. The membrane spanning regions may have much stricter
J. Paynter, J. Mercer.
structural requirements.
The blotchy mouse is a mild variant thought to be an allele of the mottled locus, but possibly of a closely linked gene. It presents as a connective tissue disorder, resembling the human disease occipital horn syndrome. RNA blots from normal and blotchy males showed that the mutant had three mRNA species detected with the MNK probe instead of the normal one. The novel mRNAs were larger than the 8.5 kb normal RNA, at about 10 and 12 kb. We believe this is due to a splicing defect, but so far we have not been able to find the mutation responsible. This
result does show however that the blotchy mutation affects the WINK gene in mice. How this RNA change results in the milder connective tissue defect remains to be established.
amount of copper released to the body from the liver and this causes a more severe copper deficiency than that in the brindled mouse. This deficiency is also seen in the females heterozygous for brindled which are tx/tx in contrast to br/+ mice which are relatively normal. These animals die, but can be saved with copper.
The brindled mutation is likely to be a single base change. A. Grimes, J. Paynter, J. Mercer. The brindled mouse is another allele of the mottled locus and is the one which most closely resembles Menkes disease. Our analysis so far has not shown any DNA or RNA changes detectable by blots. We assume therefore that the change is likely to be a point mutation affecting the coding region or translation of the RNA. We have made use of mismatch detection methods, such as the chemical cleavage and enzyme cleavage, and these have Identified a number of differences between the normal and mutant X. But so far these have proved to be polymorphisms, or changes in the ^’-untranslated region. We are continuing our search for the mutation.
Properties of the toxic milk/brindled double mutants.
Thiomolybdate treatment of the toxic milk mice. S. Gross, J. Mercer. Previous work showed that the administration of thiomolybdate, a powerful copper chelator, in food or by injection, did not lower hepatic copper concentrations in the mutants. We have now found however, that inclusion of the compound in drinking water for 1 month caused a significant reduction. Now we will be able to assess the long term affects on this treatment, which may be applicable to treatment of Wilson’s disease in humans.
Organisation and expression of the Menkes (MNK) gene in copper resistant CHO cells. M. Petris, J. Mercer, J. Camakaris.
S. Gross, S. Gazeas, J. Mercer. The interaction between these two mutations in the one animal has become more interesting now that we have some idea of the function of the molecules. As we have found previously the male double mutants die at about 10 days of age and in contrast with the brindled mice cannot be rescued by copper injection. The copper concentration in the liver of the double mutant is significantly elevated, in contrast to the brindled where it is low. One interpretation of these results is that the tx mutation results in a reduction of the
12
The Menkes (MNK) gene was shown to be expressed in cultured CHO cells, by probing Northern blots with a mouse MNK probe. Using PCR primers based on human and mouse MNK sequences PCR products were isolated using CHO cDNA and these were of the same size as PCR products from human and mouse cells. These detected MNK and mRNA in CHO, mouse and human cells. Limited sequence analysis revealed high homology between hamster, human and mouse within the regions spanned by
Northern analysis of RNA from the copperresistant mutants revealed that all three mutants produced significantly elevated levels of MNK transcript. The least copper-resistant mutant had a 5-fold increase in MNK or RNA whilst the most copper-resistant mutant had a 70-fold increase in MNK mRNA. The basis of the increase in MNK mRNA levels was shown to be gene amplification using Southern blots.
This hypothesis is supported by our findings that initial rates of Cu efflux are significantly reduced in human fibroblasts derived from patients with Menkes disease. Earlier studies had shown 64CU retention over 24 hour periods.
These results suggest that copper-resistance is associated with elevated levels of MNK gene transcript and presumably MNK protein, although no assay system has yet been developed for the latter.
dismutase. immuno-assays hence show that this enzyme is not increased suggesting that an apo form of Cu.Zn superoxide dismutase may exist which is involved in intracellular
Physiological bases of copperresistance in copper-resistant cultured CHO cells.
Copper transport in W7 mouse lymphocytes
One of the copper-resistant mutants shows abnormal cytosolic Cu distribution in that most Cu is associated with Cu.Zn superoxide
Cu storage/resistance/transport.
R.A. Farrell, J. Camakaris. P. Shen, L. Bailey, C. Economou, *R. Lambrecht, J. Camakaris. (*Australian Nuclear Science and Technology Organisation).
Further studies have confirmed an important role for a lookd Cu binding protein, and also for a 3okd protein, in processes of
Kinetic analysis using the radioactive isotopes 64CU and 67CU has revealed that the copper resistant CHO mutants accumulate significantly
intracellular Cu transport. 64CU on the lookd protein is readily exchangeable in pulse-chase studies showing this protein may function in
less Cu than wild type cells and this is due to enhanced efflux of Cu. Initial efflux rates (over
transport rather than detoxification/storage. 64CU on the 3okd protein exchanged at a very slow rate by comparison with the lookd
15 seconds) were higher in the mutants, suggesting that the altered efflux process is associated with the plasma membrane. There appears to be no effect on initial rates of Cu uptake. The enhanced efflux is not found for cells in basal media, but is clearly evident for cells grown in medium containing high levels of Cu. By contrast MNK mRNA levels are elevated in the copper-resistant mutants in both basal and high Cu media.
protein indicating that the 3okd protein may be involved in storage/detoxification of Cu. This may be of special importance in cells which do not express metallothionein as is the case for the W7 lymphocytes. The molecular weight and comparison of 64CU Western blots with anti-Cu.Zn superoxide dismutase immunoblots strongly suggests the 3okd protein is Cu.Zn superoxide dismutase.
An explanation for this data is that the MNK gene codes for a component of Cu efflux and control of efflux occurs at the mRNA and/or protein levels.
the PCR products.
13
Embryology Group
Purification of a lOOkd Cu binding protein from W7 mouse lymophocytes.
mouse lymphocytes was purified using FPLC gel filtration, FPLC ion exchange and PAGE.
The Laboratory’s focus on adhesion molecules in early embryogenesis of the nervous system has been maintained, with work to further biochemically characterise the postulated anti-adhesive chondroitin sulphate proteoglycan. Molecules with this function whose existence was suggested on theoretical
Approx. 3Ug of the purified protein was obtained. The lookd protein has a subunit molecular weight of 45kd on SDS PAGE. Larger amounts are being currently purified to permit
grounds a decade ago, have been receiving rapidly increasing attention recently. We have also explored the epithelio-mesenchymal transition of neural crest cells, a phenomenon
protein sequencing. 64CU Western blots are
that we had previously established was based on changes in cell adhesion. This work has focussed on the ways in which
R.A. Farrell, J. Camakaris. The lookd Cu binding protein from W7
being used as an assay for this protein.
Studies on cutA, a gene involved in copper transport in E.coli. D. Fong, B.T.O. Lee, (Department of Genetics, University of Melbourne) and J. Camakaris. Mutations in the cutA gene, confer copper sensitivity in the bacterium, E.coli. Using the Kohara Lambda library which spans the E.coli chromosome, the cutA gene has been localised on the E.coli chromosome (map coordinates 4392kb - 4395./kb). This has enabled sequencing of the cutA gene which has almost
Adhesion inhibiting proteoglycans: a role in neural development.
D.F. NEWGREEN
Second messengers in the control of epithelio-mesenchymal transformations.
R. Kerr, D.F. Newgreen, J. Minichiello. J. Minichiello, D.F. Newgreen. Previous work has suggested that embryonic proteoglycans (PG) play a role in the development of the pattern of the nervous system in the paraxial region. This project has built up a picture of the embryonic axial extracellular matrix as hosting a variety of PGs (Fig. A). We have isolated the largest, most heavily charged and most prevalent PG from strips of axial tissue (each measuring about 1.5mm X 0.2mm X 0.3mm) from early embryonic quails. This PG has a molecular weight overall of 1.5 x 103 kD (±10%) and, per molecule, bears about 20 chondroitin sulphate-type sugar chains of size 50 kD ( 10%), with no other long chain sugars being present. This resembles, but with some distinctions, other large connective tissue PGs, and like these PGs, the core protein is highly enriched in the amino acid glutamic acid. This
these changes are controlled, as much as on the changes themselves. A completely new avenue of research has also been started, with the arrival of Dr. Susan Bevan. This new project uses molecular genetics to explore the genetic basis of developmental changes, again focussing on the early developing nervous system. This has involved us in close collaboration with Drs. Deon Venter and Melissa Southey, Anatomical Pathology,
PG has a specific association with the extracellular matrix fibrillar molecule collagen type I, but not with the other structural matrix molecules, collagen type IV, fibronectin and laminin. Tissue culture assays of molecular
Royal Children’s Hospital, in investigating development-related changes in paediatric tumours of neural origin.
function shows that this PG interferes with embryonic neural cell attachment and movement on their preferred matrix molecule fibronectin, if collagen type I is simultaneously present. These results suggest that the PG influences development by subtly modifying the adhesion of cells in
been completed. Significant homologies have been found between cutA and rodent metallothioneins. Using selective-labelling plasmids to investigate the product of the CutA gene, three 35S methionine-labelled proteins were identified - there is a strong band corresponding to 26kd and two weaker ones at 45kd and i4kd. The 26kd band binds 64CU on Western blots supporting the motion
a complex microenvironment.
Ii
J “
that it is a Cu binding protein.
A
J»
1.0 nM
10 nM
!■ Composite gel electrophoresis of 355-labelled proteoglycans.
BMI.OmM
10.1 nM
Control O.ISDMSe
t
ilOOnM
developmental device which achieves rapid changes in cell and tissue distribution. Two of the known examples are the onset of neural crest cell migration from the neural epithelium, and sclerotome formation from epithelial somites. The former event is promoted by growth factors of the TGF-p family, presumably through intracellular second messengers. We have investigated this possible second messenger involvement in in vitro models of both the above systems. Immediate stimulation of epithelio-mesenchymal transformation could be achieved in both cases, without gene activation, either by broad spectrum kinase inhibitors, or by a cocktail of protein kinase C (PK-C) and PK-A inhibitors (Fig. B). This points to a complex phosphorylation control, via at least two kinase systems, of the cell functions governing the epithelial and mesenchymal states. This complexity is mirrored by direct protein phosphorylation studies. Two dimensional gel electrophoresis of tissues metabolically labelled with 32P revealed a complex pattern of protein phosphorylation. Most but not all of these proteins showed a marked decrease in phosphorylation, either slowly (8 hrs) during normal epitheliomesenchymal transition, or rapidly (<ihr) during transition induced by PK-C/A inhibition. The cell function altered by PK-C/A inhibition was, surprisingly, neither cell-cell or cellsubstrate adhesion, but rather cell spreading dynamics after substrate adhesion (Fig. C).
