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ADVANCES & CHALLENGES IN PKU
Welcome to Issue Six of the PKU Academy NewsletterIn this edition of the PKU Academy Newsletter we report on the highlights of the 2014 Annual Multidisciplinary European PKU Symposium held in Zagreb (Croatia) on the 6th and 7th of June 2014.
ContentsSymposium introduction ............2
New perspectives in therapy .......3
Brain function .........................4
From basic knowledge to daily management ...........................5
Oral communications ................6
Asbjørn Følling lecture ..............7
Workshop sessions ....................8
Perspectives: Harvey Levy .........9
Perspectives: Nicola Longo ....... 10
Perspectives: Aurora Martinez ... 11
PKU Academy fellowship .......... 12
ISSUE 6 · SEPTEMBER 2014
Comment from key opinion leaders in the PKU field. Report from the 2014 Annual Multidisciplinary European PKU Symposium
PKU ACADEMY NEWSLETTER
3rd Dietitians’ Workshop
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New content, online now, includes presentations and interviews from the 2014 Annual Multidisciplinary European PKU Symposium.
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We also feature three more of the ‘Perspectives’ interview features which give a personal view from the people behind the presentations and papers.
Prof Harvey L Levy, clinical biochemical geneticist and researcher, of Harvard Medical School, Boston (USA) shares his passion for the development of newborn screening along with his hopes for the future and his concerns over the possible pitfalls of genomic screening.
Prof Nicola Longo, clinical geneticist and researcher, of the University of Utah, Salt Lake City (USA) discusses the possible benefits and limitations of pegylated phenylalanine ammonia lyase as a potential treatment for PKU.
Prof Aurora Martinez, biochemical researcher, of the University of Bergen, Bergen (Norway) updates us on the search for the ‘ideal’ chaperone for the phenylalanine hydroxylase enzyme.
2 | PKU Newsletter Issue 6 . September 2014
The symposium ‘PKU: a lifetime of challenges’ took place in Zagreb (Croatia) on the 6th and 7th of June 2014.
Prof Nenad Blau1, scientific coordinator, opened the symposium and welcomed 131 delegates from 24 countries. He introduced the themes of the meeting.
�� Challenges and solutions in the management of PKU: diet, currently available medical therapy and possible future treatments
�� Pathophysiological insights into the disease, its neurocognitive repercussions for patients and strategies to counteract these events
�� Approaches to the management of special clinical conditions such as maternal PKU, adolescence and adulthood
�� The development of European guidelines for PKU management and provision of best care for PKU patients and their families
The symposium was an accredited CME event. Delegates who registered their attendance with EACCME and completed the post-event assessment materials were able to claim up to nine hours of CME credit.
The meeting was endorsed by the Croatian Pediatric Society2, on whose behalf Prof Ivo Barić3 added his welcome.
PKU in Croatia and south-eastern Europe
Dr Urh Grošelj4 gave delegates some background on the status of PKU screening and management in Croatia before comparing and contrasting PKU care across the region.
In Croatia, state-funded screening was introduced in 1978. Follow-up of patients is delivered by a multidisciplinary team, based in the capital, Zagreb. Since 1990, the Croatian PKU Association5 has worked to promote improvements in the quality of life for PKU patients and their families.
PKU screening is not, however universal across the countries of south-eastern Europe, explained Dr Grošelj. Furthermore, a number of countries are falling behind internationally established standards of care. There are great differences between countries and regions in the area which do not necessarily correlate with economic wealth or development.
Dr Grošelj presented results from a survey conducted in the months immediately prior to the symposium. The survey had been completed by professionals responsible for PKU management in the 11 countries of the south-eastern region of Europe6.
The survey found that:
�� PKU screening did not take place in four of the 11 countries and only four countries conducted routine genetic diagnosis
�� five countries were unable to provide data on PKU incidence and those that did supplied incidence data which ranged from 1:7,325 to 1:39,338
�� the number of PKU centres in each country ranged from one to six
�� the most commonly used laboratory method for assessing Phe levels was fluorimetry
�� tetrahydrobiopterin (BH4) was available in two countries
�� primary care was provided by paediatricians; dietitians were members of the care-team in only four countries
�� regular psychological assessments were conducted in six countries
�� five countries had national PKU guidelines and seven had PKU registries
�� there were patients’ PKU societies in seven countries.
It is vital, stated Dr Grošelj that we look to close the gaps in care and bring the countries and regions together. There is a need to increase the education of professionals and encourage cooperation. We need to take all opportunities to learn from other regions of the world facing similar challenges. An essential first step would be to agree minimal acceptable standards for diagnosis and treatment across the region.
2014 Annual Multidisciplinary European PKU Symposium
Prof Nenad Blau welcomed delegates to the 2014 symposium
A selection of abstracts and presentations from this event, are available to download. http://bit.ly/PKUlive2014
www.pkuacademy.org | 3
PEG-PAL on trialWork continues to investigate the potential of using recombinant Anabaena variabilis phenylalanine ammonia lyase conjugated with polyethylene glycol (rAvPAL-PEG, or PEG-PAL) as a therapy for PKU. Prof Nicola Longo7 reported that recruitment was going well in the two Phase III studies currently underway in the USA to evaluate the efficacy and safety of the compound8,9.
