I wish you an inspiring and successful learning experience here in Zagreb. Yours sincerely, Rachel Clark CEO, EXCEMED

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3 Dear conference delegate, A warm welcome to all attending the 2014 Annual Multidisciplinary European Phenylketonuria Symposium - PKU: a lifetime of challenges. I would like to inform you that as of 28 April 2014 the name of our Foundation changed to EXCEMED - Excellence in Medical Education. The name change will not impact your registration status in this or any other Foundation event. This transition marks an exciting point in the evolution of the Foundation. We are proud to have provided world-class education to thousands of healthcare professionals over the past four decades - as a result, the Foundation has become synonymous with delivery excellence and high-impact CME. As we further develop our scientific and geographical presence it is important to us that our name accurately reflects the independent nature of the education we provide; EXCEMED symbolises our enduring mission to support the best possible outcomes for patients through the medical education we offer. We take pride in our complete dedication to the provision of CME - it is our sole focus and our passion. I wish you an inspiring and successful learning experience here in Zagreb. Yours sincerely, Rachel Clark CEO, EXCEMED 1

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5 General information Venue Sheraton Zagreb Hotel Kneza Borne 2 Zagreb, Croatia Language The official language of the symposium is English. Scientific secretariat EXCEMED Salita San Nicola da Tolentino 1/b Rome, Italy Senior Programme Manager: Francesca Pellegrino Specialist Medical Advisor: Davide Mineo Tel: Fax: info@excemed.org Organising secretariat Meridiano Congress International Via Sapri, Rome, Italy Congress Coordinator: Debora Urbinelli Tel: Fax: d.urbinelli@meridiano.it This live educational symposium is endorsed by the Croatian Pediatric Society (Section for Metabolic Diseases) We value your opinion! We are continually trying to develop and improve our educational initiative to provide you with cutting-edge learning activities. During this symposium you will be asked to answer a real-time survey and after this educational event you will receive an online survey to help us to better tailor our future educational initiatives. We thank you for participating! Register to EXCEMED website: follow us on EXCEMED_EndoMet 3

6 2014 Annual Multidisciplinary European Phenylketonuria Symposium PKU: a lifetime of challenges EXCEMED live educational symposium: 2014 Annual Multidisciplinary European Phenylketonuria Symposium PKU: a lifetime of challenges 6-7 June Zagreb, Croatia Aims of the symposium Phenylketonuria (PKU) is still a challenge for clinicians and researchers. Dietary restriction of phenylalanine (Phe) remains the main therapy, with some patients being suitable for medical treatment with sapropterin. Today s research is focusing on the pathophysiology of this disease, its neurocognitive impact, its appropriate management, and possible new therapies. Ongoing work by many scientists is leading towards the development of evidence-based European guidelines that will provide the best standard of care to all patients with PKU. This symposium will review the most important research achievements in this field, and provide a unique opportunity to share knowledge and best practice in the clinical management of PKU. Learning objectives After attending this live educational symposium, participants will have up-to-date knowledge about the latest developments in research and clinical management of PKU, and will be able to: Evaluate challenges and solutions in the management of PKU, including diet and medical therapy, and possible future treatments Interpret the pathophysiological insights into the disease and its neurocognitive repercussion on patients, and compare ways to counteract such events Consider the best approaches for management of special clinical conditions, such as maternal PKU or transition age and adulthood Discuss the development of European guidelines for the management and best care of PKU Target audience Paediatricians and specialists in inherited metabolic diseases, dietitians and nutritionists, scientists and all other healthcare professionals involved in diagnosing or managing PKU. Accreditation EXCEMED ( is accredited by the European Accreditation Council for Continuing Medical Education (EACCME ) to provide the following CME activity for medical specialists. The EACCME is an institution of the European Union of Medical Specialists (UEMS), The CME 2014 Annual Multidisciplinary European Phenylketonuria Symposium - PKU: a lifetime of challenges held on 6 7 June 2014 in Zagreb, Croatia, is designated for a maximum of 9 (nine) hours of European CME credits (ECMEC ). Each medical specialist should claim only those credits that he/she actually spent in the educational activity. EACCME credits are recognised by the American Medical Association (AMA) towards the Physician's Recognition Award (PRA). To convert EACCME credit to AMA PRA category 1 credit, please contact the AMA. EXCEMED adheres to the principles of the Good CME Practice group (gcmep) 4

7 Scientific coordinators Nenad Blau University Children's Hospitals Heidelberg, Germany and Zurich, Switzerland Hulya Gokmen-Ozel Department of Nutrition and Dietetics Hacettepe University Ankara, Turkey Anita MacDonald Dietetic Department The Children's Hospital Birmingham, UK Scientific committee Ivo Barić Department of Paediatrics University Hospital Centre Zagreb Zagreb, Croatia Amaya Bélanger-Quintana Unidad de Enfermedades Metabólicas Servicio de Pediatría Hospital Ramón y Caja Madrid, Spain Anita MacDonald Dietetic Department The Children's Hospital Birmingham, UK Júlio César Rocha Centre of Medical Genetics Centro Hospitalar do Porto Porto, Portugal Friedrich K. Trefz Children s Hospital Kreis Kliniken Reutlingen GmbH Klinikum am Steinberg School of Medicine University of Tuebingen Reutlingen, Germany Nenad Blau University Children's Hospitals Heidelberg, Germany and Zürich, Switzerland Alberto Burlina Division of Metabolic Diseases Department of Paediatrics University Hospital Padua Padua, Italy Margreet van Rijn Section of Metabolic Diseases Beatrix Children's Hospital University Medical Centre Groningen Groningen, The Netherlands Francjan J. van Spronsen Beatrix Children s Hospital University Medical Center Groningen Groningen, The Netherlands François Feillet Centre de Référence des Maladies Héréditaires du Métabolisme Hôpital d Enfants, CHU Brabois Vandoeuvre les Nancy, France Valentina Uroić Department of Nutrition and Dietetics University Hospital Centre Zagreb Zagreb, Croatia 5

8 Faculty members Ivo Barić Department of Pediatrics University Hospital Centre Zagreb Zagreb, Croatia Skadi Melanie Beblo University Children's Hospital University Hospital Leipzig Leipzig, Germany Amaya Bélanger-Quintana Unidad de Enfermedades Metabólicas Servicio de Pediatría Hospital Ramón y Caja Madrid, Spain Nenad Blau University Children's Hospitals Heidelberg, Germany and Zurich, Switzerland Alberto Burlina Division of Metabolic Diseases Department of Pediatrics University Hospital Padua Padua, Italy Maureen Cleary Department of Metabolic Medicine Great Ormond Street Hospital London, UK François Feillet Centre de Référence des Maladies Héréditaires du Métabolisme Hôpital d Enfants, CHU Brabois Vandoeuvre les Nancy, France Hulya Gokmen-Ozel Department of Nutrition and Dietetics Hacettepe University Ankara, Turkey Urh Grošelj Department of Pediatric Endocrinology, Diabetes and Metabolism University Children's Hospital, UMC Ljubljana Ljubljana, Slovenia Rianne Jahja Division of Metabolic Diseases University Medical Centre Groningen Beatrix Children's Hospital University of Groningen Groningen, The Netherlands Robin Lachmann Charles Dent Metabolic Unit National Hospital for Neurology and Neurosurgery London, UK Harvey L. Levy Department of Pediatrics Harvard Medical School Boston Boston, USA Nicola Longo Division of Medical Genetics University of Utah Salt Lake City, USA Anita MacDonald Dietetic Department The Children's Hospital Birmingham, UK Aurora Martinez Department of Biomedicine University of Bergen Bergen, Norway Priscila Mazzola Department of Molecular Neurobiology Beatrix Children's Hospital University of Groningen, The Netherlands 6

9 Faculty members Andrea Pilotto Neurology Department Prof. Daniela Berg Research Group Hertie-Institut for Clinical Brain Research and German Centre for Neurodegenerative Diseases (DZNE) Tuebingen, Germany Louise Robertson University Hospitals Birmingham NHS Foundation Trust Old Queen Elizabeth Hospital Birmingham, UK Júlio César Rocha Centre of Medical Genetics Centro Hospitalar do Porto Porto, Portugal Vlado Sarnavka Division for Metabolic Diseases Department of Paediatrics University Hospital Centre Zagreb Zagreb, Croatia Valentina Uroić Department of Nutrition and Dietetics University Hospital Centre Zagreb Zagreb, Croatia Margreet van Rijn Section of Metabolic Diseases Beatrix Children's Hospital University Medical Centre Groningen Groningen, The Netherlands Francjan J. van Spronsen Beatrix Children s Hospital University Medical Centre Groningen Groningen, The Netherlands Annemiek van Wegberg University Medical Centre Groningen Radboud University Medical Centre The Netherlands Patrícia Fernanda Schuck Laboratory for the Inborn Errors of Metabolism Universidade do Extremo sul Catarinense Criciúma (UNESC) Santa Caterina, Brazil Beat Thöny Division of Metabolism Department of Paediatrics University of Zurich Zurich, Switzerland Friedrich K. Trefz Children s Hospital Kreis Kliniken Reutlingen GmbH Klinikum am Steinberg School of Medicine University of Tuebingen Reutlingen, Germany 7

10 Scientific programme 6-7 June 2014 Friday 6 June Saturday, 7 June EXCEMED opening Rachel Clark, UK Scientific coordinator welcome and introduction Nenad Blau, Switzerland and Germany L1 PKU in Croatia and southeastern Europe countries Urh Grošelj, Slovenia, Vlado Sarnavka, Croatia Session I Chairs: Alberto Burlina, Italy, Ivo Barić, Croatia Real-time session survey L2 PEG-PAL: the latest evidence Nicola Longo, USA L3 Pharmacological chaperones in PKU: where are we now? Aurora Martinez, Norway L4 BH4 in children under 4 years Harvey L. Levy, USA Revisiting real-time session survey Q&A: let s talk Coffee break Session II New perspectives in therapy Brain function Session III Oral communications Chair: Francois Feillet, France OC1 Prediction of phenotypes and BH4-responsiveness in phenylketonuria by linking data from genotypes and locus-specific database Sarah Wettstein, Switzerland OC2 Sapropterin dihydrochloride treatment in under 4 years of age Turkish hyperphenylalaninemic patients Serap Sivri, Turkey OC3 Comparing PKU patients with and without BH4 treatment: blood prolactin and serotonin concentrations Danique van Vliet, The Netherlands OC4 Cholinergic alterations are caused by hyperphenylalaninemia in rat brain Patrícia Fernanda Schuck, Brazil OC5 Three girls with microcephaly and hyperphenylalaninemia Serap Sivri, Turkey OC6 BH4 responsiveness: Ege University experience Sema Kalkan Uçar, Turkey Coffee break Chairs: Julio Cesar Rocha, Portugal, Friedrich Trefz, Germany Real-time session survey L5 Brain function in children: the lower the Phe the better the outcomes Rianne Jahja, The Netherlands L6 Brain function and adolescence: foundation for adulthood Maureen Cleary, UK L7 Optimal Phe concentrations in adults Robin Lachmann, UK L8 Nutritional changes and micronutrient supply with BH4 Skadi Beblo, Germany Revisiting real-time session survey Q&A: let s talk Lunch Session IV Parallel workshops Workshop 1: Maternal PKU - Interactive clinical case presentations Friedrich Trefz, Germany Louise Robertson, UK Workshop 2: European and USA guidelines - controversies Francjan van Spronsen, The Netherlands Alberto Burlina, Italy Nicola Longo, USA Workshop 3: Diet plus sapropterin in clinical practice - Interactive clinical case presentations Amaya Belanger-Quintana, Spain Margreet van Rijn, The Netherlands End of day 1 8