1 N'll
000 nM
I HLl H FracHon
BIM 10 mM
Conversion of epithelial cells to mesenchyme, and the reverse, is a widespread
HM^IOOnM
Effect of PK-C inhibitors on dorsal neural cell outgrowth.
Enzymology and Metabolism
These experiments establish a basis for understanding the epigenetic control of one of the most important developmental mechanisms whereby embryos attain their correct form.
Identifying genes which control development. S. Bevan, J. Minichiello, D. Newgreen, in association with M. Southey and D. Venter (Anatomical Pathology, Royal Children's Hospital). The genetic control of development depends on the differential action of specific genes at defined times in defined groups of cells. Dysfunction of some of these same genes is thought also to contribute to some paediatric tumours. To identify these genes in the neural crest system, we have focussed on specific cell groups in the neural crest lineage at a range of times throughout embryonic development. This developmental system has been chosen because it is an accessible model system for which detailed background developmental information already exists, and because the major group of paediatric solid tumours arise in its lineage. The marked molecular conservatism of early developmental stages, from the level of molecules up to the whole organism, permits us to use the most controllable, manipulatable and cheapest animal model. In this case the quail embryo. Cell biological methods have been used in the difficult task of isolating key crest cell types, firstly from non-crest cells, and secondly from crest-derived cells of different types. From fifteen of these cell populations although necessarily small in size (104-105 cells), we have extracted mRNA, and from this have constructed and amplified cDNA libraries via PCR using two different strategies, if these libraries are representative of the starting mRNA, a large number of already known gene
heterologous set of genes, including a representative growth factor, a cell adhesion molecule, an extracellular matrix-associated molecule, and several second messenger molecules: all these have given the predicted pattern of recognition in the libraries. We therefore assume that the libraries are reasonably representative. This means that the cDNA library of each defined cell population would Include both nucleotide sequences unique to that population at the time of its isolation, but also the common sequences coding for the everyday “housekeeping” proteins used by all cells indiscriminately. Preliminary comparison of neural crest lineage cells at very different points in their development, using the differential display method, confirms that the cDNA populations have both unique and common sequence. These libraries, at their present stage, therefore constitute an important resource for developmental and oncological research. Developmentally significant gene sequences would be expected among the unique sequences. The task for the coming year is to select examples based on differential display and/or subtractive hybridisation and to screen these back against the original libraries and against quail genomic libraries. From there, further studies will include sequencing for identification of new developmental molecules, and screening on human chromosome libraries to identify candidate genes at risk in the generation of neural crest-derived tumours.
D.R. THORBURN, I. FRANCIS, D.M. DANKS
Respiratory chain enzyme defects in 3-methylglutaconic aciduria.
Enzyme diagnosis of respiratory chain defects.
D.R. Thorburn, D.M. Kirby, D.M. Danks, J. Pitt (Biochemistry Department, RCH).
D.M. Kirby, M. Crawford, D.R. Thorburn
Respiratory chain enzyme defects have been described recently in several patients with mild to moderate increases in urinary 3methylglutaconate. We have investigated tissue biopsies from seven patients with 3-
(I
The mitochondrial respiratory chain is composed of at least 80 distinct proteins, 13 of which are coded for by mitochondrial DNA, and the remainder by nuclear genes, several of which are tissue-specific. Defects of the respiratory chain can cause a diverse range of clinical symptoms, which are often non-specific. This genetic, biochemical and clinical heterogeneity makes diagnosis of these conditions very difficult. We receive tissue biopsies or cell lines from most children in Australia with a suspected respiratory chain enzyme defect. Over the last 18 months we have assayed these enzymes in muscle and/or liver biopsies from over 130 such children. The samples are also studied by other methods such as histology, histochemistry, electron microscopy and DNA analysis. The enzyme results were virtually diagnostic of a defect in about 20% of patients and suggestive of a defect in a further 20%. It is probable that some of the remaining patients have a defect which was not detected by our current methods. The likely mode of inheritance is often not apparent from such analyses, and we are continuing to research novel methods that could increase our diagnostic ability and
methylglutaconic aciduria. Two cousins with Barth syndrome had markedly defective activity of complexes I to IV in skeletal muscle. A third unrelated patient with Barth syndrome had defective activity of complexes ll-rlll and IV in skeletal muscle. Three patients presented with cardiomyopathy and lactic acidosis. One of these had defective activity of complexes I, Iklll and IV in skeletal muscle, while the other two had normal skeletal muscle enzymes but an isolated complex I defect in liver. The sixth patient presented with a movement disorder and developmental regression and also had markedly deficient activity of complex I in liver, but normal activity in skeletal muscle. 3-Methylglutaconic aciduria can be a non specific marker for some respiratory chain enzyme defects. The variety of enzyme defects, tissue involvement and clinical presentations in the patients described here suggests that some, but not all, respiratory chain defects interfere with 3-methylglutaconic acid metabolism, most likely through a common mechanism.
give insight into the type of genetic mechanism involved. These methods involve functional studies of intact cells (or isolated mitochondria) using flow cytometry or measuring rates of metabolite flux such as lactate, pyruvate and ATP generation.
sequences should be present. We have therefore probed the libraries with oligonucleotide probes specific for a
17
Detection, diagnosis and management of inborn errors of metabolism. D.M. Danks, E. Treacey, D.E.M. Francis, P. McGrath, I. Francis, D.R. Thorburn, K. Seller, D.M. Kirby, E. Tsotsis, J. Pitt (Biochemistry Department, RCFi). The Murdoch Institute Enzymology and Metabolism Laboratory, together with the Metabolic Screening Laboratory of the Biochemistry Department of Royal Children’s Flospital, provide laboratory services to the whole of Victoria through the Victoria Clinical Genetics Services. These roles include the detection and definitive diagnosis of patients with inborn errors of metabolism, as well as long term management of these patients. The latter role has grown in magnitude in the last two years due to the introduction and success of newborn screening for phenylketonuria (PKU) about two decades ago. Prior to that time most PKU patients were not diagnosed until several years after birth, by which time they had suffered irreversible brain damage. Newborn screening for PKU has allowed diagnosis of these patients within a week of birth and, provided they adhere to a special diet (with a low phenylalanine content) during childhood, they attain normal intellect. Now that the first generation of “treated” PKU patients has reached child-bearing age, they need to go back on to a low phenylalanine diet prior to and during pregnancy to prevent brain damage to the foetus from high maternal phenylalanine levels. This requires careful monitoring of maternal plasma to ensure that the phenylalanine level is adequate for nutrition but well below toxic levels, and has resulted in a substantial increase in the number of amino acid samples we analyse.
Malonyl CoA decarboxylase deficiency. D.R. Thorburn, E. Tsotsis, D.M. Danks, G.B. MacPhee (Glasgow, UK), R. W. Logan (Glasgow, UK), R. Matalon (Miami, USA). Malonyl CoA decarboxylase deficiency is a rare inborn error of fatty acid metabolism characterised by developmental delay and the transient excretion of excess malonate in the urine of affected subjects during episodes of vomiting or febrile convulsions. The disorder was first described by scientists at the Murdoch Institute in 1984, and until recently only one further case had been identified, also in Melbourne by our laboratory in 1986. In the last two years, however, we have identified a further three patients. Two of these cases presented at the one hospital in Glasgow, and like the Australian cases, malonic aciduria was present only when the patients were acutely ill, and was undetectable 24 hours later. The fifth patient had a more severe clinical presentation and had consistently high urinary levels of malonate, despite having relatively high residual enzyme activity in fibroblasts. We are currently analysing the enzyme kinetics in a cell line from this patient in more detail. Given that 80% of the world cases have presented at only two hospitals, that the malonic aciduria is usually very transient and that the symptoms are non-specific, it now seems likely that this defect is substantially underdiagnosed.
of the production of energy from fat, but it
Clinical trial of Metronidazole therapy in Methylmalonic Acidaemia.
has been difficult to study because, unlike the other enzymes, it is located only in liver. By a process of deduction from biochemical
G.N. Thompson, J.V. Leonard (London, UK), J.M. Saudubray (Paris, France), A. Burllna (Padova, Italy), R. Parini (Milan, Italy), J.J. Pitt (Biochemistry Department, RCH), U. Wendei (Dusseldorf, Germany)
findings in a patient presenting with hypoglycaemia, we were able to suggest that the patient may have a defect in HMGCoA synthase. Subsequently a liver biopsy was undertaken and analysed with the help
Methylmalonic acidaemia is a condition characterised by buildup of methylmalonic acid resulting in failure to thrive, vomiting and, without treatment, early death. We have recently shown that a substantial amount of methylmalonic acid arises from bacterial metabolism in the gut. This source can be significantly reduced by administration of the antibiotic Metronidazole. While this
of the Philadelphia group, and enzyme deficiency was confirmed. Further studies are proceeding to better define the defect
therapy appears promising, its value has not been confirmed by wider testing. We have now completed a broad-based collaborative trial examining an extensive range of clinical parameters and their response to Metronidazole therapy. Patients include over 30 children with methylmalonic acidaemia in centres in Italy, France, England and Melbourne. This trial is being co-ordinated through Melbourne and the results are currently being collated.
A new defect in fatty acid oxidation: HMG-CoA Synthase Deficiency. G.N. Thompson, C.A. Stanley (Philadelphia, USA), J.J. Pitt (Biochemistry Department, RCFI), E. Treacy, G. Mitchell (Montreal, Canada). The utilisation of fat for energy production involves a number of enzymes. Defects in these enzymes have, in the last 10 years, been identified as a frequent cause of hypoglycaemia in young children, and on occasion, have led to sudden infant death, cardiomyopathy and other symptoms. FIMGCoA synthase is a key enzyme in the control
at a molecular level and to identify more simple diagnostic methods.
j-
A new pyridoxine-responsive mutation in gyrate atrophy of the choroid and the retina. G.N. Thompson, G. Mitchell (Montreal, Canada), D.M. Danks. Gyrate atrophy is a condition which causes degeneration of the retina of the eye in childhood. The defect is in the metabolism of the amino acid, ornithine, more specifically in the enzyme ornithine aminotransferase. Very rarely patients with this condition respond to administration of the cofactor for the enzyme, pyridoxine. One such patient recently presented in Melbourne and, in collaboration with Dr. Mitchell, we have identified the mutation responsible for the defect and the pyridoxine responsiveness. The mechanism of retinal damage in this condition, and therefore the best approach to the treatment, are currently unidentified. The delineation of the mechanisms surrounding this mutation will add valuable understanding in this condition.
i
Epidemiology
The value of dietary modifications in methylmalonic and propionic acidaemias. G.N. Thompson, M.J. Wilson, D.E.M. Francis, J.J. Pitt (Biochemistry Department, RCH), K. Seller. Methylmalonic and propionic acidaemias are the most common clinically important organacidopathies and recent advances in understanding of these conditions have brought about a number of new possibilities in treatment. Of great contention still is the value of supplementation of the diet with amino acid mixtures deficient in the precursors to the defective pathways. Using a range of complementary stable isotope techniques we have undertaken detailed studies in six children with these conditions to compare the effects of the supplements with conventional treatment and with carbohydrate supplementation on protein metabolism and production of the abnormal toxins. Analysis of samples is still proceeding but the studies should give definitive information on the value of these important therapeutic techniques.