Prof Longo updated delegates on the status of the four Phase II studies that have been, and are currently being, conducted. Results to date show that repeated administration of PEG-PAL is effective in reducing Phe levels to within the normal or therapeutic range in PKU patients on an unrestricted or a moderately protein-restricted diet. All patients became proficient in self-administering the drug by injection. A few patients developed hypophenylalaninemia that was resolved by lowering the drug dose. The early data show that the majority of side-effects are limited to mild-to-moderate hypersensitivity. Patients who had these types of reactions have all been successfully re-treated with PEG-PAL. There was no laboratory evidence of liver or kidney injury.
A report on the Phase I study appeared recently in the Lancet, Longo et al (2014)10 .
Hunting for chaperones
Chaperones, small chemicals capable of renaturing and restoring activity to misfolded proteins, could be important for PKU treatment, Prof Aurora Martinez11 told delegates.
Prof Martinez reported on work to find new chaperones. To date, her group had screened 10,000 candidate compounds in the search for new chaperones capable of stabilising phenylalanine hydroxylase (PAH). The screening process looked at the stability, activity, solubility and specificity for PAH binding.
Two compounds (#26 and #43) have been found that show potential as chaperones. Compared with previously screened potential chaperones, both these compounds have larger effects on PAH protein and activity (in cells) and better absorption, distribution, metabolism and excretion (ADME). Both compounds have development potential. They can both work with BH4 as they have a different mechanism of stabilisation and bind to a different site on PAH.
BH4 for children under fourBH4
is safe and effective for children under four years old, Prof Harvey L. Levy12 informed delegates, reporting on the PKU Demographic Outcomes and Safety (PKUDOS) registry, a Phase IV observational study. The study followed 97 patients aged one month to four years.
The label for sapropterin is now being altered in the USA to indicate that it can be used at ages one month to six years. However, because it has been found that it may have a more potent effect at those ages, a lower starting dose of 10 mg/kg daily is recommended. In older children and adults the typical starting dose is 10—20 mg/kg daily.
Prof Levy hoped that the clarification provided by the study would reassure parents and physicians who had been reluctant to use BH4 with children under four as this group was not part of the Phase III study on which the original label recommendations were based.
Watch Prof Levy’s presentation online. http://bit.ly/PKUlectures
1 Prof Nenad Blau (University Children’s Hospitals, Heidelberg, Germany and Zürich, Switzerland)
2 Croatian Pediatric Society, Croatian Medical Association, Šubićeva 9, 10000 Zagreb. www.hpd.com.hr
3 Prof Ivo Barić (Department of Paediatrics, University Hospital Center Zagreb, Zagreb, Croatia)
4 Dr Urh Grošelj (Department of Pediatric Endocrinology, Diabetes and Metabolism, University Children's Hospital, UMC Ljubljana, Ljubljana, Slovenia)
5 Croatian PKU Association. www.fenilketonurija.hr
6 Albania, Bulgaria, Bosnia and Herzegovina, Croatia, Kosovo, Macedonia, Moldova, Montenegro, Romania, Serbia and Slovenia
7 Prof Nicola Longo (Division of Medical Genetics, University of Utah, Salt Lake City, USA)
8 NCT01889862L - a Phase 3 study to evaluate the efficacy and safety of self-administered subcutaneous injections of BMN 165 in PKU adults who have previously been exposed to BMN165 (prism302)
9 NCT01819727 - an open-label Phase 3 study of BMN 165 for adults with PKU not previously treated w/ BMN 165 (prism301)
10 Longo N et al. Single-dose, subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria: an open-label, multicentre, Phase 1 dose-escalation trial. Lancet 2014;384:37-44
11 Prof Aurora Martinez (Department of Biomedicine, University of Bergen, Bergen, Norway)
12 Prof Harvey L. Levy (Department of Pediatrics, Harvard Medical School Boston, Boston, USA)
New perspectives in therapy
4 | PKU Newsletter Issue 6 . September 2014
Low Phe essential for better brain function in childrenThe brain develops rapidly in childhood and is vulnerable to damage resulting from high Phe concentrations, Dr Rianne Jahja13 reminded delegates. It is important to monitor neurocognitive outcomes in children so that adequate control can be maintained. Brain development continues until individuals are 20 years old, during which time there are specific periods that are critical for the maturation of different functions. It is vital to ensure that adequate Phe control is maintained for long enough to ensure fully normal brain maturation.
Although there is some evidence that 360 µmol/l is an acceptable upper limit for blood-Phe Dr Jahja recommends that Phe should be kept below 240 µmol/l as this leads to better outcomes, particularly for complex executive function and motor control.
Dr Jahja reported on the PKU-COBESO study, which looked at performance of neurocognitive tasks. This 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. For further reading see Jahja et al (2014).14
Teenage troublesThe level of adjustment to illness in early adolescence predicts disease control in the transition to adulthood, Dr Maureen Cleary15 reminded delegates.
Encouraging adolescents to maintain adherence to the PKU diet is difficult but it is vital that every effort is made to provide patients with information tailored to their developmental stage to encourage them to stay on-diet.
Of most concern for adherence is ‘middle adolescence’ when young people, as part of the process of developing as individuals, tend to reject authority (including medical professionals) and are most likely to take health risks.
What are the top tips for talking to teenagers? Don’t talk about the future, advised Dr Cleary because the prevention of long term complications is a poor motivator for compliance in these patients. Instead, focus on the present, talk about how sticking to diet will help with the exam that they want to pass or the sport they want to excel in.
What is best for adults
While the concept of ‘diet for life’ for PKU patients is intellectually appealing, in reality some patients are off-diet and leading normal lives, according to Dr Robin Lachmann16.
