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Comprehensive analysis of current PKU management practices and associated patient and parent-reported… Yuskiv, Nataliya 2020

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COMPREHENSIVE ANALYSIS OF CURRENT PKU MANAGEMENT PRACTICES AND ASSOCIATED PATIENT AND PARENT-REPORTED OUTCOMES IN CANADA by  Nataliya Yuskiv  MD, Danylo Halytsky Lviv National Medical University, 1995 M.P.H., Emory University, Rollins School of Public Health, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   October 2020  © Nataliya Yuskiv 2020   ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: COMPREHENSIVE ANALYSIS OF CURRENT PKU MANAGEMENT PRACTICES AND ASSOCIATED PATIENT AND PARENT-REPORTED OUTCOMES IN CANADA  submitted by Nataliya Yuskiv in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine  Examining Committee: Dr. Sylvia Stockler-Ipsiroglu, MD, PhD, MBA, FRCPC, Professor of Pediatrics, UBC Supervisor  Dr. Elizabeth K. Potter, PhD, Associate Professor, University of Ottawa Co-Supervisor Dr.Adele Diamond, PhD, FRSC, Professor of Developmental Cognitive Neuroscience, UBC University Examiner Dr.Anna Lehman, MD, FRCPC, MA, Associate Professor, Department of Medical Genetics, UBC University Examiner   Additional Supervisory Committee Members: Dr. Jean-Paul Collet, MD, PhD, Clinical Professor and Associate Head Research, UBC  Supervisory Committee Member  iii  Abstract  Phenylketonuria (PKU) is a genetic metabolic disorder requiring life-long treatment to prevent severe neurocognitive disability. Despite early treatment, suboptimal neurobehavioral outcomes are common in individuals with PKU. The Canadian Inherited Metabolic Diseases Research Network (CIMDRN) presented an opportunity to investigate current approaches to care and associated outcomes in children with PKU and their parents.  Four studies were conducted herein. The surveys of Canadian metabolic dietitians and physicians, described in Chapter 2, helped to understand current PKU management practices from the perspectives of healthcare providers. Data reflecting practices in metabolic treatment centres and associated outcomes were derived from the CIMDRN clinical longitudinal PKU database (Chapter 3). Finally, parent-reported child’s health and parental quality of life were analyzed in connection to the quality of metabolic control (Chapter 4).  Providers’ surveys identified overall compliance with current PKU guidelines. Variation was found in case ascertainment, diagnostic workup (neonatal BH4 loading test), monitoring and follow up tests (neuropsychological assessments), treatment practices (sapropterin and large neutral amino acids, LNAA) and organization of care in metabolic centres. The mean phenylalanine (Phe) levels were within the recommended range of 120-360 µmol/L but increased (to 331±151 µmol/L) beyond 7 years of age for children with classic PKU. Among children with classic PKU, 32% were considered in good metabolic control by the 4th week of life. This increased to 55% by age 4-5 and declined to 28% at 8-9 years.  Tiredness, lack of concentration, irritability and moodiness were reported as moderate symptoms in children. Parents reported iv  considerable guilt due to non-adherence to the diet/supplements, their child’s anxiety, and practical impact of dietary protein restriction.  Overall, healthcare providers follow the published guidelines in providing care for their patients. However, this work has identified variation in many areas of PKU nutritional and clinical management practices in Canadian metabolic centres, including diagnostic and follow up nutritional and clinical management practices. This variation has the potential to impact patients’ long-term health outcomes. The most variation was in areas of practice that were not strongly supported by empirical evidence. This knowledge will serve to further optimize health and quality of life outcomes in Canadian children with PKU.    v  Lay Summary Phenylketonuria (PKU) is a genetic disorder that leads to the accumulation of phenylalanine in the bloodstream. Dietary treatment controls phenylalanine levels and prevents severe disability. Evidence suggests that despite early and continuous treatment, suboptimal outcomes are common in children with PKU. However, current PKU management and patient outcomes in Canada have not been comprehensively described. Four Canada-wide projects were conducted in order to comprehensively describe the quality of phenylalanine control in children as well as the quality of life of parents who care for children with PKU. These studies found that different Canadian metabolic centres provide care differently to their patients in many areas. The studies also found that the quality of the phenylalanine control is not ideal in Canadian children with PKU. Finally, caregivers reported significant burden due to guilt, anxiety and the practical impact of dietary restrictions. The results of these studies identified areas of improvement for PKU care.  vi  Preface  The four projects presented in this doctoral dissertation were conducted within the framework of the Canadian Inherited Metabolic Diseases Research Network (CIMDRN). This work was done in collaboration with a CIMDRN team of investigators and under the guidance of my thesis supervisors. I was responsible for developing the project conceptual outlines, research proposals, study protocols, and two survey questionnaires for the studies described in Chapter 2. I also performed the submission for the Research Ethics Board reviews, developed the REDCap questionnaires, and activities related to the participants’ enrollment, data entry and data analysis. For the methodological approach, I received assistance from my thesis supervisor, Dr. Sylvia Stockler-Ipsiroglu, and my thesis committee members, Dr. Elizabeth K. Potter and Dr. Jean-Paul Collet. The CIDMRN and QoL data analyses described in Chapters 3 and 4 were conducted according to the data transfer agreement between UBC and the University of Ottawa on a shared CIMDRN database, housed at the Children’s Hospital of Eastern Ontario’s Research Institute.  I fully contributed to each manuscript presented in this doctoral dissertation. My supervisor and  PhD committee members contributed to the individual project designs as well as to the overall thesis review for intellectual content. My supervisor and PhD committee members guided my data analyses and interpretation of findings. The University of British Columbia Research Ethics Board approved all four studies included in this PhD thesis.  Chapter 1 gives an overview of PKU diagnostic, nutritional and clinical management practices in light of the disease etiology, pathophysiology and clinical presentation. I also described the outcomes associated with these practices and provided the explanation why vii  intermediate (surrogate) outcomes (individuals’ blood phenylalanine levels used as a quality of phenylalanine control) are of utmost importance in management of PKU.   Chapter 2. This Chapter is an overview of current nutritional and clinical management practices related to health care for children with PKU in Canada. This information was gathered with two separate surveys of Canadian metabolic dietitians and physicians who provide care for pediatric patients with PKU.  The main purpose of both surveys was to ascertain the awareness of published PKU nutritional and clinical management guidelines and the approaches of healthcare providers to PKU management.  The dietitians’ survey was developed in 2015 and distributed to metabolic dietitians in 2016, two years after the GMDI nutrition management guideline for phenylketonuria had been published. A version of this Chapter was published in a peer-reviewed journal. Yuskiv N, Potter BK, Stockler S, Ueda K, Giezen A, Cheng B, Langley E, Ratko S, Austin V, Chapman M, Chakraborty P, Collet JP, Pender A; Canadian Inherited Metabolic Diseases Research Network (CIMDRN). Nutritional management of phenylalanine hydroxylase (PAH) deficiency in pediatric patients in Canada: a survey of dietitians' current practices. Orphanet J Rare Dis. 2019 Jan 8; 14(1):7.  I developed the survey questionnaire, collected and analyzed the data and wrote the manuscript. Dr. Beth Potter, Dr. Sylvia Stockler, Ms. Keiko Ueda, Ms. Valerie Austin, Ms. Suzanne Ratko, Ms. Erica Langley, Ms. Alette Giezen, Ms. Barbara Cheng and Ms. Maggie Chapman substantially contributed to the concept and design of the study, revision of the survey questionnaire and critical review of the manuscript. Dr. Jean-Paul Collet and Dr. Pranesh Chakraborty contributed to the concept and design of the study, and critical review of the manuscript. Ms. Amy Pender contributed to the design of the study and data acquisition. The viii  survey study protocol received approval of the UBC Children's and Women's Research Ethics Board on August 8, 2015 (H15-01291).  The metabolic physicians’ survey was developed and distributed in 2017.  