Protein and amino acid metabolism in Maple Syrup Urine Disease. G.N. Thompson, K. Seller, J.J. Pitt (Biochemistry Department, RCH), G. Berry (Philadelphia, USA). Maple Syrup Urine Disease results from defective metabolism of branch chain amino acids, valine, isoleucine and leucine. Typically these children present early in the neonatal period with coma and fitting which progresses to death if treatment is not rapidly instituted. One of the unusual aspects of treatment is that leucine levels appear to fluctuate disproportionately with the other changes in protein metabolism. This would seem to suggest that, unusually, there might be some
hidden pool in the body for leucine other than protein. With stable isotope techniques we have studied protein metabolism and leucine kinetics under various conditions in patients with maple syrup urine disease to examine more the relative movements of protein and the branch chain amino acids. This project is in mid course and will be completed outside the Murdoch Institute.
Advances in Neonatal Screening: The use of tandem mass spectrometry for broad based population screening of metabolic disorders.
LJ. SHEFFIELD Study of drugs in pregnancy. L.J. Sheffield, H. McNeil, R. Batagol. A pilot study was completed over a 17 month period with the aim of documenting the drugs taken during pregnancy. This involved the co-operation of 19 community pharmacies. One hundred and six patients were enrolled and 51% completed a diary listing their drug intake. Drugs taken during pregnancy were also investigated by using computerised prescription records, but this was found to only provide limited information. This work was then extended to follow 697
G.N. Thompson, I. Francis, D. Millington (North Carolina, USA) Mass spectrometry has for a long time been the technique of choice for identification of metabolic disorders but the intensive sample preparation needed has made it unsuitable for the large numbers of samples required to be analysed in neonatal population screening programmes. Recently a new technique known as tandem mass spectrometry has been developed which obviates much of the sample preparation requirements. In collaboration with Dr. Millington, we have reviewed the presentations of about 100 patients with a wide range of metabolic disorders to examine the sensitivity and specificity of this new technique in detection of metabolic disease. Early results indicate that as many as 20 different conditions can be reliably identified by the technique on a cost-effective basis. Further development is still necessary to fully automate the procedure but, realistically, this method could become feasible for more general application in the near future.
determine what is the most useful measurement to characterised chondrodysplasia punctata and Binders syndrome.
women during pregnancy and to record their drug intake. All 697 have now delivered their babies and methods have been developed to correlate the baby’s health with drug taking during pregnancy. A method has been developed to link this with records at the Birth Defects Register kept at the Perinatal Statistics Unit. Dr. Sheffield has been working at the Birth Defects Register to help improve the accuracy of the recording of birth defects, which has recently been shown to be over 90%. This makes the Register now a very useful way of detecting birth defects.
Chondrodysplasia punctata.
Prenatal diagnosis. J. Halliday, L. Sheffield, J. Lumley, D. Danks. Jane Halliday has been completing her PhD thesis on a comparison of fetal outcome and factors influencing utilisation of amniocentesis and CVS in women 37-39 years, comparing those who do and do not have one or other of the tests. There has been quite a lot of use made of the prenatal diagnosis register which has been kept at the Murdoch Institute for the last 12 years and, by linking them with the records at the Victorian Perinatal Data Collection Unit, it has been possible to look into various aspects of prenatal diagnosis. The relationship of CVS and limb defects has been investigated and advanced maternal age has been found as a new risk factor. The data has been used to produce actual risk figures for various age groups when one considers the time of chorionic villus sampling, amniocentesis and at birth. Finally, the number and type of chromosome abnormalities found before and after 20 weeks in fetuses who have been found to be abnormal by ultrasound has been documented.
L.J. Sheffield, S. Forrest, H. Dahl, W. Hutchison in conjunction with C. Petit and A. Weil (Institute Pasteur, Paris), I. Glass (University of California, San Francisco).
Number of Amniocentesis and CVS, 1985 -1992 4500
Work is continuing on a region of the X chromosome seeking abnormalities in patients with chondrodysplasia punctata. We are using probes sent by our collaborators in Paris. Collaborations are also being carried out together with the University of Sydney, Departments of Anatomy and Orthodontics. Facial photographs are being analysed to
1 li
4000
4175
■ CVS ■ Amnio
3611
3500 2971
3000
I
m
2364
2500
1808
2000
1958
2562
44% 36%
32% 29%
22%
15%
z 1500
1985
1985
1987
1988
1989
Year
1990
1991
1992
Tissue Culture Laboratory
X-linked disease.
Marjorie Crawford, Tamara Gough, Kim Spence.
L. Sheffield, J. Halliday with S. Sherman (Emory University, USA), J. Lloyd (IMVS, Adelaide). We have continued to investigate haemophilia A by DNA studies and clotting assay studies on some well studied families from Adelaide. We are also continuing the collaborative study with Dr. S. Sherman to determine the recurrence risk of fragile X syndrome by studying pregnancies which have had prenatal diagnoses.
Polycystic kidney disease. D. Ravine, L. Sheffield, D. Banks, R. Gibson, R. Walker (Royal Melbourne Hospital), R. Richardson (Adelaide Children’s Hospital). Dr. Ravine has continued to analysis and present for publication his work on the correlation of polycystic kidney disease linked to the DNA probes on chromosome 16 and ultrasound diagnoses.
in 1993 the laboratory received 344 specimens. These were mostly established cell lines from other laboratories, biopsies to be established by us or heparinised blood for establishment of continuous lymphocyte cultures. Approximately 18% were for cytogenetic analysis, 17% were established for Orthopaedic Research and 33% for the Metabolism/Enzymology Laboratory. Although the total number of samples received was the same as in previous years, the proportion of different celt types has been changing over the last few years. There has been a reduction in the number of fibroblast lines established. In 1993 the number was 231 compared with 295 in 1992 and 254 in 1991. There has been a corresponding increase in
Congratulations to Tamara Pasque who was married in May 1993. She is now Tamara Gough. Her work on the DHPR gene continued. Three separate clones of the gene were made in order to sequence the intron/exon boundaries. This sequence was then used to study some patients with splicing mutations. She is currently trying to sequence the promoter region of the gene. Kim Spence continued to work part-time. She does an excellent job keeping us supplied with all our media requirements and testing all cell lines for the presence of mycoplasma.
L to R: Marjorie Crawford, Tamara Gough, Kim Spence.
the number of continuous lymphocyte cultures. In 1991 only 47 lines of this type were received whereas 1992 saw this increase to 66. 1993 saw a further increase to 82, many of which were established in our own laboratory. The setting up of this type of cell line by us only began in 1992. Since that time we have received 89 samples. After some initial problems our success rate for the last 40 samples is 98%. Marjorie Crawford continued her work with David Thorburn in the Metabolic Laboratory. The results from the work on lactate/pyruvate ratios in cultured fibroblasts were not reproducible enough to use the technique for diagnostic purposes and this project was suspended for the time being. Her current project involves the measurement of ATP production in fibroblasts from patients with respiratory chain defects and controls. No results are available as yet.
/
j!
List of Publications - 1993
In press previous reports, now published
Danks, D. M. - Gene therapy and related novel forms of treatment. Med J Aust 159: 276-279, 1993.
Blau, N., Heizmann, C. W., Sperl, W., Korenke, G. C, Hoffmann, G. F., Smooker, P. M. and Cotton, R. G. H. - Atypical (mild) forms of dihydropteridine reductase deficiency. Neurochemical evaluation and mutation detection. Ped Res 32: 726-733, 1992. Cole, W. G., Hall, R. K. and Rogers, j. G. The clinical features of spondyloepiphyseal dysplasia congenita resulting from the substitution of glycine 997 by serine in the alphai(ll) chain of type II collagen. J Med Genet 30: 27-35, i993Cotton, R. G. H. - Current methods of mutation detection. Mut Res 285: 125-144, 1993.
Sheffield, L. J. - DNA Diagnostic tests: presymptomatic and prenatal. Med J Aust 158: 349-352, 1993.
Danks, D. M. - The impact of molecular genetics upon clinical medicine.
Mercer, J. F. B., Livingston, J., Hall, B., Paynter, J., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemieniak, D. and Glover, T. W. - Isolation of a partial candidate gene for Menkes disease by positional cloning.
Med J Aust 158: 147-148, 1993.
Nat Genet 3: 20-25, i993-
Ferraz, O.P., Ferreira, P., Gustavson, K.-h., Halliday, J., Hockey, A., Howard-Peebles, P.N.,
Dianzani, L, Cameschella, C., Ferrero, G. B., Ramus, S., Ponzone, A. and Cotton, R. G. H. Molecular analysis of contiguous exons of phenylalanine hydroxylase: Identification of a novel PKU mutation.
Palombo, E. A., Bishop, R. F. and Cotton, R. G. H. - Intra- and inter- season genetic variability in the VP7 gene of serotype Gi (monotype Gia) rotavirus clinical isolates. Arch Virol 130: 57-69, 1993.
Jenkins, E., Kennorknecht, L., Kehkonen, M., Ladaique, P., Leisti, ]., Maddelena, A., Mazurczak, T., Mattei, J.-F., Mattina, T., Mckinley, M.J., Murphy, P., Pellissier, M.C.,
J Med Genet 30: 228-231, 1993. Du Sart, D., Kalitsis, P. and Schmidt, M. Noninactivation of a portion ofXq28 in a balanced X-autosome translocation. Am j Med Genet 42: 156-160, 1992.
Dahl, H.-H. M. - Elements of molecular genetics. Med J Aust 158: 195-201, 1993.