Without clear evidence of the long-term effects on outcome for patients resulting from elevated Phe levels in adulthood it is vital that physicians support patients in their choice regarding dietary adherence. It is important that we do as much as possible to ensure that patients are not lost to follow-up. This allows patients to be monitored and allows them to access support easily, should they wish to return to the PKU diet.
Adult patients have been shown to have white matter abnormalities on imaging, along with defects in executive function and reaction times on neuropsychological testing. However, many of these findings appear to be related to historical rather than current Phe levels. White matter legions seen on magnetic resonance imaging and neuropsychological reaction times appear to be related to Phe levels at the time of testing, but are reversible if the PKU diet is resumed. At Phe levels <800 µmol/l, adults with PKU perform similarly to controls in terms of reaction time. The clinical significance of these findings is unclear.
Free to choose a bad diet
Patients treated with BH4 have the
freedom to increase their intake of animal protein and other foods forbidden by the PKU diet. However, suddenly adding meat, fish, and dairy products to the diet presents challenges for patients, Prof Skadi Beblo17 told delegates. Patients can find it hard to develop a taste for the ‘right’ foods and have a tendency to adopt the ‘bad’ nutritional habits of their healthy peers. In Prof Beblo’s clinic it has been seen that, for patients taking BH4 the supply of macronutrients in the diet is sufficient but levels of the micronutrients: iodine, calcium and vitamin D are inadequate. Patients need to be supported and encouraged to eat a healthy diet.
Brain function
Prof François Feillet has prepared a video tutorial, ‘Nutritional treatment and biochemical follow-up of PKU: What should be measured? What should the targets be?’ Watch it online: http://bit.ly/PKUlectures
www.pkuacademy.org | 5
What do mouse models tell us? Mouse models have been and continue to be vital in the development of our understanding of PKU and the development of diet-, cell- and gene-based therapies, Prof Beat Thöny18 explained.
There are three distinct mouse models in use: PAH-enu1, PAH-enu2 and PAH-enu3. These strains were all created using random chemical mutagenesis. PAH-enu1 is a model for mild PKU/hyperphenylalanemia (HPA), the other two are models for classical PKU. PAH-enu2, the most widely used model, carries a missense mutation that completely inactivates the PAH.
Recently, heterozygous PAH-enu2/wt mice have been employed as a sensitive assay system to test antisense oligonucleotides for splice suppression therapy in the liver.
Oxidative stress in PKU
Oxidative stress caused by high Phe concentrations may be involved in the pathophysiology of brain dysfunction, said Dr Priscila Mazzola19. In vitro, high Phe concentrations (similar to those found in patients) lead to oxidative stress in neurons, although the mechanisms are not fully understood. In animal models of PKU there is oxidative stress in the brain, due to both increased production of reactive species and decreased antioxidant
capacity. Given the degree to which the brain is affected in PKU, the possibility that oxidative stress may play a part is worthy of investigation. Studies in PKU patients have found increased damage to biomolecules and lower antioxidant defences in plasma.
A number of studies on antioxidants (or redox active substances) have been conducted, showing that these substances can prevent oxidative stress in PKU. The PKU dietary formulas supply micronutrients, many of which are antioxidants. New antioxidant therapies are constantly being studied in order to prevent oxidative stress in PKU.
Oxidative stress is characterised by an imbalance in the production and removal of reactive chemical species and free radicals. These harmful substances are normally formed during cellular respiration and easily react with biomolecules. Cellular damage is prevented by the antioxidant system, a combination of enzymatic and non-enzymatic defences which convert reactive species of oxygen into non-toxic compounds.
A question of taste
Children with PKU, aged four years and over, when given the choice, prefer sweet foods over savoury foods. This, explained Prof Anita MacDonald20 , contradicts the long-held theory that early introduction of bitter-tasting Phe-free L-amino acids alters food preferences in children with PKU.
Prof MacDonald shared the results of an observational, controlled,
prospective study recently completed by her team. The study compared the taste preferences of 35 children with PKU and 35 age/gender matched (control) children aged 4—13 years.
In a blind taste test, the children tasted 10 Purée foods in random order, rating them using a 7-point pictorial hedonic scale (super yummy to super yucky) and then ranked them in preferential order. Carers completed a neophobia and food frequency questionnaire.
Both PKU and control groups rated sugar-containing foods highly. Generally, children with PKU ranked vegetables higher than controls; 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 50—100% more high energy foods including: sugar-containing drinks, sweets, chips, sweet biscuits and crisps.
13 Dr Rianne Jahja (Division of Metabolic Diseases, University Medical Center Groningen, Beatrix Children's Hospital, University of Groningen, Groningen, The Netherlands)
14 Jahja R et al. Neurocognitive evidence for revision of treatment targets and guidelines for phenylketonuria. J Pediatr 2014;164:895-899
15 Dr Maureen Cleary (Department of Metabolic Medicine, Great Ormond Street Hospital, London, UK)
16 Dr Robin Lachmann (Charles Dent Metabolic Unit, National Hospital for Neurology and Neurosurgery, London, UK)
17 Prof Skadi Melanie Beblo (University Children's Hospital, University Hospital Leipzig, Leipzig, Germany)r
18 Prof Beat Thöny (Division of Metabolism, Department of Paediatrics, University of Zürich, Zürich, Switzerland)
19 Dr Priscila Mazzola (Department of Molecular Neurobiology, Beatrix Children's Hospital, University of Groningen, The Netherlands)
20 Prof Anita MacDonald (Dietetic Department, The Children's Hospital, Birmingham, UK)
From basic knowledge to daily management
Watch Dr Mazzola's presentation online: http://bit.ly/PKUlectures
6 | PKU Newsletter Issue 6 . September 2014
Predicting BH4 responsive phenotypes
It is possible to quantify the loss of stability in the PAH enzyme and predict BH4 responsiveness using genotype scoring, explained Ms Sarah Wettstein23. This offers a robust method for patient characterisation and management.