The study was submitted to the UBC Children's and Women's Research Ethics Board as an amendment to the previously approved survey of metabolic dietitians, described above. The REB approval for the physicians’ survey was received on January 4, 2017 (H15-01291). Forty-five Canadian metabolic physicians who provide care for pediatric patients with PKU were invited to participate in a survey to ascertain their approaches to the diagnosis and management of PKU and awareness of published American College of Medical Genetics and Genomics (ACMG) and recently published European guidelines.  I was responsible for developing the initial draft of the physicians’ survey questionnaire, in collaboration with the experts in PKU: Dr.Sylvia Stockler (BC Children’s hospital – University of British Columbia), Dr.John Mitchell (Montreal Children’s Hospital - McGill University) and Dr.Michael Geraghty (Children’s Hospital of Eastern Ontario – University of Ottawa). I distributed the survey questionnaire and collected the data, performed data analysis and drafted the manuscript. Dr.Stockler, Dr.Mitchell and Dr.Geraghty substantially contributed to the concept and design of the study, revision of the survey questionnaire and critical review of the manuscript. Dr.Potter contributed to the concept and design of the study and critical review of the manuscript.  Chapter 3. This Chapter provides further exploration of the current management practices of PKU in Canada as well as patients’ intermediate outcomes that result from these management practices.  The management of PKU was assessed from another angle – specifically, from the perspective of practice-based evidence, analyzing the data collected within ix  Canadian Inherited Metabolic Diseases Research Network (CIMDRN). CIMDRN is a pan-Canadian multidisciplinary team of investigators with a vision to improve evidence-informed health care for children diagnosed with inherited metabolic diseases (IMD). The CIMDRN cohort study collected chart-abstracted clinical data for children diagnosed with one of 31 eligible IMD, who were born between 2006 and 2015 and treated at one of the 13 participating centres, including BC Children’s Hospital at UBC. With participants’ consent, clinical longitudinal data were abstracted from medical charts. The data included variables pertaining to the ongoing care and health outcomes from participant’s birth until March 31, 2017. The data were analyzed descriptively. The CIMDRN cohort study, including enrollment, data collection, and analysis, was previously approved by research ethics boards (REB) and, in particular, by the Research Ethics Board at the University of British Columbia / Children’s and Women’s Health Centre of British Columbia (UBC C&W REB) (CW14-0023 / H13-03285). Chapter 4. The results of the quality of life survey assessing the impact of PKU on the health-related quality of life of Canadian parents / caregivers who care for children with PKU were described in Chapter 4. This multi-centre, cross-sectional QoL survey study was designed and conducted as part of this PhD project. The Children’s Hospital of Eastern Ontario (CIMDRN main site) took the lead in the Canada-wide distribution of the PKU-related Quality of Life survey to the CIMDRN participants, who confirmed their willingness to be contacted for the future research. In British Columbia, the survey was distributed by the author of this thesis due to requirements of local REB. The CIMDRN PKU-QoL survey study was approved by UBC C&W Research Ethics Board (H16-02256).  The data were collected and entered into the central CIMDRN database by CHEORI research team. The data analysis and interpretation was conducted by the author of this PhD thesis under the guidance of Dr. Beth Potter, PhD.   x  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ...........................................................................................................................x List of Tables ............................................................................................................................. xvii List of Figures ...............................................................................................................................xx List of Abbreviations ................................................................................................................. xxi Glossary ..................................................................................................................................... xxii Acknowledgements .................................................................................................................. xxiv Dedication ...................................................................................................................................xxv Chapter 1: Introduction ................................................................................................................1 1.1 Prologue to the Introduction ............................................................................................1 1.2 Frequency of PKU ...........................................................................................................3 1.3 Pathophysiology and clinical presentation of PKU .........................................................4 1.3.1 Elevated and fluctuating Phe levels and their associated suboptimal outcomes .........9 1.4 Diagnosis of PKU ..........................................................................................................11 1.4.1 The use of the neonatal BH4 loading test in differential diagnosis of hyperphenylalaninemias (HPAs) .......................................................................................... 12 1.5 Classification of PKU severity ......................................................................................14 1.5.1 Classification of PKU based on untreated blood Phe levels ......................................14 1.5.2 Classification of PAH deficiency based on Phe tolerance alone ...............................15 xi  1.5.3 Correlation between genotype and phenotype and its implications for diagnosis .....16 1.6 Treatment of phenylketonuria ........................................................................................18 1.6.1 Dietary treatment .......................................................................................................18 1.6.2 Sapropterin dihydrochloride (BH4, Kuvan®) ............................................................20 1.6.3 Large neutral amino acid supplementation ................................................................21 1.6.4 Emerging treatment options .......................................................................................22 1.6.5 Treatment duration and adherence .............................................................................23 1.7 Outcomes .......................................................................................................................24 1.7.1 Intermediate treatment outcomes: determining treatment targets .............................24 1.7.2 Patient and caregiver-reported quality of life outcomes ............................................26 1.8 The evolution of clinical and nutritional PKU management practices ..........................27 1.9 Modern PKU management guidelines ...........................................................................28 1.10 Current PKU management in Canada ............................................................................29 1.11 Thesis rationale ..............................................................................................................30 1.12 Summary of objectives addressed in PhD thesis ...........................................................30 Chapter 2: Assessing the nutritional and clinical management practices of PKU ................33 2.1 Synopsis .........................................................................................................................33 2.2 Introduction ....................................................................................................................35 2.3 Methods .........................................................................................................................35 2.3.1 Development of the survey questionnaires ................................................................36 2.3.2 Sample selection and survey implementation ............................................................36 2.4 Results ............................................................................................................................38 2.4.1 Response rate and distribution of sample characteristics ..........................................38 xii  2.4.1.1 Physicians’ survey sample characteristics .........................................................38 2.4.1.2 Dietitians’ survey sample characteristics ...........................................................39 2.4.2 Reported clinical practices regarding diagnosis ........................................................40 2.4.3 