Earle, E., Voullaire, L, Hill, L, Slater, H. and Choo, K. H. - Absence of satellite III DNA in the pericentric, proximal q arm region of
Dahl, H.-H. M. - Things Mendel never dreamed
human chromosome 14: analysis of a i4pvariant. Cytogenet Cell Genet 61: 78-80, 1992.
of. Med j Aust 158: 247-252, 1993. Dahl, H.-H. M., Hansen, L. L, Brown, R. M., Danks, D. M., Rogers, J. G. and Brown, G. K. X-Linked pyruvate dehydrogenase Ela subunit deficiency in heterozygous females: variable manifestation of the same mutation. I of Inher Metab Dis 15: 835-847, 1992. Danks, D. M. - Defining the location of the Huntington disease gene. Am J Hum Genet 52: 214, 1993. Danks, D. M. - Disorders of copper transport: Menkes disease and the occipital horn syndrome. In: Royce & Steinmann (eds). Connective Tissue and its Heritable Disorders: Molecular, Genetic and Medical Aspects, New York, Wiley Liss: 487-505, 1993.
Hill, D. J., Menahem, S., Hudson, L, Sheffield, L., Shelton, M., Oberklaid, F. and Hosking, C. S. - Charting infant distress: an aid to defining colic. J Pediatr 121: 755-758, 1992.
Dost, B.A., Voeickel, M.A., Weaver, D.D. and Webb , T. - Collaborative prospective study of the Fragile X syndrome one year progress report. Am ] Med Genet 43; 355-360, 1992.
Ramus, S. J., Forrest, S. M., Pitt, D. B., Saleeba, J. A. and Cotton, R. G. H. Comparison of genotype and intellectual
Slater, H. R., Voullaire, L. E., Vaux, L. E., Bankier, A., Pertile, M. and Choo, K. H. A. Confirmation of Trisomy 22 in two cases using
phenotype in untreated PKU patients, j Med Genet 30: 401-405, 1993.
chromosome painting; comparison with t(ii; 22). Am j Med Genet 46: 434-437, 1993.
Ravine, D., Francis, R. I. and Danks, D. M. Non-specific elevation of immunoreactive trypsinogen in sick infants.
Ravine, D., Walker, R. G., Gibson, R. N., Forrest, S. M., Richards, R. L, Friend, K., Sheffield, L. J., Kincaid-Smith, P. and Danks,
activity. Brain Res Bull 29: 949-952, 1992.
D. M. - Phenotype and genotype heterogeneity in autosomal dominant polycystic kidney
Kalitsis, P., Earle, E., Vissel, B., Shaffer, L. G., McQuillan, C. and Choo, K. H. A. - A chromosome 13 specific human satellite 1 DNA
disease. Lancet 340: 1330-1333, 1992.
subfamily with minor presence on chromosome 21: further studies on Robertsonian
Purvis-Smith, S., Robinson, H., Scapagnini, U., Schaap, T., Shapiro, L.R., Smits, A.P.T., Steinbach, P., Turner, G., Uchida, LA., Van
Ponzone, A., Guardamagna, 0., Dianzani, I., Ponzone, R., Ferrero, G. B., Spada, M. and Cotton, R. G. H. - Catalytic activity of tetrahydrobiopterin in dihydropteridine reductase deficiency. Ped. Res. 33: 125-128, 1993.
Eur J Pediatr 152: 348-349, 1993. Johansen, P. A., Jennings, I., Cotton, R. G. H. and Kuhn, D. M. - Immobilization of tryptophan hydroxylase by immune absorption: A method to study regulation of catalytic
Sherman, S. L., Barbi, B., Brondrum-Neilsen, K., Brown, W.T., Carpenter, N.J., Chudley, A.E.,
Schmidt, M. - Do sequences in Xq27.3 play a role in X inactivation? Am J Med Genet 43: 273-281, 1992.
translocations. Genomics 16: 104-112, 1993. Schoeden, G., Redweik, U., Frank, G., Cotton, R. G. H. and Blau, N. - Allosteric characterization of GTP cyclohydroxylase I from E.coli. Eur J Biochem 210: 561-568, 1992.
1
Published and accepted for publication since 1992 report
Allan, G. L., Camakaris, J. and Legge, G. J. F. Elemental micro-analysis of fibroblasts by a scanning proton microprobe and application to Menkes’ disease. Biol Trace Elem Res, (in press). Bateman, J. F., Lamande, S. R., Hannagan, M., Moeller, 1., Dahl, H.-H. M. and Cole, W. G. Chemical cleavage method for the detection of RNA base changes: Experience in the application to collagen mutations in osteogenesis imperfecta. Am J Med Genet 45: 233-240, 1993. Bower, S. P. C., Hawley, 1. and Mackey, D. A. Cardiac arrhythmia and Leber’s hereditary optic neuropathy. Lancet 339: 1427-1428, 1992. Choo, K. H. A. In Situ Hybridisation Protocols. New Jersey, K. H. A. Choo (ed), Humana Press (in press). Choo, K. H. A., Trowell, H. E. and Nagy, A. Long-range map of the centromere and p-arm of human chromosome 14, in Report of the First International Workshop on Human Chromosome 14 Mapping. Cytogenet Celt Genet (in press).
Dahl, H.-H. M. and Brown, G. K. - Pyruvate dehydrogenase deficiency in a male caused by a point mutation (F205L) in the Ela subunit. Hum Mut (in press). Danks, D. M. - Carrier testing for cystic fibrosis.
Fitzgerald, J., Wilcox, S. A., Graves, J. A. M. and Dahl, H.-H. - A eutherian X-linked gene, PDHAi, is autosomal in marsupials: a model for the evolution of a second, testis-specific variant in eutherian mammals. Genomics 18: 636-642, 1993.
Howell, N., McCullough, D. A., Kubacka, 1., Halvorsen, S. and Mackey, D. A. - The sequence of the human mitochondrial DMA: the question of errors versus polymorphisms. Am J Hum Genet 50: 1333-1337, 1992.
Eujii, T., Van Coster, R. N., Old, S. E., Medori,
Temporal and tissue-specific interactions involving novel transcription factors and the
Med J Aust 159: 148-150, 1993. Danks, D. M. - Disorders of copper transport. In The Metabolic Basis of Inherited Disease Textbook. C. R. Scriver, A. L. Beaudet, W. S. Sly and D. Valle, (eds), McGraw-Hill Inc, USA, (in press).
lannello, R. C., Kola, 1. and Dahl, H.-H. M. R., Winter, S., Gubits, R. M., Matthews, P. M., Brown, R. M., Brown, G. K., Dahl, H.-H. M. and De Vivo, D. C. - Pyruvate dehydrogenase deficiency: molecular basis for intrafamilial heterogeneity. Ann Neurol (in press).
Danks, D. M. - Germ-line gene therapy: no place in treatment of genetic disease. Hum Gene Ther (in press).
Halliday, G. M., McCann, H. L., Pamphlett, R., Brooks, W. S., Creasey, H., McCusker, E., Cotton, R. G. H., Broe, G. A. and Harper, C. G. - Brainstem serotonin-synthesizing neurons in
Danks, D. M. - Menkes disease - basis of copper transport disturbance and role of metallothionein. In: Anke, M., Meissner, D., 81 Mills, C.F. (eds). Trace Elements in Man &
Alzheimer’s disease: a clinico-pathological correlation.
Animals 8, Dresden, 674-681, 1993.
HALLIDAY, J., LUMLEY, J., BANKIER, A. Karyotype abnormalities in fetuses diagnosed as abnormal on ultrasound before 20 weeks gestational age. Prenat Diag (in press)
Danks, D.M. - Potential and ethics of somatic and germ-line gene therapy. Today’s Life Sci (in press). Danks, D. M. - Whither Genetic Services? Med J Aust. 159: 221-222, 1993.
Acta Neuropathol 84: 638-650, 1992.
Halliday, J., Lumley, J., Sheffield, L. and Lancaster, P. - Limb deficiencies, chorion villus sampling and advanced maternal age. Am J Med Genet 47: 1096-1098, 1993.
Dianzani, L, Howells, D. W., Ponzone, A., Saleeba, J. A. and Cotton, R. G. H. - Two novel
Cotton, R. G. H., Jennings, 1. G., McAdam, W. J., Hutchinson, W. M. and Dahl, H.-H. M. - The
mutations in the dihydropteridine reductase gene in patients with tetrahydrobiopterin
molecular defect in dihydropteridine reductase deficiency, in International Days of Paediatrics (in press):
deficiency. J Med Genet 30: 465-469, 1993.
Cotton, R. G. H., Ramus, S. J., Saleeba, J. A., Dahl, H.-H. M., Forrest, S. M., Howells, D. W., Dianzani, L, Palombo, E. A., Bishop, R. F., Anderson, M. J., Milner, C. M. and Campbell, R.
Regional localisation of a second non-specific X-linked mental retardation gene (MRX) to Xp22. (in press)
D. - The chemical reactivity of the thymidine base in T.G mismatches is differentially influenced by a guanine or cytosine in the 5’ position., DNA & Cell Biol 12: 945-949, 1993.
Earle, E. and Choo, K. H. A. - Radioactive in situ hybridisation to replication-banded chromosomes. In: In Situ Hybridisation Protocols, New Jersey, Humana Press, (in press).
Donnelly, A. J., Choo, K. H. A., Gedeon, A. K., Kozman, H. M., Danks, D. M. and Mulley, J. C. -
Hansen, L. L., Brown, G. K., Brown, R. M. and Dahl, H.-H. M. - Pyruvate dehydrogenase deficiency caused by a 5 base pair duplication in the Ela subunit. Hum Mol Genet 2: 805807, 1993. Howell, J. M. and Mercer, J. F. B. - The pathology and trace element status of the toxic milk mutant mouse. J Path (in press).
proximal promoter of the mouse Pdha-2 gene. J Biol Chem 268: 22581-22590, 1993. Jiang, W. M., Jenkins, D., Yuan, Q., Leung, E., Choo, K. H. A., Watson, J. D. and Krissansen, G. W. - The gene organisation of the human beta 7 subunit, the common beta subunit of the leukocyte integrins HML-i and LPAM-i. Int Immunol 4: 1031-1040, 1992. Loesch, D. Z., Sheffield, L. J. and Hay, D. A. Between generation differences in ascertainment and penetrance: relevance to genetic hypothesis in Fragile X. Hum. Genet. 91: 469-474, 1993. Mackey, D. - Blindness in offspring of women blinded by Leber’s hereditary optic neuropathy. Lancet 341: 1020-1021, 1993. Mackey, D. and Howell, N. - A variant form of Leber hereditary optic neuropathy characterized by recovery of vision and a multistep mitochondrial genetic etiology. Am J Hum Genet 51: 1218-1228, 1992. Mackey, D., Nasioulas, S. and Forrest, S. Finger prick blood testing in Leber hereditary optic neuropathy. B J Opthalmol 77: 311-312, 1993. Mackey, D. A. and Buttery, R. G. - Leber hereditary optic neuropathy in Australia.