Prof Nenad Blau’s group set out to analyse data from locus-specific database PAHvdb (834 variants) and genotypes database BIOPKUdb (4,181 patients with full genotype) at www.biopku.org.
The relative frequencies of mutations, genotypes, affected gene regions and protein domains were calculated.
PAH mutations and genotypes were scored using four prediction tools. These scores were correlated with residual enzyme activity, patients’ phenotype, and BH4 response.
Ms Wettstein was awarded the prize for best oral communication at the symposium. She was presented with a voucher for €4,000 to fund her further participation in CME courses and congresses.
Sapropterin in Turkish children under four years oldSapropterin is both safe and efficacious as a treatment for HPA in children under four-years-old, said Prof Serap Sivri24 , presenting recent work in Turkey. The BH4 loading test, along with molecular studies, is useful in the detection of responders but not always in the prediction of long-term responsiveness.
In the study (single centre, cross-sectional) 44 patients (28 boys, 16 girls) were found to be responsive using a 48-hour BH4 loading test. Sapropterin treatment was for 12—45 months. Diet was liberalised in 25/30 (83.3%) of patients.
Phe tolerance increased 2.26-fold (0.88–4.23) from a median of 47.5 mg/kg/day to a median of 114 mg/kg/day (p<0.001). Phe tolerance did not change significantly in 5 patients, so sapropterin was discontinued.
Effects of sapropterin treatment on blood prolactin and serotoninRecent work shows that BH4
at 20 mg/kg might improve neuropsychological functioning by increasing cerebral neurotransmitter synthesis in both BH4-responsive and unresponsive PKU patients, reported Dr Danique van Vliet25.
The effect seems irrespective of age, but may be dependent on:
�� dose
�� availability of adequate tyrosine (Tyr) and tryptophan (Trp)
�� Phe concentration (specifically the Phe:Tyr and/or Phe:Trp ratios.)
Reduced brain dopamine and serotonin concentrations have long been considered a contributory factor in the impaired neuropsychological functioning seen in PKU patients.
European guidelines statements: have your say‘I am not here so much to talk as to listen!’ announced Ms Annemiek van Wegberg22. As we progress towards the formulation of EU guidelines we are finding that in some areas there are sufficient data for the working groups to reach consensus relatively easily. However, in other
areas the evidence-base is weak or contradictory. Consensus is much harder to reach when this is the case. The unpublished experience-base of the wider PKU community can be vital here. Ms van Wegberg asked delegates for their views and encouraged them to be part of the process.
Oral communications
Is early aging and cognitive loss a risk in PKU?
A link between high Phe levels and neurodegenerative disorders has been suggested, Dr Andrea Pilotto21 told delegates. If this proves to be the
case, it could have a bearing on the current recommended ‘safe’ Phe levels for adults and be a concern for adult patients wishing to liberalise their diet.
Early treated PKU (ETPKU) patients could have an increased risk of neurodegenerative disorders because of the complex pathophysiology of the disease. Dysmetabolic abnormalities, oxidative stress and amyloid pathology may increase the risk of incident neurodegenerative disorders such as Alzheimer’s (AD) or Parkinson’s (PD) diseases in ETPKU subjects entering their middle age. Most ETPKU patients present in early adulthood with subtle
neurological symptoms, especially in behavioural, executive, visual-spatial and motor functions. The damage to the central nervous system has been explained by several mechanisms including vitamin or neurotransmitter deficits, increased oxidative stress and white matter abnormalities.
An atypical amyloid pathology, probably secondary to Phe aggregation, has recently been 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 patients entering middle age.
www.pkuacademy.org | 7
The 2014 Asbjørn Følling Award was presented to Prof Friedrich Trefz28 who delivered this year’s Asbjørn Følling lecture.
Prof Trefz is a familiar figure in the international PKU community. He has been a key figure in a number of international studies that have furthered our understanding of maternal PKU (MPKU) and the use of BH4 therapy.
After receiving his medical degree from the University of Heidelberg (Germany) in 1972 Prof Trefz trained in pediatrics under Prof Horst Bickel. He became head of the Sektion Pädiatrische Stoffwechselkrankeheiten at the University Children’s Hospital of Heidelberg in 1985.
In 1992 he took over as head of the department of pediatrics at the University of Tübingen, Reutlingen (Germany).
In 2010 he retired from the Children’s Hospital in Reutlingen. He is still a professor of the University of Tübingen,
Germany and continues to treat patients with metabolic disorders, focusing primarily on PKU and MPKU.
Prof Trefz began his lecture by looking back at key discoveries, some which were overlooked at the time they were first noted, which have subsequently led to new approaches in the treatment of PKU.
‘What we can learn from looking back, is how PKU became a model for diagnosis, prevention and treatment of an inborn error of metabolism’ stated Prof Trefz. ‘The detection of this disease by Dr Asbjørn Følling (1888—1973) shows us the importance of looking carefully at our patients. His finding is just a ‘case report’: two handicapped children in one family without special stigmata but with an unusual chemical reaction in urine.
‘Many questions are still open and this has made the “PKU story” more attractive to basic researchers and clinicians today than it was 15 years ago. The answers to these questions are important for the treatment of our patients. I believe it will finally result in personalised medicine. For example, one patient may benefit from BH4 treatment, the other not.
