Issuu

1995 AMNUAL R€PORT

The Murdoch Institute for Research into Birth Defects

THG IMCIDGMCe Ol^ BIRTH HAMDICAP 19 3 Ih 100. THG C09T IIT TGRNT OF P€R90hAL AMD FAMILY TUFFGRIMG I9 ehORMOU9. THe C09T TO AUSTRALIA Ih T€RM9 OF GDUCATlOh AND HGALTH PROVJI9IOh I9 TQ BILLION PGR ANNUM.

.IJ

J ■S

s ■f

= o-

m MURDOCH IMSTITUTG is an )endent research organisation, a )rising 100 dedicated staff devoted SgP! vancing knowledge about genetic diseases amd birth defects.

-■i

OUR AIM is to help every child to be born healthy and with normal abilities. •v

Page i

OUR CHALLeMG€9 To find the best ways to screen for inherited diseases. "Tb conduct quality research of an international standard. Tb meet the needs of those who come to us for advice. Tb develop the most effective methods of counselling. Tb move towards treatment for genetic diseases.

WG ARG. T

one of Australia's top research centres.

'S'

a World Health Organisation reference centre for human genetics, the premier training centre for clinical geneticists in Australia, the screening centre for all Victorian newborn babies. an international model for combined clinical and research efforts.

'!!'

the originator of the POSSUM/OSSUM computer system for the diagnosis of birth defect syndromes, which is now used by specialists in over 50 countries. the Australasian screening centre for genetic diseases of cell energy.

'S'

OUR RGS'GARCH 'S'

Identification of new methods of detecting faults in genes. The body’s use of copper, and the genetic diseases causing copper deficiency (Menkes disease) and copper toxicity (Wilson's disease). The structure and function of the human chromosome; how chromosomes divide, and why this can go wrong (Down syndrome). The understanding, diagnosis and treatment of conditions affecting brain and muscle function in both childhood and adult life, including mitochondrial and metabolic disorders (such as phenylketonuria).

Page ii

Understanding embryo development and how it contributes to birth abnormalities of form (facial clefting, spina bifida and great vessel/cardiac defects) and cell differentiation (paediatric cancers like neuroblastomas and leukaemias).

Somatic gene therapy: how to treat inherited diseases in better and more natural ways by using normal copies of the genes that do not work properly in ataxias, cystic fibrosis and other genetic disorders.

The genetics of brain and nerve diseases: how they occur in some families, what happens to the genes themselves, and developing new ways to treat them.

The safety of drugs in pregnancy.

mok DOI10k9 TO TH€ moocH mme K)UMDGR9 - DOMOR9 OF niLLIOM OR nORC 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

OUR STAFF We are a recognised centre of excellence in genetics and have attracted to our staff many world class doctors, scientists, counsellors, nurses, administrators, and scholars.

Repco Corporation Limited (Ariadne) The Broken Hill Pty Ltd The Friends of the Murdoch Institute The Ian Potter Foundation The late Mr Clive Roxburgh The late Mrs L.B. Quayle The Morris Family Tust The News Corporation Limited The Percy Baxter Charitable Trust The Sidney Myer Fund

CORPORATG 9P0M90R GROUP GORPORATIONS- UMDGRTAkIMG S-UB^TAMTIAL FUTURG SUPPORT The Broken Hill Pty Ltd National Australia Bank Limited

HOW CAN YOU HELP?

BGMGFAQORS - DOMORS' OF tQSO.OOO OR MORC

You can help the Murdoch Institute reach its ^oal of helpin^> every' child to he horn healthy and with normal abilities through: - hecfucsts - annual donations - pledges over several years • named scholarships or research §ifts For further information, please telephone or write to; Professor Boh Williamson Scientific Director The Murdoch Institute Royal Children’s Flospital Flcmin^ton Road, Parhvillc 3052 Telephone; (Ot) 3 9343 5045 Fax;; (Ol) 3 9348 1391 The Murdoch Institute is a registered charity. All donations over $2.00 are taxdeductible.

The Danks Trust The Miller Foundation The Helen M. Schutt Trust

I !

TRUSTGG^ - DOMOR9 OF tQ5,000 OR nORe Arthur Andersen Foundation Coles Myer Limited Professor D.M. Danks H. 6- L. Hecht Trust J.B. Were &- Son Charitable Foundation Mrs Joan Roxburgh Mrs M.L. Griffin National Australia Bank Limited Qantas

Mr GeofF Heely (right) Executive General Manager Finauice of BHP presents Professor Bob Williamson (centre) with a cheque supporting post doctoral fellow. Dr Sharon La Fontaine with Dr Julian Mercer (left).

Page iii

DOIi/)TIOfi9 TO TH€ nuliDOCH miTure 1995 $ 470,000.00 Dame Elisabeth Murdoch 250,000.00 The Jack Brockhoff Foundation 125,000.00 Cruden Investments Pty Ltd The Scobie dr Claire Mackinnon Trust 50,000.00 50,000.00 The Miller Foundation 30,000.00 Mrs Joan Roxburgh 10,000.00 The Morris Family Trust 5,000.00 Friends of the Murdoch JB Were dr Sons Charitable Fund 3,000.00 2,888.82 Bonorge Pty Ltd Roxburgh Foundation Uncle Bobs Club 2,624.28 2,030.00 Mr Laurie Cox 2,000.00 Mrs B Stefanowski (Petra Foundation) 2,000.00 Mr dr Mrs J.K. Little 2,000.00 Mr S. Seoulis 1,953.47 Professor D. Danks 1,816.79 Professor Bob Williamson 1,750.00 Mr Grant R Stephenson 1,104.00 Arthur Robinson Hedderwicks William Angliss (Vic) Charitable Fund 1,000.00 1,000.00 Mrs Neilma Ganter 1,000.00 Mr Geoffrey Heeley Little People's Association of Australia 1,000.00 1,000.00 l.C. Edney 900.00 McMullin Nominees Pty Ltd 559.91 Mr dr Mrs E. dr M. Richards 500.00 Mr dr Mrs S.F. dr M.G. Gooley

Page iv

MrW.H. Hodgson Mr &■ Mrs K.J. 6- P.l. Redman Australian Football League Dr N.M. Cass Mrs H.M. Collis Robyna Calisenthic College McKinnon Secondary College Mr Mrs W. Ritchie Mr &- Mrs K. Troon Fionnuala Hickland Dr R. Small Becton Corporation Mowbray College Val 6- Alan Walsh Rosanne Cunningham Mrs G.A. Grimwade Mr J.A. 6- Mrs L.B. Simpson Mr &- Mrs L.R. Mills Sarah Seymour Mr C. Ritchie Mr dr Mrs N.C. Albrow Mr dr Mrs J. dr G. Ginger In memory of Dorothy Tabone; Anonymous donor D.M. Frank Catholic Regional College ' St Albans Kealba Catholic Primary School Containers Packaging Pty Ltd Catholic Regional College Mr dr Mrs P.A. dr R. Buriani

500.00 350.00 300.00 200.00 200.00 180.00

comem^

145.00 100.00 100.00 100.00 100.00 100.00

58.00 50.00 50.00 50.00 50.00 50.00 30.00 20.00 20.00 5.00 624.00 502.00 500.00 100.00 100.00

25.00 20.00

The Institute thanks all those listed for their generous support

"Changes and Challenges"

page

A Tribute to Dr Flichard Cotton

page

Our Research during 1995

page

Olive Miller Protein Chemistry Laboratory

page

Trace Element Group

page 13

Chromosome Research Group

page 16

Mouse Model Unit

page 18

Disorders of Mitochondrial Energy Generation

page 19

Metabolic Research

page 22

Embryology Group

page 23

Victorian Clinical Genetics Service

page 25

Epidemiology Unit

page 27

DNA Diagnostic Laboratory

page 30

Metabolic Laboratory

page 32

Newborn Screening Laboratory

page 33

Cytogenetics Laboratory

page 34

Murdoch Institute Staff

page 37

Victoriam Clinical Genetics Service Staff

page 39

List of Publications

page 41

Finamcial Statements

page 48

Page V

rTH€ moocH imiTure

“CH/)I1G€9 m chmencer

raeitniHC Diueaoi!

their children, but we hope that it will give them life. What could be better than insert ing a normal copy of the cystic fibrosis gene to give breath to those with this crippling disease? Or helping an ataixia victim to retain the power to walk? Or using a cancer suppressor gene to treat a child with leukaemia? The path to gene therapy will be a long and arduous one, but it is one to which we are totally committed.

Professor Bob Williamson Mr Laurie Cox, Chairman of the Board & Professor Bob \A/illiamson, Director

BOARD or DIRCaOR^ Mr L.G. Cox, Chairman Professor J. Angus Dr G.L. Barnes Mrs J. Calvert-Jones Mrs L. Cattermole Mr I. Davies Mr J.A. Fitzgerald Mr P. Griffin Mr J.S. Guest Mr WH. Hodgson, Deputy Chairman Mrs A. McFarling Mrs J. Paterson Professor D.G. Penington Professor P.D. Phelan Professor G.W. Tegear Professor R. Williamson

1995 has been a year of change, as the Murdoch Institute has faced new challenges. These have been met together by the Board and the Institute staff; hence the joint introduction to this Annual Report. The greatest change has been the retire ment of the founder of the Institute, Professor David Danks, and the arrival of Bob Williamson as Director and "David Danks Professor of Medical Genetics” of the University of Melbourne. Professor Williamson and the Institute staff are committed to building on the excellent foundations Professor Danks laid down as a world pioneer in human genetics research of the highest international standard.

mmce conMirree Mr C.P. Abbott Mr L.G. Cox Mr D.T Craig Mr P.J. Griffin Mr G.E. Heeley Mr D.E. Meikeljohn Mr F.D. Ryan

yiaORIAN CUMICAL G€li€TIC5 SeWlCG Mr L.G. Cox, Chairman Dr G.L. Barnes Professor H. Ekert Mr J.S. Guest Mr WH. Hodgson Mr G.E. Heeley Dr L.J. Sheffield Professor R. Williamson Page 1

Today human genetics is changing. The emphasis is moving from understanding the basis of childhood diseases, however sophisticated the diagnostic and biochemical skills required may be, to the even more exciting prospect of attempting to apply this knowledge for treatment. The key to this, gene therapy, uses normal human genes to replace functions which a severely ill child does not have, whether through inheritance or by accident. Gene therapy will not change people's genetic inheritance which they transmit to

The second challenge we face is to educate our clinical and scientific colleagues, and indeed all thinking people, about the impli cations of the new genetics. In the past, clinical genetics was forced to focus on severe, rare diseases. Today, with new technology and techniques, we are able to study the genetic predispositions which lead to common but severe childhood diseaises such as asthma, epilepsy, and childhood cancers, and the ways in which genes and the environment interact to cause these conditions. It follows that a whole new group of doctors, nurses and patients need to understand “the new genetics”. Our challenge is to train the medical and lay communities to cope with, and indeed welcome, the continual exciting changes resulting from our new understanding of human genetics. This year, in addition to our traditional role

Mr Laurie Cox

Professor Bob Williamson

Page 2

^ T

Another major change was the departure of Dr in teaching medical and science undergraduates, Dick Cotton to head the new Mutation Detection we have conducted three highly successful work Centre at St Vincent's Institute of Medical shops for cancer doctors and nurses. In these Research. We congratulate Dick on courses, we the exciting opportunities this change emphasise the NEUROGENETICS will afford him, and thank him most counselling sincerely for his outstanding aspects of The Murdoch Institute is moving contribution to the Institute over a oncology genetics, increasingly into the field of hrain and period of more than 25 years. Dick because we nerve diseases, as more is discovered will continue to collaborate with us in believe it is about the genetic causes of many his capacity as a Murdoch Institute important that ditions, and the neurological con Honorary Senior Fellow (we have families under general problems of mental handicap. included a tribute to Dick on page 6). stand what the To further information exchange findings will mean between our staff and neurologists, we The refurbishment of the northwest to them, to enable held three information evenings in the wing is now complete, and was them to make latter half of 1995 to discuss issues formally opened by Dame Elisabeth decisions which such as ataxia, dementia and trinucleiMurdoch on April 24 1996. Barry often have an otide repeat diseases, and to canvass Holt and his team have done a ethical dimension. the circulation of neurogenetic tremendous job transforming what The Victorian updates. At present we are was previously a jumble of play Clinical Genetics considering various means of rooms, storage areas and junk rooms Service has done maintaining contact with our into a first rate laboratory, training a commendable colleagues in the neurology facility and research area. The job in organising community, through print and launch of the new wing coincided these workshops, electronic media as well as evening with the inauguraJ Murdoch Institute which were meetings. Open Day. Many of our supporters, widely praiised. donors, families, patients and The VCGS staff collaborators were able to participate are sJso running a in this event, which presented the Institute's Graduate Diploma course in Genetic Counselling, clinical and scientific work in an informal, to meet the ever-increasing demand for properly educational manner to appeal to our wide range qualified counsellors. of friends. We look forward to seeing you at the next Open Day. As a result of the rapid changes to genetics, concerns about the ethical use of our discoveries Finally, we wish to emphasise that the Murdoch have, naturally, arisen both in the scientific and Institute has always been, and will continue to the broader community. The Murdoch Institute be, a centre for excellence in scientific research. is committed to the ethical use of scientific Maintaining excellence is not always easy; it knowledge to benefit the community, to protect requires resources above and beyond the the rights of the individual and to prevent harm average, but we believe that it is vitally important from befalling any member of our society. to aam to achieve the highest international Several of the staff are already teachers of ethics, standards of scientific endeavour. The prizes to and we particularly welcome a generous offer to humanity will be profound, and the Murdoch help us to establish a Lectureship in the Ethics of Institute is well-positioned to play a major pairt in Human Genetics, based at the Murdoch, for the the exciting period to come. We will continue to University of Melbourne.

Page 3

train the best scientists and doctors. We want every young person graduating in human genetics to know that Australia has first-rate teams to aspire to. We will strive to ensure that every medical student, every doctor, knows of the value of the new genetic approaches and how to use them. We want to ensure that children who are ill with birth defects and inherited diseases, and their families, will know that there is a place dedicated to developing new strategies for prevention and treatment of their conditions. This is our mission for the coming decade. The Murdoch Institute is changing, and reaching to meet the challenges of a changing world. We need your support more than ever to achieve our goals.

Laurie Cox Chairman

Bob Williamson Director

Page 4

Generic coun^eiunG Ano CAnCCR WORkSHOP?

pon GRADUATe DIPLONA Ih CeMCnC COUliSGLUNG

In 1995. the VCGS initiated a series of weekend Genetic Counselling and Cancer Workshops, in response to a growing demand from professionals for both current scientific knowledge in cancer genetics, and the counselling issues and techniques involved in communicating this knowledge to client groups. The workshops included updates on molecular genetics and cancer, pedigree analysis, gene mapping, mutation detection, breast and colon cancer genes, genetic counselling and family analysis, and ethical issues.

The post graduate diploma course in genetic counselling is being offered for the first time to ten fijlftime and two part-time students in 1996.

The response from participants has been enthusiastic. The workshops, with a maximum enrolment of 30, were interactive, with small groups for discussion. Two videos showing genetic counselling sessions were made by VCGS staff as teaching aides, and a booklet of papers presented is planned. Further workshops will be held throughout 1996. Our thanks to the Anti-Cancer Council of Victoria for its contribution towards the funding of the workshops. Genetic Counselling Course 1996

Back

Middle

Front

Les Sheffield, Anne Glynn, Helen Varney, Cathryn Vaux, Joanne Tilkeridis Margaret Sahhar, Judith Carrigan, Alison Boyd, Lisa Gomes, Helen Upton, Pamela Hutchins Edi Sheffield, Caroline Bowditch, Mary-Anne Aitken

Page 5 !!!!!

The course, convened by Dr Leslie Sheffield and Mrs Margaret Sahhar from the VCGS, is offered by the University of Melbourne (Faculty of Medicine) through the Murdoch Institute. The diploma fulfils Part One of a two part process of qualification and certification as a genetic counsellor in Australia. Teaching staff are drawn from the scientific, medical genetics, social work and genetic counselling staff of the Institute. The core subjects are genetics, counselling, and a four week supervised placement with a recognised genetics unit. Counselling subjects are offered in four modules (two per semester), and include training in counselling skills, interviewing in genetic counselling and genetic counselling in the community.

TmUT€ TO Dk kICHm COTTOli It is with regret that the Murdoch Institute farewells Dr Richard (Dick) Cotton as the Deputy Director of the Institute and Head of the Olive Miller Protein Laboratory. Dr Cotton has received independent funding and is moving his research group to St Vincent's Institute of Medical Research, where he will continue his work into mutation detection and cancer genetics.

made another significant breakthrough when he developed a chemical method to detect all mutations in DNA. This method and its derivative enzyme method is now considered one of the most promising techniques for identifying mutant genes. We wish him continued success as he moves to a new phase in a most distinguished career in medicail research, and thank him for all his contributions to the Murdoch Institute. We look forward to continuing fruitful collaboration with Dr Cotton, who has accepted an appointment as an Honorary Fellow of the Institute.