Howell, N., Kubacka, 1., Halvorson, S. and D., M. - Leber’s hereditary optic neuropathy: the
Aust NZ J Ophthalmol 20: 177-184, 1992.
etiological role of a mutation in the mitochondrial cytochrome b gene. Genetics. 133: 133-136, 1993.
MacPhee, G. B., Logan, R. W., Mitchell, J. S., Howells, D. W., Tsotsis, E. and Thorburn, D. R. - Malonyl coenzyme A decarboxylase , deficiency. Arch Dis Child 69: 433-436, 1993.
Matalon, R., Michaels, K., Kaul, R., Whitman, V., Rodriguez-Novo, J., Goodman, S. and Thorburn, D. - Maionic aciduria and cardiomyopathy.
Smooker, P. M., Howelis, D. W. and Cotton, R. G. H. - Identification and in vitro expression of mutations causing dihydropteridine reductase deficiency. ) Bioi Chem 32; 6443-6449, 1993.
J Inher Metab Dis i6: 571-573, 1993. Mercer, J. F. B., Grimes, A., Ambrosini, L., Lockhart, P., Paynter, J. A., Dierick, H. and Glover, T. W. - Mutations in the murine homologue of the Menkes gene in dappled and blotchy mice. Nat Genet (in press). Mercer, J. F. B., Grimes, A., Paynter, J., Lockhart, P. and Bhave, M. Molecular biology of proteins involved in copper transport. Trace Elements: Roles, Risks and Remedies. Trace Element Society 4: 185-191, 1992. Mercer, J. F, B., Livingston, )., Hall, B., Paynter, ). A., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M. and Glover, T. W. Isolation of genomic and cDNA clones of the Menkes disease gene. In: Anke, M., Meissner, D., & Mills, C.F. (eds). Trace Elements in Man
Sullivan, L. J., Makris, G. S., Dickinson, P., Mulhall, L. E. M., Forrest, S., Cotton, R. G. H. and Loughnan, M. S. - A new codon 15 rhodopsin gene mutation in autosomal dominant retinitis pigmentosa is associated with sectoral disease. Arch Ophth 111: 1512-1517, 1993. Takakubo, F., and Dahl, H.-H.M. - Analysis of pyruvate dehydrogenase expression in embryonic mouse brain: localization and developmental regulation. Develop Brain Res (in press). Takakubo, F., Thorburn, D. and Dahl, H.-H. M. A four-nucleotide insertion hotspot in pyruvate dehydrogenase Ela gene (PDHAi). Hum Mol Genet 2: 473-474, 1993.
Trowell, H. E., Nagy, A., Vissei, B. and Choo, K. H. A. - Long-range analyses of the centromeres of human chromosomes 13, 14 and 21: Identification of a narrow region containing two key centromeric DNA elements. Hum Mol Genet 2: 1639-1649, 1993. Voullaire, L. E., Slater, H. R., Petrovic, V. and Choo, K. H. A. - A functional marker centromere with no detectable alpha satellite, satellite III or CENP-B protein: Activation of a latent centromere? Am J Hum Genet 52: 1153-1163, 1993. Webb, T., Watkiss, E. and Woods, C. G. Neither uniparental disomy nor skewed Xinactivation explains Rett syndrome. Clin Genet 44: 236-240, 1993. Wilson, D. I., Cross, I. E., Goodship, J. A., Brown, J., Scambler, P. J., Bain, H. H., Taylor, J. F., Walsh, K., Bankier, A. and Burn, J. - A prospective cytogenetic study of 36 cases of Di George syndrome. Am J Hum Genet 51: 957-63, 1992.
& Animals 8, Dresden, 682-693, 1993.
Takakubo, F., Thorburn, D. R. and Dahl, H.-H. A novel mutation and a polymorphism in the X
Michalska, A. E. - Production of chimeric
chromosome located pyruvate dehydrogenase Ela gene (PDHAi).
Woods, C. G. and Danks, D. M. - The role of genomic imprinting.
animals: successes and limitations. In Kakulus, B.A., Howell, Y.M., and Roses, A.D. (eds),
Hum Mol Genet 2: 1961-1962, 1993.
Med J Aust 158: 801-802, 1993.
Duchenne Muscular Dystrophy - Animal Models and Genetic Manipulation. Raven Press: 213-221, 1992.
Thorburn, D. R., Thompson, G. N. and Howells, D. W. - A fluorimetric assay for succinic semi
Michalska, A. E. and Choo, K. H. A. - Targeting and germline transmission of a null mutation
aldehyde dehydrogenase activity suitable for prenatal diagnosis of the enzyme deficiency. ) Inher Metab Dis (in press)
Woods, C. G. and Smith, K. K. - Case of human chimerism detected by unbalanced chromosome translocation?
at the metallothioneins I and II loci in mouse. Proc Natl Acad Sci (USA) 90: 8088-8092, 1993.
Tork, I., Halliday, G. M. and Cotton, R. G. H. -
Rahman, S., Thorburn, D. R., Blok, R. B. and Dahl, H.-H. M. - Mitochondria and human
Application of anti phenylalanine hydroxylase antibody to the study of the serotinergic system in the human brain.
disease. Today’s Life Science 5: 20-28, 1993.
) Chem Neuroanat 5: 311-313, 1992.
Smooker, P., Cotton, R. and Lipson, A. Prenatal diagnosis of DHPR-deficiency by direct detection of mutation. Prenat Diag 13: 881-884, i993-
Clin Genet 44: 166, 1993.
Collaborations
Dr. J. Camakaris Department of Genetics, University of Melbourne (Dr. B.T.O. Lee) - Molecular genetic analysis of copper transport and resistance in E.coli. Department of Biological Sciences, Birmingham University, U.K. (Professor N.L. Brown) Molecular genetic analysis of copper-resistance in E.coli. Department of Chemistry and Biochemistry, North-Western University, Illinois, USA (Dr. T. O’Hailoran) - Molecular genetics and biochemical basis of copper-resistance in E.coli. Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois, USA (Dr. S. Silver) Mechanisms of copper transport in E.coli and P-type ATPases in heavy metal metabolism. Biomedicine and Health, Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW (Professor R Lambrecht)
Australia - John Mulley (X-linked mental retardation). Alfred Hospital - Ben Hock Toh (Chromosomal localisation of a mitotic spindle assembly gene). Baylor College of Medicine, Houston, U.S.A. Lisa Shaffer (Robertsonian translocation).
Dr. R.G.H. Cotton John Curtin School of Medical Research, Canberra (Dr. W.L.F. Armarego) - Crystal structure of dihydropteridine reductase. Department of Chemistry, LaTrobe University (Dr. R. Brownlee) - Pterin binding peptide structure. St. Vincent’s Hospital Institute for Medical Research (Dr. B. Kemp) - Intra molecular regulations and crystals of phenylalanine hydroxylase. Department of Biochemistry, Medical College
Dr. K.H.A. Choo
of Ohio, Toledo, Ohio (Dr. M. Ratnam) Reaction of antiidiotype antibodies with a number of pterin/folate proteins and transporters. Lafayette Clinic, Detroit, U.S.A. (Dr. D. Kuhn) Study of tryptophan hydroxylase using antibody PH8.
Collaborative Research, Inc., U.S.A. - Jen-i Mao (marker centromeres).
Oxford (Dr. C. Chen and colleagues) - Study of human brain from a range of diseases using antibody PH8.
Application of radioisotopes of copper to studies of Kinetics of Cu transport in normal and mutant cells.
Rowett Research Institute, Aberdeen, U.K. - Ian Bremner (MTl- & ll-deficient mice). Institute for Medical and Veterinary Science, Adelaide, Australia - Peter Coyle (MTl- & lldeficient mice). University of Queensland, Australia - Charles Dameron (MTl- 8i ll-deficient mice). University of Pittsburg, U.S.A. - John Lazo (MTI& ll-deficient mice). Royal Melbourne/Royal Children’s Hospital, Melbourne, Australia - Jeffrey Zajac/Garry Warne (Androgen receptor-deficient mice). Hospital for Sick Children, Toronto, Canada Lap Chee Tsui 8i Henry Heng (High-resolution centromere mapping). Adelaide Children’s Hospital, Adelaide,
Institute of Clinical Pediatrics, Turin, Italy (Dr. A. Ponzone, Dr. I. Dianzani) - Study of tetrahydrobiopterin deficient patients. Department of Gastroenterology, Royal Children’s Hospital (Dr. R. Bishop) - Chemical cleavage in the analysis of rota virus variation. University of Cologne (Professor B. Kemper) Enzymes in mutations detection.
Dr. H.H-M. Dahl
Dr. S.M. Forrest
Dept, of Genetics and Human Variation, La Trobe University, Melbourne (Prof. J. Graves) A.N.U., Canberra (Dr. S. Easteal) Dept, of Biochemistry, La Trobe University, Melbourne. ( Prof. N. Hoogenraad, Dr. T. Lithgow)
Eye and Ear Hospital, Melbourne (Dr. M. Loughnan, Dr. L. Sullivan) - Mutation detection in retinitis pigmentosa.
Genetics Lab. Dept, of Biochemistry, Oxford, U.K. ( Dr. G. Brown) Dept, of Human Genetics, erhus University, Denmark. ( Dr. L. Hansen) Charles Sturt University, Riverina, School of Science and Technology, Wagga Wagga, NSW. ( Dr. G. McKenzie). Centre for Early Human Development, Monash Medical Centre. ( Dr. I. Kola) Peter MacCallum Cancer Institute. Dr. David Woodcock, Melbourne Neuromuscular Research Centre, St.
Charles Sturt University, Riverina (Dr. G. McKenzie, Ms. L. Angel) - Methods for detection mutations in the cystic fibrosis gene. Institute for Molecular Biotechnology, Jena, Germany (Dr A. Rosenthal) - Mutation detection in the LI CAM gene causing X-linked hydrocephalus. Department of Medicine, Cambridge Univerity (Dr. S. Kenwick) - Structure and mutations in LI CAM gene.
Dr. J.F.B. Mercer School of Veterinary Studies, Murdoch
Biochemistry, La Trobe University, Melbourne (Professor N Hoogenraad).