‘PKU is a disease model for inborn errors of metabolism. History shows the progress on different diagnostic levels which we now call genomics, proteomics and metabolomics. Now we are beginning to understand the interactions of metabolite, protein and mutations in the gene.
‘Only by continuing our close co-operation between different specialisations within the field and by developing new computerised models and networks will we provide further rapid progress to help our patients.’
21 Dr Andrea Pilotto (Neurology Department, Prof. Daniela Berg Research Group, Hertie-Institut for Clinical Brain Research and German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany)
22 Ms Annemiek van Wegberg (University Medical Center Groningen, Radboud University Medical Center, The Netherlands)
23 Sarah Wettstein (c/o Prof Nenad Blau, Division of Metabolism, University Children’s Hospital, Zürich, Switzerland)
24 Prof Serap Sivri (Department of Pediatrics, Section of Metabolism, Faculty of Medicine, Hacettepe University Hospital, Ankara, Turkey)
25 Dr Danique van Vliet (Division of Metabolic Diseases, University Medical Center Groningen, Beatrix Children's Hospital, University of Groningen, Groningen, The Netherlands)
26 Dr Patrícia Fernanda Schuck (Laboratory for the Inborn Errors of Metabolism, Universidade do Extremo sul Catarinense Criciúma (UNESC), Santa Caterina, Brazil)
27 Prof Uçar Kalkan (Pediatric Metabolism Department, Ege University Medical Faculty, İzmir, Turkey)
28 Prof Friedrich K. Trefz (Children’s Hospital, Kreis Kliniken Reutlingen GmbH, Klinikum am Steinberg School of Medicine, University of Tübingen, Reutlingen, Germany)
It has been suggested that BH4, also a cofactor for Tyr and Trp hydroxylase, could also have a role in improving brain neurotransmitter synthesis beyond its effect through lowering Phe concentrations.
Hyperphenylalaninemia causes cholinergic alterations in rat brainPhe induces cholinergic alterations in rat brains, said Dr Patrícia Fernanda Schuck26. Since cholinergic imbalance is associated with progressive neurologic decline in learning and memory functions it is possible that acetylcholinesterase (AChE) alterations might contribute to the intellectual deficiency observed in HPA patients. Dr Schuck presented results from ongoing work to investigate the in vitro, in vivo and in silico effects of Phe on AChE activity and its expression.
Microcephaly and hyperphenylalaninemiasScreening for maternal PKU and mild hyperphenylalaninemia (MHP) is essential if the offspring has early-onset microcephaly not attributable to PKU, said Prof Serap Sivri24. This is especially the case in a country, such as Turkey, where there is a high incidence of the disease but relatively late adoption of national screening.
BH4 responsivenessProf Uçar Kalkan27 reported the efficacy of BH4 treatment in terms of increased Phe tolerance in patients treated at the Ege University (İzmir, Turkey.) BH4 administration in HPA increased phenylalanine tolerance in BH4-responsive patients.
Sarah Wettstein was awarded the prize for best oral communication.
Asbjørn Følling lecture and award
8 | PKU Newsletter Issue 6 . September 2014
European and US guidelines — controversies
The workshop led by Prof Francjan van Spronsen30, Dr Alberto Burlina31 and Prof Nicola Longo32 looked at the principal challenges to reaching consensus in the formulation of regional, Europe wide and worldwide guidelines for the management of PKU.
The workshop explored issues relating to:
�� defining PKU and the development of a simplified classification scheme
�� defining ‘expert PKU’ centres
�� defining the Phe level at which PKU treatment should be started.
At present, classification of PKU is usually based on pre-treatment Phe or Phe tolerance. A simplified classification is under consideration:
�� patients who need strict dietary treatment (PKU)
�� patients who do not need any treatment (mild HPA)
�� patients who may be treated with BH4, either alone or in a combination with diet (BH4-responsive PKU.)
The definition of an expert centre is usually based on the number of patients (experience) and proven research from that centre. But, what should the minimum number of patients be for a centre to be expert, >50, >200 or somewhere in-between?
And finally, what is the basis for the optimal Phe level? Are there parameters to include in follow-up beside absolute Phe level? What is the role of Tyr supplementation?
Maternal PKU — Interactive clinical case presentationsThis workshop, led by Prof Friedrich Trefz28 and Ms Louise Robertson29 looked at the importance of managing maternal PKU (MPKU) and focused on practical actions that can be taken to improve outcomes.
Prof Trefz reminded participants that untreated MPKU can lead to a number of birth defects including: heart malformations, oesophageal atresia, renal agenesis, Potter syndrome, anencephalus and cleft palate. Ideally, dietary treatment should start before conception. For planned pregnancy, women should be encouraged to use contraception until they have achieved Phe levels <360µmol/l.
Ms Robertson focused on the care needed by the mother. Throughout pregnancy, support and education is essential.
It is vital to ensure that the diet provides adequate energy for appropriate weight gain and that the protein demand is met by Phe-free L-amino acids. Phe levels need to be monitored closely (at least twice a week). Adequate folic acid supplementation should be provided. Breastfeeding should be encouraged.