Physicians’ approaches to classification of PKU severity .........................................43 2.4.4 Dietitians’ approaches to classification of PKU severity ..........................................44 2.4.5 Nutritional management practices of PKU ................................................................45 2.4.6 Clinical management practices ..................................................................................48 2.4.7 Frequency of communication and clinical follow up with dietitians.........................51 2.4.8 Physicians’ approaches to the utilization of neuropsychological assessments ..........52 2.4.9 Clinical practices of pharmacological treatment with sapropterin among physicians ……………………………………………………………………………………53 2.4.10 Medical formulas, foods and supplements from dietitians’ perspectives ..............56 2.4.11 Monitoring of dietary Phe intake ...........................................................................57 2.4.12 Monitoring adherence to the medical formula and low protein foods ...................58 2.4.13 Use of published PKU management guidelines ....................................................60 2.5 Discussion ......................................................................................................................61 Chapter 3: Observed PKU management practices and intermediate outcomes ....................64 3.1 Synopsis .........................................................................................................................64 3.2 Background ....................................................................................................................66 3.3 Methods .........................................................................................................................67 3.3.1 CIMDRN data compilation ........................................................................................67 3.3.2 Key PKU management practices selected for the analysis of clinical data ...............68 3.3.3 Definition of PKU severity ........................................................................................70 xiii  3.3.4 Assessment of PKU severity from the perspective of peak Phe levels .....................71 3.3.5 Frequency of Phe monitoring and communication ....................................................71 3.3.6 Assessment of Phe levels and quality of Phe control ................................................72 3.3.7 Mean Phe levels and quality of Phe control as intermediate outcomes of PKU treatment ................................................................................................................................73 3.3.7.1 Mean blood Phe levels .......................................................................................74 3.3.7.2 Quality of Phe control ........................................................................................74 3.3.8 Age .............................................................................................................................76 3.4 Statistical Analysis .........................................................................................................76 3.5 Results ............................................................................................................................77 3.5.1 Sample characteristics ...............................................................................................77 3.5.2 Diagnosis of metabolic phenotype in treating centres ...............................................79 3.5.3 Frequency of communication ....................................................................................81 3.5.4 Intermediate treatment outcomes ...............................................................................83 3.5.4.1 Phenylalanine levels beyond neonatal period ....................................................83 3.5.4.2 Quality of Phe control during the first month of life .........................................87 3.5.4.3 Quality of Phe control beyond neonatal period .................................................89 3.5.5 Sensitivity analysis of the quality of Phe control utilizing a different definition of the quality of Phe control among children with classic PKU. .....................................................91 3.6 Discussion ......................................................................................................................93 Chapter 4: The quality of life of parents who care for children with PKU in Canada .........97 4.1 Synopsis .........................................................................................................................97 4.2 Introduction ....................................................................................................................99 xiv  4.3 Methods .......................................................................................................................100 4.3.1 Health-related QoL measures: .................................................................................101 4.3.2 Measures of metabolic control and mean blood Phe levels .....................................104 4.3.3 Statistical analysis ....................................................................................................104 4.4 Results ..........................................................................................................................105 4.4.1 Sample characteristics .............................................................................................105 4.4.2 Parent-reported child’s health status and symptoms ...............................................106 4.4.3 Impact of child’s PKU on parental health-related QoL ...........................................108 4.4.4 Reported PKU-QoL in parents of children with severe and milder PKU ...............109 4.4.5 Child’s health status as predicted by the quality of Phe control. .............................111 4.4.6 Overall impact of PKU on parental QoL as predicted by the quality of Phe control ………………………………………………………………………………….113 4.5 Discussion ....................................................................................................................116 Chapter 5: Discussion ................................................................................................................118 5.1 Variation in care and its impact on health outcomes ...................................................118 5.2 Observed variation in PKU diagnostic practices .........................................................119 5.2.1 Variation in classification of PKU severity .............................................................119 5.3 Potential implications for patient care .........................................................................121 5.3.1 Over-restriction of natural protein consumption .....................................................121 5.3.2 Averting trial of sapropterin ....................................................................................122 5.3.3 Potential impact of over-diagnosis of PKU severity on caregivers’ quality of life .122 5.3.4 Impact of uncertainty in PKU classification on PKU research ...............................123 5.4 The use of the neonatal BH4 loading test in Canadian treating centres ......................123 xv  5.4.1 Potential implications on patient care ......................................................................125 5.5 Variation in follow up practices ..................................................................................126 5.5.1 Frequency of Phe monitoring and clinic visits ........................................................126 5.6 Neuropsychological assessments .................................................................................128 5.7 Quality of Phe control in Canadian pediatric population ............................................130 5.8 The quality of life of a parent who provides care for child with PKU ........................132 Chapter 6: Conclusion ...............................................................................................................137 6.1 Summary of findings and contributions to the practice-based evidence .....................137 6.2 Study limitations ..........................................................................................................138 6.2.1 Limitations of healthcare providers’ survey study ..................................................138 6.2.2 Limitations of clinical data analysis ........................................................................139 6.2.3 Limitations of the QoL study ...................................................................................139 6.3 Recommendations for the optimization of PKU care and future research ..................140 Bibliography ...............................................................................................................................143 Appendices ..................................................................................................................................166  Variation in clinical management practices between countries ..........................166 A.1 Historical variation in clinical management practices between countries. ..............166 A.2 Comparison of current key recommendations of ACMG/GMDI and European PKU guidelines. ............................................................................................................................168  Assessment of nutritional and clinical management practices of PKU...............169 B.1 Survey questionnaire to assess nutritional management practices (dietitians’ survey) …………………………………………………………………………………….169 xvi  B.2 Survey questionnaire to assess clinical management practices (physicians’ survey) …………………………………………………………………………………..188 B.3 Factors influencing prescription of medical formula ...............................................206  Overview of the indices of Phe control and classification of PKU severity in treating centres .........................................................................................................................207 C.1 Overview of the indicators of Phe control reported in the published literature .......207 C.2 Criteria used to determine the severity of PKU as derived from the medical charts by centre where criteria were recorded  ....................................................................................210 C.3 PKU monitoring and communications by treating centrea: classic PKU. ...............212 C.4 PKU Monitoring and Communications: milder forms of PKUa ..............................213    xvii  List of Tables Table 2.1 Sample characteristics of physicians’ survey ............................................................... 38 Table 2.2 Sample characteristics of metabolic dietitians’ survey. ................................................ 39 Table 2.3 Selected reported practices related to diagnosis ascertainment among physicians treating pediatric PKU .................................................................................................................. 42 Table 2.4 Selected reported PKU nutritional follow up practices among dietitians treating pediatric PKU................................................................................................................................ 46 Table 2.5 Clinical PKU management practices (reported by physicians). ................................... 49 Table 2.6 Reported frequency of clinic visits and provider-family communications. .................. 52 Table 2.7 Reported practices on treatment with sapropterin dihydrochloride (reported by physicians). ................................................................................................................................... 54 Table 2.8    Monitoring dietary Phe intake (reported by dietitians). ............................................. 57 Table 2.9 Reported practices on monitoring adherence to the diet (reported by dietitians). ........ 59 Table 3.1 Key practices identified by surveys and further complemented by the analysis of CIMDRN clinical PKU data ......................................................................................................... 69 Table 3.2 Characteristics of PKU cohort with complete minimum data (n=215). ....................... 78 Table 3.3 Maximum (peak) blood Phe levels at any point in time among children diagnosed with classic PKU as reported in their medical chart (n=92). ................................................................ 79 Table 3.4 Median of individuals’ maximum (peak) blood Phe levelsa during the first month of life (28 days) and after the first of month of life by PKU severity. .............................................. 80 Table 3.5 Average Frequency (rate)a of PKU monitoring and communication by the severity of PKU and age. ................................................................................................................................ 82 Table 3.6 Meana (SD) of age-specific blood Phe levels by PKU severity.. .................................. 86 xviii  Table 3.7 Lifetime meana (SD) of blood Phe levelsb by severity of PKU and by treating centrec,....................................................................................................................................................... 86 Table 3.8 Proportion of newborns with more than 60% of their Phe levels within therapeutic range 120-360μmol/L during the first fours weeks of life. ........................................................... 88 Table 3.9 Proportion (%) of children with “good” and “limited” quality of Phe control in different age groups by PKU severitya. ........................................................................................ 90 Table 3.10 The proportion of children with classic PKU having various quality of Phe control given as percent of Phe levels within and outside therapeutic range (120-360 μmol/L) by age. . 91 Table 4.1 PKU-QoL sample characteristics. ............................................................................... 106 Table 4.2 Child’s health status and symptoms as reported by parents. ...................................... 107 Table 4.3 Parent-reported health status and symptoms in children with severe and milder PKU..................................................................................................................................................... 107 Table 4.4 PKU-QoL domain scores in the study sample. ........................................................... 108 Table 4.5 Reported parental PKU-QoL scores by severity of PKU. .......................................... 110 Table 4.6 Child health status by PKU severity, age, mean Phe levels and quality of Phe control (n=61) a. ....................................................................................................................................... 111 Table 4.7 Univariable associations between mean Phe levels, quality of Phe control, age, PKU severity and child health status (n=61)a. ..................................................................................... 112 Table 4.8 The association between mean Phe levels/quality of Phe control and parent-reported child’s health status, adjusted for age and severity of PKU. ...................................................... 113 Table 4.9 Overall impact of PKU by PKU severity, age, mean Phe levels and quality of Phe control (n=62)a. ........................................................................................................................... 114 xix  Table 4.10 Univariable associations between mean Phe levels, quality of Phe control, age, PKU severity and the overall impact of PKU on QoL (n=62)a. .......................................................... 114 Table 4.11 The association between mean Phe levels/quality of Phe control and the overall impact of PKU on parental QoL, adjusted for age and severity of PKU. ................................... 115 Table 5.1 Comparison of Canadian, European (132) and Australian PKU-QOL surveys (171).134                   xx  List of Figures Figure 1.1 Overall structure ofthe doctoral dissertation. .............................................................. 32 Figure 2.1 Basis for classifying severity of PKU by metabolic physicians. ................................. 44 Figure 2.2 Basis for classifying severity of PKU by metabolic dietitians. ................................... 45 Figure 3.1 Distribution of mean blood Phe levels in classic PKU by age. ................................... 84 Figure 3.2 Distribution of mean blood Phe levels in mild PKU by age. ...................................... 84 Figure 3.3 Distribution of mean blood Phe levels in mild HPA by age. ...................................... 85 Figure 3.4 Proportion (%) of newborns with more than 60% of their Phe levels within treatment range 120-360μmol/L during the first fours weeks of life by PKU severity. ............................... 88 Figure 3.5 Proportion (%) of children with “good” Phe control a beyond the first month of life by PKU severity and age category. .................................................................................................... 90 Figure 3.6 The distribution (%) of children with classic PKU with “limited low”a and “limited high”b quality of Phe control by age. ............................................................................................ 