University, W.A. (Professor J. McC. Howell) Copper toxicosis in sheep. CSIRO Division of Animal Production, Prospect, N.S.W. (Dr. K. Wand) - Expression of metaliothionein genes in normal and transgenic sheep.
Institute of Reproduction and Development, Monash Medical Centre, Melbourne (Dr R lanneilo).
Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, U.S.A. (Dr. T. Gover) - Cloning the Menkes gene.
Biozentrum der Universitat Basel, Switzerland (Dr. T. Lithgow).
CSIRO Division of Animal Production, Prospect, N.S.W. (Dr. K. Wand) - Expression of metallothinonein genes in normal and transgenic sheep.
Vincent’s Hospital, Melbourne (Professor E. Byrne). Human Genetics Group, A.N.U., Canberra (Dr. S. Easteal).
Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, U.S.A. (Dr. T. Gover) - Cloning the Menkes gene.
Dr. L.J. Sheffield Pharmacy, Royal Women’s Hospital, Melbourne (Mr. R. Batagol) - Study of drugs in pregnancy. Victorian Perinatal Data Collection Unit (Dr. ). Lumley). Department of Allergy, Royal Children’s Hospital (Dr. D. Hill) - Treatment of colic. Department of Anatomy, University of Sydney (Dr. A. Howe, Professor W. Webster) Chondro-dysplasia punctata. Department of Orthodontics, University of Sydney (K. Titus) - Chondrodysplasia Punctata Research. Institute of Medical and Veterinary Science, Adelaide (Dr. J. Lloyd) - Haemophilia A. Department of Paediatrics, Emory University, Atlanta, Georgia, USA (Dr. G. Sherman) Genetics of Haemophilia A. Institute Pasteur, Paris (C. Petit and A. Weil) and University of California, San Francisco (I. Glass) - Chondrodysplasia Punctata Research. Department of Orthodontics, University of Sydney (K. Titus) - Chondrodysplasia Punctata Research.
Dr. D.R. Thorburn Department of Anatomical Pathology, Royal Children’s Hospital (Dr C.W. Chow) - Liver histochemistry and electron microscopy in respiratory chain disease. Department of Biochemistry, Royal Children’s Hospital (Mr James Pitt) - Respiratory chain enzyme defects in 3-methylglutaconic aciduria Department of Haematology & Oncology, Royal Children’s Hospital (Dr Simon Bol) - Flow cytometry of cells and mitochondria. State Neuropathology Service, Department of Pathology, University of Melbourne (Dr X. Dennett)’ - Muscle enzyme histochemistry in respiratory chain disease. Neurology Department, St Vincent’s Hospital (Dr E. Byrne) - Respiratory chain disease in adults and children.
93
The Murdoch Institute for Research into Birth Defects
for research into birth defects Design: Nikki Flood, Seldon Hunt, Swinburne Design Centre
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Financial Statements
The Murdoch Institute for Research into Birth Defects Limited and its controlled entities -
Financial Statements and Reports 31st December 1993
A.C.N. 006 566 972
at the University of Melbourne. He represents the National Health & Medical Research Council (NHMRC).on the Institute’s Board. Director since 1991.
University of Melbourne. Director since 1986.
Mr. I. Davies, F.A.I.M., F.C.H.S.E. Directors' Report The Directors present their report together with the accounts of the Murdoch Institute for Research into Birth Defects Limited “the Company” and the consolidated accounts of the economic entity, being the Company and its controlled entities, for the year ended 31 December, 1993 and the auditors’ report thereon.
Dr. G.L. Barnes, M.D., Ch.B., F.R.A.C.P. Non-Executive Director Age 52 Director of the Department of Gastroenterology, Royal Children’s Hospital. Co-ordinator of Research, Royal Children’s Hospital Research Foundation. Dr. Barnes represents the Hospital on the Institute’s Board. Director since 1986.
Directors The Directors of the Company in office at the date of this report are:
Mr. L.G. Cox, B.Com., A.S.A., F.S.I.A. Chairman (Non-Executive Director) Age 55 Chairman of the Australian Stock Exchange Limited.
Mrs. J. Calvert-Jones Non-Executive Director Age 55 Chairman of the Herald and Weekly Times Limited. Chairman of the Advisory Council for Children with Impaired Hearing (Vic). Mrs Calvert-Jones represents Cruden Investments Pty Ltd on the Institute’s Board. Director since 1986.
Chairman of Potter Warburg Limited. Director since 1986 - appointed Chairman 1993.
Dr. R.G.H. Cotton, B.Ag.Sci., Ph.D., D.Sc. Mr. W.H. Hodgson Deputy Chairman (Non-Executive Director) Age 64 Formerly Deputy Managing Director of the National Australia Bank Limited, an appointment which completed a long career with the Bank. Mr Hodgson is a Director of various public and private companies. Director since 1986.
Professor J.A. Angus, B.Sc.(Hons), Ph D. Non-Executive Director Age 45 ' Professor Angus holds the chair of Pharmacology
Non-Executive Director Age 53 Chief Executive Officer, Royal Children’s Hospital .Mr. Davies was formerly the Chief Executive Officer of the Princess Margaret Hospital for Children in Perth. Director since 1991.
Mr. J.A. Fitzgerald Non-Executive Director Age 62 Mr. Fitzgerald is a public relations consultant. He is Corporate Affairs adviser to some of Australia’s largest corporations. Director since 1986.
Mr. P. Griffin, B. Comm. (Melb) Non-Executive Director Age 54
Professor D.M. Danks, A.O., M.D., B.S., F.R.A.C.P.
Mrs. McFarling is a successful public relations advisor. She is the President of the Friends of the Murdoch Institute, the successful Fundraising Auxiliary of the Institute. Director since 1988.
Mr. A.S. Murdoch Non-Executive Director Age 52 President of the Royal Children’s Hospital. Appointed during 1993.
Professor P.D. Phelan, B.Sc., M.D., B.S., F.R.A.C.P. Non-Executive Director Age 57 Professor Phelan is the Stevenson Professor of Paediatrics at the University of Melbourne and a distinguished thoracic physician. Director since 1986.
Chairman of the Institute’s Finance Committee. Mr. Griffin is an investment banker and
Professor A.J. Pittard, Ph.D.,. Dip.Pharm., D.Sc., F.A.A.
a director of N.M.Rothschild & Sons (Aust) Pty Ltd Group companies, various public and private companies, industry and community groups. Appointed during 1993.
Non-Executive Director Age 61
Deputy Scientific Director , Age 53 Senior Principal Research Fellow, National Health 8. Medical Research Council. Dr. Cotton acts for the NHMRC in the Assigners ■ and Regional Grants Interviewing Committees. Director since 1986.
Mrs. I. McFarling Non-Executive Director Age 56
Mr, J.S. Guest, A.M., O.B.E., V.R.D., B.Sc., M.B., B.S., F.R.C.S., F.R.A.C.S. Non-Executive Director Age 77 Mr. Guest is a distinguished Melbourne surgeon and the Chairman of the jack Brockhoff Foundation. Director since 1986.
Professor Pittard is a professor of Microbiology at the University of Melbourne. He represents the NHMRC on the Board. Director since 1993.
Professor G.B. Ryan, A.C., M.D., B.S., Ph.D., F.R.C.P.A., F.R.A.C.P. Non-Executive Director Age 55 Dean of the Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne. Director since 1986.
Scientific Director Age 62 Executive Director of the Victorian Clinical Genetics Services. Professor of Paediatric Research,
Mr. R.N. Walford, Dr. P.M. Gray and Mrs. C.M. Searby retired as Directors of the Company during the year.
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Directors' Meetings
Principal Activities
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The number of directors’ meetings and number of meetings attended by each of the directors of the'Company during the financial year are:
Director
No of Meetings ’ Attended
The principal activities of the econornic entity during the course of-the financiai year were to ' promote and undertake medical research into the understanding, prevention and treatment of birth defects, and to provide services for the diagnosis and treatment of genetic diseases and other birth defects. No significant'change in the nature of these activities occurred during the year.
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No of Meetings Held *
Dividends ,
,
.
Mr. L.G. Cox' • Mr. W.H. Hodgson Professor J.A. Angus Dr. G.L. Barnes , Mrs. J. Calvert-Jones Dr: R.G.H. Cotton Professor D.M. Danks Mr. I. Davies Mr. ).A. Fitzgerald Mr. P. Griffin
■ Mr. ).$,■ Guest . Mrs,; I. McFarling ■ • Mr. A.S. Murdoch Professor P.D. Phelan Professor A.J. Pittard , Professor G.B. Ryan Mrs. C., Searby; Mr. N. Walfdrd Dr. P.M. Gray
6
6. 6 6 .6 6
3 4 3 5 6 6 6
The Company is a company limited by guarantee. As such it has-no share capital, and no dividends are paid. . ,
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Consolidated Result
6 6 6 6
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6 6
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5 6 6 6
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The consolidated net surplus of the economic entity for the financial year ending :^i December 1993 was $112,401. (1992 $268,478). No provision is required for taxation as the Company.and its controlled entity are exempt from Income Tax. ...
Review of operations ■
A review of the operations of the Company and its controlled entity during 1993 has been, included in the Report of the Board. .
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Significant changes in State of affairs
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In the opinion of the Directors there were no significant changes in the state of the economic entity’s affairs during the financial year under review not otherwise disclosed in the report or the consolidated accounts.
/ * Reflects the number of meetings held during the time the director held office during the year.
Events Subsequent to Balance Date There has not arisen in the interval between the end of the financial year and the date of this report any item, transaction or event of a material arid unusual nature likely, in the opinion of the
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Directors of the Company, to affect significantly the operations of the economic entity, the results of those operations, or the state of affairs of the economic entity, in subsequent financial years.
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Likely future developments and expected results The scope of the alterations to the 10th floor of the main building of the Hospital planned for the near future has been reduced. It is anticipated that refurbishment of the area which will accommodate the Cytogenetics Laboratory, the DNA Diagnostic Laboratory and the Neonatal Screening Laboratory will commence late in 1994 at an estimated cost of $1 million.