11 Scientific programme 6-7 June 2014 Saturday 7 June Saturday, 7 June Session V From basic knowledge to daily management Session VII Asbjørn Følling and PKU awards PKU Academy fellowship Chairs: Hulya Gokmen-Ozel, Turkey, Valentina Uroić, Croatia Real-time session survey L9 PKU mouse models: what do they tell us? Beat Thöny, Switzerland L10 Oxidative stress: the evidence in PKU Priscila Mazzola, The Netherlands L11 Feeding development and nutritional issues in PKU: latest research Anita MacDonald, UK L12 Genes involved in early aging and cognitive loss: is PKU also at risk? Andrea Pilotto, Germany Revisiting real-time session survey Q&A: let s talk Coffee break Chairs: Amaya Bélanger-Quintana, Spain, Anita MacDonald, UK Asbjørn Følling lecture and award Friedrich Trefz, Germany EXCEMED award for best oral communication in PKU To be announced at the meeting PKU Academy fellowship 2012: final results Priscila Mazzola, The Netherlands PKU Academy fellowship 2013: winner presentation Patrícia Fernanda Schuck, Brazil Closing remarks Anita MacDonald, UK End of the symposium Closing lunch Session VI European guidelines Chair: Francjan van Spronsen, The Netherlands European guidelines statements: have your say Annemiek van Wegberg, The Netherlands 9

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13 Biosketches

14 Biosketches Ivo Barić Department of Paediatrics University Hospital Centre Zagreb Zagreb, Croatia Ivo Barić was born in 1959, in Zagreb, Croatia. He graduated from the School of Medicine in Zagreb. He is paediatrician and head of the Division for Metabolic Diseases, University Hospital Centre Zagreb, which is part of the national referral centre for genetic and metabolic diseases in children. He is full professor of paediatrics. During his entire paediatric career his special interest has been inborn errors of metabolism. He led or participated in several research projects. As a fellow he spent ten months in 1991 in Heidelberg, Germany, dealing with molecular genetics of phenylketonuria, and in as Alexander von Humboldt fellow he was in Marburg dealing with diagnostic tests for glutaric aciduria type 1. His major scientific contribution is the first report of the S-adenosylhomocysteine hydrolase deficiency in humans. For that work he was awarded the Horst Bickel Award in He is author of 55 articles cited in Current Contents. He was president of the Croatian Society of Human Genetics. He is a council member of the Society for the Study of Inborn Errors of Metabolism and communicating editor for the Journal of Inherited Metabolic Disease. He is a member of the European Union Committee of Experts for Rare Diseases. Skadi Melanie Beblo University Children's Hospital University Hospital Leipzig Leipzig, Germany Amaya Bélanger-Quintana Unidad de Enfermedades Metabólicas Servicio de Pediatría Hospital Ramón y Caja Madrid, Spain Dr Bélanger-Quintana studied medicine at the Universidad Autónoma de Madrid and did her paediatric residency at the Hospital Ramón y Cajal de Madrid, Spain. The Metabolic Unit of this hospital is of national reference, and she soon became interested in the field, working with them first as a fellow in an investigation level and soon after as full-time paediatrician. She is interested in the expansion of knowledge on metabolic diseases both among paediatricians and other medical specialists, being one of the paediatric resident tutors and giving classes both in medical school and doctorate settings. Within the metabolic field she has worked mainly on aminoacid disorders, especially on PKU, developing her doctoral degree on this disease. She is currently a member of several national and international inborn errors of metabolism associations, the PKU European Scientific Advisory Board, European Nutritionists Expert Panel and KAMPER Advisory Board. Nenad Blau University Children's Hospitals Heidelberg, Germany and Zurich, Switzerland Professor Blau is currently a senior consultant in biochemical genetics at the Children s Hospital in Heidelberg, Germany, and senior lecturer in biochemistry and metabolic disorders at the University of Zürich, Switzerland. His research is focused on inborn errors of neurotransmitter and BH4 metabolism and application of ipsc technology in the elucidation of new disorders. He has established a locus-specific database for PAH and genotypes database for PKU ( 12

15 Biosketches Alberto Burlina Division of Metabolic Diseases Department of Paediatrics University Hospital Padua Padua, Italy Alberto Burlina was born in 1955, at Motta di Livenza, Italy. In 1979 he graduated in Medicine at the University of Padova, and State board examination. Authorized to practice Medicine in Italy, National Board certification in Paediatrics in In 1984, he became a Clinical Research fellow at the Department of Clinical Chemistry, Hospital for Sick Children in Toronto. Since 1989 he works at the Metabolic Unit of the Department of Paediatrics, University of Padova and recently became Head of this service. He is an author and coauthor of more than 100 research publications and book chapters. His research interests include clinical and biochemical characterisation of inborn errors of intermediary metabolism, metabolic diseases in adulthood, and clinical application of tandem mass spectometry. Maureen Cleary Department of Metabolic Medicine Great Ormond Street Hospital London, UK Dr Cleary has been working as a consultant in the specialty of inborn errors of metabolism since 1998 initially in Manchester, UK, and currently, since 2002, in Great Ormond Street Hospital for Children NHS Trust/ Institute of Child Health, London, where she holds the posts of Consultant in Metabolic Medicine and Senior Lecturer. From 2008 to 2011 she was in Singapore where she was Senior Consultant at KK Women s and Children s Hospital and associate professor at the Duke-NUS Medical Faculty. She sees children with a range of metabolic disorders in both acute and chronic settings and has been principle investigator for many research studies including PKU and lysosomal storage disorders. François Feillet Centre de Référence des Maladies Héréditaires du Métabolisme Hôpital d Enfants, CHU Brabois Vandoeuvre les Nancy, France François Feillet is a professor of paediatrics at the Reference Centre for Inborn Errors of Metabolism, Children's University Hospital of Nancy, France. He received his MD in 1990 and has been a paediatrician since He received his PhD on metabolic consequences of malnutrition in He has been professor of paediatrics since He was paediatric intensivist for 13 years and is now the head of one of the six national reference centres for Inborn Errors of Metabolism in France. He treats patients with metabolic diseases from birth to adulthood. At a national level, Professor Feillet is a member of the Paediatric Nutrition Committee and of the scientific committee of the French Society of Paediatrics. He is a member of the national board of the society for systematic newborn screening and he was president of the national board of the French Society for IEM until At an international level, he is a member of various European and international advisory boards and working groups on both phenylketonuria and other metabolic disorders. 13

16 Biosketches Hulya Gokmen-Ozel Department of Nutrition and Dietetics Hacettepe University Ankara, Turkey Dr Hulya Gokmen-Ozel, Associate Professor in Nutrition and Dietetics at Hacettepe University, is a pediatric dietician. She received her MSc in 1998 and PhD in Between 2008 and 2009 she attended several research and development projects in the field of inherited metabolic diseases at Birmingham Children s Hospital. She was awarded a grant by The Scientific Technical Research Council of Turkey as a postdoctoral fellow in PKU. She is now involved in teaching and training activities of the dietetic students and patients/families. She is a member of numerous national committees, the Society for the Study of Inborn Errors of Metabolism (SSIEM), SSIEM Dietitians Group Steering Committee and European Expert Panel in PKU. Her main aim is to improve dietetic management of the patients with inherited metabolic diaseases in Turkey. Urh Grošelj Department of Paediatric Endocrinology, Diabetes and Metabolism University Children's Hospital, UMC Ljubljana Ljubljana, Slovenia Dr Groselj was born in 1980 in Ljubljana, Slovenia. After obtaining his MD degree in 2007 at the Faculty of Medicine, University of Ljubljana, Slovenia, he also obtained an MA degree in bioethics in 2008 at the University of Leuven, Belgium. In 2012 he finished his PhD at the Faculty of Medicine, University of Ljubljana with the thesis entitled, Analysis of the phenylalanine hydroxylase gene and assessment of the correlation with the phenotype in patients with phenylketonuria. Since 2009 he has been a resident in pediatrics at the Department of Paediatric Endocrinology, Diabetes and Metabolism, University Children s Hospital, Ljubljana. From 2012 he has been a teaching assistant of paediatrics at the Faculty of Medicine, University of Ljubljana. Since 2012 he has served as a member of the National Committee on Medical Ethics. His current main clinical and research interests include inborn errors of metabolism, especially phenylketonuria, mild hyperphenylalaninemia and familial hypercholesterolemia, and also neonatal metabolic screening. Altogether, he has coauthored over 100 publications; some of which have also appeared in SCI journals as Molecular Genetics and Metabolism, Journal of Inherited Metabolic Disorders, Clinical Biochemistry and Slovenian Medical Journal. Rianne Jahja Division of Metabolic Diseases University Medical Centre Groningen Beatrix Children's Hospital University of Groningen Groningen, The Netherlands After completing the Research Master s in Clinical Child and Adolescent Studies with specialty in Developmental Psychopathology, Rianne Jahja started in May 2011 as a PhD student at the Department Metabolic Diseases of the Beatrix Children s Hospital, University Medical Centre Groningen, The Netherlands. Her supervisors are Professor Dr Francjan van Spronsen and Dr Stephan Huijbregts who are well known in and are strongly involved in the field of phenylketonuria. At the moment she is conducting a Dutch multicentre study, the PKU-COBESO study, which aims to examine COgnition (executive functions), BEhaviour and SOcial functioning in early and continuously treated patients with PKU. She is also in collaboration with other national and international centres to expand the knowledge of cognition, behaviour, social factors and treatment guidelines in PKU. 14