92  xxi  List of Abbreviations  ACMG American College of Genetics and Genomics ADHD H/I Attention deficit hyperactivity disorder hyperactive-impulsive type AHRQ Agency for Healthcare Research and Quality BBB Blood brain barrier BH4 Tetrahydrobiopterin CIMDRN Canadian Inherited Metabolic Diseases Research Network DHPR Dihydropteridine reductase DSM-IV Diagnostic and Statistical Manual of Mental Disorders FDA U.S. Food and Drug Administration GMDI Genetic Metabolic Dietitians International GMP Glycomacropeptide HPA Hyperphenylalaninemia HRQoL Health-related quality of life IQ Intelligence quotient IQR Interquartile range LNAA Large neutral amino acids NIH National Institutes of Health OMIM Online Mendelian Inheritance in Man database PAH Phenylalanine hydroxylase PAL Phenylalanine ammonia lyase PCD Pterin-4a-carbinolamine dehydratase Phe Phenylalanine PKU Phenylketonuria QoL Quality of life ROCFT Rey-Osterrieth Complex Figure Test SEE Standard error estimate UK United Kingdom US United States  xxii  Glossary   BH4 (tetrahydrobiopterin) Naturally-occurring compound that serves as a cofactor for PAH and other enzymes   BH4 loading test The administration of oral BH4 with subsequent measurements of Phe levels using the standard protocol with two objectives: (a) to differentiate between PAH deficiency and disorders of BH4 metabolism and (2) to identify patients who would benefit from the treatment with sapropterin dihydrochloride (Kuvan ®, BH4).    Hyperphenylalaninemia Elevated phenylalanine concentrations  in bloodstream.   Low protein foods  Special foods with low protein content specifically made for PKU and other inborn errors of amino acid metabolism: special low protein pasta, breads, ice cream, cookies, flour and other foods. The main purpose is to provide variety in the diet and to supplement total energy intake.    Medical formulas Amino acid mixtures, also known as medical formulas or medical food.   mg/dL Unit of  measure of phenylalanine concentration in the bloodstream; used in some Canadian metabolic centres.   µmol/L  Unit of  measure of phenylalanine concentration in the bloodstream, commonly used in the Canadian metabolic centres    Mutations and variants Mutations are events that result in changes of genomic DNA. Variants are deviations from the human genome reference sequence observed in a specimen. The difference is subtle; the two terms are often used interchangeably.    Mutations in “trans” Referring to two heterozygous variants on opposite homologous chromosomes.   PAH deficiency Deficiency of Phenylalanine hydroxylase: enzyme that converts phenylalanine into tyrosine and caused by mutations in PAH gene.   PKU diet Natural protein-restrictive diet (also known as phenylalanine-restrictive diet). xxiii    Protein-restricted diet (PKU diet, low protein diet, phe-restricted diet) Vegan or vegetarian-like diet partially or completely exclusive of meat, milk, cheese, eggs, nuts, or bread, supplemented with a synthetic, phenylalanine-free amino acid mixtures containing essential amino acids, vitamins, fats, trace elements, and minerals.   Recommended Phe range  American College of Medical Genetics recommends maintaining treated phenylalanine levels within 120-360μmol/L range for all ages.    Sapropterin dihydrochloride (Kuvan®)   Synthetic form of BH4, supplemental treatment to phenylalanine-restricted diet.      xxiv  Acknowledgements  I have been fortunate to have mentors who helped me to accomplish these collaborative Canada-wide projects. I am grateful for the amazing learning experience with the Canadian Inherited Metabolic Diseases Research Network that provided me with the opportunity to collaborate in various projects. I am grateful for the most helpful and supportive mentors: Dr. Potter, Dr.Stockler and Dr.Collet who were my PhD supervisors and who provided the most valuable and helpful guidance and mentorship on research methodology and clinical aspects of care for children with PKU.   I am grateful to metabolic dietitians who helped so very enthusiastically to develop and finalize the survey to assess current nutritional management practices in Canada, and to metabolic physicians who provided invaluable expertise for the development of the metabolic clinicians’ PKU survey. I am grateful for the amazing research team of the University of Ottawa for their support and continued kindness, especially Michael Pugliese and Kylie Tingley who helped to navigate through CIMDRN database.  Wholeheartedly, I am grateful for my early career mentors and remarkable researchers, Dr. Godfrey P. Oakley Jr. and Dr. Wladimir Wertelecki, who directed me to a fascinating world of medical genetics research. Special thanks are owed to my parents Alicia and Ihor, who have supported me throughout my years of education. I am forever grateful for my precious girls Oksana and Ivy who are the light of my life.   xxv  Dedication  This work is dedicated to Miss Ivy Holmes, beautiful soul, amazing, joyful and bright child.   1  Chapter 1: Introduction  1.1 Prologue to the Introduction Phenylketonuria (PKU; OMIM 262600) is an autosomal recessive inborn error of phenylalanine (Phe) metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). PKU is one of the most frequent and well-studied among rare metabolic conditions and is rightfully regarded as a prototype of inherited metabolic disorders. In 1934 Norwegian biochemist, Dr.Følling, discovered the presence of phenylpyruvic acid in the urine of two young siblings presenting with severe intellectual disability. To confirm his hypothesis on the association between phenylpyruvic acid and intellectual disability, Dr.Følling tested an additional 430 patients with intellectual impairment in the Oslo area. He subsequently identified eight patients with a similar biochemical abnormality to the first pair of siblings (1). This remarkable discovery enabled worldwide diagnosis, ultimately leading to the development of a successful treatment. PKU was the first genetic metabolic disease for which the biochemical origin was discovered in 1934 (2) (1) and deficient PAH enzyme was identified in 1953 (3). Almost seventy years ago it was the first genetic metabolic disorder for which effective treatment (a semi-synthetic, protein-restrictive diet) was discovered (4).  PKU newborn screening was successfully established and applied in many countries for the first time in history (5). It is also the first genetic metabolic disorder for which the unfavorable prognosis of progressive mental and neurological deterioration was eliminated with early and continuous treatment (6). Rightfully, PKU is often referred to as a medical success story. Today, virtually all patients in developed countries are diagnosed via newborn screening and the majority are prescribed lifelong Phe-restrictive dietary treatment, supplemented with amino acid mixtures and low 2  protein foods (7) (8). For some, sapropterin dihydrochloride and large neutral amino acids are available as a pharmacological treatment option (9). There are newer and emerging treatments such as glycomacropeptide (a whey protein free of Phe) and phenylalanine-ammonia lyase and gene therapy (10) (11)  While current treatment advances should result in optimal long-term outcomes for all PKU patients, unfavorable outcomes are common despite early and continuous treatment (12). Variability in clinical practice is one of the plausible causes (13) (14). Identified inconsistencies in management practices, along with the need to streamline practices towards optimized outcomes, prompted the development of evidence-based PKU management guidelines in both North America (7) (8) and Europe (15). PKU management and some associated outcomes were assessed in European countries before the guideline development (14) (16), yet there were no similar published studies in Canada.  Thus, the main purpose of this dissertation is to describe Canadian management practices within and outside of published guidelines and their associated outcomes in Canadian children affected by PKU. This knowledge will identify the weakest areas in management practices, highlighting where improvements could be made.  In the Introduction I have aimed to draw a wholesome yet concise picture of pediatric PKU including: PKU etiology and pathophysiology, clinical presentation, diagnosis, treatment, clinical and nutritional management practices, and associated outcomes.  First, I would like to make some clarifications. PKU-related literature revealed remarkable heterogeneity in PKU-related terminology. For example, various terms were used for Phe-free amino acid supplements including: medical formula, amino acid formula, medical food, amino acid mixture, Phe-free medical formula, and Phe-free L-amino acid supplement. Classification of severity also varied in the published literature, as the diagnoses “classic” 3  (classical) PKU, “severe PKU/PAH deficiency”, and “PKU requiring treatment” all encompassed severe PAH enzyme deficiency. Moreover, there is no clarity regarding the name of the disease itself. Dr.Følling called the condition “imbecillitas phenylpyruvica” reflecting the association between the accumulated metabolite and corresponding phenotype. Years later, “Phenylketonuria” was introduced by Dr. Lyonel Penrose to reflect consistent findings of Phe metabolites in the urine of affected children who also presented with intellectual impairments.  