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Directors' Interests and Benefits Since the end of the previous financial year, no Director of the Company has received or become entitled to receive any benefit (other,than a benefit included in the aggregate amount of remuneration received or due and receivable by Directors shown in the consolidated accounts) because of a contract made by the Company, its controlled entities, or a related body corporate with the Director or with a firm of which the Director is a member, or with an entity in which the
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Director has a substantial interest. Dated at'Melbourne this 13th day of April 1994
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Signed in accordance with a resolution of the Directors:
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LAURENCE G. CO; : Directors
C WILLIAM H. HODGSON 4
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The Murdoch Institute for Research
The Murdoch Institute for Research
into Birth Defects Limited
into Birth Defects Limited
and its controlled entities
and its controlled entities
A.C.N. 006 566 972
A.C.N. 006 566 972
Balance Sheets
Profit and Loss Accounts
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for the year ended 31 December 1993
j
as at 31 December 1993
11
Note Note 1993 Operating Profit before Income tax Income tax attributable to Operating Profit
2
1993
1992
$
- $
$
$
112,401
268,478
34.827
194.304
1
Operating Profit after Income Tax
112,401
Accumulated Funds at beginning of the financial year
268,478
34.827
194.304 L- -
8,917,161
1993
1992
$
$
$
$
69,513
243.744 288,827
64,188
138,953-
4 5
418,408
176,160
95.243
615,994
887,935
280,454
636,841
6
13,848
16,175
13.848
1.117.763
1,436,681
CURRENT ASSETS Cash Receivables Investments ‘ Inventories TOTAL CURRENT ASSETS
8,771.183
8.947.675
8,875,871
NON CURRENT ASSETS Receivables Investments Plant & Equipment TOTAL NON CURRENT ASSETS
4 5 7
y Total available-for appropriation Aggregate of amounts transferred to reserves Accumulated funds at the end of the financial year
9,029,562
9,039,661
8,982,502
(122,500)
3
9.029,562
8,982,502
8,917.161
9,076,175 .
TOTAL ASSETS
(122,500)
CURRENT LIABILITIES Creditors and Borrowings Provisions TOTAL CURRENT LIABILITIES
/ 8,947,675
The Company^
1992
The Company
Consolidated 1992
Consolidated 1993
8 9
534.650 .
16,175 . 887,212
1,160,000
1,232,500
1,160,000
1,232,500
8,115,072
7,607,926
8,115,072
7,607,926
229,482
237.053
214,219
219,015
9.504.554
9.077.479
9,489,291
9,059,441
10,622,317
10,514,160
10,023,941
9,946,653
644.885 585.282
688,150
450,613
471.288
504.411
365,634
302,426
1,230,167
1,192,561
816,247
773.714
I
• I
The profit and loss accounts are to be read in conjunction with the attached notes to and forming part of the financial statements set out on pages 14 to 25.
\
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.8
\
1
■Ift
Note
Consolidated 1993 $■
NON CURRENT LIABILITIES Creditors and Borrowings TOTAL NON CURRENT LIABILITIES
8
137,396
The Company 1992
1992
1993
$
$
$
137,396
179,174 179,174
TOTAL LIABILITIES
1,367,563
1,371,735
816,247
773,714
NET ASSETS
9,254,754
9,142,425
9.207,694
9,172,939
9,029,562
8,917,161
8,982,502
225.192 9,254,754
225,264
225.192
8,947,675 225,264
9,142.425
9,207,694
9.172,939
MEMBERSHIP FUNDS Accumulated Funds Reserves TOTAL MEMBERSHIP FUNDS
3
The balance sheets are to be read in conjunction with the attached notes to and forming part of the financial statements set out on pages 14 to 25.
■)
' The Murdoch Institute for Research into Birth Defects Limited and its controlled entities A.CN. 006 566 972
I
Statements of Cash Flows for the year eroded 31 December 1993
CASHFLOWS FROM OPERATING ACTIVITIES Payments to suppliers and employees Government Grants received Donations received Other receipts Interest received Patient Fees received
.• *.
The Company
Consolidated 1993
1992
1993
1992
Inflows (Outflows)
Inflows (Outflows)
Inflows (Outflows)
Inflows (Outflows)
$
$
$
$
(5,642,054)
(5,908,604)
(3,621,479)
3,236.609
3.435.276
1,701,209
922,048
984.858
922,048
356,117
320,822
356.117
314.215
(662.145)
(853.433)
NET CASH PROVIDED/(USED) BY OPERATING ACTIVITIES
(642,105)
(893.497)
1992
Inflows
Inflows
(Outflows)
$
$
16,628
1,606,119
210,953
1,606,119
NET INCREASE IN CASH HELD
(645,517)
752,686
(431.152)
.712.622
CASH AT THE BEGINNING OF THE REPORTING PERIOD
1.131.679
378.993
775.794
63.172
CASH AT THE END OF THE REPORTING PERIOD (NOTE io(l))
486.162
1.131.679
344,642 .
775.794
NET CASH PROVIDED BY INVESTING ACTIVITIES
(4.044.404) 1.845.227 984.858 320.822
The Company1993
(Outflows)
c.
9.657
455.478
. Consolidated . , 1992 Inflows Inflows 1993
(Outflows)
(Outflows) "
$
(Note lo(ii)) The statements of cash flows are to be read in conjunction with the attached notes to and forming part of the financial statements set out on pages 14 to 25.
CASH FLOWS FROM INVESTING ACTIVITIES Interest received Dividends received Proceeds on sale of investments Payment for investments Payment for property, plant and equipment
. ;
683,773
511.246
683,773
506,226
169,786
242,604
169,786
242,604
7.550,970
9.255.291
7.550.970
(8,082,714)
(8,385,830)
(7.883,369)
9.255.291 , (8.385.830)
(132,660)
(189,719)
(132,660)
(189,719)
s
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The Murdoch Institute for Research into Birth Defects Limited and its controlled entities A.C.N. 006 566 972
V-
Notes to and Forming Part of the Financial Statements
Principles of Consolidation
for the year ended 31 December 1993
The consolidated financial statements combine the financial statements of the Murdoch Institute for Research into Birth Defects Limited, being the chief entity and its controlled entity Victorian Clinical Genetics Services Limited for the twelve months ended 31 December 1993.
1. STATEMENT OF SIGNIFICANT ACCOUNTING POLICIES
All balances and transactions between the chief entity and the controlled entity have been eliminated..
The''Significant policies which have been adopted in the preparation of these financial statements are:
Investments Basis of Preparation
investments are stated at the iower of cost or recoverable amount. Dividends are brought to account as received.
The financiai statements have been drawn up in accordance with applicable Accounting Standards, the Corporations Law and Schedule 5 to the Corporations Regulations. They have been prepared on the basis of historical costs and do not take into account changing money values or, except where stated, current valuations of non-current assets.
Property, Plant and Equipment
The accounting policies have been consistently applied by the entities in the economic entity and are consistent with those of the previous year..
items of property, plant and equipment are recorded at cost. The depreciable amounts of all items of property, plant and equipment are depreciated over their useful lives commencing from the time the asset is held ready for use. The , straight line method of depreciation is used.
The carrying amounts of all non-current assets are reviewed at least annually to determine whether they are in - excess of their recoverable amount at balance date. If the carrying amount of a non-current asset exceeds the recoverable amount, the asset is written down to the lower value. In assessing recoverable amounts the relevant cash flows have not been discounted to their present value.
Prepayments Items of expenditure having a benefit or relationship to more than one accounting period are amortised over the periods to which they relate.
The financial statements of the Company and its controlled entities reflect all entries affecting the Company and its controlled entities and include transactions that were specifically recorded in the bank accounts of the Company and its controlled entities.
Employee Entitlements The provision for employee entitlements relates to amounts expected to be paid to employees for long service leave and annual leave and is based on legal and contractual entitlements. Current wage rates are used in the calculation of the provisions.
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Income Tax
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The eompany and its controlled entities are exempt from income tax under section 23(e) of the Income Tax Assessment, Act 1936 ■ Consolidated 1993
2. OPERATING PROFIT
.
The Company 1992
$
1993
1992
$
$
1993
Operating Profit before Income tax has been determined after; (a)- ■ V
CREDITING,AS REVENUE Grants - NHMRC GrantsH& CS Grants - Other ,
mbtldtis '
'
■■ IntmeSt ■ ■ ■
•
•
Dividends Net Gain on sale of investments Possum/Ossum Sales. Income - Other
$
1.299.014
1,248,015
1,299,014
1.879.221
1.803.586
245.997
216,160 , . 295,063
140.725
295.063
-140,725
92 2-, 648
984,85-8
922,048
520.903
683.773
,506,226
169,786
242.60A
■i69,786
174,747
121.979
114,869 .
154.868
714,468
411.050
.
Proceeds from Sale of Investments Less: Cost of Investments NET GAIN SALE OF INVESTMENTS
1,248,015
y '
■
CHARGING AS EXPENSE ■ Salaries'& Wages Employee entitlements • Laboratory Consumables Refurbishment CoSts Repairs & Maintenance Travel . Clinical Research Patient Care Services Central Services & Administration Depreciation
Building Development Fund Social Work Fund BALANCE AT END OF YEAR
121,979
,114,869
'154,868
258,990
120,342
OSSUM project
5.945.796-
3.832,402
2,460,985
2.562,098
2,460.985
.77,624 .
15.571
•59.961
13,684
596.731
545.059
596,731
,545.059
■
.
9.255.291
7.376,223
9.133.312
174.747
121,979
-174.747
121.979
222,50b 2,692
222.500
222,500 2,692
222,500
2.764
215.192
225.264
225,192
225.264
7.550.970 .
7.376.223
9.255.291 _ ,
9.133.312
2,764
2,764
2,764 -
2.764
(72) 2,692
2,764 ■ 2,764
18,898 69.523
53.252
69.523
103.697
67.123
103.697
67.123
3.690
7.193
3.090
7,193
, 1,805.437
1,589.238
486,128
473.110
, 286,198
140.232 -
89,861
137.457 77.306
263,050 87,086 258.542
3.797,575
3.873.358
77,306
$
:
2,562,098
,53.252
5,823,380
-1992
$
7,550,970
MOVEMENTS IN RESERVES-SOCIAL WORK FUND Balance at beginning of year 2,764 Transfer to revenue (72) BALANCE AT END OF YEAR' 2,692
4,067^662
5,935.781
18,898
i9'93
$
683,773 '
s; .