Working alongside Professor David Danks, Dr Cotton played a major role in the establishment of the Murdoch Institute and its development from the Genetics Research Unit of the Royal Children's Hospital Research Foundation. He joined the Research Foundation in 1968 supported by the Elizabeth Sweet Fellowship of the University of Melbourne to work on inborn errors of metabolism. Soon after he airrived, he began to study phenylalanine hydroxy lase, the enzyme at fault in phenylketonuria. Over the subsequent years he hais made major contributions to the understanding of this most complex of enzymes amd he is acknowledged ais one of the world leaders in this subject. Dr Cotton's other most significant research has been in the discovery of the production of monoclonal antibodies, during his postdoctoral work at Cambridge University. This discovery led on to the work for which Cesar Milstein aind his colleagues received a Nobel Prize. In 1986/1987, Dr Cotton

Dr Richard Cotton

Page 6

Y’

roun fie^mcH oumc 1995 Human genetics has entered a very exciting era. Continual advances in the field have opened up new horizons previously only dreamt of The use of moleculair genetic techniques to study common multifactoriaLl diseaLses as well as catastrophic single gene disorders, with a view to intervention by screening, prevention and treatment, has become paramount. The development of gene therapy will be a particular focus at the Murdoch; not only does it have enormous potential for treating the children we see at the hospital, it will also weave together several strands of the Institute's research. What could be a better way of delivering the normal gene to treat Menkes disease or cystic fibrosis than to package it in a humam artificial chromo some? Working together, our reseao-ch staff are ideally positioned to make great advamces in pioneering genetic endeavours for the benefit of those in most need, children with serious disease. 1995 saw a change of policy from one of our main funding bodies, the Commonwealth Government's National Health and MedicaJ Research Council (NH&-MRC). Traditionally, the Murdoch has been one of five research institutes in Australia to receive "block grant" funding from the NH&-MRC, giving a one-line grant to cover work over a five year period. Although the inflexibility of the system was

Page 7

irksome, particularly to senior staff who were unable to compete with their opposite numbers in Universities and other Institutes, the guarantee of funding and the status of the award made up for these restrictions in large measure. This year, the NH&-MRC advised us that the block'grant funded institutes would now have to submit them selves to program-based assessments in competition with all other research groups, and that these assessments would then be considered as a whole to determine whether block funding would continue as before. Whilst this request has demanded a great deal of extra work, in the end it has proved an interesting and useful exercise. Although we must wait until mid-1996 to know the outcome of our application, I believe that the process has served to sharpen our vision for next five years, and to crystallise the ways in which we can best work together to achieve common obiectives. I also believe it will strengthen our arguments for a revi sion of the block grant system so that it becomes more flexible, gives more authority amd independence to senior scientists as well as to the Director, and yet retains the element of strategic planning which is so welcome. I agree with the Directors of other Institutes that the NH&-MRC must find ways to offer long term strategic resources to the best of Australian biomedical science, without fe&r or favour; it is more important

to fund the best than to have artificial "protected areas" or to sway funding to meet fashion in research. At the Murdoch Institute, we are pursuing a single theme: the advancement of human molec ular genetics in the quest to understand and treat genetic birth defects. Our key programs will include Human Chromosome Structure and Function (Dr Choo), Heavy Metal Transport and Genetics (Dr Mercer), Genes, Development and Dysmorphology (Dr Newgreen), and Analysis, Mapping and Somatic Gene Therapy for Neurogenetic and Mitochondrial Diseases (Drs Dahl, Forrest, Gardner, Thorburn and myself). To support these programs, two smaller support units will be formed; the Transgenic Mouse Model Group (Ms Fowler) and Linkage Analysis, Mutation Detection and Information Science. The Institute will also extend its research into the increaisingly important field of community genetics under Drs Halliday, Rogers and Williaimson. In addition, we hope to become the centre for studies of ethics in human genetics at the University of Melbourne's Faculty of Medicine.

Directorship such a pleasure. The Murdoch Institute has many strengths. Excellent staff, engaged in world-class research, striving together to achieve credible and meaningful goals. Close integration of research, clinical and pathology services under one roof fruitful collaborations within and outside the Institute, and a common commitment to excel lence. The continuation of these strengths represents our goal and our pledge to our colleagues, supporters and patients who read this annual report.

Professor Bob Williamson, April, 1996

May I add a personal note? It is never easy to take over the helm of an Institute which was led by a strong and internationally renowned Director from its inception. David Danks hais helped me in many ways, as have Anne Cronin and Barry Holt, all clinical and scientific staff of the Institute, and all its friends at the Royal Children's Hospital and elsewhere, who have become my friends. I thank you all for making my transition to the

Page 8

tf.

roud€ m€R pi^oTen cHenmv iMomopv R.G.H. Cotton, Group Leader; I.G. Jennings, Senior Research Assistant; Postdoctoral Scientist; R.Youil, Research Assistant; O. Horaitis, Research Assistant; M. Knight, PhD Student; S. Ramus, PhD Student; J. Babon, MSc Student; T. Gough, J. CrosS'Foletta, Editorial Assistant Human Mutation; Post'doctoral Scientist S. Hufton, (until May, 1995) The Protein Laboratory focuses on the biochemical genetics of conditions such as phenylketonuria (PKU) and its variants, in which certain key enzymes are inactive. Our research sums to determine the reasons why the enzymes are inactive, and to develop diagnostic methodology for these diseases. One of our major objectives has been to identify the structure ofphenylalanine hydroxylase (PAH), the enzyme defective in PKU. No enzyme of this class has ever had its structure defined. Enzymes related to PAH are crucial in the functioning of the nervous system, and the elucidation of their structure may lead to new drugs for neurological disease. Our other msun activity has been the development and refinement of cheap and efficient methods for detecting mutations in genes. We have already produced a

Page 9

useful chemicad cleavage method, and have recently succeeded in defining an improved method based on enzyme cleavage of mismatch. Such methods are becoming more important as continual advances are made in our understanding of the role of genetics in serious diseases common in the community. Mutation detection will become increasingly important in the diagnosis of conditions such as cancer. With Dick Cotton’s departure, there will be changes in the Olive Miller Protein Chemistry Laboratory, to ensure that it can meet the challenges of the future in collaboration with the Mutation Detection Centre which Dick will direct at the St Vincent's Institute of Medical Research. A detailed knowledge of mutations will be required before the Murdoch Institute can begin its attempts at gene therapy for ataxia, cystic fibrosis, epilepsy and other serious disezLses affecting children. We are pleased that the Olive Miller Laboratory, with its proud history and traditions, will still play the major role in defining the precise reasons why many different clinical symptoms and outcomes are seen for the same disease depending on the mutation; we need these data ais much as ever, and are ever grateful for the support of the Miller family in meeting this need.

found that the PAH genotype did not correlate with the intellectual phenotype. Several families contained affected siblings with the same genotype and different phenotypes. It was shown that the siblings with different phenotypes had the same diet in infancy and early childhood and therefore diet was not causing the changes in phenotype. We proposed that another gene must be modifying the effect of the genotype. Analysis of CA repeats on chromosome 12 of these siblings were used to show that the modifying gene was not closely linked to the PAH gene on chromosome 12. The tyrosine hydroxy' laise gene was chosen as a candidate gene for modifying the phenotype of PKU patients as this enzyme is expressed in the brain and can also convert phenylalanine to tyrosine. Testing the siblings for a common polymorphism in the tyrosine hydroxylase gene established that this is not the modifying gene.

Of these two proteins, only the phosphorylated form gave crystals large enough to be analysed and a full data set has been collected. However, a heavy metal derivative of this crystal, essential for the complete three dimensional structure determination, has yet to be found. More recently, expression of a new construct of PAH with a C'terminal deletion has resulted in crystals of both the phosphorylated and dephosphorylated forms. This should enable us to determine the structural changes caused by phosphorylation, one of our original aims.

9TRUaUR€-(^UNaiOM AMALV9I9 OF PAH

Rania Horaitis has completed the cloning and nucleic acid sequencing of the two pterin mimicking antibodies which we previously isolated and characterised. This has led to the identification of the amino acids sequences of the complementarity'determining regions on the antibody chains. Synthesis of peptides corresponding to these regions and subsequent PAH binding assays should allow identification of the critical mimicking peptide or peptides.

Structurc'function analysis of PAH has concentrated on the determination of the three' dimensional structure by X'ray crystallography in collaboration with Drs B. Kemp and M. Parker at St Vincent's Institute of Medical Research. Initial attempts involved purification of fuILlength PAH produced in a baculovirus expression system and subsequent production of pure phosphorylated and dephosphorylated PAH.

Simon Hufton hais mapped an important region in PAH involved in the formation of PAH tetramers, using the yeaist two hybrid system, which is an in vivo assay of protein'protein interaction. By introducing a mutation in an amino acid at the extreme C'terminus, interaction between PAH subunits was abolished, ie. PAH was transformed from a tetramer to a monomer.

PHenvutaoMuiiiA wu) PKU is caused by mutations in the pheny' lalanine hydroxylase (PAH) gene, and results in mental retardation if left untreated. In a study of 55 untreated PKU patients, we

from left to right I.G. Jennings, J. Babon, S. Ramus, R.G.H. Cotton, R.Youil, J. Cross-Foletta, O. Horaitis.

Page 10

ILLGGITIMATe TRAM^aiPTIOM

the NADH^mediated reduction of quinonoid dihydrobiopterin and is an essential component of the pteridin dependent aromatic amino acid hydroxylating system which ultimately produces neurotransmitters.

Illegitimate transcripts are mRNA from a tissue specific gene, in a tissue where it is not normally expressed. These transcripts, although present at a very low level, were successfully used to The aim of the DHPR project was to produce a identify mutations in the PAH gene in PKU genomic map of this gene. At this point three patients. Illegitimate transcripts were also used to clones have been isolated that cover the whole screen for mutations in the GTP cyclohydrolase gene. cDNA primers were used to gene in three sequence from exons into introns patients with Human Mutation using RTPCR of patients showing Segawa's disease deletions as a means of predicting (dopa responsive Human Mutation is now puhllsbin^ the location of intron/exon dystonia), and a its seventh volume of papers on broad boundaries. So far, six introns have patient with GTP aspects of mutation research. The been found and their boundaries cyclohydrolase editors, Dick Cotton and flai^ sequenced. deficiency. Kazazian (Philadelphia), with the

assistance ofJulie Cross Foletta and This project ailso included There are two the 55 worldwide communicating sequencing intron/exon boundaries forms of Segawa's editors, continue to review and select of patients to detect splicing diseaise. The hl^h {Quality papers for publication. mutations. One patient has been recessive condition The publishers, M^lley-Liss in iNew found to have a splicing mutation to is caused by York, have been sta^^ered by the this point. 1995 was spent trying to mutations in the (Quality and c(uantity of research in map the GC'rich 5' end of the gene tyrosine hydroxylase this field (290 manuscript submittals where a putative intron may exist gene, whilst the in 1995). They are presently and continuing to sequence genomic dominant one is deciding the most appropriate me thod DNA of patients suspected of having due to a mutation in of making part of the Journ al splicing mutations. To sequence the the GTP cyclohy^ available through the Internet. 5' end we have employed many drolase gene. As ■ strategies including subcloning, the dominant form extended PCR and altering sequence has incomplete ing conditions, none of which have been penetrance it is often difficult to establish which successful. In 1996, we aim to sequence this form of the disease a patient has from a family unknown region. history. A previously unpublished mutation was identified in the GTP cyclohydrolaise gene in one of the Segawa patients.

nUTATION Daecnotl AMD DATAOAtet

ceiiOMic mucTuce oa humam WHVDROPTeRIDIMe CeDUQA^C Dihydropteridine reductase (DHPR) deficiency is a rare, severe neurological disorder which results in death in the first years of life. DHPR catalyses

Page 11

The laboratory continued with its mutation detection work throughout 1995, and Dr Cotton completed his book. Mutation Detection, which is to be published by Oxford University Press in 1996. Numerous individuals nationally and internationally were helped in their projects on mutation detection.

Dr Cotton ran the third in a series of workshops entitled “Mutation Detection” in Visby, Sweden, in May 1995. The meeting was sponsored aind run by HUGO for the first time with major financial assistance from FEES. Susan Ramus and Sue Forrest attended from the Institute. Progress has been made in the field of mutation databases. Up'tO'date listings of mutations in genes are not available on the Internet at present. Dr Cotton, together with Drs McKusick and Scriver, has led efforts to eliminate this problem. Following an evening meeting at Visby, funds were obtained from the March of Dimes (USA) to host a 24'hour meeting in Minneapolis, organised by HUGO Europe. Database curators, journal editors, society representatives, publishers, and systems experts were invited to attend the meeting, which approved the formation of an Alliance of Database Curators and the formation of working parties covering software and content, nomenclature, central databases and the Alliance of Curators.

A new nuTATioM DcreaioM mcthod - enZYNG MI9riATCH CLGAViAGG 1995 began on a high note with the publication of our first report on a novel way to detect mutations in DNA, using the- enzyme T4 Endonuclease VII to detect and cleave at small deviations in DNA. Since this preliminary report, we have shown that “Enzyme Mismatch Cleavage" (EMC) method has a very high detection rate, having identified 81 out of 81 known mutations tested. The real test, however, came with the use of EMC for detecting unknown mutations. Using the EMC method, we searched a cohort of 84 different brain tumours for mutations in the p53 gene, which is commonly involved in many cancers. We found numerous mutations, including four which had not been described previously. Although the method is currently useful for

research work only, we have gained sufficient confidence in its use in our laboratory to provide diagnosis in special cases in various genes, including the p53 gene and the HCS gene, which is involved in the metabolic pathway. 1995 also saw the introduction of a significant modification to the conventional method. By converting the system from 'liquid' to 'solid' phase we have not only improved on the detectability of mutations, but we now have the capacity to increase our throughput many fold ' which is particularly important for diagnostics. We have also recently completed a major study to identify mutations within the breast cancer gene, BRCA1. This case control cancer genetic study is the first of its kind in Australia. The data collected will provide valuable information on the scope and prevalence of BRCA 1 mutations within the Australian female population. In addition, we have suggested a possible strategy for conducting the screening for mutations (particularly those for cancer). A study of this magnitude would not have been possible in our time frame if other existing mutation detection techniques had been used. In 1996, we plan to refine this method and to distribute the EMC method in kit form to accommodate the high level of interest it has generated both nationally and internationally.

The use of a solid phase to selec tively bind the DNA molecules m '

ftiff-I fIf. A

S' -■

>4 of(heteroduplexes) leads

Cleavage (EMC) technique.

This increase in the signal to noise ratio leads to results typified by those shown opposite in dupli cate, with a clear signal for each of 5 mutations, and should lead to the technique being more widely used throughout the

world.

Page 12

mc€ aeneiiT croup Croup Leader; BHP Postdoctoral Fellow; Research Officer; Andrew Grimes, Research Assistant; Stephen Firth, Research Assistant; Paul Lockhart, Research Assistaint; Jenny Paynter, Research Officer; Sharon Horton, Loreta Ambrosini, PhD Student; PhD Student; Michael Petris, Michael Theophilos, Helen M. Schutt PhD Student; Alice Franek, Honours Student; Senior Associate, Jim Camakaris, University of Melbourne. Julian F. B. Mercer Sharon La Fontaine,

encoded by the Menkes gene (MNK). By using an antibody raised against the N-terminal portion of the protein, which had been obtained by expression in E.coli, we have been investigating the intracellular location of the protein. Over the last few years we have achieved a major breakthrough with the isolation of the gene affected in Menkes disease, and this has led to further exciting studies which are beginning to unravel the complex process of copper transport. Now we are able to study the mutations which cause the disease in affected families, and this may lead to better diagnosis and treatment. We are also investigating ways of correcting the defect in Menkes disease by putting a normal gene into cells from patients. Over the next few years we intend to use the mouse models of both Menkes and Wilson diseases to study the possibility ofgene correction in whole animals. This process will enable us to study the normal function of the gene and may lead ultimately to gene therapy

This year has seen exciting progress in our understanding of the role of the protein

The Trace Elements Group studies the manner in which the essential trace element, copper, is carried i; around the body and within / cells. Although copper is vital for life, it is also potentially toxic, and must be transported carefully to avoid damage to cells. Our particular interest is to elucidate the molecular basis of the genetic diseases which disrupt copper transport. The two main diseases we study are Menkes and Wilson diseases. from left to right Menkes is a genetic copper Paul Lockhaut, Jenny Paynter, Loreta Ambrosini, back deficiency, whilst Wilson is a Stephen Firth, copper toxicosis disease. Julian F. B. Mercer, Andrew Grimes, front Sharon La Fontaine, Sharon Horton.