Eventually, the abbreviated term “PKU” was devised in the late 1950s. However, “PAH deficiency” is technically more correct as it depicts the root of the metabolic disturbance more precisely (7). Yet, “PKU” has been used in clinical practice for decades and my communications with healthcare providers showed that the term “PAH deficiency” is not yet routinely used. Therefore, “PKU” is the term used most throughout this dissertation, as it is the term used most commonly by the metabolic community worldwide. Finally, I would like to emphasize that inherited metabolic diseases is a rapidly advancing field of medicine. PKU management is an ongoing dynamic process trying to continuously adapt to the new changes in evidence.   1.2 Frequency of PKU   While a rare metabolic genetic disorder, PKU is one of the most frequently occurring inherited errors of metabolism. Overall prevalence in Europe is approximately one case per 10,000 livebirths (17), with a higher incidence in Ireland – 1:4,500 (18), Sicily – 1:2,700 (19), and Turkey - 1:2,600 (20). Higher prevalence is observed in geographically and culturally isolated populations; the reported prevalence in the Karachay ethnic group from North Caucasus is one case per 332 livebirths (21). The lowest overall prevalence is observed in Japan 4  (1:125,000), and Finland (below 1:100,000) (22).  In South America PKU prevalence ranges from 25,000 to 50,000 (23) while North America reports one case per 15,000 livebirths in both the US and Canada  (24) (25).   1.3 Pathophysiology and clinical presentation of PKU  Phenylalanine hydroxylase (PAH, phenylalanine-4-monooxydase) catalyzes the conversion of phenylalanine to tyrosine in the liver with the help of co-factor tetrahydrobiopterin (BH4) and two regenerating enzymes (pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR)) in the presence of molecular oxygen and iron. Reduced PAH enzyme activity leads to the accumulation of Phe in the bloodstream (hyperphenylalaninemia, HPA) with subsequent elevation of Phe concentration in the brain (26). To cross the blood brain barrier (BBB), Phe competes with other large neutral amino acids  (LNAA) for a carrier protein, responsible for LNAA uptake into the central nervous system (27). This affects synthesis of dopamine and norepinephrine, leading to the interruption of dopaminergic and noradrenergic neurotransmission  (28), (29).  Phe also directly affects myelin metabolism causing diffuse changes of cerebral white matter, though the mechanism of this damaging effect is not clear (30), (31), (32).  Evidence shows that the prefrontal cortex, responsible for “executive functioning” including inhibition, working memory, and cognitive flexibility, is especially sensitive to the deficit of dopamine, arising from the moderate reductions of tyrosine reaching the brain. Diamond et al. investigated the cognitive neuropsychological functioning of early and continuously treated children with PKU in a 4-year longitudinal study.  The authors found that tasks requiring working memory and inhibitory control were more difficult for PKU children 5  with blood Phe levels between 360 and 600 μmol/L compared to PKU children with lower Phe levels, matched controls, their own siblings, as well as children from the general population. This impairment was observed for all age groups and was inversely correlated with mean blood Phe levels. The deficits, selectively affecting working memory and inhibitory control, specifically pointed to the dorsolateral prefrontal cortex, responsible for executive functioning and highly sensitive to even small decreases in tyrosine with subsequent selective loss of dopamine. Other cognitive functions dependent on other neural systems appeared to be spared (33). Another study by Diamond et al. showed that the retina is also sensitive to even small tyrosine fluctuations due to rapid dopamine turnover (34). In their study of visual contrast sensitivity, the exposure to phenylalanine was defined as mean blood Phe levels 6 weeks preceding the test, 2 years preceding the test, during the first year of life and during the first month of life. Interestingly, mean Phe levels during the first month were significantly associated with contrast sensitivity performance, while Phe measurements later in life were not. Children whose Phe levels had been high during the first month of their lives (1,050 -1,470 μmol/L) were less sensitive to contrast at the two highest spatial frequencies than were PKU children whose Phe levels had been lower during their first month (336 - 636 μmol/L). This means that substantially elevated Phe levels during the first month of life might impact the visual system.   Notably, impaired contrast sensitivity might make it more difficult for children to see printed material under conditions of low contrast, impeding academic performance if not recognized and properly addressed (34).   The above studies indirectly confirmed the hypothesis that the amount of tyrosine reaching the eye and brain is reduced due to moderately elevated plasma Phe levels.  The reason for this is that phenylalanine and tyrosine compete for the same transporter proteins to cross the 6  blood-brain barrier, resulting in large amounts of Phe entering the brain.  Some authors also argue that the phenylalanine to tyrosine ratio in blood is more important for executive functioning, rather than the blood levels of Phe alone (35)  In most untreated cases of severe enzyme deficiency, clinical symptoms emerge around 4-6 months of age. Children with untreated PKU often fail to attain early developmental milestones, commonly raising parental concerns.  Later in the natural progression of severe PKU children frequently develop epilepsy, ataxia, other neurological symptoms (such as Parkinson-like features) microcephaly and intellectual disability. Severe behavioural disturbances, including psychotic, autistic, aggressive disorders and stereotyped behaviour, are also among the most consistent findings in untreated or late-treated individuals with PKU (36). Other symptoms and signs include skin disorders (eczema), musty body odor, depigmentation of the skin and hair, and strongly aromatic urine (37).  Surprisingly, published literature on the well documented natural progression of PKU is limited. One study reported 46 patients with PKU who were initially assessed and diagnosed with profound intellectual disability in 1968-69 and re-assessed twenty years later. The baseline assessment classified twenty-two individuals as mildly to moderately impaired and three as having normal to borderline intelligence using the Binet scale. Follow up assessment revealed significant neurological sequelae such as ataxia, tremor and poor gait in three patients. Epilepsy was present in 12 patients, limb spasticity in four, and facial palsy in one. Both initial and follow up average blood Phe levels were above 1,000 µmol/L, with observed comorbidities similar to the non-PKU population of comparable age. At the follow up assessment twenty patients resided in institutions and 26 were living at home (38). Another study compared children with PKU who were diagnosed and treated early due to the confirmed diagnoses in their older siblings. It 7  concluded that individuals with persistently higher Phe levels are more likely to develop severe intellectual impairment, pointing to a somewhat dose-dependent effect of Phe on brain function (39).  While intellectual disability is a hallmark of untreated PKU, there are published reports of individuals with untreated severe hyperphenylalaninemia that escaped intellectual disability; Vliet et al. (40) described 59 cases. Yet, all 59 individuals did present with one or more neurological symptoms including tremor, abnormal reflexes, and movement disorders. In addition, there were reports describing female patients diagnosed with PKU after giving birth to children with Phe embryopathy due to maternal hyperphenylalaninemia (41). Another study screened umbilical cord blood samples of 435,000 women. Out of those, 22 women with impaired Phe metabolism were identified.  Two out of 22 individuals were diagnosed with classic PKU and six were diagnosed with milder forms of PKU with mean blood Phe ranges from 595 to 992 μmol/L and 165 to 540 μmol/L.  Twelve individuals agreed to participate in neuropsychological assessment. The assessment revealed average or above average ranges of intelligence with IQ scores >90 (42) in all but one participant.  Another population-based study identified three individuals with Phe concentration over 1,200 μmol/L in 250,000 blood specimens collected from the “normal” population (43) (44).  The fact that some untreated patients with significantly elevated blood Phe levels have retained normal, or nearly normal, cognitive abilities despite PKU-specific treatment suggests the presence of protective factors. These are not fully understood. It was hypothesized that a protective mechanism involves Phe transport and acts at the blood-brain barrier and/or within the brain itself.  