29 Louise Robertson (University Hospitals Birmingham NHS Foundation Trust, Old Queen Elizabeth Hospital, Birmingham, UK)
30 Prof Francjan J. van Spronsen (Beatrix Children’s Hospital, University Medical Center Groningen, Groningen, The Netherlands)
31 Prof Alberto Burlina (Division of Metabolic Diseases, Department of Pediatrics, University Hospital Padua, Padua, Italy)
32 Dr Amaya Bélanger-Quintana (Unidad de Enfermedades Metabólicas, Servicio de Pediatría, Hospital Ramón y Caja, Madrid, Spain)
33 Dr Margreet van Rijn (Section of Metabolic Diseases, Beatrix Children's Hospital, University Medical Center Groningen, Groningen, The Netherlands)
Find out more about the role of the ESPKU in the formulation of European guidelines. Watch the Perspective interview with President of the society, Mr Eric Lange. http://bit.ly/PKUperspective
Prof Trefz has created a video tutorial for PKU Academy, ‘Maternal PKU: who to treat, and how to treat.’ Watch the presentation online. http://bit.ly/PKUlectures
Diet plus sapropterin in clinical practice —Interactive clinical case presentationsThe workshop lead by Dr Amaya Bélanger-Quintana32 and Dr Margreet van Rijn33 focused on adapting the PKU diet for use with sapropterin. Among the points discussed, participants considered that:
�� it is important to reassess Phe tolerance before beginning treatment with sapropterin as the recorded Phe tolerance of the patient may not have been tested for some time and may have increased
�� sapropterin treatment is much more than a certain number of tablets each day; the correct balance of medicine and the best diet for the patient must be identified
�� patients taking sapropterin require counselling by healthcare professionals using an individual approach
�� changing conditions might require that the treatment needs to be re-evaluated
�� a small amount of Phe mixture can help get better control
�� sapropterin does not change compliance issues and may even require more attention from patients and caretakers to achieve a better outcome.
Workshop sessions Delegates at the 2014 Annual Multidisciplinary European PKU Symposium participated in three workshop sessions to explore current practice in PKU management
www.pkuacademy.org | 9
Prof Harvey Levy12 of the Boston Children’s Hospital and Harvard Medical School, Boston (USA) is widely associated with the development and advancement of newborn screening. He addressed delegates at the 2014 Annual Multidisciplinary European PKU Symposium on use of BH4 in children under four years old.
As well as being a practising clinical biochemical geneticist, Prof Levy is an active researcher. His recent publications, including Levy (2014)34 and Landau et al (2014)35 have continued his long association with screening for inborn errors of metabolism (IEM), asking the questions, ‘Where is newborn screening going?’ and ‘Where should it be going?’ We asked him the same questions.
A genomic future‘It is quite clear’, Prof Levy told us, ‘that we now have the ability to take the newborn blood specimen; the filter paper containing circles saturated with blood we collect to test for PKU and other metabolic disorders, and to go further with it. Thanks to new developments in genomic sequencing we now have the opportunity to use that blood specimen to look at all of the different base pairs of the baby’s DNA.
‘This is both an exhilarating and a frightening possibility. With genomic screening you can theoretically identify virtually any genetic disease as well as variations within those disorders that may be important for the baby.
‘But in the course of looking at all of the different areas of the DNA
there is a risk that we could also misinterpret what we see. We could think that the baby has a disease because we find a variation in the DNA that in actuality is simply a harmless difference. We do not yet know all of the normal variations within the world’s population of individuals.'
Eliminate the false-positiveHe added: ‘The promise is that we will be able to identify far more diseases by newborn screening than we are now identifying, many of which are important to identify because we might be able to use early treatments to prevent the complications. Even when we don’t have treatments it would still be of great benefit to the family to have a diagnosis as early as possible in the course of the child’s life.
‘On the other hand we may be saying things to families about their children that are totally untrue because we don’t really know the implications of many of these variations. This could cause anxiety, uncertainty and possibly harm, including unnecessary treatment. These promises and concerns are being examined in a multicentre research effort funded by the National Institutes of Health (NIH) in the USA. Four centres are involved. Boston is one of them. We will be examining these questions for five years. Hopefully, at the end we will all know much more about what to expect if genomic sequencing is added to newborn screening.’
Aside from the ethical considerations arising from misdiagnosis (false-positive
results), surely, we thought, there is also a danger that genomic screening could generate an overwhelming quantity of data.
Data overload
Prof Levy commented that ‘Genomic sequencing has the potential to generate an extraordinary amount of data, so we’ll need to decide how wide we open the “aperture.” Perhaps we will initially focus our search on just a few genes but then later open up the aperture wider to examine many more genes. Questions such as “How many genes should we look at?” and “Which genes should we look at?” are encompassed within our study’Are there benefits to the genomic approach that go beyond screening, we asked. ‘If we understand disorders in terms of both their basic genomic aspects and their biochemical aspects we will have more information on which to base our thinking for new therapies,’ Prof Levy answered.
‘If we know, for example, precisely, how an enzyme works, we might be in a position to develop therapies capable of restoring activity to that enzyme in a patient with an IEM, thus concerting a disease to a non-disease.'
Tailored treatments‘My hope for the future in PKU therapy is really that we will not only have better treatments for PKU but also that we’re going to have a much better understanding of PKU and other IEMs.
‘When I first entered the field of biochemical genetics, we knew that these diseases were inherited, that they had some sort of genetic basis. But we had no idea what the genetic basis was or what the genes looked like. Today, we can identify the mutations of just about every individual with an IEM and we can even begin to look at using this information to design, develop and tailor treatments. I am more excited about working with PKU and other IEMs today than when I first began. It’s what keeps me going.’