Page 13

This work was facilitated by the use of the copper resistant Chinese Hamster ovary cells developed in Jim Camaikaris' lab (see Collaboration with the University of Melbourne insert box). In collaboration with that group we showed that the copper resistance was achieved by the amplification of the Menkes gene, and this W21S accompanied by a similar increase in the amount of MNK. The resistant cells were able to efflux copper more efficiently. Thus MNK is clearly involved in the efflux of copper from the cell, and increasing the amount of this protein allows the cell to maintain a lower intracellular copper concentration in the face of high extra' cellular copper. The gene amplification was easily observed by FISH analysis, carried out by our collaborator, Tom Clover in Michigan. Since the amount of protein was increased in the resistant cells, the determination of its intracellular location was much easier than in the parental CHO cells which express MNK at very low levels. We found that normally the protein is located primarily in the transGolgi network. This location is consistent with the need for MNK to deliver copper into the Golgi for incorporation into the copper'dependent enzyme, lysyl oxidase. The most surprising result was that in the presence of high copper, the intracellular location alters, with the protein being dispersed through' out the cell. In collaboration with Janetta Culvenor of the Department of Pathology, University of Melbourne, we have EM evidence that the protein relocates to the plasma membrane. This is an important finding and it helps to explain how the cell can maintain copper levels in a safe range. Copper is only effluxed when the levels become excessive, below these dangerous levels.

the efflux mechanism is not activated. We are proposing that copper is transported in MNK' containing vesicles, which fuse with the plasma membrane from where copper is effluxed. When copper levels are lowered, MNK is returned to the transGolgi network. Cu2 MD Cu2+ lysyl oxidase /

Cu+GSH

MD >

.U,+^ 6-

Golgi

SOD

Cu+-MT

Pathway of copper in fibroblasts and the location of the Menkes disease protein MNK. The block to copper transport in Menkes disease is indicated by the red arrows.

The discovery of the copper'induced movement of MNK and associated vesicles raises questions about the nature of the signals involved. We are planning to investigate the molecular basis of the signalling process by in vitro mutagenesis of a construct which expresses MNK. We have been working on the production of the construct for some time now, and it has proved very difficult because of the instability of the Menkes cDNA in E.coli. We are confident that the major obstacles have been overcome, and anticipate answering a number of interesting questions concerning the targetting signals and mechanism of action of the Menkes protein. The cDNA construct of the Menkes cDNA will also be transfected into cells from Menkes patients and from mouse models of Menkes disease to determine if the copper efflux can be corrected by the construct. These experiments will lead to introduction of the cDNA into trans' genic mice. These studies will give an idea of the feasibility of gene therapy for Menkes disease and allow more detailed study of the action of the gene in the whole animail.

Page 14

days after birth, dies before 10 days gestation. Work is continuing on the mouse models of The heterozygote brindled females, which are Menkes disease. The most severely affected quite healthy on a normal genetic background, mutant of the mottled locus (the murine are affected to varying degrees in the absence of homologue of the Menkes gene), is termed MT Some are relatively unaffected, but most are dappled. Previously we published the data runty and some die in fetal demonstrating a life. We suspect that the Southern blot abnormal Collaboration with the UMurersity of copper which accumulates ity in this mutant. This Melbourne. in cells in which the differed from the report brindled mutation is from Jane Gitschier's Dr Jim Gatnakari,s, our Senior Associate, expressed, is toxic in the group in San Francisco. developing systems for absence of MT and bis ^roup are We have now directly studying copper uptake. compared DNA from The group is also “dappled” mice from the ATP dependent uptake of copper by vesicles investigating the copper two labs and this has been demonstrated in membrane prepa transport defect in a mouse confirms that they are rations from tbe copper resistant cell bne model of Wilson disease indeed different. We CUR3. Tbe advanta^ of tins cell line is (WD), the toxic milk have now shown that mouse (tx). We have tbat AINK bas been amplified by copper our "dappled” is actually selection and so tbe activity is inucb ea.sier isolated the Wilson disease another mutant of to mea.sure than in tbe parental cell line.s. gene homologue from the Mo9H, with a very normal mouse and the similar phenotype to that This system will be used to study tbe charac mutant. Sequence analysis reported for dappled. teristics of tbe copper transport by A1NK hats demonstrated a point We have shown that d indeed to provide tbe first bioebemical mutation in a highly an this mutant mouse dies proof tbat it is indeed a P-type ATPa.se. conserved region of the in late gestation, and the Vesicles from liver extracts are also being gene in the mutant. We defective foetuses see if^^ilson ATPase activity consider that it is likely to assesse d to observed suggest that be the causative mutation, can be measured by ibis system. the death may be and this conclusion is related to abnormal Copper uptake is also being investigated in supported by the recent connective tissue. Work cultured mouse lympbocHe.s. An uptake sysmapping of the tx mutation is continuing on the tern bas been found which is not blocked by to the same chromosomal detailed characterisation protein synthesis inhibitors, but is energy location as the Wilson of the mutant in this dependent. Such a .system may function to homologue by other groups mouse. sequester copper in vesicles to minimise tbe [Reed, 1995, Rauch, copper tlepcndent free radical damage to 1995]. This shows that The role of metallothplasma membranes. the tx mouse is a true ionein (MT) in copper model of WD, and we can transport is being proceed with confidence to investigated by using use the mouse to study various treatment MT “knockout” mice. Of particular interest are strategies for this disease. the experiments in which the brindled mutation (the brindled mouse is very similar to patients with Menkes disease), has been bred into the MT minus mouse. The results are dramatic; the affected male, which normally survives to 15

Page 15

CHkONOfOne k€fmCH GROUP Andy Choo, Liz Earle, Kellie Tainton, Desiree du Sart, Paul Kalitsis, Damien Hudson, Helen Trowell, Michael Cancilla, John Martyn, Richard Saffery,

Group Leader; Scientific Officer; Research Assistant; PhD Scholar; PhD Scholar; PhD Scholar; Postdoctoral Scientist; Postdoctoral Scientist; Postdoctoral Scientist (till Nov '95); Postdoctoral Scientist (from Nov '95).

Each of our body cells carries over 100,000 genes. For ease of handling the cells have packaged these genes onto 46 different chromosomes. When the cells decide to divide, each of these chromosomes will first make a replica copy of itself, then some special structure on the chromosomes will perform the task of separating the replica copies so that the two new daughter cells will end up with one copy each. One aim of our work is to investigate how this process of chromosome separation is achieved by the cells. The study of this process is important for two reasons. First, it will help us understand why the separation sometimes goes awry and results in patients whose cells carry an extra unwanted chromosome, as is the case with Down syndrome. The second reason is that it will permit us to isolate the

pieces of DNA that we need to put together an artificial chromosome. The construction of a human artificial chromosome (which will make it the 47th chromosome in the cell) is the second sum of our work. We believe that all the key components of a human chromosome have now been identified by our own work and the work of other investigators. Our present task is to stitch all these components together to create a much smaller version of the human chromosome. Once we are successful in producing such an artificial chromosome, we will use it as a vehicle for human gene therapy. In this therapy, we will incorporate essential genes into the artificial chromosomes and deliver the chromosomes into patients to correct their gene defect. A number of gene'delivery vehicles are currently in use by various laboratories around the world but these all have major drawbacks. We believe

from left to right back Michael Cancilla, Richard SafFery, Damien Hudson middle Paul Kalitsis, Helen Trowell, Joanne Hill, Kellie Tainton front Desiree du Sart, Liz Earle, Andy Choo, Kerry Fowler

Page 16

that the concept of using human artificial chro' mosomes for gene therapy is far superior to other existing methods.

The centromere work has taken on a major thrust to clone and delineate the functional DNA element found in a human marker chromosome discovered by us in which the fully active centromere is devoid of the vast amount of repetitive DNA normally present on a centromere (Voullaire et a/, 1993). Using a combination of fluorescence in situ hybridisation (FISH) of DNA probes and amti'Centromere antibody staiining of the marker centromere (see figure), we have now narrowed the centromere DNA to a region of less tham 200 kb. We will continue to use these techniques to "walk" further into the centromere DNA. To help us pinpoint the exact location of the critical centromere DNA element, we have also initiated the construction of a detailed restriction map of the entire 200 kb region. In addition to the DNA work, we have begun functional studies on two of the known centromere'binding proteins, CENP'B and CENP'C, by gene knockouts or through the production of structurally altered mutant proteins. These studies will be performed in cell culture and in transgenic mice. All the necessary constructs have now been prepared and transfected into mouse embryonic stem cells. Cell colonies that are heterozygous for a CENP'B and CENP'C knockout event have been produced. These cell lines appear to be karyotypically normal, and we are awaiting results for the homozygous knockout events and the effect of the structural mutations on mitosis and meiosis Two approaches will be used for the develop' ment of HACs. The first will involve the design and construction of new vectors that are capable of existing episomally in human cells. The critical human centromere DNA plus some of its flanking sequences (derived from our marker chromosome in the centromere work) will be cloned into these new vectors to create a HAC in vitro. In the

Page 17

second approach, we will use the technique of targetted chromosome fragmentation to reduce the size of our marker chromosome to less than 1 megabase, thus creating a HAC in vivo. Important groundwork for both of these approaches, including the completion of our first vector system and the establishment of gene tar' getting technique (Michalska and Choo 1993), has already been established in our laboratory. Once we have achieved a stable HAC in human cells, we will explore its utility in the investigation of the biological properties of human chromo' somes and centromeres. Concurrently, we will test the suitability of the HACs &s a safe and effective vehicle for gene therapy. Such tests will initially be performed in laboratory animals, and if successful, is expected to lead to clinical trials in human subjects. References Voullaire, L.E., Slater, H.R., Petrovic, V. and Choo, K.H.A. (1993). A functional marker centromere with no detectable alpha satellite, satellite III or CENP'B protein: Activation of a latent centromere? Amer. J. Hum. Genet. 52:1153'1163. Michalska, A.E. and Choo, K.H.A. (1993). Targetting and germline transmission of a null mutation at the metalloth' ioneins I and II loci in mouse. Proc. Natl. Acad. Sci. U.S.A. 90: 8088'8092.

wo/re nooa urn Kerry Fowler, Sophie Gazeais, Robyn Breslin, Joanne Hill,

Senior Research Officer; Senior Technical Officer; Technical Assistant; (part'time) Research Assistant.

The development and testing of suitable gene therapy strategies relies on good animal models of human disease, amd there is growing pressure to ensure that all therapeutic strategies are tested on precise transgenic models. The Murdoch Institute has long recognised the immense value of transgenic animal technology for molecular genetic work. Over the last five years, we have established this technology in'house, under the direction of Dr Andy Choo's group. As the transgenic unit's work has expanded and been incorporated into almost every project in the Institute, the support group has been accorded a more independent status. Transgenic technology is of great signifi' cance at the Murdoch Institute. We use it to pursue studies on the function of genes such £LS those which encode centromere binding proteins (which may have a critical role in meiosis as well as mitosis); mutations which cause neurogenetic disorders such as ataxia and epilepsy; metabolic disorders such as Menkes’ and Wilson’s diseases; chondrodysplasia punctata; mammalian homologues of avian growth factor/receptor genes; and even to create mouse models for mitochondrial diseases.

Localisation of a cosmid clone (arrowed) to the short arm side of the marker centromere

The major research projects which are

currently in progress are: ^ Gene targeting by homologous recombination in transgenic mice: metallothionein I and II The use of existing mouse models for Menkes disease (mottled mouse mutant) and Wilson's disease (toxic milk mouse mutant) to validate gene therapy strategies Centromere and human artificiaJ chromosome studies

OTGRMAL COLIABORATIOM9 Given the scarcity of expert transgenic facilities, we have been asked to collaborate with other groups in developing models for human inherited disease. Whenever possible, we attempt to meet their needs, as this represents an efficient use of our resources and allows us to participate in a wide range of research programs. Two examples of these collaborations are: Kennedy’s disease mouse model (with Garry Warne’s group. Endocrinology, Royal Children's Hospital Research Foundation) Connective Tissue Disease mouse models (with John Bateman, Orthopaedic Molecular Biology Research Unit, Department of Paediatrics and Royal Children's Hospital Research Foundation)

OTHGR f[}me DIRGaiOM9 Developing "transgenic" mouse models for mitochondrial disease (with Henrik Dahl and David Thorburn) Transgenic Models for Ataxia and Epilepsy to study Gene Therapy A Transgenic Mouse to Study the Early Roles of TGF'P Superfamily Members in Development Transgenic mice to study the human disease chondrodysplasia punctata

(CDP)

Page 18

r DI90ftD€k^ Of niTOCHommL enencv GOiemioii

Group Leader; Helen M. Schutt Postdoctoral Fellow; Wendy Hutchison, Scientific Officer; PhD Student; Laraine Peters, PhD Student; Sarah White, Honours Student. Viviana Lieu, Henrik Dahl, Rozanne Blok,

Disorders caused by deficiency in energy production, the sO'Called mitochondrial disorders, are highly variable diseases, which often affect brain and muscle function. They are much more prevalent than previously thought. Mitochondrial dysfunction is seen in several paediatric syndromes with a total incidence of at least I in 5000 births, and can also be a contributing factor in many iatC'Onset disorders, such as diabetes, Alzheimer's disease, Parkinson's disease and general dementia. The Mitochondrial Group studies the molecular causes and genetic peculiarities of these conditions in collaboration with the Metabolic Laboratory. Defects in mitochondrial dysfunction can be caused by changes in genes coded for on one of the 46 nuclesur chromosomes or on the small mitochondrial DNA (mtDNA). Our studies ofpatients from both groups have led to a better under'

Page 19

standing of these diseases and therefore to better counselling of affected families. The clinical presentations are still very complex smd unpredictable, however, and treatment is ususdiy unsatisfactory. We have therefore continued our detailed research, focusing on three areas; -*■ steps towards the development of a mouse model for mtDNA diseases without which full understanding and treatment of these diseases will be very difficult; the study of how mtDNA changes are transmitted from mother to child as this is different from other inherited diseases; and -*■ the possibility of correcting the effect of a defective gene by introducing a normal gene into the affected cells. Our understanding of mitochondrial disor ders is hampered by the lack of suitable animal models. In collaboration with Kerry Fowler, we are trying to make transgenic mice with known mtDNA mutations. This is difficult because the DNA has to be introduced into the mitochondria and a suitable selection system is not available. However, using the megaprimer PGR approach we have engineered mutations into mouse mtDNA, can microinject mitochondria into oocytes, and are now focusing on transformation of mitochondria with engineered mutant mtDNA. We are also trying a more direct approach to correct human mtDNA disorders. We have synthesised a universal codon equivalent of the human mitochondrial ATPaseb gene (nuclear and mitochondrial genes use slightly different genetic codes).

Mutations in the ATPase6 gene can cause NARP or Leigh syndrome. This nuclear gene homologue has been provided with a mitochondrial import sequence that should direct the protein into the mitochondria. The construct has been cloned into a mammalian expression vector and we are now investigating if this artificial gene can correct the respiratory chain defect when transfected into patient cell lines. The pyruvate dehydrogenase complex (PDHC) is an essential and rate limiting enzyme complex connecting glycolysis with the TCA cycle. It is also a main target for insulin action. More importantly, PDHC deficiency is a major cause of primary lactic acidosis in infants and young children. In nearly all cases the defect is in the PDHC E1 a subunit. This year we have focused on the methylation pattern of the PDH E1 a promoters using the recently developed bisulfite method. This showed that the testis-specific PDH E1 a promoter is methylated in somatic tissues but not in spermatogenic cells. The somatic PDH E1 a is not methylated on the active X chromosome, or in spermatogenic cells. Methylation is observed on the inactive X chromosome, but the degree of methylation varies between CpG dinucleotides. However, correctly expressed PDHA2 transgenes in somatic tissues from transgenic mice are sometimes methylated and sometimes completely unmethylated, raising interesting questions about the relationship between methylation and gene expression.

In addition, we have developed methods for analysing common mutations and deletions. These techniques are now being used for routine analysis of patients with possible mitochondrial DNA (mtDNA) mutations by the DNA Diagnostic Laboratory. They have also led to the identification of a number of new mtDNA mutations, especially deletions. In collaboration with Dr David Thorburn, we have conducted the largest study on genotype/phenotype correlation in patients with Leigh syndrome. The understanding and treatment of mitochondrial disorders are insufficient, partly due to the unusual genetics. Maternal inheri tance and the consequences of mtDNA mutations in tissues are often not obvious due to heteroplasmy and tissue variation. Mutations accumulate during life and a mechanism must exist in oocytes that ensures that mutant mtDNAs are not passed on to offspring. The bottleneck hypothesis proposes that at an early stage during oocyte development the number of mtDNAs is dramatically reduced so that only oocytes with non-deleterious mtDNA survive. We have studied oocytes from a woman whose three sons were affected by the mtDNA nt8993 mutation. The asymptomatic mother has a low mutant level in her blood, but the individual oocytes have extreme levels of either normal or mutant mtDNA. This cannot be explained by the bottle neck hypothesis alone. We have suggested that one mtDNA during oocyte development is preferentially amplified which would explain many of the unusual features of mitochondrial

from left to right Stephen Wilcox, Henrik Dahl, Amelia Osborn, Sarah White, Laraine Peters, Wendy Hutchison, Moira Graham

Page 20

poor respiratory chain substrate galactose. Our current work is focusing on cell lines from patients with isolated or combined defects of complex IV or with complex I defects.

inheritance. ,:3.

#•••!!

nermDuc ke'^mcH •••••

David Thorburn

li * ; During development ofan oocyte there is a genetic bottleneck in mitochondrial numbers allowing substantial changes in the proportion ofmutant (red) and normal (blue) mitochondria.

In collaboration with Dr Leslie Sheffield we have also initiated a search for mutations in patients with chondrodysplasia punctata (CDP), a disease characterised by a flattened nose and midface and often accompanied by short stature, deafness and intellectuaJ handicap. Dr Sheffield has collected the largest group of CDP patients worldwide. CDP is in some cases caused by mutations in the arylsulfatase E gene. This gene is in a cluster of three arylsulfatase genes (D, E and F) on the x chromosome. Partial analysis of arylsulfatase D and E genes has identified two polymorphisms in the E gene, and at least two new mutations have been detected. We plan to improve mutation detection for these genes, to finish the analysis of the three arylsulfate genes in our patients and to investigate patients with the clinically related Binder syndrome, fetal warfarin and hydantoin syndrome and Larsen syndrome.