17 Biosketches Robin Lachmann Charles Dent Metabolic Unit National Hospital for Neurology and Neurosurgery London, UK Dr Robin Lachmann is one of two consultants at the Charles Dent Metabolic Unit, London, UK, where over 1,300 adult patients with a wide range of inherited metabolic diseases are treated. After qualifying in 1990, Dr Lachmann continued his training in Internal Medicine and Metabolic Medicine, as well as performing PhD research on herpes simplex virus-mediated gene delivery to the brain and postdoctoral work on glycosphingolipid lysosomal storage disorders. Harvey L. Levy Department of Pediatrics Harvard Medical School Boston Boston, USA Dr Levy has devoted his career of over 40 years to metabolic disorders, primarily phenylketonuria (PKU). He began his career training in pediatrics in Boston, New York, and Baltimore, USA. Returning to Boston he completed his pediatric training and then began training in metabolic disorders under Dr Mary Efron at the Massachusetts General Hospital, USA. Subsequently, he joined the faculty at that hospital and the Harvard Medical School, became consultant in newborn screening to the Commonwealth of Massachusetts, and then chief of biochemical genetics in the New England Newborn Screening Program and director of the PKU/Metabolic Program at Boston Children s Hospital. Dr Levy is senior physician in medicine and genetics at Boston Children s Hospital and professor of pediatrics at Harvard Medical School. He also serves as Chairman of the Workgroup on Newborn Screening Diagnosis and Follow-up and is the former chairman of the steering committee of the Newborn Screening Translational Research Network, both for the American College of Medical Genetics. Dr Levy has published over 400 medical articles and book chapters on metabolic disease, most of which are on PKU. He was the 2012 Asbjørn Følling Memorial Lecturer at the SSIF Symposium in Rome. Nicola Longo Division of Medical Genetics University of Utah Salt Lake City, USA Dr Longo received his MD and PhD in molecular biology and pathology from the University of Parma School of Medicine in Italy. He trained in pediatrics, medical and biochemical genetics at Emory University in Atlanta, Georgia, USA. Dr Longo is board certified in medical genetics and clinical biochemical genetics. He is currently professor of pediatrics and pathology at the University of Utah, USA, chief of the Division of Medical Genetics, director of the Metabolic Service in the Department of Pediatrics, director of the Training Program in Clinical Biochemical Genetics and medical co-director of the Biochemical Genetics and Newborn Screening Laboratories at ARUP Laboratories in Salt Lake City. His research concerns the molecular bases of disorders of membrane transport and fatty acid oxidation. He has an active clinical research program directed toward the development of new therapies for patients with metabolic disorders. In the past few years, his clinical research has focused on the psychometric evaluation and development of new therapies for patients with phenylketonuria. 15

18 Biosketches Anita MacDonald Dietetic Department The Children's Hospital Birmingham, UK Anita MacDonald is consultant dietitian in inherited metabolic disorders at Birmingham Children s Hospital, UK, and an honorary professor in dietetics at Plymouth University, UK. She has a wealth of experience in paediatric dietetics, having worked as a clinical dietetic specialist for 35 years. She is a fellow of the Royal College of Paediatrics and Child Health (RCPCH). Dr MacDonald obtained her PhD in phenylketonuria (PKU) in 1999 and is actively involved in all aspects of IMD nutritional research, as well as teaching and development. She has extensive research experience and more than 300 publications. She has lectured in more than 30 different countries on topics relating to nutrition, and is currently one of the leads for the Inherited Metabolic Disease module of the Paediatric Group of the British Dietetic Association/University of Plymouth. She is a member of numerous UK (including chair of Dietetic Group, BIMDG) and international Committees (including the chair of the European Expert Nutrition Panel in Phenylketonuria (ENEP), editorial board member of PKU Academy). She is a member of three European IMD guideline groups. Aurora Martinez Department of Biomedicine University of Bergen Bergen, Norway Aurora Martinez obtained her PhD in biochemistry in1988 (University of Basque Country, Bilbao, Spain), and was postdoctor in at the University of Bergen, Norway, where she became associate professor in 1995 and full professor in1997. She directs the research group Biorecognition, contributing to the development and application of biochemical, biophysical and computational methods, and high through-put screening of small compounds, applied to investigations of stability and function of aromatic amino acid hydroxylases, notably phenylalanine hydroxylase (PAH), and other biomolecular proteins and networks of clinical relevance, with special focus on the genotype-phenotype correlations and therapeutic approaches in phenylketonuria (PKU) and other rare diseases. In this field, Professor Martinez has contributed with important publications characterizing molecular mechanisms behind tetrahydrobiopterin (BH4) responsive PKU and the effect of pharmacological chaperones on PAH and tyrosine hydroxylase (TH) function and stability, showing that pharmacological chaperones have therapeutic potential in the correction of misfolding diseases. Professor Martinez has been council member in international scientific committees for the European Molecular Biology Laboratory and the Human Frontier Science Program ( ); member of the Norwegian Academy of Sciences and Letters since 2007; and partner of the K.B. Jebsen Centre for Neuropsychiatric Diseases. She has also authored 130 peer-reviewed publications since For publications and additional information see Priscila Mazzola Department of Molecular Neurobiology Beatrix Children's Hospital University of Groningen, The Netherlands Priscila Mazzola has been studying oxidative stress and exercise effects in PKU since she was an undergraduate student in physical education. She has a Master s degree in biochemistry from the Federal University of Rio Grande do Sul (UFRGS), Brazil. In 2012, she won the PKU Academy Fellowship to carry out a project with PKU patients and PKU mouse model, part in Brazil, part at the University of Groningen in The Netherlands. Nowadays she is a double-degree PhD student at the University of Groningen and UFRGS. 16

19 Biosketches Andrea Pilotto Neurology Department Prof. Daniela Berg Research Group Hertie-Institut for Clinical Brain Research and German Centre for Neurodegenerative Diseases (DZNE) Tuebingen, Germany Dr Pilotto graduated from the University of Padova School of Medicine, Italy, in In 2010 he started his residency programme at the Neurology Department of Brescia, Italy, under the supervision of Professor Padovani. His special research interests are clinical, neuroimaging and genetic aspects of neurodegenerative disorders, especially presenile dementia and parkinsonism. In 2013 he joined the research group of Professor Berg, in Tuebingen, Germany, for a research fellowship focused on early neurodegenerative biomarkers. Since 2014 he has been collaborating with Professor Trefz and Professor Freisinger on a project with middle-aged ETPKU patients, with a special focus on possible neurodegenerative aspects, in Tuebingen, Germany. Louise Robertson University Hospitals Birmingham NHS Foundation Trust Old Queen Elizabeth Hospital Birmingham, UK Louise Robertson is a specialist dietitian working in inherited metabolic diseases. After obtaining her degree in 2002 Louise gained experience in a wide range of dietetics. In 2007 she was employed to help set up the new adult Inherited Metabolic Diseases centre at the Queen Elizabeth Hospital in Birmingham, UK. She is on the committee for the dietitian s group of the British Inherited Metabolic Diseases Group and sits on the medical advisory panel of the Galactosaemia Support Group UK. Júlio César Rocha Centre of Medical Genetics Centro Hospitalar do Porto Porto, Portugal Júlio César Rocha was nutritionist at the Faculty of Food and Nutrition Sciences, University of Porto, Portugal, in 2001; and post-graduate in clinical nutrition there in A PhD student in metabolism: clinics and experimental (Faculty of Medicine, University of Porto), his main working experience in the field of PKU includes: nutritionist at the Centre of Medical Genetics, National Institute of Health, Porto; and tutoring/teaching group classes in the disciplines of biochemistry I and II at the Faculty of Medicine, University of Porto. Vlado Sarnavka Division for Metabolic Diseases Department of Paediatrics University Hospital Centre Zagreb Zagreb, Croatia 17

20 Biosketches Patrícia Fernanda Schuck Laboratory for the Inborn Errors of Metabolism Universidade do Extremo sul Catarinense Criciúma (UNESC) Santa Caterina, Brazil Beat Thöny Division of Metabolism Department of Paediatrics University of Zurich Zurich, Switzerland Beat Thöny, PhD, is a clinical biochemist (FAMH) and professor of clinical biochemistry at the University of Zurich. He obtained his master and PhD in natural sciences from the Swiss Federal Institute of Technology in Zurich, Switzerland, and is currently head of research in the Metabolic Division and responsible for the diagnosis of genetic, neurotransmitter and tetrahydrobiopterin disorders in Division of Clinical Chemistry and Biochemistry at the University Children s Hospital in Zurich, Switzerland. He is author of numerous research publications with a main research interest in gene characterization and novel molecular therapies for metabolic liver and neurometabolic (brain) diseases. Friedrich K. Trefz Children s Hospital Kreis Kliniken Reutlingen GmbH Klinikum am Steinberg School of Medicine University of Tuebingen Reutlingen, Germany Professor Dr med. Friedrich Trefz received his medical degree from the University of Heidelberg in After receiving his degree he had a training in the biochemical laboratory at the Childrens Hospital in Heidelberg, and in 1975 he started his training in pediatrics under Prof.Dr.Horst Bickel. In 1983 he received his degree in Paediatrics, in 1985 he became the head of the Sektion Pädiatrische Stoffwechselkrankeheiten at the University Children s Hospital of Heidelberg. Later in 2009 he received an additional degree in Paediatric Diabetes and Endocrinology. Professor Dr med Trefz has received many grants from the German Federal Ministry of Education and Research, from he was pricipal investigator of the International Maternal PKU study together with Prof. Richard Koch (Los Angeles), and Prof. Harvey Levy (Boston). He also was principal German investigator in the International Sapropterin Study from 2005 to In 1992 he moved from Heidelberg to Reutlingen and became the head of the department of pediatrics at the Klinik für Kinder und Jugendmedizin at Klinikum am Steinenberg, Reutlingen, School of Medicine, University of Tuebingen. In 2010 he retired from the Childrens Hospital in Reutlingen and is now a paediatrician and medical director of the outpatient medical centre for Women, Children and Adolescents, Kreiskliniken Reutlingen, in Gammertingen, Germany. He is still a professor of the University of Tuebingen, Germany. Beside of general pediatrics he is still treating patients with metabolic disorders focused on phenylketonuria and maternal phenylketonuria. 18