(b)
■
1992
3. RESERVES
■ 984.^58 ■
174,747 ,
, the Company
Consolidated
258.542 5.677,318
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7m
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4. REGEIVABLES
3,292,079
3.570,542
3.292,079
3.570,542
2,880,874
3.570.542 ' 3,570,542 2,234,344
2,880,874
2,234,344
TOTAL NON-CURRENT INVESTMENTS
8,115,072
7,607,926
8,115,072 -
7,607,926
TOTAL INVESTMENTS
8,731,066
8,495,861
8,395.526
8,244.767
market value of listed INVESTMENTS
3.381.939
2.943.984
3,046.399
2,692,390
13.848
16,175
13.848
16,175
523,499
390,839
495.748
363,088
294,017
153,786
281,529
144,073
229,482
237,053
214,219
219,015
1993
$
$
$
$
$
332,733
216,327
50,000 40,485
85,675
Z2,500 288,827
’
85,675 176,160
22,743 72,500
-Unlisted
1,232,500
1,160,000
3,292,079 3,292,079
95,243
Interest in Trusts 1,160,000
I
Government Bonds - Listed on a prescribed stock exchange
\
1,232,50b
5. INVESTMENTS AT COST
! !
99.308 100,038 416,648
887.935
280,454
636,841
615,994
887,935
280,454
636,841.
6. INVENTORIES Raw Materials & Stores - at cost
NON CURRENT
I
$
1992
418,408
Shares - Listed oh a prescribed stock exchange - Unlisted
$
1993
CURRENT Amount Owing by Controlled Entities
CURRENT Bank'Bill Bond ■ Short Term Deposit TOTAL CURRENT INVESTMENTS
1992
1992
$
NON CURRENT Prepayments
1993
1992
1993
- Debtors Prepayments
The Company
Consolidated
The Company
Consolidated
7. PLANT & EQUIPMENT 1,942,119
1,942,119
1,803,040
1,803,040
1,942,119
1,942,119
Plant 8i Equipment -at cost Accumulated Depreciation
1,803,040
TOTAL
1,803,040
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Consolidated 1993
Consolidated'
The Company 1992
1993
1992
$
$
$
8. CREDITORS & BORROWINGS CURRENT Royal Children’s Hospital Sundry Creditors TOTAL
The Company
1993
1992
1993
.1992
$
$
$
$
138,953
10. NOTES TO THE STATEMENTS OF CASH FLOWS 504,711
547,202
282,132
140.174
140.948
168,481 ■
122,945
644.885
688,150
450.613
471.288
348,343
NOTE: Royal Children's Hospital This is a suspense account which is used for payments to creditors. The Hospital pays the creditors on behalf of the Institute,and the Institute . subsequently reimburses the Hospital. NON-CURRENT Special Purpose Funds
Note
137.396
(i) RECONCILIATION OF CASH. For the purposes of the Statements of Cash Flows, the entity considers cash to include cash on hand and at bank and short term deposits at call. Cash as at the end of the reporting period as shown in the Statements of Cash Flows is reconciled to the related items in the Balance Sheets as follows: CASH SHORT TERM DEPOSIT
179.174
243.744
64,188
416,649
887.935
280,454
636,841
486,162
1.131.679
344.642
775.794
(10 RECONCILIATION OF NET CASH PROVIDED BY OPERATING ACTIVITIES TO OPERATING PROFIT AFTER INCOME TAX. Operating Profit after income tax 112,401
268,478
34.827
194.304
212,731
162,361
209,956
159.586
100,975
287,937
63,208
153.968
(174.747)
(121,979)
(174.747)
, (121,979)
(169,786)
(242,604)
(169,786)
(242,604)
(511,246)
(683,773)
(506,226)
(683,773)
69.513
5
•
9. PROVISIONS CURRENT Annual Leave Long Service Leave TOTAL
198,696
190,692
118.934
106,471.
386,586
313.719 504.411
246,700
195.955
365.63'4
302,426
585,282
Add/(Less) Non Cash Items Depreciation & Amortisation Amounts set aside to Provisions Add/(Less) Items classified as Investing Activities Gain on sales of investments Dividends received Interest received
.21
li
1993
1992
1993
$
$
$
69,250
(80,917)
Add/(Less) Changes in Assets & Liabilities (lncrease)/Decrease in Debtors (179,581) (lncrease)/Decrease in Grant Receivable (increase)/Decrease in inventory 2.327
Consolidated,
The Company
Consolidated Note
$
$
$
112,187
78,949
112,187
78.949
Number of Directors of the holding company whose total income from the Company or related bodies corporate falls within the following bands:
No.
No.
No,
Na
$0 - $9,999
17
16
17
16
$10,000 - $19,999 $70,000 - $79,999 $90,000 - $99,999 The individual remuneration received by the seventeen Directors in the band $0 - $9,999 was nil.
1
45,743 Total income received or receivabie by the Directors of the Company from the Company or related bodies corporate excluding amounts inciuded under
2,623 1,656
• 2.327
■
(35,043)
(20,675)
(346,103)
85,989
85.989
lncrease/(Decrease) in Provisions increase/(Decrease) in Reserve NET CASH PROViDED BY
(20,104)
(272,366)
(140,284)
OPERATiNG ACTiViTiES
(662,145)
(72)
1,656
retirement payments.
(72)
(853.433)
(642.105)
(893.497)
11,REMUNERATION OF AUDITORS Amounts received or due and receivable by the Auditors: for Auditing the accounts. For other Services TOTAL
$
■
)2. DIRECTORS' REMUNERATION
(411,005)
increase/(Decrease) in Creditors increase/CDecrease) in Grants
1992
1992 $
- .The Company 1992 1993
1993
13,000
13,000 ^
5.000
5,000
13,000
13,000
5>ooo
5>o6o
1
V.
1 1
1
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I Directors of the holding company in office at any time during the year: Professor J.A. Angus Dr. G.L. Barnes Mrs. J. Calvert-Jones Dr. R.G.H. Cotton Mr. L.G. Cox Professor D.M. Danks
Mr. I. Davies Mr. |. Fitzgerald Mr. P. Griffin Mr. J.S. Guest Mr. W.H. Hodgson Mrs. I. McFarling
14. LIABILITY OF MEMBERS
Professor P.D. Phelan Professor A.J. Pittard Professor G.B. Ryan
members of the company is 80.
Mrs. C. Searby Mr. N. Walford Mr. A.S. Murdoch •
15. RELATED PARTY DISCLOSURES
In accordance with the Articles of Association, in the event of the company being wound up, members may be called upon to make a subscription not exceeding ten dollars. As at the 31 December 1993 the number of
Dr. P.M. Gray During the year the Company was owed $50,000 by its controlled entity Victorian Clinical Genetics Services Limited. No interest is charged and the amount is repayable at the discretion of the Company.
Dr. P.M. Gray, Mrs C. Searby and Mr. N.. Walford retired during the year.
Consolidated 1993 1992
$
$
The Company 1992
$
$.
13. SUPERANNUATION BENEFITS
12,173
The Company and its controlled entities contribute various percentages of employees’ gross salaries to an employee superannuation fund. Employee contributions are based on various percentages of their gross salaries. All employees are entitled to join the fund. Fund members are entitled to benefits on retirement, disability or death. The fund provides defined benefits based on years of service and final average salary. The Company and other controlled entities are under no legal obligation to make up any shortfall in the fund’s assets to meet payments due
Superannuation contributions paid in respect of Directors The Directors believe that the provision of full particulars would be unreasonable.
16. SUPERANNUATION COMMITMENTS
1993
12,035
12.173
12,035
to employees. An actuarial assessment of the fund as at 31 December 1991 was carried out by Mr. C J White FIA, FIAA. The assets of the fund are sufficient to meet all benefits payable in the event of the fund’s termination, or the voluntary or compulsory termination of employment of each employee of the Company and other controlled entities.
}
The Murdoch Institute for Research into Birth Defects Limited
I
and its controlled entities <_ •
•r
A.C.N. 006 566 972
1 }
• >
Statement by Directors
INDEPENDENT AUDITORS' REPORT TO THE MEMBERS OF THE MURDOCH INSTITUTE FOR RESEARCH iNTO BIRTH DEFECTS LIMITED
f ■i
i.ln the opinion of the Directors of the Murdoch institute for Research into Birth-Defects Limited:
SCOPE
(a) the financial statements set out on pages 8 to 25 are drawn up so as to give a true and-fair view ■ ' of the results and cash flows for the financial year ended 31 December 1993, and the state of affairs
We have audited the financiai statements of The Murdoch Institute for Research into Birth Defects Limited for the financial year ended 31 December 1993, consisting ofthe profit and' loss accounts, balance sheets, statements of cash flows, accompanying notes, and statement by directors set out on pages 8 to 26. The financial statements comprise the accounts ofthe Company and the consolidated accounts ofthe economic entity, being the Company and its controlled entities. The Company’s directors are responsible for the preparation and presentation ofthe
at 31 December 1993, of the Company and the economic entity; ■ , • (b) the consolidated accounts have been made out in accorda‘nce with Divisions 4A and '4B of Part 3:6 of the Corporations Law; and (c) at the date of this statement, there are reasonabie grounds to believe that the company>iii be ebie' to pay its debts as and when they fali due.
financial statemehts and the information they contain. We have conducted an independent audit of these financial statements in order to express an opinion on them to the members of the company.
2. The financial statements have been made out in accordance with applicable Australian Accounting Standards.
Our audit has been conducted in accordance with Australian Auditing Standards to provide reasonable assurance as to whether the financial statements are free of material misstatement. Our procedures included examination,,bn a test basis, of evidence supporting the amounts and other disclosures in the financial statements, and the evaluation of accounting,policies and significant accounting estimates. These procedures have been undertaken'to form an opinion as to whether, in all material respects, the financial statements are presented fairly in accordance with Australian accounting standards and statutory requirements so as to present a view which is consistent with our understanding ofthe company’s and the economic entity’s financial position and the results of their operations.
i'
Dated at Melbourne this i3th day of April 1994.
1
' Signed in .accordance with a resolution of the Directors;
. The audit opinion expressed in this report has been formed on the above basis.
Audit Opinion In our opinion, the financial statements of The Murdoch Institute for Research into Birth Defects Limited are property drawn up: ■ . . (a) so as to give a true and fair view of:
LAURENCE G. COX
: Directors
'
i) the state of affairs of the Company and the economic entity at 31 December 1993 and the results and cash flows ofthe Company and the economic entity for the financial year ended on that date; and
'.'i
ii) the other matters required by Divisions 4, 4A and'4B of Part 3.6 ofthe Corporations Law to be dealt with in the financial statements;
.5
1
.1
(b) in^accordance with the provisions of the Corporations Law; and (c) in accordance with applicable Australian Accounting Standards. Dated at Melbourne this 13th day of April 1994.
KPMG Peat Marwick Chartered Accountants
R. Douglas - Partner y
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The Murdoch Institute for Research into Birth Defects
Design: Nikki Flood, Seldon Hunt, Swinburne Design Centre
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