^)!!=Page21

Unit Head

Our current aind future research efforts are focused on defining the genetic basis of dis orders of mitochondrial energy generation. This task is very complex because more than 100 genes are involved, and different patients will have mutations in different genes. Our first job is to define which patients are likely to have mutations in the same gene so that we can define groups of patients large enough to find the gene by linkage analysis, as was done by Julian Mercer two years ago for Menkes disease. The main focus of our research work is determining the genetic basis of disorders of the mitochondrial respiratory chain. Approximately 1 in 5000 children will suffer from severe respiratory chain dysfunction. Further incentive for research comes from the increasing evidence that mitochondrial DNA (mtDNA) mutations or respiratory chain enzyme defects contribute to adultonset diseases such as diabetes, heart dis ease, and neurodegenerative conditions such as Parkinsonism and Alzheimer's dis ease. It is only in the laist two years that we have been able to measure respiratory chain complexes I to IV accurately in cultured cell lines from patients with respiratory chain defects. We have invested considerable effort in characterising patient fibroblasts to determine the exact type of defect. As well as enzyme measurements this has involved functional studies such as ATP synthesis and establishing conditions under which patient cell lines fail to grow in the presence of the

Now that we have well characterised cell lines, our current and future studies involve a combination of somatic cell genetics (in conjunction with Kerry Fowler and Marjorie Crawford) and molecular biology, particu larly linkage analysis (in conjunction with Henrik Dahl). We have begun using the cybrid method to determine whether patients have nuclear or mtDNA-encoded defects. This is done by enucleating patient cell lines and fusing the resulting cytoplast with a special rho cell line that lacks mtDNA (see diagram). If the resulting cybrid or "cytoplasmic hybrid" is still defective then the defect has been transferred with the mtDNA so the patient had an mtDNA mutation. We expect these studies will confirm that most of the patient cell lines have nuclear-encoded defects. This infor mation will be vaJuable for genetic coun selling and provides the basis for further studies aimed at defining nucleau" gene defects. A number of the families are consanguinous, and it is likely that we will be able to perform linkage analysis using homozygosity mapping. karyoplast Patient cell line \ enucleated ^

J cytoplast

rho° cell line

Liybrid patient mtDNA normal nDNA

Cybrid analysis of cells with an OXPHOS defect

Page 22

emRVOLOGv group Don Newgreen, Susan Bevan, Catherine Hearn Richard Kerr Joseph Minichiello Tom Edwards

Group Leader; Postdoctoral Scientist; PhD Student; PhD Student; Research Assistant; Honours student.

The Embryology Group focuses on the morphogenesis (development ofphysical form) and differentiation (development of specific cell types) of a crucial embryonic organ system called the neural crest. The neural crest is of great clinical significance since it is disproportionately involved in birth abnormalities of form (dysmorpho' geneses like facial clefiing, spina bifida and great vessel/cardiac defects) and of cell differentiation (paediatric cancers like neuroblastoma). As psirt of a strategy to study gene regulation of neural crest development, and consequently, ofgene involvement in neural crest abnormalities, we have meticulously microdissected and isolated about 30 specific populations of neural crest cells from different staged embryos. From these we have made cDNA libraries, with each library containing copies of only those gene sequences that are being used by the cells at a specific stage. The moleculatr genetics of development of the neural crest is of great theoreticaJ, general and clinical interest. Developmental genes have been sought by homology and

Page 23

by differential expression, but whichever strategy is used, it is preferable to start with material which is enriched for RNA transcripts specific to the cells being investigated. The usual source of transcripts is a cDNA library, but libraries have heretofore been constructed from convenient but inappropriate sources containing mixtures of cells of various lineages, and often from different stages than those being investigated. Fundamentd to studies on developmental gene expression is the harvesting of cell populations that are pure and defined by spatial, temporal amd cell lineage criteria. We have adapted old and devised new techniques to purify neural crest cell and lineage-related populations from avian embryos, including enzyme-assisted microdissection, cell dissociation, Percoll gradient centrifugation, differential cell adhesion, immunoselection, and magnetic bead separation. Obtained so far are: dorsal neural tube/premigratory neural crest, ventral neural tube, epidermal ectoderm, early migration-stage neural crest, neural tube minus migratory neural crest, late migration/localization-stage neural crest, differentiation-stage Schwann cells, sympathetic neurons and sensory ganglion cells, and melanocytes. From these we have created a systematic series of cDNA Richard Kerr, Joseph Minichiello,

Susan Bevan, Don Newgreen, Catherine Hearn

libraries representing crucial stages in the development of this lineage. Given the extraordinary conservatism of developmental processes, these will be important tools for the exploration of the molecular genetics of normal development of the neural crest, amd will be useful in the study of neurocristopathies in humans. The neural crest is an archetypal example of epithelium^mesenchyme transformation (EMT): this process gives cells the capacity to migrate and invade surrounding tissues. The neural crest is a particularly exaggerated model of this common normal morphogenetic process and also is a model of metaistatic cancer cell behaviour. We have therefore selected this process for more detailed study. Our tissue culture assays of neural crest EMT have pointed to a control by TGF-S family growth factors. Using our cDNA libraries of stages when EMT is occurring, we have studied the expression of this family of growth factors, and their receptors. This has revealed that at least three growth factors, including a new family member, and four receptors, including two new members, are active at this time. Clearly, the growth factor control of neural crest EMT is more complex than was at first thought. The cell biology of EMT has been further tested, particulairly the role of phosphorylation of proteins that control cell-to-cell adhesion, cell -toextracellular matrix adhesion, and cell shape. All of these cellular properties change in concert at EMT, and our results using drugs which alter various phosphorylating enzymes have allowed us to propose a new model of how these changes are orchestrated. In addition, our studies on the large chondroitin sulphate proteoglycan which reduces neural crest cell adhesion to extracellular matrix, have revealed an unexpected role in cell-to-cell adhesion (and hence in EMT), since it triggers a rapid decompaction of neural cells that are otherwise tightly bound to each other. This observation may clarify a controversy in which these proteoglycans were held by some to

stimulate, and by others, to inhibit cell migration. Other highlights of 1995 included: Morphogenetic events are usually played out in a complex four-dimensional stage and are consequently extremely difficult to analyse. We have devised highly consistent, simple assays to model and measure the important event of EMT We have isolated and partially characterised an extracellular matrix molecule that negatively regulates neural crest cell adhesion/migration. -*■ Our analysis of highly orchestrated simultaneous changes of cell phenotype in EMT of the neural system show that the apparently unitary process can be dissociated into sub-routines, and that the normal tight co-ordination can be achieved epigenetically. We have proposed a new model which, compared to the current model, represents a radically different interpretation of the regulation of this basic and complex process

of EMT We have correlated changes in multiple adhesion molecules with neural crest cell migration in the gut of normal rat embryos and in mutant embryos with a Hirschsprung's disease phenotype. Wotein'P+ , li Protein'P'

iSsr'fi '

Signal(s)?

t t I Proteasfe|

4' v T EGM + Integrin tt EChWntegtii

--I

Jlir"'"

Epithelial State

• Illl

Mesenchymal State

The cadheriri'based celhcell adhesion system, the integrim based extracellular matrix adhesion system and the acf/hbased cytoskeleton tremsmodulate each others functions to regulate the complex events of EMT.

Page 24

rwaom cufiim cetiaia ^€r\iic€ 1995 has been a year of change, growth and expansion. The retirement of Professor David Danks was marked by a two day scientific meeting in March 1995 in his honour. Guest speak ers included Professor Victor McCusick (Baltimore), Professor Charles Scriver (Montreal), Professor Malcolm Ferguson-Smith (Oxford), interstate colleagues (who had been trained by Professor Danks) and members of staff from the Murdoch Institute. We wish him a long and enjoyable retirement. Professor Bob Williamson, Director of the Murdoch Institute and Executive Director of the VCGS, brought with him new energy and enthusiasm. His keen interest in education stimulated a number of new activities coordinated by Dr Leslie Sheffield and Mrs Margaret Sahhar. These include the establishment of a Post-Graduate Diploma in Genetic Counselling under the auspices of Melbourne University, weekend workshops on cancer genetics and counselling, as well as a large number of lectures to medical students (Monash and Melbourne Universities), and medical and nursing colleagues at a broad range of scientific meetings. The training of doctors has increased significantly this yeau*. Dr Martin Delatyki is training in Clinical Genetics, and has just

Page 25

Our laboratory diagnostic services and Newborn Screening Program have had a busy year. There are plans to establish a serum screening program for pregnant women to identify those at high risk of having a baby with Down syndrome and neur^ al tube defects.

been joined by Dr Heidi Peters. In conjunction with the Royal Children's Hospital, the VCGS has launched six^month training positions for Paediatrics Trainees. Dr Karen Dunn will take up the first of these posts in early 1996. Several medical students will also be spending their elective term in the VCGS in 1996. Education and close liaison with a large number of patient support groups has continued under the guidance of Margaret Sahhar. This is a very important part of our support activities, and will play a vital role in community education, which we hope to expand in the near future. Professor Williamson hais forged closer ties this year with the Department of Paediatrics, the Research Foundation and other departments of the Children's Hospital, as well as with colleagues in adult medicine, including cancer and neurology. New clinics have been established by Dr Mac Gardner at the Royal Melbourne Hospital and the St Vincent's Hospital providing counselling services for patients with neurological conditions and inherited cancers. The Monash Familial Breast Cancer Clinic was begun under the guidance of Dr Agnes Bankier. Further services will need to be established in these areas. We have continued to provide a network of outreach clinics in country centres in Victoria, with Dr John Rogers running counselling services for Tasmania.

from left to right Sue Mansie Margaret Sahhar

We have farewelled Dr Jim McGill from the Metabolic Service. He returns to Queensland after a two year appointment here. Dr Maureen Cleary is in the middle of a two year visit from Manchester. We are actively seeking to recruit a doctor trained in the care of inborn errors of metabolism to run this service on a long term basis.

Agnes Bankier Clinical Director

T'tl

Leslie Sheffield Director of Education Training

6?^

from left to right Margaret Olsen, Mary-Anne Young, Ann Robertson, Linda Warwick

♦

from left to right Maureen Cleary, Karen Dunn, Les Sheffield, John Rogers, Mac Gardner, Heidi Peters. Seated Agnes Bankier, Martin Delatycki.

Page 26

CLINICAL & GPIDCmOLOGICAL RCSCARCH

€PID€niOLOCV urn Les Sheffield, Jane Halliday, Carole Webley, Ron Batagol,

Medical Geneticist / Epidemiologist; Epidemiologist; Research Assistant; Research Assistant.

We have been active in a number of areas of research, primarily focused on issues that relate to the public health aspects of genetics and therefore those which affect the broader community. One of our main interests has been in the area of prenatal diagnosis of genetic disorders where we have continued to monitor the diagnostic tests, amniocentesis and chorion villus sampling, in women of advanced maternal age (37 years and older) and in women with a particular risk of fetal abnormality because of a family history of am inherited disorder.

Another field of work that began in 1995 was an involvement in the development of the cancer genetics service for Victoria. With the identification of a number of cancer predisposition genes, there has been a growing interest in provision of such a service and the VCGS has had a major role to play. The VCGS has developed a plan to offer a blood test to all pregnant women in Victoria which can detect the majority of babies with Down syndrome or neural tube defects. The test will be carried out in the VCGS neonatal screening laboratory and women tested will be offered counselling by VCGS genetic counsellors. This will ensure there is a programme incorporating laboratory amd counselling in a properly coordinated manner. Such coordination and counselling is essential for the success of the programme and is not currently available in Victoria. The programme is planned to start in 1996 and the epidemiology unit is actively involved in planning the service. As an epidemiologist with a broad overview

-■m

• '•f

m from left to right

Carole Webley, Jane Halliday

Page 27

of the subject, Jane Halliday was seconded to the Anti-Cancer Council of Victoria (ACCV) for four months in 1995. She worked as a project officer, to prepare a report on Cancer Genetic Testing and Counselling in Victoria and was therefore absent from the Murdoch Institute for this time. The project was funded by a grant from the Department of Health &- Community Services. After interviewing over 70 experts in related fields, the report was presented to the Genetics Advisory Committee of the Victorian Cooperative Oncology Group of the ACCV and the Department of H&-CS in July. The report was very well received, and led to the formation of four working parties to address the recommen dations outlined. Since this work was completed there have been many developments in this new field of genetics, which are being overseen by the national and State-wide organisations and committees that have formed and are focused on the development of a cancer genetics service, particularly for breast and colorectal cancer. Finally, the dual role of Jane Halliday as half-time Director of the Perinatal Data Collection Unit (PDCU), Department of Health Community Services, has led to a collaborative study being undertaken that examined the uptake of genetic counselling by parents of a child with a congeni tal abnormality. It is hoped that this association with the PDCU will lead to further collaborative projects on birth defects.

DOCUneMTIMG THG €FF€a9 OF DRUG9 TAkGM DURIMG PRGGMAHCY Using methodology we have previously developed, we have run a pilot study of 6 different classes of drugs on the fetus. This was done by ascertaining patients who contact 2 obstetric drug information services and enrolling them in a study to monitor outcome of pregnancy. We have decided to change the methodology and obtain patient consent to be in the study by referral to a special clinic for counselling from a genetic counsellor about the effects of the drugs taken during pregnancy as well as requesting consent to follow up the baby at the birth defect register. We have been fortunate in receiving a research grant from the ANZ Trust for this work.

9TUDY OF CHOMDRODY9PlAqA

punaATA Last year we were part of the team that discovered 3 new genes on the X chromosome one of which was found to cause some cases of chondrodysplasia punctata. We have been developing methods to test our other patients for mutations in all 3 genes. So far we have confirmed a mutation in one additional patient and suspect mutations in a further two. Initially we used one mutation analysis method and have now switched to a much more efficient method and expect more rapid progress.

PRGMATAL DIAGM09I9 Follow-up of early amniocentesis Objective : To determine whether there were any risks associated with having this test at an earlier gestational age, compared with the established gestational age of 15 - 16 weeks. The ability to link data between the Murdoch Institute and the Perinatal Data Collection Unit

Page 28

facilitated this research. It was possible to track the pregnancy outcome of all women having amniocentesis at 14 weeks gestation or earlier. We found that spontaneous loss (miscarriage) rates were higher than at 15^ 16 weeks gestational age. This was as expected, because of the natural loss rates being higher in the earlier stages of pregnancy. An unexpected finding was that women who have the earlier test have an increased risk of developing a genito^urinary tract infection. This study has been completed and is being written up for publication.

nOlilTODIMG Of PDetlAtAL DIAGIiCCTIC

tettt Objective: To continue the monitoring of all prenataJ diagnostic tests done in Victoria. This has been done since the mid 1970s and a report is circulated to all practitioners in the field and has been a useful source of research material for many. Data was again collated from the four cytogenetics laboratories who receive fetal samples for testing. There were 2365 amniocentesis and 1351 chorion villus samplings done on Victorian women in 1994. In 1995 these numbers increased to nearly 3000 amnios and over 1600 CVS. The high utilisation rates by older women, as well as the effectiveness of ultrasound, has ensured a high detection rate of fetal abnormalities such as Down syndrome in our population. We will be continuing to collect and report these data and hope to include in future annual reports the results of the forthcoming VCCS maternal serum screening program for Down syndrome and neural tube defects.

CAMCGR GGNeria Objective : To evaluate the psychological impact of predictive genetic testing for familial adenomatous polyposis (FAP). It is unknown at this stage how people will react to the new'found knowledge of their individual risk of carrying a cancer predisposing mutation. It is important to try and determine how this information will affect them. There continues to

^pi!!!!!F^ge 29

be considerable interest by affected families in testing for FAP, due to the availability of the protein truncation test by the diagnostic laboratory of the VCGS. The laboratory has looked for mutations in 39 affected individuals. Relatives who then undergo presymptomatic testing are then asked to participate in an evaluation study of the pyschological effects of this testing. Questionnaires, before, 2 weeks after and six months after the test are completed by the person tested. So far we have received 17 completed questionnaires. The survey results will be analysed as part of an international collaboration, eis well as for local information. We hope to extend our experience with this type of evaluation to other cancer genetic counselling and testing programs.

BIRTH oefeas Use of two Statewide registers to determine the rate of uptake of genetic counselling in Victoria when a baby is born with an abnormality. Objective: To see how often parents of a baby born with a specific birth defect were accessing the available genetic counselling service provided by the VCGS.This study found that, overall, only 17% of parents were having genetic counselling. However, when results were analysed on the basis of need for genetic counselling, it was found that over 40% in the "high need' category were seen by the VCGS. There was no particular disadvantage for mothers born in non^English' speaiking countries (reflecting language and cultural barriers), but maternal age was important, with older and teenage women being seen more often (proportionately) than those between the ages of 20 and 35 years. This study has provided information useful in planning of future genetic services and will be followed by an interview'based study to determine whether other counselling services have been available to these families.