21 Biosketches Valentina Uroić Department of Nutrition and Dietetics University Hospital Centre Zagreb Zagreb, Croatia Valentina Uroić, MSc in Nutrition, graduated from the Faculty of Food Technology and Biotechnology at the University of Zagreb, Croatia. She has been working as clinical dietitian at the Department of Nutrition and Dietetics of the University Hospital Centre Zagreb, as a part of the hospital nutrition team since Her special interest is diet therapy for metabolic diseases and inherited metabolic disorders in children and adults, as she works in inpatient and outpatient settings educating patients. She is the co-author of a chapter in a textbook called, 'Diabetes mellitus in adults,' an edition of the School of Medicine, University of Zagreb. She has also completed a professional course entitled, 'Management of diabetes mellitus', in Petersfield, UK. She has given several lectures on nutrition in hereditary metabolic disorders and is educated through a series of conferences and scientific meetings. She completed professional training entitled 'Metabolic University' at the Children s Hospital Colorado, in Denver, Colorado, USA. At the moment, she is involved as a student in a postgraduate PhD Study of Nutrition at the Faculty of Food Technology and Biotechnology, University of Zagreb. Margreet van Rijn Section of Metabolic Diseases Beatrix Children's Hospital University Medical Centre Groningen Groningen, The Netherlands After certification as dietitian her professional career started (and never ended) in the University Medical Hospital of Groningen, The Netherlands. Being involved in different departments of the hospital, from 1980 onwards her focus narrowed to paediatrics, which became the most important part of her activities. Metabolic diseases offered great chances to get involved in high-tech dietetics. The forum of information exchange expanded from national to international contacts and also became progressively more disease specific. Experience in clinical practice does not constitute scientific evidence, but rather is the source of research questions. The next step was a research project, resulting in her PhD titled From experience to evidence. Nowadays she is active as clinical and research dietitian, focused in particular on metabolic diseases for all age groups, with a special interest and expertise in phenylketonuria and glycogen storage diseases. She is also involved in national and international educational activities and working groups for metabolic dietitians and clinicians. Francjan J. van Spronsen Beatrix Children's Hospital University Medical Centre Groningen Groningen, The Netherlands Francjan J. van Spronsen finished his PhD in 1996 on phenylketonuria and his general pediatric training in He started in the field of metabolic diseases in 1983 as a MD student-phd student and after some years of general pediatrics he rejoined the metabolic section. At a national level, he chairs the Dutch Advisory Committee on Neonatal Screening for inherited metabolic diseases, is a member of the Dutch Committee on Neonatal Screening, chairs the Scientific Advisory Board of the European Society of PKU and allied disorders and is an active member of various European and international advisory boards and working groups on various inborn errors of metabolism. His papers focus on metabolic and cerebral processes in especially phenylketonuria, urea cycle defects and tyrosinemia type I. He has coordinated various undergraduate MD programs, and vice-chaired the exam committee for M.D. students of the University of Groningen. He is married and has seven daughters and one son. 19

22 Biosketches Annemiek van Wegberg University Medical Centre Groningen Radboud University Medical Centre The Netherlands Annemiek van Wegberg graduated as a dietitian in 2009 and as a clinical epidemiologist in She has worked as a paediatric metabolic dietitian since January 2010 at RUNMC (Radboud University, Nijmegen Medical Centre, The Netherlands). In February 2013 she started as the project assistant in the development of European guidelines on PKU at UMCG (University Medical Centre of Groningen, The Netherlands). 20

23 Abstracts

24 L1. PKU in Croatia and southeastern Europe countries Urh Grošelj Department of Pediatric Endocrinology, Diabetes and Metabolism University Children's Hospital, UMC Ljubljana Ljubljana, Slovenia Vlado Sarnavka Division for Metabolic Diseases, Department of Paediatrics University Hospital Centre Zagreb Zagreb, Croatia Background: Management of phenylketonuria (PKU) varies widely throughout the countries and regions. Nevertheless, all the existing guidelines on PKU screening and management agree, that the neonatal PKU screening is necessary, for the treatment to be started as early after birth as possible, and that PKU dietary treatment with regular monitoring of blood phenylalanine levels is necessary at least throughout childhood and adolescence. However, in contrast to most other parts of Europe, very scarce data exist on PKU management in the region of southeastern Europe. Aims: To assess the current state of PKU screening and management in the region of southeastern Europe (i.e. Balkan Peninsula countries). Materials and methods: The survey was performed involving the identified professionals responsible for the PKU management in the 11 countries from the southeastern region of Europe (Albania, Bulgaria, Bosnia and Herzegovina, Croatia, Kosovo, Macedonia, Moldova, Montenegro, Romania, Serbia, Slovenia). The questionnaire was designed to assess the characteristics regarding PKU management in three main areas: PKU screening, nationwide characteristics, characteristics of the PKU management in the responding centre. It consisted of 56 questions in total. The distribution and collection of the questionnaires (via ) took place place from December 2013 to March Results: Responses from participants from 11 countries were included; the countries cumulative population is approx million. PKU screening was not yet introduced in 4 of 11 countries. Reported PKU incidences ranged from 1/7325 to 1/39338 (and were not known for 5 countries). National PKU guidelines existed in 5 of 11 countries, and 7 of 11 countries had PKU registry (registries included 10 to 194 patients). The number of PKU centres in each country varied from 1 to 6. Routine genetic diagnostics was reported in 4 of 11 countries. the most commonly used laboratory method to assess phenylalanine levels was fluorimetric. Tetrahydrobiopterine was used in only 2 of 11 countries. Most frequently, paediatricians were caring for the patients. A dietitian was a member of the PKU team in only 4 of 11 countries, while regular psychological assessments were performed in 6 of 11 countries. A patient's PKU society existed in 7 of 11 countries. Conclusions: Assessment of the current state of PKU screening and management in the region of southeastern Europe showed the region to face certain important challenges. Neonatal PKU screening should be introduced throughout the region. Furthermore, PKU management was in many aspects falling behind internationally established standards of care. 22

25 L2. PEG-PAL: the latest evidence Nicola Longo Division of Medical Genetics, University of Utah Salt Lake City, USA Phenylketonuria (PKU) is caused by impaired activity of phenylalanine hydroxylase, a tetrahydrobiopterin-dependent enzyme that converts phenylalanine to tyrosine. The resultant accumulation of phenylalanine (Phe) can lead to neurocognitive dysfunction, ameliorated by a special low-phe diet initiated early in life. Compliance with diet worsens as patients become older. In our patients, increased levels of Phe were associated with decreased perceptual reasoning in children and psychiatric distress in adults. Recombinant Anabaena variabilis phenylalanine ammonia lyase conjugated with polyethylene glycol (ravpal-peg) converts Phe to ammonia and trans-cinnamic acid. In a Phase 1 trial, ravpal-peg at 0.1 mg/kg significantly reduced Phe levels (36%-97% reduction) after a single injection in patients with PKU and was well tolerated, with injection-site reactions as the most frequent adverse event. All patient developed antibodies against PEG after a single injection and some patients reacted to PAL as well. In the Phase 2 clinical trial (open-label, multi-center), repeated administration of ravpal-peg was effective in reducing Phe levels in the normal or therapeutic range in patients with phenylketonuria on an unrestricted or moderately protein-restricted diet. Reduction of PKU levels was not immediate, but required several weeks of therapy. Side-effects consisted of injection site reactions and, in some cases, generalised reactions. A few patients developed hypophenylalaninemia that produced no symptoms and resolved by lowering drug dosage. All patients became proficient in drug self-administration. A Phase 3, randomised, double-blind, placebocontrolled, discontinuation study to evaluate the efficacy and safety of subcutaneous injections of ravpal-peg self -administered by adults with PKU was recently initiated. These data show that novel therapies in addition to diet can help in controlling Phe levels in patients with PKU. 23

26 L3. Pharmacological chaperones in PKU: where are we now? Aurora Martinez Department of Biomedicine, University of Bergen Bergen, Norway Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism caused by mutations in phenylalanine hydroxylase (PAH). As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant PAH to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilisation of PKU-mutants in vitro, in cells, and in mice by small compounds that have been found either by using tetrahydrobiopterin (BH4), the PAH cofactor, as query structure for shape-focused virtual screening or by high-throughput screening of commercially available compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH4. Furthermore, PAH shares a number of ligands, such as cofactor, amino acid substrates and inhibitors, with the other aromatic amino acid hydroxylases: the neuronal/neuroendocrine enzymes tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatised to avoid unwanted interactions with these other enzymes. After derivatisation, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU. 24

27 L4. BH4 in children under 4 years Harvey L. Levy Department of Pediatrics, Harvard Medical School Boston Boston, USA BH4 has been a very important addition to the treatment of phenylketonuria (PKU), in some affected individuals lowering their blood phenylalanine levels and increasing tolerance for dietary protein, thus allowing more natural food in the diet. However, BH4 as sapropterin, the synthetic form of BH4 now available as a drug, has often been limited in children less than 4 years of age. This is a result of the labeling which states that clinical trials have involved subjects who were 4 years of age and older. While current labeling in the USA does not prohibit the use of sapropterin in children below the age of 4 years it states that safety and efficacy have not been established by clinical studies in patients less than 4 years of age, and this has had the effect on some physicians and parents of not wanting to give it to children less than 4 years old. Recent studies and anecdotal experience have indicated that sapropterin is not only safe in children younger than 4 years but may also be more effective in lowering the blood phenylalanine level than at older ages. As a consequence sapropterin is being relabeled in the USA to indicate that it can be used at ages 1 month to 6 years but, because it may have this more potent effect at those ages, the starting dose should be 10 mg/kg daily instead of the mg/kg daily recommended in older children and adults. There would now seem to be no reason to withhold sapropterin from those at any pediatric age because of concern over safety or efficacy. 25

28 L5. Brain function in children: the lower the Phe the better the outcomes Rianne Jahja Division of Metabolic Diseases, University Medical Centre Groningen, Beatrix Children's Hospital University of Groningen, Groningen, The Netherlands The main objectives are to review evidence concerning the importance of lower phenylalanine (Phe) levels and to review treatment guidelines in relation to neurocognitive outcomes. Untreated phenylketonuria (PKU) is known to cause, among others, severe mental retardation, neurological problems, motor deficits, and behavioural problems during childhood. All of these can be largely prevented by early introduction of dietary Phe restriction following neonatal screening. However, neurocognitive outcomes are still suboptimal: average IQ in treated PKU is 8 to 10 points lower than normal, while patients perform less accurately and more slowly than controls on several neuropsychological tasks. Cognitive impairments in treated PKU have been associated with concurrent blood Phe levels and, even more strongly and consistently, with lifetime blood Phe. Notwithstanding the known positive effects of decreasing blood Phe levels, the upper target Phe level varies not only between countries, but also from centre to centre. For the first decade of life, recommendations for upper target Phe levels usually vary between 240 and 360 µmol/l. A study by Burgard et al. (1997) among 22 French patients, 23 German patients and healthy controls demonstrated that PKU children with concurrent Phe below 360 µmol/l performed as well as controls on information processing speed, concluding that it would be safer to keep Phe levels below 360 µmol/l for the first 10 years of life. A distinction of Phe below and above 360 µmol/l was also made by Griffiths et al. (2000) in a group of 57 children, where those with annual lower Phe had an IQ 10 points higher. De Sonneville et al. (2010) showed that children with concurrent Phe below 360 µmol/l performed better on attentional processing, but also that lifetime Phe was a better predictor. There is only one study by Schmidt et al. (1996) proving that 240 µmol/l is the best upper target limit for a better development of IQ. The recommended guidelines, however, are not based on studies comparing outcomes at Phe levels below 240 µmol/l, between 240 and 360 µmol/l, and above 360 µmol/l. Our PKU-COBESO study showed that 6- to 15-year-old PKU patients with mean lifetime Phe below 240 µmol/l outperformed those with higher mean Phe, and also outperformed those with mean lifetime Phe levels between 240 and 360 µmol/l, on neurocognitive tasks (Jahja et al., 2014). As the brain develops rapidly in childhood, and is therefore vulnerable for high Phe concentrations, it is important to monitor neurocognitive outcomes and preferably to maintain Phe below 240 µmol/l. 26