Using magnetic resonance spectroscopy, Weglage et al. (45) observed various brain Phe levels in patients presenting with high blood Phe concentrations. The authors concluded that 8  cerebral Phe levels are major contributors to the development of clinical outcomes and are more important than blood Phe concentrations. While brain Phe levels would be the ideal biomarker for monitoring PKU, neither brain Phe nor the BBB kinetics are measured routinely in treating centres; instead, concentrations of Phe in the bloodstream serve as the primary surrogate biomarker to monitor intermediate treatment outcomes in clinical practice (7) (10). Suboptimal neurocognitive outcomes in early and continuously treated patients.  While early diagnosis and treatment eliminate the most severe manifestations of PKU, suboptimal cognitive and behavioural outcomes can still occur. Frequently identified areas of weakness include abstract thinking, executive functioning, attention, processing speed, irritaibility, and impulsivity. Problems in school performance, decreased autonomy, as well as psychiatric disorders are therefore more frequently observed (46) (12) (47) (48)(49) (50) (12) (47) (51).  A symptom of impaired executive functioning could be poorer treatment compliance, potentially worsening executive functioning in and of itself (52) (53). Gassio et al. (54) reported lower IQ scores in 26 adolescents with early and continuously treated PKU when compared to gender and age-matched controls. The most affected areas were fine motor, problem solving, executive function and attention. In addition, evidence points to a high prevalence of attention deficit and hyperactivity symptoms. Similarly to “isolated” ADHD, low dopamine levels in the prefrontal cortex and striatum are shown to be common in PKU (55) (56) (57). While studies on the prevalence of ADHD in the PKU population are lacking, one study indicated a two times greater prevalence when compared to the general population (58). Arnold et al. (59) found that the use of stimulants for treating ADHD is significantly higher compared to gender- and age-matched controls which indirectly pointed to a high prevalence of ADHD in the PKU population. 9  Processing speed may also interfere with executive deficits, but only limited evidence is available to date. A slower information processing speed (60) (61) and IQ scores averaging 10 points lower than controls (54) (62) was identified in several studies. Taking the above, one can imagine the challenges in school performance that are reported in children with PKU.  While data on the visual impact of PKU are limited, decreased contrast sensitivity was detected in patients with PKU compared to normal controls in Adel Diamond’s study (33), and deficiencies were also found in more complex visual constructive tasks assessed by the Rey-Osterrieth Complex Figure Test (ROCFT) (60). The preliminary results of the study conducted at BC Children’s Hospital also pointed to the lower contrast sensitivity in PKU patients compared to normal controls. Moreover, there was some preliminary evidence that the contrast sensitivity improves with lowered blood Phe levels (63).  Looking to psychiatric disorders, Burton et al. (47) assessed PKU patients for symptoms of psychiatric distress including somatization, obsessive-compulsive behaviours, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, and psychoticism. As many as thirty-two percent of all assessed patients screened positive for one or more of the above conditions.   1.3.1 Elevated and fluctuating Phe levels and their associated suboptimal outcomes Elevated blood Phe levels have been suggested as a major contributor to the development of suboptimal neurocognitive and behavioural outcomes. Romani et al. (62) assessed performance across various cognitive domains in relation to “lifetime” mean blood Phe levels. Performance was then assessed in relation to the historic and current average blood Phe levels in children younger than 10 years, adolescents (11-17 years), and adults.  “Good” control was 10  defined as average blood Phe less than 650 μmol/L and “poor control” as average Phe higher than 950 μmol/L.  The comparison was also made with age- and gender-matched controls. Consistent with results from other studies, adults with lower blood Phe levels performed better in all cognitive domains except inhibitory control when compared to the group with higher Phe. Yet, when compared to the control group, individuals with lower Phe performed less successfully in almost all domains suggesting that even accepted levels of Phe might not be entirely protective against disease manifestations.  Timing and duration of exposure to elevated blood Phe levels are also of great importance. Romani et al. (62) found that sustained attention, verbal memory and visuomotor coordination were better predicted by recent metabolic control, while processing speed tasks were more impacted by long-term blood Phe levels. Some argue that short-term elevation of brain Phe is largely responsible for disruption of neurotransmitter metabolism resulting in reversible neurobehavioural deficits, while long-term exposure to high blood Phe levels disrupts the myelination processes (64) (31).   While evidence is currently inconsistent, some authors suggest it is the fluctuation of (not necessarily merely the elevated) blood Phe levels that contribute to suboptimal neurocognitive outcomes. Some studies suggest that Phe fluctuation is in fact a stronger predictor of cognitive performance than other indices of Phe control. Measuring the physiological variability of plasma Phe within 24-hour periods in sixteen children between the ages of 1 and 18, MacDonald et al. (65) demonstrated Phe median fluctuation could be up to 155 μmol/L with the highest levels occurring in the morning, and the lowest levels occurring between 6 PM and midnight. Hood et al. (66) examined a number of indicators of blood Phe control to determine which index best predicted neurocognitive outcomes in forty seven school-age children with early and 11  continuously treated PKU. The indicators were calculated during the lifetime and as a secondary analysis during developmental epochs. Three out of six indicators reflected Phe variability. Results indicated that mean lifetime blood Phe levels exceeded the recommended range and increased with age. Indices of Phe variability indicated considerable instability over the lifetime (66). The correlation between Phe variability and cognitive and executive tasks was stronger compared to mean Phe, especially in older children. Concluding that Phe fluctuation might be a strong predictor of neuropsychological outcomes, this study recommended both average and variability in Phe be carefully monitored and controlled (66).  The overall harmful effect of Phe on brain function (whether elevated or fluctuating) is well established. Likewise, the presence of milder neuropsychiatric deficiencies in individuals with PKU suggest this is most likely due to poor Phe control.   1.4 Diagnosis of PKU As mentioned, clinical symptoms of hyperphenylalaninemia are absent in newborns. In order to detect hyperphenylalaninemia before clinical presentation, in 1963, Dr. Guthrie developed a universal newborn screening method based on a bacterial inhibition test using dried blood spots collected on filter paper (5). Today in Canada, screening is conducted nationwide using tandem mass spectrometry (MS/MS) technology with dried blood spots (sometimes called “blood dots”) that are typically collected on filter paper from a heel prick taken within the first few days of life; two mass spectrometers linked ‘in tandem’ can analyze components of complex mixtures without requiring prior chemical separation.  This method allows for early detection and treatment of about 30 inherited metabolic disorders, and virtually 100% of PKU cases in 12  Canada are detected within approximately the first week of life. Elevated NBS Phe triggers an urgent referral to the nearest metabolic centre. Interestingly, early diagnosis and treatment sometimes prevent accurate classification of PKU severity (67) (7). Peak levels of bloodstream Phe are commonly used in defining the metabolic phenotype of PKU indicating severity. Historically the blood dot was taken around the 5th day of life. By that time newborns were fed breast milk or regular formula allowing blood Phe levels to reach their maximum at the time of the screening, thus reflecting actual metabolic severity. In 2003 - 2004, for normal vaginal deliveries, the average Canadian postpartum hospital stay had shortened to approximately 2 days and NBS heel prick blood spots were accordingly collected earlier, before discharge. Screening within the first two days of life potentially does not allow sufficient time for Phe to reach its highest level, resulting in a milder severity diagnosis. Moreover, not all patients are diagnosed via newborn screening as it is not well established in some developing countries (68) (69). In cases of late diagnosis, children (or adults) may present with some (or all) clinical presentations of long-term hyperphenylalaninemia. Considering global migration, awareness of undiagnosed and untreated PKU should always be present as a differential diagnosis for developmental delays or intellectual disability.   