VCGf PWG/yOfT/C lMCmTOI^I€9 DMA DIAGM09TIC lABORATORV Sue Forrest Ivan Biros Karina Forshaw Steven Nasioulas Janet Shaw

Scientist'in'charge Scientific Officer Medical Scientist Medical Scientist Medical Scientist

The DNA Diagnostic Laboratories of the Victorian Clinical Genetics Service are located at the Murdoch Institute and Monash Medical Centre said are responsible for all diagnostic DNA testing for Victoria and Tasmania. Week by week, new tests become available, and we feel that we should try to offer DNA testing, even if only a few children are sJfected by a particular disease. Prevention through prenatal diagnosis requires accurate DNA testing, and we know that it will only be possible to offer gene therapy if we have identified the mutations causing a disease. For all of these reasons, the DNA Diagnostic Laboratory will continue to play a centred role in clinical research as well as in service. This year has seen some changes in the Murdoch laboratory with the introduction of new staff (Dr Ivan Biros) and new testing methodologies. The laboratory has taken over the mitochondrial DNA testing which

had previously been performed by the enzymology group. Our major focus for the year has been improvement of the methodolo©/ for identifying mutations in families with familial polyposis colon cancer which accounts for about 1 % of colon cancer. The gene that causes this disease is known as APC and is very large, so searching for the different mutation in each family is a very time consuming task. Steven Nasioulas has set up a new technique called the protein truncation test. Protein from the APC gene can be made synthetically in a tube and can then be checked for changes by separation on a gel according to size. Many of the alterations in the APC result in a shortened protein product which is easily visible on the gel. Thus we have identified the family specific change in over half of the 70 FAP families in Victoria. We are then in a position to offer presymptomatic tests to members of these families to determine whether or not they have inherited the defective gene before they develop symp' toms of the cancer. So far, thirty people

from left to right Sue Forrest, Ivan Biros, Karina Forshaw, Steven Nasioulas, Janet Shaw

Page 30

have requested presymptomatic testing.

1 !

Our work on cystic fibrosis (Karina Forshaw) particularly with respect to prenatal diagnosis continues to increase. We have also developed better methods for carrier detection in faunilies with Duchenne muscular dystrophy where the affected male has a deletion in a particular part of the dystrophin gene (Janet Shaw). Identification of couples at risk for having a child with thalassemia means the Monash laboratory must keep up to date with the vairying types of thalassemia mutations that are now present in the cosmopolitan Melbourne population and develop diagnostic tests to quickly ascertain their presence or absence (Jan Brasch, Andrea Twomey and Kathy Garafolo). Both laboratories have spent some time during the year becoming conversant in a number of different mutation detection technologies to ensure the transfer of these methods from Dr Cotton's skilled laboratory staff. Our interest in adult neurological disorders continues to grow particularly with the expansion of the VCGS into clinics in adult hospitals. We have now set up tests for most of the common trinucleotide repeat disorders of adult onset including Huntington's disease, dentatorubralpallidoluysian atrophy and spinal cerebellar ataixia types 1 and 3. We have also organised three monthly neurogenetic clubs for neurologists, geneticists and scientists to examine such disorders in more detail. It has been a busy year and no doubt with the burgeoning amount of research in the field of cancer genetics, some more of our time will need to be devoted to this area in 1996.

lijfePage31

UCG9 DI/)GI109TIC lMCmTORI€9 NCTABOLIC lABORATORV David Thorburn, Ivan Francis, Denise Kirby, Erin Oldaker,

Scientist-in^charge, Enzymology; Scientist-in-charge, Metabolism; Research Officer; Research Assistant.

The Metabolic Laboratory provides a diagnostic service and conducts research in the field of Inborn Errors of Metabolism. This term covers children with inherited disorders affecting the breakdown and re-use of fat, protein and sugars. Many of these conditions are severely disabling but in some cases, affected children can live a normal life if their condition is recognised early enough. They can then be put on special diets or given medication, for example to prevent the accumulation of natural compounds that may otherwise build up to very high (toxic) levels. We currently monitor over 100 such children from around Victoria to ensure their "metabolic control" is adequate to prevent long-term disability. Our major research interest is in disorders of energy generation in children, for which we act as the Australasian referral centre and are referred 50 - 100 new cases each year suspected of such disorders. All our organs, but particularly brain, heart, muscle and

liver require energy to perform normally, and so any or all of these orgams can be affected when our cellular power plants (known as mitochondria) do not work properly. Much of our work over the last four years has been aimed at improving our ability to recognise these disorders, and while we have had considerable success, there is still much scope for improvement. For further details of our research work, please refer to page 22. We also maintain an interest in other inborn errors of metabolism and have achieved over 40 new diagnoses this year. Thirty of these were respiratory chain defects, and there were also six cases of phenylketonuria, four cases of succinic semialdehyde dehydrogenase deficiency (a disorder of GABA metabolism) plus isolated cases of several other inborn errors.

from left to right Denise Kirby, Erin Oldaker, David Thorburn

Page 32

veer DIAGI109TIC iMOMTome^ NGWBORM raeeMiMG lABORATORV Ivan Francis, Nick Tzanakos, Len Bonacquisto, Nella Napolitano, Mona El-Masri,

Scientist-in-charge; Medical Scientist; Medical Scientist; Trainee Medical Scientist; Trainee medical Scientist.

The Newborn Screening Laboratory (NSL) tests every newborn (Victoria has about 65,000 births per year) for three serious diseases; phenylketonuria (PKU), congenital hyperthyroidism (CH) and cystic fibrosis (CF). Before their discharge from hospital, new born babies are given a heelprick, and the “dried blood” or Guthrie spots are sent by ordinary mail to the NSL, where about 300 specimens are tested each day for the three disorders. PKU is an inherited disorder of amino acid metabolism, and occurs in 1/13000 births (5 per year in Victoria). The availability of screening and effective early dietary treatment means that PKU infants can now look forward to a normal life; before newborn screening they faced a life time of institutional care with severe mental handicap. Untreated cases of CH were called cretins, and suffered from severe mental impairment and stunted growth.

Page 33

CH occurs in 1/3200 births (20 per year in Victoria). Treatment by thyroid hormone replacement (one tablet per day) is completely effective, and is continued throughout life. CF is the most common lifc'limiting genetic disorder among Caucasians, occurring in 1/3100 births (22 per year in Victoria). Newborn screening has made the early diagnosis of CF possible, thereby allowing appropriate therapy to be employed, before debilitating malnourish' ment and life-threatening respiratory disease has taken hold. The future of newborn screening is linked closely to developments in genetic research. Advances in genetic knowledge and gene therapy will provide opportunities for screening and early intervention in disorders which at present are considered untreatable. This has the potential to make a major contribution to the quality of life of future generations. We are pleased that the new born screening laboratory is ailready able to help several hundred Victorians avoid severe handicap and lead normal lives through treatment; we will participate in putting each new advance into practice.

from left to right

back Mona El-Masri, Nella Napolitano front Len Bonacquisto, Ivan Francis, Nick Tzanakos

UCGS' DIAChO^TIC lMOI^TORI€9 CYTOGGNGTICS' lABORATORV Howard Slater, Vida Petrovic, Scientists:

Technicians:

Cytogeneticist in Charge; Deputy; Lucille Voullaire; /knne Robertson; Sue Dale; Louise Hills; Melissa Curtis; Julie Davies; David Francis; Marie Thorpe; Ralph Oertel; Cathryn Vaux; Julie Garratt; Trent Burgess; Tracy Vankuyk; Selena Bourke; Lynda Phillips; Ian Brooks; Bo Jezierski

The VCGS Cytogenetics Laboratory provides a postmatal chromosome analysis service to hospital and private clinicians throughout Victoria and Tasmania. This includes detection of congenital and acquired (cancer) abnormalities. The laboratory also performs DNA testing for some syndromes formerly analysed cytogenetically, notably the Fragile X syndrome which is also tested prenatally. Approximately 4500 tests are performed

SLnnusJIy. Our new laboratory space has facilitated closer collaboration with the other clinical and scientific research teams, to our mutual benefit. Extensive development work is carried out in molecular cytogenetics where DNA techniques are combined with traditional chromosome analysis to detect submicroscopic mutations in syndromes such as the Velocardiofacial syndrome and Williams syndrome. Research activities capitalise on the opportunities recognised in the many unusual cases which are uncovered in the laboratory's routine work. Several collaborations are in progress both in-house and with other institutions in a wide variety of areas including Fragile X, Prader-Willi syndrome and childhood leukaemia. The diagnostic work of the cytogenetics laboratory continues to expand in quantity but also in the range of testing offered. The numbers of referrals made to the laboratory from Victorian and Tasmanian paediatricians, pathologists, haematologists and general practitioners increased by 10%, continuing the trend of the last few years. A major initiative was made last February when the laboratory extended its activities into the analysis of paediatric leukaemias which in Victoria are all treated by the Dept of Haematology and Oncology in the Royal Children's Hospital. The laboratory has provided a service for the analysis of paediatric solid tumours for several years amd it was a natural progression to provide am "in-house" service for leukaemiais. One

Page 34

year on, this new aspect of our work is well established and we are now looking at ways to improve diagnosis in this particularly difficult area by application of our molecular cytogenetics expertise. The distinction between cytogenetics and DNA testing continues to blur and considerable effort is spent in development work aimed at improving and facilitating diagnostic procedures which are inadequate when approached by standard chromosome analysis. In previous years the laboratory has abandoned cytogenetic testing of Fragile X, Prader-Willi and Angelman syndromes in favour of DNA testing. We have played a valuable role in assisting other laboratories to take this approach by training visiting staff. A major development success this year was the introduction of a PCR screening test for Fragile X syndrome in boys with developmental! delay, the reward of a full year's rather frustrating effort. The underlying reason for the delay is the long stretch of triplet CCG repeat in the Fragile X

from left to right back Ian Brooks, Ralph Oertel, David Francis, Trent Burgess middle Anne Robertson, Julie Garratt, Cathryn Vaux, Marie Thorpe, Louise Hills, Wali Drummond front Lynda Phillips, Vida Petrovic, Howard Slater, Melissa Curtis, Bo Jezierski

Page 35

mutation which is difficult to copy using DNA polymerases. We are particularly pleased with this test which is faster and cheaper than the older Southern Blot test and is available only in very few other laboratories worldwide. It will provide a model for rapid DNA testing for other triplet expansion diseases. We have also introduced testing for several microdeletion syndromes which were previously extremely difficult to diagnose using standard techniques. These tests use Fluorescence in situ Hybridisation(FISH) which uses DNA probing directly onto chromosomes. A major project was completed with Dr Martin Delatycki, our genetics fellow, which screened children suspected of having the Velocardiofacial syndrome. This is a new area of diagnosis and is stimulating a great deal of interest as the deletion in this syndrome includes a gene or genes important for embryological development of the heart. We have diagnosed about 40 children, a few with inherited deletions for what was previously thought to be an extremely rare syndrome. It is

estimated that 5% of all cardiac abnormalities in children are the result of mutations in the VCFS gene(s). Another microdeletion syndrome whose diagnosis hais been transformed is Williams syndrome, another disorder involving heart defects. The laboratory made the first prenatal diagnosis of Prader-Willi syndrome in a case referred to us from the cytogenetics laboratory at the Royal Women's Hospital, where a mosaic trisomy 15 was detected during routine age-related screening on chorionic villus sampling. Our Prader-Willi syndrome DNA test which we have used for two years to diagnose the disorder in neonates and young children was used in this prenatal setting. We are constantly confronted with interesting cases, some of which have definite research value and implications. In some situations we are able to investigate these ouselves and in others, set up collaborations with other research groups within and also outside the Murdoch Institute. Looking back over recent years at the most important breakthroughs in genetics research, many were triggered by the fortuitous discovery and exploitation of single cytogenetics cases. The importance of such cases cannot be overstated and it is extremely important that we try to identify opportunities for research as they appear. Two such cases discovered several years ago in the VCGS have produced notable advances through collaborations.

adds another twist to the peculiar genetics of this disorder. Another interesting finding with research implications was the discovery in two individuaJs of a chromosome abnormality regarded as a unique tumour marker of Ewing's sarcoma in a completely different tumour type. Publication of this finding is allowing other centres to re'evaluate the status and significance of the marker. The future direction of the laboratory will to a large extent be driven by the constant pressure to convert research advances into clinical applications. The challenge will be to prioritise our efforts and to apply our resources to the benefit of the largest groups of patients.

In collaboration with Dr Danuta Loesch at LaTrobe University, retrospective analysis of our routine Fragile X testing data revealed a previously unreported sex difference in the size of mutations produced in mother to daughter and mother to son transmissions where the latter were strikingly larger. This fascinating finding

Page 36

moocH miTUTC Scientific Director: David Danks, A.O., M.D., B.S., F.R.A.C.P. (to 31/03/95) Robert Williamson, Ph.D.,FRCPath., Hon. MRCP, Hon.M.D. (Turku) (from 01/04/95) Deputy Scientific Director: Richard Cotton, B.Ag.Sci., Ph.D., D.Sc. (to 31/12/95) Business Manager: Anne Cronin, B.Sc., B.Bus.(Acc.), A.S.A., C.PA.

Laboratory Manager: Barry Holt, B.App.Sci.(M.T), A.A.LM.L.S. Scientists (Senior) Richard Cotton, B.Ag.Sci., Ph.D., D.Sc. (to 31/12/95) Jim Camakaris, B.Sc.(Hons.), Ph.D. K.H. Andy Choo, B.Sc.(Hons.), Ph.D. HanS'Henrik Dahl, Ph.D. Julian Mercer, B.Sc.(Hons.), Ph.D. Donald Newgreen, B.Sc.(Hons.), Ph.D. Les Sheffield, B.Med.Sci., M.B., B.S., M.Sc., D.C.H., F.R.A.C.P.

Page 37

Research Fellows Susan Forrest, B.Sc.(Hons.), D.Phil.(Oxon.) David Thorburn, B.Sc.(Hons.), Ph.D. Clinical Scientists 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. Mac Gardner, M.B., Ch.B., M.Sc. F.C.C.M.G. Postdoctor3J Fellows Susan G. Bevan, Ph.D. Rozanne Blok, B.Sc.(Hons.), Ph.D. (Helen M. Schutt Fellow) (to 28/07/95) Michael Cancilla. B.Sc.(Hons.), Ph.D. Jane Halliday, B.Sc.(Hons.), Ph.D. Simon Hufton B.Sc. (Hons.), Ph.D. (to 04/08/95) Sharon La Fontaine. B.Sc. (Hons.), Ph.D. John Martyn, B.Sc.(Hons.), Ph.D. (to 31/10/95) Richard Saffery, B.Sc. (Hons.), Ph.D. Helen Trowell, B.Sc.(Hons.), Ph.D. RimaYouil, M.Sc., Ph.D. (to 31/12/95) Clinical Fellows Martin Delatycki, M.B., B.S., F.R.A.C.P. (Part 1) Catherine Rose, M.B., B.S. Scientific Officers and Research Assistants Leanne Bailey, B.Sc. (Hons.) (to 12/95) Hilary Brooks, B.Sc. (Hons.) Marjorie Crawford, A.R.M.l.T Elizabeth Earle, A.A.I.M.L.S. Stephen Firth, M. App. Sci. Kerry Fowler, M.App.Sci., M.Sc. Tamara Gough, B.App.Sci. (to 28/07/95) Andrew Grimes, B.App.Sci. Joanne Hill, B.App.Sci.

Ourania Horaitis, B.Sc. (to 31/12/95) Sharon Horton, B.Sc., Grad.Dip.Diet. Wendy Hutchison, B.App.Sci.(App.Biol.) Ian Jennings, B.Sc. (to 31/12/95) Denise Kirby, B.Sc.(Hons.) Melanie Knight B.Sc. (Hons.) (to 31/12/95) Paul Lockhart, B.Sc. (Hons.) (to 28/04/95) Henny Miller Joseph Minichiello, M.Sc. Erin Oldaker B.App.Sci. Jenny Paynter, B.Sc.(Hons.) Kaye Seller, B. App. Sci. (to 07/04/95) Janet Shaw, B.Sc. (Hons.) Kellie Tainton B.Sc. (Hons.) EfFie Tsotsis, B.Sc. (to 24/02/95) Carole Webley

Administrsition Accountant: Hilary Lloyd, B.Ec., A.S.A. Personnel Assistant: Debbie Zombolas Personal Assistant to the Director: Lee Jackson from left to right Hilary Lloyd, Christine (to 13/04/95) Keenan, Debbie Zombolas Personal Assistant to the Director: Elizabeth Krahling, B.A., M.Def Studies

Ph.D. Scholars Loreta Ambrosini, B.App. Sci. Jeffrey Babon B.Sc.(Hons.) (to 31/12/95) Desiree Du Sart, B.App.Sci. Catherine Hearn, B.Sc. (Hons.) Damien Hudson, B.Sc.(Hons.) Paul Kalitsis, B.Sc. Richard Kerr, B.Sc. (Hons.) Laraine Peters, B.Sc.(Hons.) Michael Petris B.Sc.(Hons.) Susan Ramus, B.Sc. (Hons.) Michael Theophilos B.Sc.(Hons.) Sarah White B.Sc. (Hons.)