29 L6. Brain function and adolescence: foundation for adulthood Maureen Cleary Department of Metabolic Medicine, Great Ormond Street Hospital London, UK Phenylketonuria (PKU) treatment usually requires life-long adherence to a low phenylalanine Phe diet in order to protect brain development and function. It is now recognised that brain development continues beyond early childhood and good biochemical control of PKU is necessary to achieve the best cognitive outcome. Adolescence is a time when adherence to medical therapy is challenging and poorer biochemical control is often seen at this age in individuals with PKU. In order to promote better adherence, the PKU team should consider several issues including: phase of adolescence, peer support, family dynamic, consistency of professional input, suitability of the clinic setting. The desired Phe levels in this age group remains a controversial issue; the evidence of brain development and cognitive function and the need for maintaining good Phe levels throughout childhood and adolescence will be discussed. 27

30 L7. Optimal Phe concentrations in adults Robin Lachmann Charles Dent Metabolic Unit, National Hospital for Neurology and Neurosurgery London, UK The concept of diet for life is intellectually appealing, but in reality we know that many adult patients are off diet and leading normal lives, and that the metabolic control of those who stay on diet deteriorates as they get older. Adult patients with phenylketonuria (PKU) have been shown to have white matter abnormalities on imaging and defects in executive function and reaction times on neuropsychological testing. However, many of these findings seem to relate to historical rather than current phenylketonuria (PKU) levels. White matter lesions on MRI and reaction time appear to be related to Phe levels at the time of testing, but are reversible if dietary treatment is re-instituted. There is no difference in reaction times between subjects with PKU and controls at Phe levels of 800 mcmol/l or less. The clinical significance of these findings is unclear. Real life practice shows that, even after periods of excellent metabolic control (as with preconception and pregnancy diets), the vast majority of adult patients choose higher Phe levels over the rigours of diet, and continue to lead productive lives. 28

31 L8. Nutritional changes and micronutrient supply with BH4 Skadi Melanie Beblo University Children's Hospital, University Hospital Leipzig Leipzig, Germany Abstract not available at the time of printing. 29

32 L9. PKU mouse models: what do they tell us? Beat Thöny Division of Metabolism, Department of Pediatrics, University of Zurich Zurich, Switzerland Originally, three distinct mouse models for classical human phenylketonuria (PKU) or hyperphenylalaninemia (HPA) were generated and described in the BTBR genetic background, the so called Pah-enu1, Pah-enu2 and Pah-enu3. Since then, they have proved to be invaluable assets in the investigation of PKU pathophysiology and the development of diet-, cell- and gene-based therapies for PKU. These animals were the product of a random chemical (N-ethyl-N-nitrosourea, ENU) mutagenesis project undertaken in the laboratory of Dr William Dove at the University of Wisconsin-Madison, USA. While the Pah-enu1 is a model for mild PKU/HPA, the other two are models for classical PKU. The most widely used model is probably the Pah-enu2 mouse which carries a missense mutation that completely inactivates the PAH protein. PKU mice are hyperphenylalaninemic, hypopigmented, mildly growth-retarded and cognitively impaired relative to wild-type or heterozygous mice. Affected females are fertile, but in a phenomenon that is quite similar to human maternal PKU, many of the offspring have structural defects including congenital heart lesions. Fertility is also limited in hyperphenylalaninemic Pah-enu2 homozygous sires on the original BTBR genetic background; crossbreeding of the Pah-enu2 mutation onto a different inbred background such as C57Bl/6 has resulted in more facile breeding and greater success at colony maintenance (B. Thöny, personal observation). The Pah-enu2 mouse is thus an outstanding model for human PKU, one of the most common inborn errors of metabolism. Investigations of phenylalanine (Phe) metabolism, novel dietary treatment, therapeutic liver repopulation, enzyme substitution therapy, and various gene transfer experiments using this mouse model have been extremely informative to study liver and brain abnormalities and pathology under elevated blood L-Phe levels. Besides the Pah-enu2 mouse, the heteroallelic mutant Pah-enu1/2 and also the very mildly affected Pah-enu1 mice strains became important specifically for pathophysiological studies of BH4-cofactor-responsivenes and/or chemical pharmachaperone loading to stabilise the hepatic PAH The most recent application is the use of heterozygous Pah-enu2/wt mice (with normal blood L-Phe levels) as a sensitive assay system to test antisense oligonucleotides for splice suppression therapy in the liver. In my presentation, I will focus on the Pah-enu2 mouse and the recent development in the field of hepatic gene therapeutic 4 and gene suppression testing 5. References 1. Lagler FB, Gersting SW, Zsifkovits C, et al. New insights into tetrahydrobiopterin pharmacodynamics from Pah enu1/2, a mouse model for compound heterozygous tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. Biochem Pharmacol 2010;80: Sarkissian CN, Ying M, Scherer T, et al. The mechanism of BH4-responsive hyperphenylalaninemia - as it occurs in the ENU1/2 genetic mouse model. Hum Mutat 2012;33: Gersting SW, Lagler FB, Eichinger A, et al. Pah-enu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo. Hum Mol Genet 2010;19: Viecelli HM, Harbottle RP, Wong SP, et al. Treatment of phenylketonuria (PKU) using minicircle-based naked-dna gene transfer to murine liver. Hepatology 2014; in press. 5. Gallego-Villar L, Viecelli HM, Pérez B, et al. A sensitive assay system to test antisense oligonucleotodes for the splice suppression therapy in the mouse liver 2014; submitted. 30

33 L10. Oxidative stress: the evidence in PKU Priscila Mazzola Department of Molecular Neurobiology, Beatrix Children's Hospital University of Groningen, The Netherlands Oxidative stress is characterised by an imbalance in production and removal of reactive species and free radicals. These harmful substances are normally formed during cellular respiration and they easily react with biomolecules as DNA, proteins and lipids. Oxygen is essential for proper energy production in mitochondria; however, many oxygen-derived toxic compounds are formed during this process. Cellular damage is prevented by the antioxidant system, which consists of enzymatic and non-enzymatic defenses. These defenses are able to convert reactive species of oxygen into non-toxic compounds, avoiding biomolecule damage. However, when production of reactive species is high and/or the defenses are not working properly, oxidative stress takes place. Oxidative stress has been related with several pathological conditions and also in phenylketonuria (PKU). In vitro, high phenylalanine concentration (similar to those found in patients) leads to oxidative stress in neurons, although the mechanisms are not yet completely elucidated. PKU animal models have shown oxidative stress in the brain, due to both increased production of reactive species and decreased antioxidant capacity. As this organ is the most affected in PKU, it has been stated that oxidative stress may be involved in the pathophysiology of the brain dysfunction. Studies in PKU patients have found increased biomolecule damage and lower antioxidant defenses in plasma. Due to the hard dietary compliance, even early-treated PKU patients often show high blood phenylalanine, potentially leading to oxidative stress. In this way, many studies on antioxidants (or redox active substances) have been conducted, showing that these substances can actually avoid oxidative stress in PKU. As the PKU diet is quite restricted, the metabolic formula is an important source of micronutrients for patients. Some of these compounds also act as antioxidants, playing an important role in improving antioxidant capacities. Following strict dietary treatment along with the metabolic formula shows beneficial outcomes on oxidative stress parameters. New antioxidant therapies are constantly being studied in order to prevent oxidative stress in PKU, thus avoiding its related issues. 31

34 L11. Feeding development and nutritional issues in PKU: latest research Anita MacDonald Dietetic Department, The Children's Hospital Birmingham, UK Background/Objectives: In phenylketonuria (PKU), little is known about the effect of dietary restriction and L-amino acid (bitter taste) exposure on food preference development. This observational, controlled, prospective study aimed to determine the flavour preferences of children with PKU compared with healthy control children. Methods: 35 children with PKU and 35 age/gender matched control children aged 4-13 years, tasted 10 blinded puree foods (apple, banana, strawberry, custard, broccoli, cauliflower, carrot, sweet potato, lemon, coffee) in random order, rating them using a 7-point pictorial hedonic scale (super yummy to super yucky) and then ranking them in preferential order. Carers completed a neophobia and food frequency questionnaire. Results: Both PKU and control groups rated sugar containing foods highly. Children with PKU rated sweet potato (p=0.007), carrot (p=0.008) and custard (p=0.001) higher whilst apple was rated higher by control children (p=0.02). Generally children with PKU ranked vegetables higher than controls and the controls ranked fruit higher than children with PKU. Children with PKU had more overall neophobia (uncomfortable in unfamiliar environments) and were untrusting/fearful of new foods. Compared with control children, those with PKU consumed % more high energy: sugar containing drinks, sweets, chips, sweet biscuits, crisps, and butter/margarine. Conclusions: Children aged 4 years with PKU preferred sweet foods compared with savoury foods. This study did not support the theory that the early introduction of bitter tasting phenylalanine-free L-amino acids altered food preferences in children with PKU but they were suspicious of new foods and flavours. 32

35 L12. Genes involved in early aging and cognitive loss: is PKU also at risk? Andrea Pilotto Neurology Department, Prof. Daniela Berg Research Group, Hertie-Institut for Clinical Brain Research and German Centre for Neurodegenerative Diseases (DZNE), Tuebingen, Germany Presentation main objective: to discuss the risk of neurodegenerative disorders in early-treated phenylketonuria (ETPKU) patients. Neurodegenerative disorders and genetics: Alzheimer s (AD) and Parkinson s (PD) diseases are the most common neurodegenerative disorders, affecting an increasing number of individuals in the increasingly older population of many societies. Mutations within several genes have been associated with rare monogenic AD and PD presenile forms. On the other hand, relatively common genetic variations have been linked to an increased risk of sporadic AD and PD cases. Early neurodegeneration: mechanisms and biomarkers: Oxidative stress, mitochondrial dysfunction, vitamins and neurotransmitter deficits have been consistenly associated with cognitive dysfunction and neurodegeneration. An underlying neurodegenerative process could be detected early by olfactory dysfunction, cerebrospinal fluid (CSF) and imaging biomarkers. Neurological and psychiatric involvement of ETPKU subjects: Most ETPKU patients present in early adulthood subtle neurological symptoms, especially in behavioural, executive, visuo-spatial and motor functions. The damage to the central nervous system (CNS) has been explained by several mechanisms including vitamins or neurotransmitter deficits, increased oxidative stress and white matter (WM) abnormalities. Increased neurodegeneration risk in ETPKU: An atypical amyloid pathology probably secondary to phenylalanine (Phe) aggregation has been recently reported in PKU brains. A pilot study to assess the risk of early neurodegeneration (EN-ETPKU Study) will evaluate the presence of neurodegenerative biomarkers in ETPKU subjects entering their middle age. Conclusion: Dysmetabolic abnormalities, oxidative stress and amyloid pathology increase the risk of incident neurodegenerative disorders such as AD or PD in ETPKU subjects entering their middle age. Results of this study may have an impact on PKU therapy beyond the age when it is currently stopped. 33