34 Levy HL. Newborn screening: the genomic challenge. Mol Genet Genomic Med 2014;2:81-4
35 Landau YE et al. Genomics in newborn screening. Pediatr 2014;164:14-9
PerspectivesHarvey Levy
Prof Harvey Levy shares his hopes for the future of newborn screening
Watch the online video of this interview. http://bit.ly/PKUperspective
10 | PKU Newsletter Issue 6 . September 2014
Work to evaluate clinically the use of pegylated recombinant phenylalanine ammonia lyase (PEG-PAL) as a potential therapy for PKU is ongoing. Prof Longo provided an update on the Phase III clinical studies looking at this novel enzyme substitution therapy when he spoke at the 2014 Annual Multidisciplinary European PKU Symposium.
Prof Longo7 is both a physician and a research scientist. He sees patients with various metabolic disorders, including PKU from the State of Utah but also the neighbouring states of Idaho, Wyoming and Nevada. He leads the Division of Medical Genetics at the University of Utah in Salt Lake City (USA). Over recent years, his work to develop PEG-PAL as a therapy for PKU has attracted interest from around the world.
What is PEG-PALWe asked what makes PEG-PAL different from Sapropterin, the only drug so far to be licensed for use with PKU patients.
‘Sapropterin is a very nice therapy because in those patients who are responsive, their high blood-Phe levels are reduced to more acceptable levels,’ the professor said. ‘However, not all patients are responsive, as this is dependent on their PAH genotype.
‘Also, even with responsive patients, the levels do not reduce to the level we would consider normal in a person without PKU. Enzyme substitution therapies such as PEG-PAL are promising because they
are not dependent on a specific genotype and this greatly widens the number of people who may be amenable to the therapy. The second difference is that sapropterin is an oral medication and PEG-PAL is an injectable one. Obviously it is usually easier to take an oral medication.
‘A third difference is that a PKU patient taking sapropterin will synthesise Tyr from Phe, as a product of the transformation of Phe. With PEG-PAL there is no generation of Tyr so we might need to supplement Tyr for patients treated with the enzyme.’
To substitute or to replaceThis raises the question, why substitute? Why not synthesise PAH? ‘In theory we could just give PAH back to patients, Prof Longo explained. ‘We don’t do this because it is a formidable task to synthesise PAH and to make it active. Bacteria make enzymes by different mechanisms to humans and we really don’t understand how to achieve the necessary supramolecular structure or how to involve the cofactor which stabilises the enzyme.’
Does the use of a non-human enzyme pose an increased risk of triggering an immune response, we wondered.
Prof Longo explained: ‘Because PEG-PAL is an enzyme derived from bacteria we have to expect a degree of immune response, but there are similar enzymes in humans like histidine ammonia lyase (HAL) which have some degree of homology. The principle of using foreign proteins to reduce
the level of some chemical in the body has also been proven previously. Pegloticase for example, licensed for the treatment of gout, is a pegylated bacterial enzyme. While every patient with PKU will respond to PEG-PAL we still do not know the degree to which the immune response of a patient taking the therapy will be an issue.
‘How tolerable will it be to patients? How will the patient’s immune response limit the action of the enzyme? We hope, through the Phase III study to learn how patients adapt to the administration of the foreign protein over time. We know from other cases that the human body can develop tolerance to the administration of foreign proteins over time but we don’t yet know whether this will be the case with PEG-PAL. We might find that we have to match PEG-PAL to patients according to their immune reaction, just as we have to match sapropterin to the patient’s PAH genotype. The Phase III study should allow us to understand this better.’
Assessing the unassessable PEG-PAL is currently being investigated for safety and efficacy with regard to controlling blood-Phe levels. But, how important is it going to be to the lives of patients? The extent of the effect of reduced blood-Phe on the executive function of adult patients is much harder to quantify and the value of aggressively controlling blood-Phe levels in adults is still debated.
Professor Longo pointed out that: ‘If we can demonstrate an effect of lowering the Phe level on executive function or on other adult intellectual functioning in such patients then we would have data to show that even in adults a reduction in Phe level is beneficial.
‘We will need to develop better tests of executive function that are less affected by confounding factors, like inattention, stress and mood. The tests will also need to be simple to use and not inconvenient for the patients or the physicians so that they can be repeated often. This is a challenge but something that we must continue to work on with our psychologist colleagues.’
PerspectivesNicola Longo
Prof Nicola Longo discusses the potential of PEG-PAL therapy and his hopes, one day, for a PKU cure
Watch the online video of this interview. http://bit.ly/PKUperspective
www.pkuacademy.org | 11
Prof Aurora Martinez11 provided delegates at the 2014 Annual Multidisciplinary European PKU Symposium with an update on the search for new and better chaperone candidates. Pharmacological chaperones are chemicals with the ability to restore some, or all function to an enzyme affected by a gene mutation. These have great potential as therapies for PKU and other IEMs, caused by missense mutations.
Prof Martinez began working with PAH as a postdoctoral researcher. Today, now a Professor of Biomedicine at the University of Bergen (Norway) and director of the Biorecognition research group she continues to work with this challenging protein. She told us: ‘I joined a group that was already working on PKU and initially I was looking at the expression of the protein, trying to understand the effect of the mutations on the action of the enzyme; the relationship between genotype and phenotype.’
Misfolding holds the key'We realised that most mutations that lead to IEMs do so by causing a misfolding of the protein, which destabilises it and results in a loss of activity,' Prof Martinez explained. ‘PAH is a very complex protein that is responsible for a complex reaction. Mutations can cause conformational changes that have a significant effect on the enzyme function and lead to faster degradation of the enzyme itself.'
How then, we asked, did this evolve into a hunt for pharmacological chaperones and why are these important.