Secretsuries: Debbie Davis Valerie Mehl Kristine Yeomans

Receptionists: Fiona Keltie Olga Wilson

Accounts Clerk: Christine Keenan Cathy McMillan Photography/Graphic Design: Michele Winsor Kati Hidegh

Technical Assistants Leonie Anastasiou Alison Blake Evelyn Boyer Robyn Breslin Sophie Gazeas Moira Graham Michelle Guneratne Tiffany Symes Editorial Assistant, Human Mutation Julie Cross, B.Ed. (to 31/12/95) Dietitian: Dorothy Francis, SRD

from left to right Lorraine White, Fiona Keltie, Margot Latham, Kristine Yeomans, Olga Wilson, Debbie Davis

Page 38

r v/ao)P//)/y CLimm G€ii€nc9 reev/ce Executive Director: David Danks, A.O., M.D., B.S., F.R.A.C.P. (to 31/03/95) Robert Williamson, Ph.D.,FRCPath., Hon. MRCP Hon.M.D. (Turku) (from 01/04/95) Director of Clinical Services: Agnes Bankier, M.B., B.S., F.R.A.C.P. Director of Education and Training: Les Sheffield, B.Med.Sci., M.B., B.S., M.Sc., D.C.H., FR.A.C.P. Clinical Geneticists: John Rogers, M.B., B.S., D.C.H., F.R.A.C.P. Mac Gardner, M.B., Ch.B., M.Sc., F.C.C.M.G. Metabolic Physician James McGill, M.B., B.S., F.R.A.C.P. Maureen Cleary, M.B., Ch.B., M.R.C.P.

(UK) Scientists - DNA Diagnosis Susan Forrest, B.Sc.(Hons.), D.Phil.(Oxon.) Scientist in charge Ivan Biros, B.Sc., Ph.D. Janice Brasch, B.Sc.(Hons.), M.Sc. Karina Forshaw, B.App.Sci. Kathy Garofalo Steven Nasioulais, B.Sc. (Hons.) /Vidrea TA/omey, B.Sc.(Hons.)

Page 39

Scientists ' Cytogenetics Howard Slater, B.Sc., Ph.D., Dip.R.C.Path., H.G.S.A.C.C. ' Scientist in charge Selena Bourke, B.Sc. Trent Burgess, B.Sc.(Hons.) Melissa Curtis, B.Sc. Sue Daile, B.Sc.(Hons.) Julie Davies, B.Sc., H.G.S.A.A.C. David Francis, B.Sc.(Hons.) Julie Garratt, B.Sc.(Hons.) 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. Marie Thorpe, B.Sc.(Hons.) Tracy Vankuyk, B.Sc. (Hons.) Cathryn Vaux, B.Sc. Lucille Voullaire, M.Sc., H.S.S.A.C.C. Technical Assistant Lynda Phillips Laboratory Assistant lam Brooks Bozena Jezierski Jia Fang Shi (to 20/07/95) Neonatal Screening Laboratory Ivan Francis, B.Sc., Dip.Comp.Sci. Scientist-in^Charge Leonard Bonacquisto, B.Sc.(Hons.), Dip.Comp.Sci. Mona El'Masri

Nella Napolitano Nick Tzanakos, B.App.Chem.

Administrative Assistant Sharon Vandersluis

Genetic Clinic Co'ordinators/Genetic Counsellors Sue Casanelia, S.R.N. Clara Gaff, B.Sc.(Hons.), Ph.D. Margaret Olsen, Dip.App.Biol. /knn Robertson, S.R.N. Linda Warwick, 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

from left to right Sharon Vandersluis, Jo Wells

Secretary Lorraine White

Business Manager Anne Cronin, B.Sc., B.Bus.(Acc.), A.S.A., C.PA. Laboratory Manager Barry Holt, B.App.Sci.(M.T), A.A.I.M.L.S.

Project Officer Kelley Gardner, R.N. Assoc. Sci. (Hons.)

Page 40

U9T Of PUBUO^TIOliS' -

1-480, 1995.

1995

Earle, E. and K.H.A. Choo. Radioactive in situ hybridisation to replica tion-banded chromosomes. In: In Situ Hybridisation Protocols, (ed) K.H.A. Choo. New Jersey: Humana Press, Tbtowa, USA, 147-158, 1995.

IM PRGS'S' PPGMIOU9 RGPORT, NOW PUBLI9HGD Alexander, I.E., G.P. Tauro, and A.B. Bankier. Fetail brain disruption sequence in sisters. EurJPediatr 154: 654-657, 1995. Balnaves, M.E., L. Bonacquisto, I. Francis, J. Glazner, and S. Forrest. The impact of newborn screening on cystic fibrosis testing in Victoria, Australia. J Med Genet 32: 537-542, 1995. Bankier, A. Syndromes of the month - Menkes disease. JMed Genet 32: 213-215, 1995. Bankier, A., C.G. Keith, and I.K. Temple. Absent iris stroma, narrow body build and small facial bones: a new association or variant of SHORT syndrome. Clin Dysmorph4: 304-312, 1995. Blok, R.B., D.R. Thorburn, G.N. Thompson, and H.-H.M. Dahl. A topoisomerase II cleavage site is associat ed with a novel mitochondrial DNA dele tion. Hum Genet 95: 75-81, 1995. Choo, K.H.A. In situ hybridisation protocols. In: In situ hybridisation protocols, (ed), K.H.A. Choo. New Jersey: Humana Press, Tbtowa, USA,

Page 41

Fong, S.T., J. Camakaris, and B.T.O. Lee. Molecular genetics of a chromosomal locus involved in copper sensitivity in Escherichia coli K-12. Mol Micro 15: 1127-1137, 1995. Franco, B., G. Meroni, G. Parenti, J. Levilliers, L. Bernard, M. Gebbia, L. Cox, P. Maroteaux, L. Sheffield, G.A. Rappold, G. Andria, C. Petit, and A. Ballabio. A cluster of sulfatase genes on Xp22.3 mutations in chondrodysplasia punctata (CDPX) and implications for Warfarin embryopathy. Cell 81: 15-25, 1995. Halliday, J., J. Lumley, aind L. Watson. Comparison of women who do and do not have amniocentesis and chorion villus sam pling. Lancet 345: 704-709, 1995. Halliday, J.L., L.F. Watson, J. Lumley, Danks. D.M., and L.J. Sheffield. New estimates of Down syndrome risks at chorion villus sampling, amniocentesis and live birth in women of advanced maternal age from a uniquely defined population. PrenatDiag 15: 455-465, 1995.

Hahn, S.H., M.S. Tanner, D.M. Danks, and W.A. Gahl. Normal metallothionein synthesis in fibroblasts obtained from children with Indian childhood cirrhosis or copper-associated childhood cirrhosis. Biochem Med Metab Biol 54: 142-145, 1995.

Page, S.L., W.C. Earnshaw, K.H.A. Choo, and L.G. Shaffer. Further evidence that CENP'C is a necessary component of active centromeres. Studies of a dic(X; 15) with simultaneous immunofluescence and FISH. Hum Mol Genet 4: 289-294, 1995.

Han, J.Y., K.H.A. Choo, and L.G. Shaffer. Molecular characterisation of 17 rob (I3ql4q) by fluorescence in situ hybridisation, narrowing the region containing the breakpoints. Am J Hum Genet 55: 960-967, 1994.

Penny, L., M. DellAquila, M. Jones, J. Bergoffen, C. Cunniff, J. Fryns, E. Grace, J. Graham, B. Kousseff, T. Mattina, J. Syme, L. Voullaire, L. Zelante, J. Zenger-Hain, O. Jones, and G. Evans. Clinical and molecular characterisation of patients with distal 1 Iq deletions. Am J Hum Genet 56: 676-683, 1995.

Hewitt, B. and A. Bankier. Prenatal ultrasound diagnosis of BeckwithWiedemann syndrome. Aust &- NZ J of Obst &■ Gynaecol 34: 488 490, 1994. Hill, D.J., I.L. Hudson, L.J. Sheffield, M.J. Shelton, S. Menahem, and C.S. Hosking. A low allergen diet is a significant intervention in infantile colic: Results of a community based study. J Allergy Clin Immunol 96: 886-892, 1995. Howe, A.M., A.H. Lipson, L.J. Sheffield, E.A. Hatan, J.L. Halliday, F. Jensen, and W.S. Webster. Prenatal exposure to phenytoin and facial devel opment. Am J Med Genet 58: 238-244, 1995. Lazo, J.S., Y. Kondo, E. Woo, A. Michalska, K.H.A. Choo, and B.R. Pitt. Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice deficient in metallothionein I and II genes. J Biol Chem 270: 5506-5512, 1995. Lee, C.S., M.C. Southey, H. Slater, A.W. Auldist, C.W. Chow, and D.J. Venter. Primary cutaneous Ewing's sarcoma / peripheral primitive neuroectodermal tumors in childhood: a molecular, cytogenetic and immunohistochemical study. Diag Molec Path 4: 174-181, 1995.

Philcox, J.C., P. Coyle, A. Michalska, K.H.A. Choo, and A.M. Rofe. Is metallothionein synthesis necessary for inflam mation mediated hepatic zinc uptake? Biochem J 308: 5453-546, 1995. Ramus, S.J. and R.G.H. Cotton. Mutations Ivs4ntl, 47 del CT and G148S identi fied in the phenylalanine hydroxylase gene by RTPCR of illegitimate transcripts and chemical cleav age of mismatch. Hum Mut 6: 250-251, 1995. Ramus, S.J., E. Treacy, and R.G.H. Cotton. Characterization of phenylketonuria alleles in untreated PKU patients from Victoria, Australia: origin of alleles and haplotypes. Am J Hum Genet 56: 1034-1041, 1995. Shaffer, L.G., C. McCaskill, J.Y Han, K.H.A. Choo, D.M. Cutillo, A.E. Donnenfeld, L. Weiss, and D.L. Van Dyke. Molecular characterisation of de novo secondary Trisomy 13. Am J Hum Genet 55: 968-974, 1994. Slater, H., J.H. Shaw, G. Dawson, A. Bankier, and S.M. Forrest. UPD 13 - No indication of maternal or paternal imprinting of genes on chromosome 13. J Med

Genet 32: 493, 1995. Slater, H., T. Tiong, S. Konstantakopoulos, C. Shan, V. Petrovic, L. Voullaire, and G. Kannourakis. Cytogenetic and DNA analysis of two neuroectO' dermal tumours (Ewing's Sarcomas) without a simple t(l 1;22). Cancer Genet £r Cytogenet 83: 1247, 1995. Smooker, P.M., J. Christodoulou, R.R. Mclnnes, and R.G.H. Cotton. A mutation causing DHPR deficiency results in a ffameshift and a secondary splicing defect. J Med Genet 32: 220-223, 1995.

Woods, C.G., M. Leversha, and J.G. Rogers. Severe intrauterine growth retardation with increased Mitomycin-C sensitivity; a further chro^ mosome breaikage syndrome. J Med Genet 32: 301^305, 1995. Woods, C.G., S. Treleaven, F.R. Betheras, and L.J. Sheffield. 'Disorganisation4ike syndrome' with 47,XXY and unilateral narrowing of the common iliac artery. Clin Dysmorphology 4: 82-86, 1995. Youil, R., B. Kemper, and R.G.H. Cotton. Screening for mutations by enzyme cleavage of mismatch using T4 endonuclease VII. PNAS 92: 87-91, 1995.

Smooker, P.M. and R.G.H. Cotton. The molecular basis of dihydropterine reductase deficiency. Hum Mut 5:279-284, 1995. Takakubo, F., D.R. Thorburn, R.M. Brown, G.K. Brown, and H.-H.M. Dahl. A novel mutation (P316L) in a female with pyru vate dehydrogenase El alpha deficiency. Hum. Mut. 6: 274-275, 1995. Treacy, E., D. Johnson, J. Pitt, and D.M. Danks. Trimethylaminuria, fish odour syndrome, a new method of detection and treatment with Metronidazole. J Inher Metab Dis 18: 306-312, 1995. Woods, C.G., G. Black, and G. Norbury. Male neonatal death and progressive neuropathy in females in a family with an unknown X-linked condition. J Med Genet 32: 191-196, 1995. Woods, C.G. and D.M. Danks. DNA diagnostic tests: clinical applications. Aust Pres 18: 76-79, 1995. Woods, C.G. and D.M. Danks. DNA diagnostic tests: approach and techniques. Aust Pres 18: 45-48, 1995.

Page 43

PUBLI9HGD AND ACCGPT€D FOR PUBUCATION ?INC€ 1994 RGPORT Babon, J., R. Youil, and R.G.H. Cotton. Improved strategy for mutation detection - a mod ification to the enzyme mismatch cleavage method. NucI Acids Res 23: 5082-5084, 1995. Bankier, A. Genetic counselling. In: Clinical Paediatric Surgery., Blackwells, (in press). Berkovic, S.F., A. McIntosh, R.A. Howell, A. Mitchell, L.J. Sheffield, J.L. Hopper. Familial temporal lobe epilepsy. Ann Neurol (in press). Blok, R.B., D.R. Thorburn, D.M. Danks, and H-H.M. Dahl. A mitochondrial DNA deletion in a patient with mitochondrial cytopathy but without ragged red fibres. Biochem Mol Med 56: 26-30, 1995.

Blok, R.B., D.R. Thorburn, D.M. Danks, and Dahl. MtDNA deletion in a patient with symptoms of mitochondrial cytopathy but without ragged red fibres. Biochem Mol Med 56: 26'30, 1995. Blok, R.B., D.R. Thorburn, G.N. Thompson, and H.-H.M. Dahl. A novel mitochondrial DNA deletion in KearnsSayre syndrome associated with a putative topisomerase II cleavage site. Hum Genet 95: 75-81, 1995. Brown, N.L., S.R. Barrett, J. Camakaris, B.TO. Lee, and D.A. Rouch. Molecular genetics and transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid PRJ1004. Mol Microbiol (in press). Camakaris, J., M. Petris, L. Bailey, P. Shen, P. Lockhart, T.W. Glover, C.L. Barcroft, J. Patton, and J.F.B. Mercer. Gene amplification of the Menkes (MNK;ATP 7A) P-type ATPase gene of CHO cells is associat ed with copper resistance and enhanced copper efflux. Hum Mol Genet 4: 2117-2123, 1995. Chan, D., W.G. Cole, J. Rogers, and J.F. Bateman. Type X collagen multimer assembly in vitro is pre vented by a Gly618 to Val mutation in the al (X) NCI domain resulting in Schmid metaphyseal chondrodysplasia. J Biol Chem 270: 4558-4562, 1995. Cotton, R.G.H. Detection of mutation in DNA. Bull Hum Genet Soc of Australia 7: 13-16, 1994. Dahl, H.H.M. Pyruvate dehydrogenase El alpha deficiency: Males and females differ yet again. Am J Hum Genet 56: 553-557, 1995.

Danks, D.M. Paediatricians' reports to general practitioners: Should parents receive copies? J Paed &- Child Health 30: 190, 1994. Dierick, H.A., L. Ambrosini, J. Spencer, T.W. Glover, and J.F.B. Mercer. Molecular structure of the Menkes disease gene (ATP7a). Genomics 28: 462469, 1995. Distante, S., S. Nasioulas, Somers. G.R., D.J.S. Cameron, M.A. Young, S.M. Forrest, and R.J.M. Gardner. Familial adenomatous polyposis in a 5 year old child: a clinical, pathological and molecular genet ic study. J Med Genet (in press). du Sart, D. and K.H.A. Choo. Simultaneous fluorescence in situ hybridisation and anti-centromere antibody staining of normal and "stretched" metaphase chromosomes. In: Molecular Biotechniques, (eds) R. Rapley and J.M. Walker. Tbtowa, NJ, USA: Humana Press, (in press). Duband, J-L., F. Monier, M. Delannet, and D. Newgreen. Epithelium-mesenchyme transitions during the development of the neural crest. Acta Anat (in press). Forrest, S., R. Cotton, U. Landegren, and E. Southern. How to find all those mutations. Nat Genet 10: 375-376, 1995. Fowler, K.J., F. Walker, W. Alexander, M.L. Hibbs, E.C. Nice, R.M. Boher, G.B. Mann, C. Thumwood, R. Maglitto, J.A. Danks, R. Chetty, A.W. Burgess, and A.R. Dunn. A mutation in the epidermal growth factor recep tor in waved-2 mice has a profound effect on receptor biochemistry resulting in impaired lacta tion. PNAS 92: 1465-1469, 1995.

Page 44

Giunta, C., R. Youil, D. Venter, C.W. Chow, G. Somers, A. LafFerty, B. Kemper, and R.G.H. Cotton. Rapid diagnosis of germline p53 mutation using the enzyme mismatch cleavage method. Diagn Mol Path (in press). Gupta, S.D., B.T.O. Lee, J. Camakaris, and H.C. Wu. Identification of cutC and cutF (nIpE) genes involved in copper transport in Escherichia coli. J Bacteriol (in press). Hufton, S.E., I.G. Jennings, amd R.G.H. Cotton. Structure and function of the aromatic amino acid hydroxylases. Biochem J 311: 353-366, 1995. Hutchinson, W. and H'H.M. Dahl. Detergent enhanced sequencing of heat'dena' tured plasmid DNA. Bio Tech 19: 554'556, 1995.

Kedinger, M. and D. Newgreen. Development features and disesises affecting the gut. In: Birth Defects-Perspectives from Contemporary Development Biology, (ed) P. Thorogood. John Wiley £r Sons,( in press).