36 European guidelines statements: have your say Annemiek van Wegberg University Medical Centre Groningen, Radboud University Medical Centre The Netherlands Currently European guidelines on PKU are being developed. Five working groups were formed in October These five working groups each focus on a different topic; nutritional treatment and biochemical / nutritional follow up; neurocognitive outcome including imaging; psychosocial outcome and adherence; adult and maternal and late treated PKU; diagnosis of PKU and drugs in PKU. The working group members include pediatric metabolic physicians, an adult metabolic physician, a pediatric neurologist, a biochemist, metabolic dieticians and (neuro)psychologists. The members come from several countries, namely Denmark, France, Germany, Italy, the Netherlands, Poland, Spain, Swiss and United Kingdom. All working groups formulate key questions, search evidence and reformulate these questions in to recommendations. Literature search, critical appraisal and evidence grading are done according to the SIGN (Scottish intercollegiate guidelines network) method. Recommendations with no or low level of evidence are being discussed with all participants of all working groups during plenary sessions until consensus is reached (Delphi method). Unfortunately it is quite rare that evidence for a particular recommendation is of high quantity, clear and not ambiguous. Especially for practical recommendations little or no evidence is available. Here consensus is needed. To this aim input of professionals can be extremely helpful. During this session the opinion of professionals will be asked regarding topics of the European guidelines with no or little evidence available. 34

37 Oral communications

38 OC1. Prediction of phenotypes and BH4-responsiveness in phenylketonuria by linking data from genotypes and locus-specific database Sarah Wettstein 1, Jarl Underhaug 2, Belen Perez 3, Brian D Marsden 4, Wyatt W Yue 4, Aurora Martinez 2, Nenad Blau 5,1 1 Division of Metabolism, University Children's Hospital, Zürich, Switzerland; 2 Department of Biomedicine, University of Bergen, Bergen, Norway; 3 Centro de Diagnóstico de Enfermedades Moleculares, Centro de Biologia Molecular UAM-CSIC, CIBERER, IDIPAZ Universidad Autonoma de Madrid, Madrid, Spain; 4 Structural Genomics Consortium, University of Oxford, Oxford, OX3 7DQ, UK 5 Division of Inborn Metabolic Diseases, University Children's Hospital, Heidelberg, Germany Background: Management of phenylketonuria (PKU) patients depends on the individual phenotype. The variability in metabolic phenotypes in PKU is caused by different variants within the phenylalanine hydroxylase (PAH) gene leading to variable impairment of PAH activity. In addition, it has been shown that genotypes are useful in predicting cofactor tetrahydrobiopterin (BH4) responsiveness in PKU. Objectives: To analyse data from locus-specific database PAHvdb (834 variants) and genotypes database BIOPKUdb (4181 patients with full genotype). To establish genotype-phenotype correlations and to develop models predicting phenotype and BH4-responsiveness in PKU. Both databases can be found at: Methods: First we calculated the relative frequencies of mutations, genotypes, affected gene regions and protein domains. Subsequently, PAH mutations and genotypes were scored using four prediction tools: FoldX (estimating protein damage using an empirical force field), SIFT Blink (predicting protein function based on the degree of AA residue conservation), PolyPhen-2 (impact of an AA substitution on protein structure and function), and SNPs3D algorithm (functional effects of non-synonymous SNPs based on structure and sequence analysis). FoldX, PolyPhen-2, SIFT and SNP3d values were correlated with residual enzyme activity, patients phenotype, and BH4 response. The 3D atomic environment of each mutation was visualised using the interactive isee concept. Results/Discussion: Amongst the 4181 patients (15.1% HPA, 24.4% mild PKU, 41.3% classic PKU, 19.2% N/A) we observed 1543 different genotypes and 486 different mutations. Exon 7 (22.9%) and intron 10 (9.4%) were the most affected sites, respectively. The 8362 mutations were predominantly located in the catalytic domain (60.9%), followed by intronic sites (20.1%), the regulatory (14.0%) and least the oligomerization domain (4.9%). The most frequent genotype was c g>a/c g>a (3.3%). BH4 response was known for 2128 patients (44.4% responders). Using genotype scoring both phenotype and BH4-responsiveness were estimated offering a robust method for patients' characterisation and management. 36

39 OC2. Sapropterin dihydrochloride treatment in under 4 years of age Turkish hyperphenylalaninemic patients Sivri HS 1, Ünal Ö 1, Gökmen-Özel H 2, Özgül K 1, Yücel D 1, Hişmi B 1, Dursun A 1, Tokatlı A 1, Coşkun T 1 1 Hacettepe University, Faculty of Medicine, Department of Pediatrics, Section of Metabolism, Ankara, Turkey 2 Hacettepe University, Faculty of Dietetics, Ankara, Turkey Background: Sapropterin (BH4) decreases blood phenylalanine (Phe) concentration and increases dietary Phe tolerance by enhancing phenylalanine hdroxylase activity. In responders, early introduction of BH4 treatment allows more natural protein and bioavailable microand macronutrient intake at the time when the brain develops fast. If started in the newborn period, breastfeeding may continue successfully. Unfortunately, the use of BH4 under 4 years of age has not yet been approved in most countries. Studies on efficacy and safety of sapropterin in this age group are scarce in the literature. Aims: The purpose of this study was to evaluate efficacy and safety of sapropterin treatment in hyperphenylalaninemic (HPA) children under 4 years and to check if genotype analyses are of help with the prediction of responsiveness. Materials and methods: This was a single-centre cross-sectional study. Patients with HPA, younger than 4 years of age when treatment was started, were included. A 48-hour BH4 loading test was used for evaluation of responsiveness. The test was found positive in 35 patients. In 9, sapropterin treatment was started prior to initiation of Phe-restricted diet. Clinical features were evaluated in all and molecular analyses were performed in 25 patients. Results: A total 44 patients (28 boys, 16 girls) were found responsive. Treatment was started at 1.9 years (median). Duration of treatment was median 26.7 months (12-45 months). Diet was liberalised in 25/30 patients (83.3%). Phe tolerance increased median 2.26-fold ( ) from median 47.5 mg/kg/day to median 114 mg/kg/day (p<0.001). Phe tolerance did not change significantly in 5, so sapropterin was discontinued. In 9 (20.5%), sapropterin was started prior to initiation of Phe-restricted diet. In 25, whose molecular data is available, mutations on 49 alleles were detected. Frequent mutations were p.e390g (12.2%), IVS10-11G>A (10.2%), p.a300s (8.2%), and two novel ones: p.y417c and p.f121s. Conclusions: This was one of the preliminary studies on sapropterin treatment that included relatively more patients under 4 years. We found sapropterin as a safe and efficacious therapeutic option in this age group. BH4 loading test along with molecular studies are useful in the detection of responders but not always in the prediction of long-term responsiveness. 37

40 OC3. Comparing PKU patients with and without BH4 treatment: blood prolactin and serotonin concentrations Danique van Vliet 1,2, Karen Anjema 1, Martijn J. de Groot 1,2, Rianne Jahja 1, Geertje Liemburg 1, Ido P. Kema 3 M. Rebecca Heiner-Fokkema 3, Francjan J. van Spronsen 1 1 University of Groningen, University Medical Center Groningen, Beatrix Children s Hospital, Groningen, The Netherlands 2 University of Groningen, Center of Behavior and Neurosciences, Department of Molecular Neurobiology, Groningen, The Netherlands 3 University of Groningen, University Medical Center Groningen, Department of Laboratory Medicine, Groningen, The Netherlands Background: Reduced brain dopamine and serotonin concentrations have been hypothesised to contribute to impaired neuropsychological functioning in phenylketonuria (PKU) patients. Tetrahydrobiopterin (BH4), being a cofactor for tyrosine and tryptophan hydroxylase, has been suggested to directly improve brain neurotransmitter synthesis beyond its effect through lowering blood phenylalanine (Phe) concentrations in BH4-responsive PKU. Aims: To further investigate whether BH4 has a positive effect on brain neurotransmitter synthesis, this study compared blood prolactin (as a parameter of brain dopamine) and blood serotonin concentrations (as a parameter of peripheral serotonin synthesis) in early treated PKU patients on BH4 treatment compared to dietary treatment alone. Patients and methods: In total, 92 blood samples of 27 PKU patients with BH4 (10 males, 17 females, age: 3 years, 9 months to 38 years) and 326 blood samples of 62 PKU patients without BH4 (27 males, 35 females, age: 11 months to 44 years) were compared at different blood Phe, tyrosine, tryptophan, Phe:tyrosine, and Phe:tryptophan levels. Prolactin concentrations were investigated in PKU males only. Possible correlations with blood prolactin or serotonin concentrations were investigated by multivariate linear regression analysis. Results: At Phe >600 μmol/l, prolactin in PKU males without BH4 positively correlated to Phe (p=0.000), while negatively correlated to tyrosine (p=0.046). No correlation was found with age. Prolactin was significantly lower in PKU males on BH4 compared to PKU males without BH4 at high tyrosine ( μmol/l; p=0.035), low Phe:tyrosine (ratio <4; p=0.019) and almost significantly lower at Phe μmol/l (p=0.061). Moreover, in PKU males receiving BH4 treatment, prolactin positively correlated to Phe:tyrosine ratios (p=0.000), while negatively to BH4 dose (p=0.022). Without BH4 treatment, serotonin was significantly lower at Phe >600 μmol/l compared to levels between μmol/l (p=0.012). Serotonin was significantly higher in patients using BH4 compared to patients without BH4 at high tryptophan (50-80 μmol/l; p=0.003), low Phe:tryptophan (ratio 1-6; p=0.005), and low Phe ( μmol/l; p=0.042). Discussion: These results suggest that BH4 may increase cerebral dopamine and peripheral serotonin synthesis in PKU patients. Such an effect seems irrespective of age, but may be dose dependent. Moreover, it may require adequate tyrosine and tryptophan availability and not too high Phe concentrations and Phe:tyrosine or Phe:tryptophan ratios. Therefore, we hypothesise that BH4 at 20 mg/kg might improve neuropsychological functioning in both BH4-responsive and unresponsive PKU patients, regardless of age, by increasing cerebral neurotransmitter synthesis. 38