The Professor replied: ‘We found that it is possible to recover part of the lost activity by using small compounds; these are the chaperones that recover the protein. They can renature and stabilise the native conformation and delay degradation, increasing the lifetime of the enzyme.’
Success of sapropterinProf Martinez began by saying ‘One of the best known small molecule chaperones is the naturally occurring cofactor BH4. Exogenous BH4, is now licensed as sapropterin for use as a therapy for PKU. However, it needs to be remembered that BH4 is itself a co-substrate in the reaction, so it has other effects. It can accelerate the reaction. All reactions in the body have their own homeostasis so if you try to increase the concentration of a compound above a threshold level there will be other reactions that will try to degrade it.
‘In addition,' she continued ‘the chaperone effect of BH4 is only seen in 20—30% of patients, as it is dependent on the nature of their genotype and the resulting misfolding. There are many patients with misfolding that cannot be helped using BH4. For these two reasons, BH4
although effective, is not the ideal chaperone and so we continue to look for alternatives.’
The perfect chaperoneHow, we asked, was the search going. Prof Martinez replied: ‘We studied BH4 for many years, as this really was the “Proof of concept” for pharmacological chaperones. From this, we started our search for other compounds, not found in vivo, that could have a better chaperone effect without affecting the reaction in other ways. We had a first generation of compounds that worked very well but had limitations. We found there were issues with solubility and specificity. They also affected other aromatic amino acid hydroxylases.
‘For our second generation compounds we changed our approach, this time screening for compounds that could work with BH4, looking for synergistic effects. This time we were screening for compounds that would bind to different parts of PAH to those which BH4 binds, in the hope that the two compounds together would have a greater stabilising effect. If this were achieved, the new chaperone could potentially stabilise PAH while allowing BH4
to act normally so the reaction would proceed as it should.
‘We are now in the final steps of in vitro studies where we have combined a formulation of compounds with BH4 and we are seeing that this is in fact possible. With these second generation compounds we have been looking for effects on other hydroxylases from the start.
‘There are a lot of things to take into account at each step of the investigational process and many more steps to go before we reach even the preclinical stage but it is promising and I hope that we will see new pharmacological chaperones that are soluble, effective and specific.’
What then were her hopes for the future for PKU treatment? ‘I think it is possible, given the different therapeutic approaches currently being investigated, that we would be able to better adapt to the three categories of patients, for example: the very mild, the mild and the classic PKU, and each of these three classes will have a therapy. And the diet will always be there for all.’
PerspectivesAurora Martinez
Prof Aurora Martinez tells us why chaperones are still in fashion
Watch the online video of this interview. http://bit.ly/PKUperspective
12 | PKU Newsletter Issue 6 . September 2014
PKU Academy fellowshipThe PKU Academy in collaboration with EXCEMED has developed a public competition for the award of a fellowship in the field of PKU. The fellowship is given to one young graduate in medicine, specialised or specialising in metabolic disorders, or to scientists or dietitians, with at least one year of experience in the PKU field. It supports planned research projects.
The 2013 award was awarded to Dr Patrícia Fernanda Schuck26. She received €24,000 to develop her research project proposal over the next two years. In accepting the award Dr Schuck remarked, ‘I’m starting my scientific career so it’s very important to have some financial support. In Brazil it is not that easy to get funding when you are at the beginning of your career so this award will help me a lot by supporting my research. I will be able to buy reagents and equipment that I would have struggled to fund in other ways.’
Dr Priscila Mazzola19, winner of the 2012 Fellowship presented her final results to delegates and thanked the PKU Academy for their support. Her study took place in two parts, looking at the effect of:
1. Acute exercise on metabolic and hormonal levels in treated PKU: comparing eight treated PKU patients with 17 controls.
2. Regular exercise on amino acid and oxidative stress levels in PKU mice: comparing 16 wild-type (WT) and 16 PAH-enu2 C57BL/6 mice. The 16 mice of each type were assigned to either and exercise (Exe) group or a sedentary (Sed) group. The Exe mice had access to a running wheel, the Sed group did not.
Excellence in Medical Education
Editorial Board MembersProf Nenad Blau, Prof Anita MacDonald, Prof Francjan van Spronsen
PKU Academy Newsletter
EditorsFrancesca Pelligrino,Howie Watkins
ContributorsAlison Eden, Francesca Nardecchia, Howie Watkins
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Dr Priscila Mazzola, winner of the 2012 fellowship presented her final results to delegates
We welcome your feedback or requests for topics in future issues. Please contact us at [email protected]
Find out more about the PKU academy fellowship: http://bit.ly/PKUfellowship
Dr Mazzola reported that:
1. In pre-exercise, PKU patients showed lower levels of total cholesterol, both high- and low-density lipoproteins (HDL and LDL), and higher adiponectin concentration. In both PKU patients and controls total cholesterol, HDL and LDL increased after exercise; in the control (but not in PKU group) adrenaline increased and glucose decreased.
2. The SedPKU mice showed increased oxidative stress and impairments in co-ordination and balance compared with SedWT mice. ExePKU mice showed increased antioxidant potential levels in comparison to SedPKU mice. Plasma Phe and branched chain amino acid levels were negatively correlated with the daily distance ran only for the ExePKU mice.
She concluded that HPA along with diet composition might change acute exercise outcomes and that in the mouse model, oxidative stress and exercise increased antioxidant systems in the brain. Exercise seemed to modify plasma amino acid levels.
2012 fellowship award
Dr Patrícia Fernanda Schuck, winner of the 2013 fellowship