Pedagogos, E., G. Flanagan, D.M.A. Francis, G.J. Becker, D.M. Damks, and R.G. Walker. A case of craniomamdibular dermatodysostosis associated with focal glomerulosclerosis. Pediatr Nephrol 9: 354^356, 1995.

Kondo, Y, E. Woo, A.E. Michalska, K.H.A. Choo, and J.S. Lazo. Metallothionein null cells have increased sensitive ty to anticancer drugs. Proc Am Assoc Cancer Res 36: 1944'1950, 1995.

Philcox, J.C., P. Coyle, A. Michalska, K.H.A. Choo, and A.M. Rofe. Endotoxin'induced inflammation does not cause hepatic zinc accumulation in mice lacking metal' lothionein gene expression. Biochem J 308: 543' 546, 1995.

Loesch, D.Z., R. Huggins, V. Petrovic, and H. Slater. Expansion of CGG repeat in Fragile X depends on sex of the offspring: genetic inferences. Am J Hum Genet 57: 1408'1413, 1995. Maw, M.A., D.R. Allen'Powell, R.J. Goodey, I.A. Stewart, D.J. Nancarrow, N.K. Hayward, and R.J.M. Gardner. The contribution of the DFNBI locus to neU' rosensory deaffiess in a caucaisian population. Am J Hum Genet 57: 629'635, 1995.

Rahman, S., R.B. Blok, H'H.M Dahl, D.M. Danks, D.M. Kirby, C.W. Chow, J. Christodoulou, and D.R. Thorburn. Leigh syndrome: clinical features and biochemical and DNA abnormalities. Annals of Neurol (in press). Ramus, S.J. and R.G.H. Cotton. Polymorphism in the 3' untranslated region of the phenylalanine hydroxylase gene detected by enzyme mismatch cleavage: evolution of haplo' types. Hum Genet (in press).

Jones, C., L. Penny, T. Mattina, S. Yu, E. Baker, L. Voullaire, W.Y Langdon, G. Sutherland, R.I. Richards, and A. Tunnacliffe. The fragile site FRAl IB, in the proto'oncogene CBL2, is associated with a chromosome deletion syndrome. Nature 376: 145'149, 1995.

McDougall, P, M. Loughnan, J. Rogers, and G. Silk. The death of a child, in Paediatric Handbook 5th Ed, (ed) D. Efron, Assoc Ed: T Nolan, Blackwell Science Pty Ltd 1995.

Jones, C., L. Penny, T. Mattina, S. Yu, E. Baker, L. Voullaire, W.Y. Langdon, G.R. Sutherland, R.I. Richards, and A. Tunnacliffe. Association of a chromosome deletion syndrome with a fragile site within the protO'Oncogene CBL2. Nature 376: 145'149, 1995.

Michaud, J., G.N. Thompson, L.C. Steel, C. Obie, G. Fontaine, K. Schappert, G.C. Keith, D. Valle, and G.A. Mitchell. Pyriodoxinc'responsive gyrate atrophy of the choroid and retina: clinical and biochemical cor' relates of the mutation A226V. Am J Hum Genet 56: 616'622, 1995.

Rudzki, Z., S.E. Rogers, L.J. Sheffield, and J.V Lloyd. Detection of carriers of haemophilia A: use of bioassays and restriction fragment length polymor' phisms (RFLP). Aust NZ J of Med (in press).

Newgreen, D.F. and J. Minichiello. Control of epithelio'mesenchymal transformation. I. Events in the onset of neural crest cell migra' tion are separable and inducible by protein kinase inhibitors. Dev Biol 168: (in press).

Takakubo, F., P. Cartwright, N. Hoogenraad, D.R. Thorburn, F. Collins, T. Lithgow, and H.'H.M. Dahl. An amino acid substitution in the PDH El alpha gene, affecting mitochondrial import of the pre' cursor protein. Am J Hum Genet 57: 772'780, 1995.

Jones, L.N., D.J. Peet, D.M. Danks, A.P. Negri, and D.E. Rivett. Structural defect in the fibre cuticle of hair from patients with maple syrup urine disease. J Invest Dermatol (in press).

Page 45

Takayama, K., D.M. Danks, E.P. Salazar, J.E. Cleaver, and C.A. Weber. DNA repair characteristics and mutations in the ERCC2 DNA repair and transcription gene in a trichothiodystrophy patient. Hum Mut (in press). Treble, N.J., F.O. Jensen, A. Bankier, J.G. Rogers, and W.G. Cole. Development of the hip in multiple epiphyseal dysplasia. J Bone Joint Surg Br 72: 1061'1064, 1994. Youil, R. and R.G.H. Cotton. Detection of unknown mutations. International Pediatr (JournaJ of Miami Children's Hospital) 10: 1995. Youil, R., B. Kemper, and R.G.H. Cotton. Detection of 81 of 81 known mouse beta'globin promoter mutations with T4 endonucleaLse VII ' the EMC method. Genomics (in press).

Ramus, S.J., D.B. Pitt, and R.G.H. Cotton. Genotyp^phenotype correlations: Intellectual phenotypes in untreated PKU patients. International Pediatr. (Journal of Miami Children's Hospital) 10: 92'95, 1995.

Page 46

rme moocH mme fon ke^mcH iiiTO mm D€F€a9 Lm€D

A.C.N. 006 566 972 and its controlled entity Financial Statements and Reports 31 St December 1995

Page 47

Page 48

r Dii^eaoii^ nepokT

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 entity, for the year ended 31 December, 1995 and the auditors' report thereon.

DIRGQOR^ The Directors of the Compamy 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 57 Chairman of Macquarie Corporate Finance Limited. Director since 1986 - appointed Chairman 1993. Mr. W.H. Hodgson Deputy Chairman (Non-Executive Director) Age 66 Formerly Deputy Managing Director of the National Australia Bank Limited, an appoint ment 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

f'l

Page 49

Chief Executive Officer, Women's and Children's Healthcare Network. Mr. Davies was formerly the Chief Executive Officer of the Royal Children's Hospital. Director since 1991. Age 47 Professor Angus holds the Chair of Pharmacology at the University of Melbourne. He represents the National Health Medical Research Council (NHMRC) on the Institute's Board. Director since 1991. Dr. G.L. Barnes, M.D., Ch.B., F.R.A.C.P. Non-Executive Director Age 54 Scientific Director, Royal Children's Hospital Research Foundation. Senior Gastroenterologist, Royal Children's Hospital. Dr. Barnes represents the Hospital on the Institute's Board. Director since 1986. Mrs. J. Cadvert'Jones Non-Executive Director Age 57 Chairman of the Herald amd 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.

Mr. J.A. Fitzgerald Non-Executive Director Age 64 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 56 Chairman of the Institute's Finance Committee. Mr. Griffin is an investment banker and a director of N.M.Rothschild Sons (Aust) Pty Ltd Group companies, various public and private companies, industry amd community groups. Director since 1993. 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 79 Mr. Guest is a distinguished Melbourne surgeon and the Chairman of the Jack Brockhoff Foundation. Director since 1986.

Mrs. L. Cattermole, B.Sc. Non-Executive Director Age 47 Chairperson of the Women's and Children's Healthcare Network. Appointed during 1995.

Mrs. I. McFarling Non-Executive Director Age 58 Mrs. McFarling is a successful public relations advisor. She is the President of the Friends of the Murdoch Institute, the Fundraising Auxiliau'y of the Institute. Director since 1988.

Mr. 1. Davies, B.H.A., F.A.I.M., F.C.H.S.E. Non-Executive Director Age 55

Mrs. J. Paterson Non-Executive Director Age 38 Appointed during 1996.

Professor D.G. Penington, A.C., M.A., D.M., B.Ch., F.R.C.P., F.R.A.C.P., F.R.C.P.A. Non-Executive Director Age 66 Professor Penington was formerly the ViceChancellor, the University of Melbourne. He is a director of various public and private compainies. Appointed during 1996. Professor P.D. Phelan, B.Sc., M.D., B.S., F.R.A.C.P. Non-Executive Director Age 59 Professor Phelan is the Stevenson Professor of Paediatrics at the University of Melbourne and a distinguished thoracic physician. Director since 1986. Professor G.W. Tregear, B.Sc., PhD., F.R.A.C.I. Non-Executive Director Age 55 Associate Director, Howard Florey Institute of Experimental Physiology and Medicine. Professor Tregear represents the NH&-MRC on the Institute's Board. Director since 1994. Professor R. Williamson, PhD., F.R.C. Path., Hon. M.R.C.P., Hon.M.D. (Turku) Scientific Director Age 57 Executive Director of the Victorian Clinical Genetics Services. Professor of Medical Genetics, University of Melbourne. Appointed during 1995. Dr. R.G.H. Cotton, Professor D.M. Danks, Mr. A.S. Murdoch, Professor G.B. Ryan and Professor P. Smith retired as directors of the Company during the year. Professor P.D. Phelan retired as a director of the Company on 12th April 1995 and was reappointed on 14th February 1996.

Page 50

DiR€aoR9 neeriMG?

defects. No significant change in the nature of these activities occurred during the year.

The number of directors' meetings and number of meetings attended by each of the directors of the Company during the financial year are:

Director

Mr. L.G. Cox Mr. W.H. Hodgson Professor J.A. Angus Dr. G.L. Barnes Mrs. J. Calvert'Jones Mrs. L. Cattermole Dr. R.G.H. Cotton Professor D.M. Danks Mr. I. Davies Mr. J.A. Fitzgerald

Mr. P. Gnffin Mr. J.S. Guest Mrs. I. McFarling Mr. A.S. Murdoch Professor P.D. Phelan Professor G.B. Ryan Professor P. Smith Professor G.W. Tregear Professor R. Williamson

No of Meetings Attended

No of Meetings Held *

5 5 5 5

6 6 6 6

3 2

6 T 6

4 6 2

4 4 5

6 6 6 6 6 4*

0 5*

5 5

5* 6 5*

* Reflects the number of meetings held during the time the director held office during the year.

PRIMCIPAL AaNITIG9 The principal activities of the economic entity during the course of the financial 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

Page 51

DNIDGND9 The Company is a company limited by guarantee. As such it has no share capital, and no dividends are paid.

C0M90UDATGD RG9ULT The consolidated operating loss of the economic entity for the financial year ending 31 December 1995 was $546,520. (1994 $388,015). No provision is required for taxation as the Company and its controlled entity are exempt from Income Tax.

end of the financial year and the date of this report aoiy item, transaction or event of a material and unusual nature likely, in the opinion of the 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.

LIkGLY f^UTURG DGVlGLOPnGMT9 AMD G^GQGD RG9ULT9 The area redeveloped for the use of the Cytogenetics Laboratory, the DNA Diagnostic Laboratory and the Neonatal Screening Laboratory will be completed early in 1996 and ready for occupancy. Building on the expertise of the Institute’s new Scientific Director, Professor Bob Williamson, it is proposed to set up a research group in 1996 to study the delivery of genes to cells. The study will focus on gene tar^ geting of cells of the central nervous system.

RGMIGW Of OPGRATIOM9 A review of the operations of the Company and its controlled entity during 1995 has been included in the Report of the Board.

9IGMIAICAMT CHAMGG9 IM 9rATG Of AAf¥ilR9 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.

DIRGaOR9 IMTGRG9r9 AMD BGMGHT9 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 bene^ fit included in the aggregate amount of remunera^ tion received or due and receivable by Directors shown in the consolidated accounts) because of a contract made by the Company, its controlled entity, 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 Director has a substantial interest.

Dated at Melbourne this 24th day of April 1996. Signed in accordance with a resolution of the Directors:

LAURENCE G. COX : Directors

GVIGMT9 9UB9GQUGMT TO BAIAMGG DATG There has not arisen in the interval between the

ROBERT WILLIAMSON

Page 52

TH€ MURDOCH miTUTC KDR RCTCARCH IHTO BIRTH DCfCaT

me MURDOCH mme fOR rctcarch mo birth Defeat

UMITCD m ITT coliTRoacD cmnv

UMITCD m ITT COtITROLLCD OITIJV

Profit and Loss Accounts for the year ended 31 December 1995

Balaifice Sheets as Sit 31 December 1995 Note

Note

Operating Loss before Income Tax

Income tax attributable to Operating Loss

Consolidated

The Company

1995

1994

1995

1994

(546,520)

(388,015)

(556,439)

(434,991)

Operating Loss after Income Tax

(546,520)

(388,015)

(556,439)

(434,991)

Accumulated Funds at beginning of the financial year

8,606,547

9,029,562

8,512,511

8,982,502

Adjustment to accumulated funds at the beginning of the financial year due to initial adoption of an Accounting Standaird AASB1028, Accounting for Employee Entitlements.

8,060,027

8,641,547

7,956,072

8,547,511

(185,087)

(94,391)

Aggregate of amounts transferred from reserves

257,550

Total available for appropriation Aggregate of amounts transferred to reserves

Accumulated funds at the end of the financial year

8,132,490 4

8,641,547

8,119,231

35,000

8,132,490

8,606,547

8,547,511 35,000

8,119,231

8,512,511

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 56 to 63.

Page 53

Consolidated 1994 1995 $ $

The Company 1994 1995 $

270,517 715,917 887.862

28,686 628,829 809.668

234,684 328,210 562 535

20,771 350,291 505.141

1 874 296

1 467.183

1125.429

876203

1,015,000 5,903,686 1 297.463 8.216.149

1,087,500 7,705,604 2.25.564 9 018 668

1,015,000 5,903,686 1.287.750 8 206.436

1,087,500 7,705,604 213.076 9.006.180

10 090 445

10485.851

9.331.865

9 882 383

996,640 743.629 1.740.269

871,552 683 676

756,220 410.177 1 166.397

680,528 429.152 1109.680

CURRENT ASSETS

Cash Receivables Investments TOTAL CURRENT ASSETS NON-CURRENT ASSETS Receivables Investments Property, Plant &- Equipment TOTAL NON-CURRENT ASSETS

5 6

5 6 7

TOTAL ASSETS CURRENT LIABILITIES Creditors and Borrowings Provisions TOTAL CURRENT LIABILITIES

8 9

NON-CURRENT LIABILITIES Creditors and Borrowings Provisions TOTAL NON-CURRENT LIABILITIES

8 9

1 555.228

49,600 165 444

63,884

215.044

63 884

43.595

TOTAL LIABILITIES

1 955 313

1 619.112

1.209.952

1 109.680

NET ASSETS

8,135,132

8,866,739

8,121,873

8,772,703

MEMBERS' FUNDS Accumulated Funds Reserves TOTAL MEMBERS' FUNDS

8,132,490 2.642 8,135,132

8,606,547 260.192 8,866,739

8,119,231 2 642 8,121,873

8,512,511 260.192 8,772,703

43 595

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 56 to 63.

Page 54

THC MURDOCH IHTTITUTC K)R RCTCHRCH IHTO BIRTH DCfCaT UMITCD mo ITT COMTROaCD CHTITV

TH€ MUHDOCH imTUT€ fOR RE^mCH IMTO BIRTH D€F€aT UniT€D m ITT COIiTROIl€D OITIJV

Statements of Cash Flows for the year ended 31 December 1995

Notes to smd Forming Psurt of the Financial Statements for the year ended 31 December 1995

Consolidated 1994 Inflows (Outflows)

1995 1994 Inflows Inflows (Outflows) (Outflows) $ $

(6,400,451) (5,715,812) 3,111,705 3,099,527 1,149,756 1,000,928 629,712 1,141,120 8,887 12,840 662.326

(4,098,747) (3,445,812) 1,431,905 1,439,527 1,149,756 1,000,928 648,680 579,408

1995 Inflows (Outflows) $ CASHFLOWS FROM OPERATING ACTIVITIES Payments to suppliers and employees Government Grants received Donations received Other receipts Interest received Patient fees received NET CASH PROVIDED/(USED) BY OPERATING ACTIVITIES (Note lO(ii)) CASH FLOWS FROM INVESTING ACTIVITIES Interest received Dividends received Proceeds on sale of investments Payment for investments Payment for property, plant and equipment

The Company

(834,112)

(465,350)

2,886

(865,520)

(425,949)

STATGMGMT Of 9IGMIflCAMT ACCOUMTIMG POLICIG9

The significant policies which have been adopted in the preparation of these financial statements are: Basis of Preparation (a) The financial 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 his' torical costs and do not take into account changing money values or, except where stated, current valuations of non-current assets. The accounting policies have been consistently applied by the entities in the economic entity and are consistent with those of the previous year except as disclosed in Note (2). 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. The financial statements of the Company and its controlled entity reflect all entries affecting the Company and its controlled entity and include transactions that were specifically recorded in the bank accounts of the Company and its controlled entity.

79,847 263,404 207,573 84,109 7,315,112 8,841,731 (5,358,304) (8,389,932) (1.218.7051 (1 17.4291

249,615 65,314 84,109 207,573 6,906,112 8,241,731 (4,884,304) (7,789,970) (1.218.705) (117.4291

NET CASH PROVIDED BY INVESTING ACTIVITIES

1.085.616

621.790

1136.827

607.219

NET INCREASE IN CASH HELD

251,504

156,440

271,307

181,270

CASH AT THE BEGINNING OF THE REPORTING PERIOD

642.602

486.162

525.912

344.642

CASH AT THE END OF THE REPORTING PERIOD (Note 10(i))

894,106

642,602

797,219

525,912

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 56 to 63.