41 OC4. Cholinergic alterations are caused by hyperphenylalaninemia in rat brain Schuck PF 1, Macan TP 1, Oliveira MB 1, Citadin S 1, Kist LW 2, Pereira EG 1, Marques S 1, Souza CT 1, Streck EL 1, Ferreira GC 3 Bogo MR 2 3, Budni J 1, Zugno AI 1 1 Universidade do Extremo Sul Catarinense, Brazil 2 Pontifícia Universidade Católica do Rio Grande do Sul, Brazil 3 Universidade Federal do Rio de Janeiro, Brazil Background: Accumulation of phenylalanine (Phe) in tissue and body fluids is the hallmark of phenylketonuria (PKU). Brain injury is a clinical characteristic of PKU patients, although the pathophysiology of this damage is poorly understood. Aim: The aim of the present work was to investigate the in vitro, in vivo and in silico effects of Phe on acetylcholinesterase (AChE) activity and its expression. Materials and methods: For the in vivo experiments, animals received a single subcutaneous administration of 0.9% saline (control group) or 5.2 μmol/g Phe plus 0.9 μmol/g p-chlorophenylalanine (HPA group). One hour after the administration, the animals were euthanised by decapitation; brain structures were isolated and homogenised. AChE activity and its relative RNA expression, PKA and PKC content were determined. Also analysed was the interaction between Phe and AchE by molecular dynamics. Results: It was observed that animals subjected to acute HPA presented an increase of AChE activity in striatum. On the other hand, AChE mrna expression was not altered by HPA. In order to verify the mechanisms by which AChE activity is increased, the content of PKA and PKC proteins was assayed and no difference was found between groups. Also evaluated was the molecular interactions between Phe and AChE, the crystallographic structure of AChE was used to dock ACh in the AChE binding site in the presence or absence of phenylalanine. The affinity of ACh to AChE is decreased in the presence of Phe, probably due to steric hindrance. Conclusions: Taken together, our results suggest that Phe induces cholinergic alterations. Since cholinergic imbalance is associated with failure and progressive neurologic decline in learning and memory functions, it is tempting to speculate that AChE alterations might contribute to the intellectual deficiency observed in HPA patients. Financial support: Universidade do Extremo Sul Catarinense (UNESC), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), For Women in Science Grant (L Oreal/ABC/UNESCO). 39

42 OC5. Three girls with microcephaly and hyperphenylalaninemia A.Tokatli, Y. Yildiz, A. Dursun, T. Coskun, HS. Sivri Hacettepe University Faculty of Medicine, Department of Pediatrics, Division of Metabolism Ankara, Turkey Case series: Here, we present three Turkish girls with microcephaly and hyperphenylalaninemia, who shared another comorbid condition. Patient A was a 22-day-old premature girl born at 32 weeks of gestation who was found to have a blood phenylalanine level (BPL) of 1698 μmol/l and put on a phenylalanine-restricted diet. Despite good metabolic control, she had delayed developmental milestones. Microcephaly became evident as she thrived and her mother s BPL was found to be 1056 μmol/l. Patient B presented at two months of age due to Guthrie test positivity. Upon examination she was found to have microcephaly and bilateral pes equinovarus. Her and her mother s BPLs were 906 and 984 μmol/l, respectively. Patient C presented with a BPL of 144 μmol/l and microcephaly. At 14 months of age, while her developmental milestones were within normal limits, her mother s BPL was found to be 364 μmol/l. The mother and patient C were both found compound heterozygotes for R241H/A403V mutations on the PAH gene. Conclusion: These three cases demonstrate the necessity of screening for maternal PKU and MHP if the offspring has early-onset microcephaly not attributable to PKU, especially if the mother s date of birth precedes the onset of national newborn screening in a country with high PKU prevalence, like Turkey. 40

43 OC6. BH4 responsiveness: Ege University experience Kağnıcı M 1, Atik Altınok Y 1, Köse M 1, Canda E 1, Kalkan Uçar S 1, Habif S 2, Çoker M 1 (*) : Pediatric Metabolism Department, Ege University Medical Faculty, İzmir, Turkey (** ) : Clinical Chemistry Department, Ege University Medical Faculty, İzmir, Turkey Background: Defects in either phenylalanine hydroxylase or the production or recycling of tetrahydrobiopterin (BH4) may cause hyperphenylalaninemia (HPA). Pharmacological doses of BH4 can reduce blood phenylalanine (Phe) levels in some patients with PKU and can be used solely or in adjunction to diet therapy in treatment of HPA. The most frequent definition of BH4 responsiveness is a reduction of 30 % in blood phe level after 20 mg/kg BH4 administration. Aims: To evaluate patients with HPA in terms of BH4 responsiveness, rate of decline in Phe levels and changes in daily consumption of phenylalanine. Materials and methods: BH4 loading test was performed in 45 patients (33 PKU, 12 HPA). Following a six day Phe load (1st: 5 mg/kg/day, 2nd: 10 mg/kg/day, 3rd to 6th: 15 mg/kg/day), BH4 was administrated at 5th and 6th days at a dosage of 20 mg/kg/day. BH4 responsiveness was evaluated at 4, 24, 48 and 72 hours after administration. To be confirmed as a responder, there had to be at least a 30% drop in blood Phe levels. Results: The results for BH4 responsiveness and increase of Phe tolerance are given in Table 1. Table 1: Evaluation of BH4 responsiveness and increase of daily Phe tolerance Conclusions: BH4 administration in hyperphenylalaninemia increased phenylalanine tolerance in our BH4 responsive patients. Acknowledgements: We would like to thank Dr Nenad Blau and Dr Beat Thöny for his long time collaboration to analyse pterin metabolites in our patients. 41

44

45 Workshops

46 Workshop 1. Maternal PKU - Interactive clinical case presentations Friedrich K. Trefz Children s Hospital, Kreis Kliniken Reutlingen GmbH Klinikum am Steinberg, School of Medicine Louise Robertson University Hospitals Birmingham NHS Foundation Trust Old Queen Elizabeth Hospital, Birmingham, UK University of Tuebingen Reutlingen, Germany Abstract not available at the time of printing. 44

47 Workshop 2: European and USA guidelines - controversies Francjan J. van Spronsen Beatrix Children s Hospital University Medical Centre Groningen Groningen, The Netherlands Alberto Burlina Division of Metabolic Diseases Department of Pediatrics University Hospital Padua, Padua, Italy Nicola Longo Division of Medical Genetics University of Utah Salt Lake City, USA Abstract not available at the time of printing. 45

48 Workshop 3. Diet plus sapropterin in clinical practice - Interactive clinical case presentations Amaya Bélanger-Quintana Unidad de Enfermedades Metabólicas Servicio de Pediatría, Hospital Ramón y Caja Madrid, Spain Margreet van Rijn Section of Metabolic Diseases Beatrix Children's Hospital, University Medical Centre Groningen Groningen, The Netherlands Background: The availability of BH4 in the form of sapropterin dihydrochloride raises new questions in the management of phenylketonuria (PKU) patients. BH4 effectively lowers blood phenylalanine (Phe) levels in a proportion of patients with PKU. A very small number of patients who respond to BH4 are able to discontinue their Phe-restricted diet completely. In most responsive patients, BH4 is used as an adjunctive therapy to a low-phe diet. Existing dietary guidelines for PKU were published before the approval to treat BH4 responsive PKU patients. New guidelines and possibly adapted supplementations are required for managing the changes in diet, keeping blood Phe concentrations within target ranges and ensuring adequate nutrition in these patients. A strict evaluation of dietary intake in combination with metabolic control is crucial in judging the long-term effectiveness of new treatment options such as BH4. Furthermore, the PKU patient has a need for information about the protein and nutritional content of higher protein foods, the principles of healthy food choices, weight control and illness management, and understanding that this new treatment regimen still requires compliance (medication intake, food intake and regular monitoring of blood Phe concentrations). Aim of the workshop: How the existing guidelines need to be extended due to introduction of BH4 How to adapt the dietary management to optimise nutritional intake Judging effectiveness of an expensive treatment not only in terms of blood Phe concentrations 46

49 Asbjørn Følling lecture Friedrich K. Trefz Children s Hospital, Kreis Kliniken Reutlingen GmbH, Klinikum am Steinberg School of Medicine, University of Tuebingen, Reutlingen, Germany What we can learn from looking back is how phenylketonuria (PKU) became a model for diagnosis, prevention and treatment of an inborn error of metabolism. The detection of this disease by Dr Asbjoern Foelling ( ) first shows us the importance of looking carefully to our patients. It is just a case report : two handicapped children in one family without special stigmata but with an unusual chemical reaction in urine. In the following a number of medical doctors and scientists were involved in the elucidation of this disease, an exciting story Professor Harvey Levy presented to us in 2012 very carefully at the SSIF meeting ( In my lesson I will focus on the people I personally met and who influenced my view of what PKU is like and where we should go in the future. After description of the first PKU patients by Foelling, later it was evident that phenylpyruvic acid in urine derived from highly elevated phenylalanine which could not be metabolised. The next diagnostic level became the protein. It was a challenge elucidating the enzyme phenylalanine hydroxylase or better said the phenylalanine hydroxylating system in the early 1950s. Seymour Kaufman ( ) and his team working at the NIH in Bethesda, Maryland, USA, is one of the key persons not only describing the enzyme and the natural cofactor tetrahydrobiopterin but also discussing in numerous publications the aetiology of possible brain damage in untreated PKU patients. He also investigated the genetics of PKU when he analysed the residual enzyme activity of patients and parents in liver biopsies finding that there was a deviation from gene dosage. He also first discussed that negative inter allelic complementation in a multimeric enzyme may be responsible for this discrepancy and that biopterin may be able to enhance residual enzyme activity of an altered mutated enzyme 25 years before Kure first published in 1999 the response of pharmacologic doses of tetrahydrobiopterin to decrease blood phenylalanine in patients with deficient phenylalanine hydroxylase. Basic research on one side and clinical trial on the other side of the Atlantic Ocean: after several others also had the idea of treatment PKU by diet, Horst Bickel ( ) was the first one who started a low phenylalanine dietary treatment during his time at the Birmingham Children's Hospital, UK, in The result of treatment in a one case study is shown on YouTube with commentary by Harvey Levy ( and demonstrating the effect of phenylalanine intoxication. What we also can learn from PKU is the importance of co-operation. Without the brilliant idea of a bacterial inhibition assay for mass screening of elevated phenylalanine by Robert Guthrie ( ), PKU treatment would not have become such a success story for the prevention of mental retardation in today worldwide ca. 50,000 patients. Horst Bickel and Robert Guthrie became very good friends and both together insisted in their countries and many others to install early neonatal diagnosis and treatment in the late 1960s. Another co-operation also resulted in an important step forward on the metabolic/protein level: Hans Christoph Curtius, a biochemist, and his group in Zürich worked together with the groups at the Children's Hospital in Heidelberg and Munich, Germany. Based on the work of Seymour Kaufman the first patients with a defect in the synthesis of the cofactor tetrahydrobiopterin could be treated by use of the newly synthesised tetrahydrobiopterin from Dr Schircks, a postgraduate fellow in the Zurich group. At this time I joined the group in Heidelberg and was excited how a patient with atypical PKU responded to BH4. Until the approval of sapropterin dihydrochloride in 2007 this was the only cofactor available worldwide. What we also can learn from PKU is the importance of clinical studies. It was Samuel Bessman ( ) (otherwise a very brilliant scientist) who questioned that a low phenylalanine diet may be helpful. To prove that this was wrong and a low phenylalanine diet will prevent mental retardation and not tyrosine supplementation, Richard Koch ( ) initiated a prospective collaborative (multicentre) NIH study in the USA and later on the NIH International Maternal PKU Study. The importance of studies like these and careful follow up of patients is demonstrated by unpublished malpractice treatment with an amino acid mixture produced in the seventies which missed the essential amino acid tryptophan. Irreversible central nervous defects were observed in many patients. The GCP dietary treatment studies today are the basis for developing guidelines for maternal PKU on a high evidence level we just are working on, together with the ESPKU under the responsibility of Francjan van Spronsen. 47

50 Asbjørn Følling lecture Friedrich K. Trefz Children s Hospital, Kreis Kliniken Reutlingen GmbH, Klinikum am Steinberg School of Medicine, University of Tuebingen, Reutlingen, Germany The next level of understanding the disease was the DNA level. In 1983 Savio Woo cloned the gene for phenylalanine hydroxylase and later on described several mutations responsible for the classic phenylketonuria (PKU) phenotype. Charles Scriver, who also realised very early on the importance of databases, initiated the PKU mutation database with today more than 800 mutations. Much work had to be invested to elucidate the biopterin pathway: Nenad Blau and his group in Zurich continued the work of Curtius and his coworker Niederwieser and described in many papers the pathway of biopterin and the enzymes and genes involved. After elucidation of the three diagnostic 'levels' metabolic, protein and DNA, it was time to investigate the interactions between them and develop new therapies. After first enthusiasm about the possibility of genotype-phenotype correlation it came out that the conditions were much more complex than expected. In vitro experiments especially by Ania Muntau and Aurora Martinez and their groups investigated the complex interactions between BH4, phenylalanine and normal and mutant phenylalanine to better describe the chaperonine effect of BH4 on stabilisation of an otherwise unstable mutant protein. The new treatment option with sapropterin dihydrochloride in 2007/2008 was another milestone in the history of PKU. The first time pharmaceutical companies entered the world of PKU and many paediatricians and patients realised that a treatment with an approved drug is something different to a low phenylalanine diet requiring no drug approval studies. Many questions are still open and this has made the 'PKU story' more attractive to basic researchers and clinicians than 15 years ago. Who will benefit from this new treatment and what is the role of a combined drug and dietary treatment? Can we better monitor the quality of treatment by using modern methods like high resolution nuclear magnetic resonance spectrometry? How can we better prevent maternal PKU syndrome? Are adult PKU patients older than 50 years at risk of developing neurodegenerative disease? Is enzyme replacement with PEG-phenylalanine ammonia lyase an option for treatment especially in adult patients? What are the perspectives of gene therapy? How can we better establish international co-operation by using modern communication techniques? How we can better store our knowledge and individual clinical experiences in a 'cloud' beside of conventional publishing and use 'big data' and data mining? Can we better describe pharmacologic effects of BH4 by developing pharmacodynamic models? Is the rapid development of computerised metabolic pathways by bioinformatics helpful to elucidate the effects of phenylalanine on many enzymes? Can we further develop chaperones to treat, e.g. patients with the p.r408w mutation (a mutation most prominent in Russia and other Eastern European countries)? The answers to these questions are important for the treatment of our patients. It will finally result in a personalised medicine: one patient may benefit from BH4 treatment the other not. Health economic considerations may also play a role in our treatment decisions, and a better knowledge about vulnerable brain phases as in infancy, maternal PKU, the late treated patient and the 'elderly' PKU patient. A careful clinical, biochemical and genetic investigation in every patient is essential. Frequent monitoring of blood phenylalanine, e.g. using home monitoring and rapid measurement by tandem mass spectrometry, fast feedback by dietitians and doctors via modern communication is essential. The treatment must further on be managed in a multidisciplinary specialised team. In summary: PKU is a disease model for inborn error of metabolism. History shows the progress on different diagnostic levels called today genomics, proteomics and metabolomics. We start understanding the interactions of metabolite, protein and mutations in the gene. Only the continuation of our close co-operation and developing new computerised models and networks will provide further rapid progress in favour for our patients. I have to thank my colleagues from the European PKU Advisory Board who gave me the opportunity in many meetings to discuss new ideas, develop new methods, publish a number of reports, and especially the opportunity to speak in honour and memory of Asbjoern Foelling. 48

51 PKU Academy fellowship 2012: final results Priscila Mazzola Department of Molecular Neurobiology, Beatrix Children's Hospital University of Groningen, The Netherlands Background: Even treated phenylketonuria (PKU) patients can show increased levels of phenylalanine and its harmful implications as oxidative stress and imbalance of amino acids. Exercise has shown antioxidant effects in the brain of an animal model of hyperphenylalaninemia, as well as in many other diseases and aging. Also, acute exercise increases peripheral catecholaminergic system that in turn increases oxidation of bioenergetic sources (lipids, glucose, amino acids), leading to adaptations when performed regularly. This study was performed in two parts, aiming to verify (1) acute exercise effects on metabolic and hormonal levels in treated PKU patients, and (2) regular exercise effects on amino acid and oxidative stress levels in PKU mice. Methods: (1) Eight treated PKU patients (median phenylalanine 677 µmol/l) and 17 controls were studied. Participants performed a maximum oxygen consumption test in order to determine their aerobic target zone. Then the participants carried out an exercise session of 30 min within this submaximal aerobic intensity. Blood sampling was taken in fasting state and immediately after exercise, for evaluation of lipid profile, glucose, catecholamines and adiponectin. (2) Sixteen wild-type (WT) and 16 Pah(enu2) (PKU) C57BL/6 mice from both sexes were placed solitarily in cages with (exercise, Exe) or without (sedentary, Sed) running wheels for 24-h/day during eight weeks. The animals activity was tracked throughout the experiment. After seven weeks, the animals performed the balance beam test that assesses motor coordination and balance and one week later they were euthanised. Plasma and total brain were collected for amino acid levels and oxidative stress parameters analyses. Data were analysed with two sided t-test. Results: (1) In pre-exercise, PKU patients showed lower levels of total cholesterol, HDL and LDL, and higher adiponectin concentration. In both PKU and controls total cholesterol, HDL and LDL increased after exercise, but only in the control (and not in PKU) group adrenaline increased and glucose decreased. (2) SedPKU group showed increased oxidative stress and impairments in co-ordination and balance in comparison to SedWT. ExePKU showed increased antioxidant potential levels in comparison to SedPKU. Plasma phenylalanine and BCAA levels were negatively correlated with the daily distance ran only for the ExePKU group. Conclusions: (1) Hyperphenylalaninemia along with diet composition might change acute exercise outcomes. (2) This PKU mouse model showed oxidative stress and exercise was able to increase antioxidant system in the brain. Exercise seems to modify plasma amino acid levels. 49

52 Notes 50

53 Disclosure of faculty relationships EXCEMED adheres to guidelines of the European Accreditation Council for Continuing Medical Education (EACCME) and all other professional organisations, as applicable, which state that programmes awarding continuing education credits must be balanced, independent, objective, and scientifically rigorous. Investigative and other uses for pharmaceutical agents, medical devices, and other products (other than those uses indicated in approved product labeling/package insert for the product) may be presented in the programme (which may reflect clinical experience, the professional literature or other clinical sources known to the presenter). We ask all presenters to provide participants with information about relationships with pharmaceutical or medical equipment companies that may have relevance to their lectures. This policy is not intended to exclude faculty who have relationships with such companies; it is only intended to inform participants of any potential conflicts so participants may form their own judgements, based on full disclosure of the facts. Further, all opinions and recommendations presented during the programme and all programme-related materials neither imply an endorsement, nor a recommendation, on the part of EXCEMED. All presentations solely represent the independent views of the presenters/authors. The following faculty provided information regarding significant commercial relationships and/or discussions of investigational or non- EMEA/FDA approved (off-label) uses of drugs: Ivo Barić No potential conflict of interest Amaya Bélanger-Quintana Honoraria & consultation fees from Nutricia, Nestle, Merck Serono Nenad Blau Grants and contracts from Merck Serono and BioMarin Pharmaceutical Inc Alberto Burlina Member of a company advisory board: Merck Serono Maureen Cleary Honoraria & consultation fees from: Merck Serono. Member of Merck-Serono advisory board François Feillet Honoraria & consultation fees from: Merk Serono, Genzyme, BioMarin Hulya Gokmen-Ozel Honoraria & consultation fees from Enep group Urh Grošelj No potential conflict of interest Rianne Jahja Honoraria & consultation fees for the MEMG in December 2013 Robin Lachmann No potential conflict of interest Harvey L. Levy Grants and contracts from BioMarin Pharmaceutical Inc Nicola Longo Grants and contracts from Biomarin Pharmaceutical Inc. (to the University of the Utah). Member of a company advisory board: Biomarin Pharmaceutical Inc Anita MacDonald Grants and contracts from Nutricia, Vitaflo, Merck Serono Aurora Martinez Grants from Novoseeds (NovoNordisk ) Priscila Mazzola No potential conflict of interest Andrea Pilotto No potential conflict of interest Louise Robertson No potential conflict of interest Júlio César Rocha Member of the European Nutritionist expert panel in PW - Merk Serono Vlado Sarnavka No potential conflict of interest Beat Thöny No potential conflict of interest Friedrich K. Trefz Honoraria & consultation fees from: Merk Serono Valentina Uroić No potential conflict of interest Margreet van Rijn Honoraria & consultation fees from: Danone, Nutircia, Merk Serono Francjan J. van Spronsen Honoraria & consultation fees from: Merk Serono, Nutricia Grants and contracts from: Merk Serono, Nutricia Annemiek van Wegberg No potential conflict of interest 51

54 Disclosure of faculty relationships The following faculty has provided no information regarding significant relationship with commercial supporters and/or discussion of investigational or non-emea/fda approved (off-label) uses of drugs as of 27/05/2014 Skadi Melanie Beblo Patrícia Fernanda Schuck All EXCEMED programmes are organised solely to promote the exchange and dissemination of scientific and medical information. No forms of promotional activities are permitted. This programme is made possible thanks to educational grants received from: Arseus Medical, Besins Healthcare, Bristol-Myers Squibb, Celgene, Centre d Esclerosi Multiple de Catalunya (Vall d Hebron University Hospital), Centre Hépato-Biliaire (Hôpital Paul Brousse), Croissance Conseil, Cryo-Save, Datanalysis, Dos33, Esaote, Ferring, Fondazione Humanitas, Fundación IVI, GE Healthcare, GlaxoSmithKline Pharmaceuticals, IPSEN, Italfarmaco, International Society for Fertility Preservation, Johnson & Johnson Medical, K.I.T.E., Karl Storz, Lumenis, Merck Serono Group, PregLem, Richard Wolf Endoscopie, Sanofi-Aventis, Stallergenes, Stopler, Teva Pharma, Toshiba Medical Systems, Université Catholique de Louvain (UCL), University of Catania. 52

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56 Improving the patient's life through medical education EXCEMED - Excellence in Medical Education Representative Office Salita di San Nicola da Tolentino 1/B Rome, Italy. T: , F: Headquarters 14, Rue du Rhone, 1204 Geneva, Switzerland Copyright EXCEMED, All rights reserved