Principles of Consolidation (b) 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 1995. All balances and transactions between the chief entity and the controlled entity have been eliminated. (c) Investments Investments are stated at the lower of cost or recoverable amount. Dividends are brought to account as received. (d) Property, Plant and Equipment Items of property, plant and equipment are recorded at cost. The depreciable amounts of all items of plant and equipment are depreciated over their estimated useful lives commencing from the time the asset is held ready for use. The straight line method of depreciation is used. Items of plant and equipment costing less than $5,000 are not capitalised. Leasehold improvements are to be amortised over 15 years - that being the same period of time as rent prepaid to the Royal Children's Hospital from the time that the laboratories and offices are ready for use. No amortisation has been charged during this financial year as the laboratories and offices were not ready for use until early 1996.

Page 56

(c)

Company: a decrease of $ 10,506).

Prepayments

Items of expenditure having a benefit or relationship to more than one accounting period are amortised over the periods to which they relate.

Revenue Recognition ' Patient Fees

if)

From the beginning of the financial year, patient fees have been separately disclosed as operating revenue of the economic entity. In the past patient fees were accounted for in the specific purpose funds. The amount disclosed as revenue was the transfer from the specific purpose funds, net of relevant expenses.

Employee Entitlements

Wages, Salaries and Annual Leave The provisions for employee entitlements to wages, salaries and annual leave represents the amount which the economic entity has a present obligation to pay resulting from employees’ services provided up to balance date. The provisions have been calculated at nominal amounts based on current wage and salary rates and includes related on-costs.

The financial effect has been to increase patient fees by $215,868. Comparatives Where necessary, comparative information has been reclassified to achieve consistency in disclosure with current financial year amounts and other disclosures.

Long Service Leave The liability for employee entitlements to long service leave represents the present value of the estimated future cash outflows to be made by the employer resulting from employees' services provided up to the balance date.

Consolidated 1994 1995 $ -$

Liabilities for employee entitlements which are not expected to be settled within twelve months are discounted using the rates attaching to national government securities at balance date, which most closely match the terms of maturity of the related liabilities.

In determining the liability for employee entitlements, consideration has been given to future increases in wage and salary rates, and the economic entity’s experience with staff departures. Related on^costs have also been included in the liability. (g)

(a)

Income Tax

Revenue Recognition

Fee revenue comprises revenue earned from the provision of services to entities outside the economic entity. Fee revenue is recognised when the fee in respect of services provided is receivable. (i)

CREDITING AS REVENUE Grants - NHMRC Grants ' H&-CS Grants ' Other Donations Interest - other persons Dividends ' other persons Net Gain on sale of investments Patient Fees POSSUM Sales Income ' Other

Doubtful Debts

The collectibility of patient fees is assessed at year end and a specific provision is made for any doubtful debts.

OP€RATIMG PROFIT

Operating Loss before Income tax has been determined after:

The Company and its controlled entity are exempt from income tax under section 23(e) of the Income Tax Assessment Act 1936.

(h)

The Company 1995 1994 $

Proceeds on sale of investments

CHAMGGS IM ACCOUMTING POLICV (b)

Provision for Employee Entitlements Employee entitlements have been calculated in accordance with AASB1028, Accounting For Employee Entitlements for the first time. The adoption of this Standard has resulted in a change in the method of calculating the economic entity’s provision for long service leave. In the past the economic entity provided only in respect of employees with more than ten years service. The economic entity now provides for long service leave in respect of all employees, based on the present value of the estimated future cash outflow to be made resulting from employees’ services up to the balance date and having regard to the probability that employees as a group will remain in the entity’s employ for the period of time necessary to qualify for long service leave. The financial effect of the change has been to decrease accumulated funds at the beginning of the financial year by $185,087 in the consolidated accounts (the Company: $94,391). The effect on the current year has been to increase the provision for employee entitlements by $6,141 in the consolidated accounts (the Company:a decrease of $ 10,506) resulting in a corresponding increase in the deficit of $6,141 in the consolidated accounts (the

CHARGING AS EXPENSE SalariesAVages, inch superannuation Employee entitlements Laboratory Consumables Refurbishment Costs Repairs &- Maintenance Travel Clinical Research Patient Care Services Central Services 6- Administration Depreciation POSSUM Costs

1,256,156 2,047,686 182,643 1,024,756 275,351 84,109 219,890 777,570

1,260,302 1,787,091 210,492 1,125,928 87,659 207,573 42,293 561,702

1,256,156 171,386 182,643 1,024,756 252,501 84,109 219,890

1,260,302 163,249 210,492 1,125,928 67,794 207,573 42,293

129,431 240.564

129,431 294 331 3,615,203

132,207 263.663

6,238,156

132,207 286.121 5,701,368

3,473,501

7,315,112

8,841,731

6,906,112

8,241,731

4,461,622 280,185 576,714 1,403

4,077,872 98,394 546,080 32,069 61,246 63,216 9,758 329,205 562,253 121,346 187.944 6,089,383

2,484,894 170,103 576,714

2,461,137 63,518 546,080 32,069 61,246 63,216

37,098 79,283 2,304 320,219 752,628 141,806 13L414 6,784,676

1,403

37,098 70,853 2,304 557,828 139,031

9,758

131.414

364,953 118,571 187.944

4,171,642

3,908,492

•• J

Page 57

Page 58

Consolidated 1995 1994 $ $

MOVEMENTS IN RESERVES SOCIAL WORK FUND Balance at beginning of year Transfer to revenue Balance at end of year BUILDING DEVELOPMENT FUND Balance at beginning of year Transfer (to)/from revenue Balance at end of year

257,500 2.642 2.642

2,692 (501 2,642

257,500 (257.5001

2.692

260,192

2,692 2,692

222,500 35 000

Z642 2,642

2,692

2,642

257,500 (257.5001

257,500

257,500 2 692 260,192

NON-CURRENT Shares ' Listed on a prescribed stock exchange Government Bonds - Unlisted Interest in Trusts TOTAL NON-CURRENT INVESTMENTS

1,435,052

2,158,051

1,435,052

2,158,051

1,437,489 3 031 145 5 903 686

3,619,541 I 928 012 7.705.604

1,437,489 3.031.145 5 903 686

3,619,541 1 92.8 012 7.705.604

TOTAL INVESTMENTS

6,791,548

8,515,272

6,466,221

8,210,745

MARKET VALUE OF LISTED INVESTMENTS

1,795,965

2,456,731

1,795,965

2,456,731

2,692 2,692

222,500 35 000

257,500

PROPGRTY, PLAMT & GQUIPMGMT 1,078,233

Leasehold Improvements

1,078,233

Plant &■ Equipment - at cost Less: Accumulated Depreciation Written Down Value TOTAL

776,399 (557,169) 219,230 1,297,463

640,927 (415,363) 225,564 225,564

748,648 (539,131) 209,517 1,287,750

613,176 (400,100) 213,076 213,076

161,188 534,558 300,894 996,640

29,900 486,933 354,719 871,552

161,188 327,589 267,443 756,220

29,900 326,721 323,907 680,528

RGCGNABLG9 CURRENT Patient fees due Less provision for doubtful debts Accrued Income Amount Owing by Controlled Entity Debtors - Sundry Prepayments Sundry Receivable NON-CURRENT Prepayments

The Company 1995 1994 $ $

RG9GRV1G9 BUILDING DEVELOPMENT FUND SOCIAL WORK FUND Balance at end of year

Consolidated 1994 1995 $ $

The Company 1995 1994 $ $

130,844 (15,600) 115,244 125,000

125,000

513,511 82,162 5,000 715,917

415,971 87,858

167,776 73,272 82,162 5,000

628,829

328,210

350,291

1,015,000

1,087,500

1,015,000

1,087,500

137,433 87,858

CURRENT Unearned Income Royal Children's Hospital Sundry Creditors TOTAL

IMVIG9TriGNT9 AT C09T CURRENT Bank Bill Commercial Bonds Short Term Deposit TOTAL CURRENT INVESTMENTS

Page 59

64,274 200,000 623 588

95,752 100,000 613 916

887,862

809,668

562 535 562,535

505.141 505,141

CRGDITOR9 & BORROWIMG9

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

49,600

63,884

Page 60

Consolidated 1994 1995 $ $

PROVII9IOM9

11.

CURRENT Annual Leave Long Service Leave

252,305 491.324 743,629

(i)

165,444

112,938 297 239 410,177

113,368 315.784 429,152

270,518 623.588 894,106

28,686 613.916 642,602

RECONCILIATION OF NET CASH PROVlDED/(USED) BY OPERATING ACTIVITIES TO OPERATING PROFIT AFTER INCOME TAX. Operating Profit/(Loss) after Income Tax (546,520) (388,015) Add/(Less) Non Cash Items Depreciation &■ Amortisation 214,306 193,846 Amounts set aside to Provisions 40,311 112,596 Add/(Less) Items classified as Investing Activities Gain on sales of investments Dividends received Interest received

(219,890) (84,109) (263,404)

Add/(Less) Changes in Assets 6- Liabilities (lncrease)/Decrease in Debtors - Sundry (97,539) (Increase)/Decrease in Patient Fees Due (115,244) (Increase)/Decrease in Accrued Income 125,000 (Increase)/Decrease in Prepayments 2,174 (Increase)/Decrease in Amount owing and controlled entity (Increase)/Decrease in Inventory (Increase/(Decrease) in Creditors 33,341 (Increase/(Decrease) in Grants in Advance 4,363 (Increase/(Decrease) in Accrued Expenses 66,570 6.529 (Increase/(Decrease) in Unearned Income NET CASH PROVIDED/(USED) BY OPERATING ACTIVITIES

Page 61

1994

14,000

16,000

6,000

9,000

239,357

116,985

239,357

116,985

No.

15 1

1 1

R€MUM€PATION Of^ AUDITORS Amounts received or due and receivable by the Auditors: For auditing the accounts.

Directors’ Income Total income received or receivable by the Directors of the Company from the Company or related bodies corporate.

RECONCILIATION OF CASH. Note 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:

SHORT TERM DEPOSIT

1995

43,595

MOTG9 TO THG STATGriGMT9 Of CA9H aOW9

CASH

(ii)

210,667 473.009 683,676

The Company 1995 1994 $ $

12. DIRGaOR9 R€MUMGRATIOM

NON-CURRENT Long Service Leave

10.

Consolidated

The Company 1995 1994 $ $

The number of Directors of the Company whose total income from the Company or related bodies corporate falls within the following bands:

$10,000 $100,000 $130,000 -

234,684 562 535 797,219

20,771 505.141 525,912

$9,999 $19,999 $109,999 $139,999

The individual remuneration received by sixteen of the Directors in the band $0 - $9,999 was nil. (556,439)

(434,991)

211,531 (69,771)

191,071 68,601

(42,293) (207,572) (76,292)

(219,890) (84,109) (249,615)

(42,293) (207,572) (65,314)

(46,949)

64,162

(46,949)

(125,000) 11,527

125,000 5,696 (167,776)

(125,000) (2,183)

13,848 (90,121) 3,591

868 4,363

13,848 45,758 3,591

145,584 29 900

63,931 6529

145,584 29.900

(465,350)

(865,520)

(425,949)

Superannuation contributions paid in respect of directors are included in totaJ income received The above amounts (including the comparatives) are disclosed in accordance with a ASC Class Order 94/1529 dated 13/10/94 The names of each person holding the position of director of The Murdoch Institute for Research into Birth Defects Limited during the financial year: Professor J.A. Angus Dr. G.L. Barnes Mrs. J. Calvert'Jones Mrs. L. Cattermole Dr. R.G.H. Cotton Mr. L.G. Cox

Professor D.M. Danks Mr. I. Davies Mr. J. Fitzgerald Mr. P. Griffin Mr. J.S. Guest Mr. W.H. Hodgson

Mrs. 1. McFarling Mr. A.S. Murdoch Professor P.D. Phelan Professor G.B. Ryan Professor P. Smith Professor G. Tregear Professor R. Williamson

Dr. R.G.H. Cotton, Professor D.M. Danks, Mr. A.S. Murdoch, Professor G.B. Ryan and Professor P. Smith retired during the year. Professor P.D. Phelan retired as a director on 12th April 1995 and was reappointed on 14th February 1996.

(834,112)

Page 62

ia uABiuTY Of neMBeii^

TH€ MURDOCH ItimUTC KDR RCTCMCH IHTO BIRTH DCfCaT UMITCD m ITT COMTROLLCD CtlTITV

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 1995 the number of members of the company is 75.

Statement by Directors

14. RGLATGD PARTY DI9CL09URG9 I. Transactions with Directors: Some directors made donations to the Company during the year. Transactions with the controlled entity: Financial assistance is provided by the Company to the controlled entity by way of infrastructure and over heads. At 31 December 1995 the controlled entity owed the Company $ 167,776

In the opinion of the Directors of The Murdoch Institute for Research into Birth Defects Limited:

(a) the financial statements set out on pages 53 to 63 au-e drawn up so as to give a true and fair view of the results and cash flows for the financial year ended 31 December 1995, and the state of affairs at 31 December 1995, of the Company and the economic entity; (b) the consolidated accounts have been made out in accordance with Divisions 4A and 4B of Part 3.6 of the Corporations Law; and (c) at the date of this statement, there are reasonable grounds to believe that the company will be able to pay its debts as and when they fall due. The financial statements have been made out in accordance with applicable Accounting Standards. 2.

15. 9GGMGhT IMFORnATIOM The Company and its controlled entity operates predominantly in a single industry, being research into causes, diagnosis and treatment of genetic diseases and other birth defects, within a single geographical segment, being Australia.

Dated at Melbourne this 24 th day of April 1996.

Signed in accordance with a resolution of the Directors:

16. PARTICULAR? IM RGIATIOM TO COMTROLLCD CMTITV The Company is the only member of its controlled entity, Victorian Clinical Genetics Services Limited. No consideration was paid for this ownership, but in the event of the controlled entity being wound up, the Company may be called upon to pay an amount not exceeding ten dollars. The directors of the controlled entity are appointed by the Company. LAURENCE G. COX Contribution to Consolidated Profit/fLoss) : Directors 1995 $

1994 $

(444,015)

(434,991)

(lQ2.5Q5f (546,520)

46.976 (388,015)

ROBERT WILLIAMSON The Murdoch Institute for Research into Birth Defects Ltd Controlled entity: Victorian Clinical Genetics Services Limited

Page 63

Page 64

INDePeNDCMT AUDITOR9 RGPORT TO THG HGUBGRT Of THG MURDOCH IliTTITUTG fOR RG9GARCH IMTO BIRTH DGfGQT UMITGD

\s/ Scope We have audited the financial statements of The Murdoch Institute for Research into Birth Defects Limited for the financial year ended 31 December 1995, consisting of the profit and loss accounts, balance sheets, statements of cash flows, accompanying notes, and statement by directors set out on pages 53 to 64. The financial statements comprise the accounts of the Company and the consolidated accounts of the economic entity, being the Company and its controlled entity. The Company's directors are responsible for the preparation and presentation of the financial statements 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. 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, on 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 Accounting Standards and other mandatory professional reporting requirements (Urgent Issues Group Consensus Views) and statutory requirements so as to present a view which is consistent with our understanding of the Compamy's and the economic entity's financial position and the results of their operations, and their cashflows. The audit opinion expressed in this report has been formed on the above basis.

MURDOCH ON THE WEB The Instilute now has ils own home pa^e, at http://www.rch.unimelh.edu.au/murdoch/ A former iMurdoch staff-memher, Tvonne Mamey, designed the pa§e as part of her computer studies. The pa^e includes information about the Institute, staff vacancies, courses on offer, staff hinail addresses and details of the POSSUM/OSSUM datah ase.

Audit Opinion In our opinion, the financial statements of The Murdoch Institute for Research into Birth Defects Limited are properly drawn up: (a)

so as to give a true and fair view of; the state of affairs of the Company and the economic entity at 31 December 1995 and the results and i) cash flows of the Company and the economic entity for the financial year ended on that date; and the other matters required by Divisions 4, 4A and 4B of Part 3.6 of the Corporations Law to be dealt ii) with in the financial statements;

(b)

in accordance with the provisions of the Corporations Law; and

(c)

in accordance with applicable Accounting Standards and other mandatory professional reporting requirements.

Dated at Melbourne this 24th day of April 1996. Chartered Acc'^i^

The Murdoch Institute for Research into Birth Defects Limited Royal Children's Hospital Fiemington Road PARKVILLE VICTORIA 3052 ACN 006 566 972 Postal Address; Post Office Box 1100 PARKVILLE 3052

KPMG

Telephone: Facsimile;

(03) 9345 5045 (03) 9348 1391

R. Douglas ' Partner

Page 65

Page 66

Acknowledgements The Murdoch Institute for Research into Birth Defects Limited acknowledges the following donations:

Color scanning hy Wilke Color.

Printing by Pac-Rim Direct

Pac-Rim Direct

Produced by Anne Cronin, Kati Hidegh, Libby Kraihling, Michele Winsor

Page 67

■■V

1 i

•>

•-,'1

:'f

'M •'■Si;

rltii!: S5

I I

«!&*

for Research into Birth Defects

The Murdoch Institute