1.4.1 The use of the neonatal BH4 loading test in differential diagnosis of hyperphenylalaninemias (HPAs)  One of the goals of newborn screening is to promptly identify a newborn child with elevated blood Phe level. After this, the diagnosis of PAH deficiency needs to be confirmed. Several conditions present with elevated Phe in blood in neonatal period and thus these conditions need to be differentiated. The BH4 loading test is especially helpful in two ways. 13  Firstly, in the newborn, the test helps to distinguish between PAH enzyme deficiency and BH4 cofactor deficiencies, as all these conditions present with hyperphenylalaninemia. Secondly, it assesses responsiveness to treatment with sapropterin thus this application of BH4 loading test is described later in the “treatment” paragraph. To better describe the role of the neonatal BH4 loading test in the algorithm of differential diagnosis of HPAs, a brief overview of potential causes of HPAs is presented below.  Hyperphenylalaninemia (HPA) is detected via PKU newborn screening (NBS) and could result from several metabolic genetic or non-genetic conditions that require further investigation. Among inherited metabolic disorders, HPA is commonly caused by a phenylalanine hydroxylase (PAH) deficiency or, much less frequently (in 1-3% cases), by impaired BH4 synthesis or regeneration including an autosomal-recessive form of GTP-cyclohydrolase I deficiency (GTPCH), 6‐pyruvoyl tetrahydropterin synthase (PTSP), sepiapterin reductase, dihydropteridine reductase (DHPR),  and pterin‐4α‐carbinolamine dehydratase (PCD) (70) (71) (72) (73). In addition, recently described DNAJC12 deficiency leads to the misfolding of the PAH enzyme, also presenting with hyphenylalaninemia (74). Other causes of elevated, and most frequently transient, Phe concentrations in the blood in the neonatal period may be due to: liver immaturity, protein overload from cow’s milk, total parenteral nutrition, possible heterozygosity for PAH deficiency in premature babies, or maternal HPA (75).  Historically, BH4 loading was used for differential diagnosis of hyperphenylalaninemias and was convenient in diagnosing biopterin variants. The development and availability of newer diagnostic methods now allows BH4 deficiencies to be primarily differentiated through the analysis of pterin metabolites in urine, dihydropteridine reductase in blood spots, and genetic tests (76). Current guidelines recommend all HPA-positive newborns be tested for pterins 14  (neopterin, biopterin and primapterin) from dried blood spots (DBS) or urine, and dihydropteridine reductase (DHPR) in DBS in order to confirm the diagnosis. The BH4 loading test is positive in all BH4-deficient patients, however it does not always differentiate between mild PKU with substantial residual activity of the PAH enzyme (BH-4 responders) and truly BH4-deficient patients.   1.5 Classification of PKU severity  Two parameters have traditionally been used in the classification of metabolic severity: Phe levels in plasma prior to treatment (“untreated” or “pre-treatment” Phe levels), and Phe tolerance (maximum consumption of Phe while maintaining therapeutic blood Phe levels). With advances in genetic testing, mutation analysis is also used to aid diagnosis and classification of hyperphenylalaninemia. Different algorithms for defining and establishing PKU severity classification are described here.  1.5.1 Classification of PKU based on untreated blood Phe levels Untreated maximum Blood Phe levels were traditionally used to determine the metabolic severity of PAH deficiency. This is logical, because the concentration of Phe in blood depends on the degree of the loss of enzyme function. One of the earliest proposed classifications was published in 1980 and included three different metabolic phenotypes of PKU. They were: classic PKU with Blood Phe levels of >1,200 µmol/L, variant PKU with levels of 600-1,200 µmol/L, and mild hyperphenylalanemia (HPA) with blood Phe levels of less than 600 µmol/L (77). Fourteen years later Dr. Guldberg proposed a more detailed classification including four metabolic phenotypes of PKU: classic PKU presenting with blood Phe levels of >1,200 µmol/L, 15  moderate PKU with levels within 900-1,200 µmol/L, mild PKU with levels of 600-900 µmol/L, and mild HPA with untreated blood Phe levels of less than 600 µmol/L (78).  Recently the Scientific Review Committee of The National Institute of Health recommended yet another classification based on “untreated” blood Phe levels to reflect the lack of evidence on treatment for the Phe range between 360 and 600 µmol/L: classical PKU with maximum pre-treatment blood Phe levels of >1,200 µmol/L, moderate PKU with levels within 900-1,200 µmol/L, mild PKU with levels within 600-900 µmol/L, mild HPA-gray zone with blood Phe levels within 360-600 µmol/L, and mild HPA-no treatment (NT) with levels within 120-360 µmol/L (recommended therapeutic range) (79). While evidence strongly suggests patients with high pre-treatment blood Phe levels be treated, in cases of mild HPA there is no robust evidence finding dietary treatment especially beneficial or harmful; as a consequence, this type of PKU falls into the “gray zone” of uncertainty (80), (7).   1.5.2 Classification of PAH deficiency based on Phe tolerance alone Another classification scheme proposed by Guttler and Guldberg in 1996 (81) (19) defined PKU severity based on the amount of daily Phe intake that would still allow blood Phe levels to stay within the therapeutic range. The four categories were: (1) classic PKU characterized by very low tolerance of 250-350 mg of dietary Phe per day; (2) moderate PKU with tolerance of 350-400 mg of dietary Phe per day; (3) mild PKU with affected individuals tolerating 400-600 mg of dietary Phe per day and (4) mild hyperphenylalaninemia (MHP) where affected infants had plasma Phe concentrations less than 600 µmol/L (10 mg/dL) on a normal diet.  16  Similarly to untreated blood Phe levels, Phe tolerance is not a fully reliable criterion in defining severity for two main reasons. First, recent evidence suggests that Phe tolerance could not be reliably determined by age 2 (82). Second, Phe tolerance is only determined under controlled conditions; accurate assessments of natural protein tolerance are not always feasible in clinical settings and many patients are found to have underestimated Phe tolerance (83) (84) (85).  Regardless, properly assessed Phe tolerance is likely the more reliable determinant of the PKU metabolic phenotype when compared to frequently fluctuating blood Phe levels.   1.5.3 Correlation between genotype and phenotype and its implications for diagnosis  The phenylalanine hydroxylase (PAH) gene spans 90 kb and is located on the long (q) arm of chromosome 12, region q22–24.1. PKU is inherited in autosomal-recessive Mendelian fashion, where two PAH alleles are responsible for the expressions of the residual activity of the PAH enzyme in the liver. Generally, it is the type of gene variant that determines the residual activity of PAH; residual activity of PAH determines Phe blood concentrations, and Phe accumulation in the blood and brain determines clinical phenotype.  Due to the multitude of PAH variants and the high prevalence of compound heterozygosity (86), the hallmark of PKU is a marked allelic heterogeneity. For example, the online database BIOPKU (87) has described and catalogued information on PAH variants and their associated metabolic phenotypes. BIOPKU houses more than a thousand variants of the PAH gene and over 2,500 different genotypes. Published evidence indicates that residual activity of the PAH enzyme is not simply a product of mean expression of two PAH alleles.  While it is possible to predict the level of PAH residual activity and BH4 responsiveness in a single 17  underlying variant, the interallelic relationships remain complex and not completely understood (88). The proteins arising from different alleles influence each other, a phenomenon termed interallelic complementation (89) (90). The complexity of molecular interaction between two alleles makes the predictions of genotype-phenotype correlations inconsistent (91). Optimal predictions are observed for the “extremes” of the phenotypic spectrum: the most severe and mildest forms of PAH deficiency. For example, in a recent study, 2,589 different genotypes (588 variants) were investigated.  Using an allelic phenotype value (APV) algorithm, the genotype-based phenotype prediction was calculated to be 99.2% accurate for classic PKU, 89.5% for mild hyperphenylalaninemia and only 46.2% for mild PKU phenotype (92). Thus, contrary to early expectations, gene testing does not always provide straightforward answers when attempting to define PKU severity. This lack of predictability in a seemingly straightforward single-gene disorder highlights that phenylalanine hydroxylase is embedded within a complex system where genetic polymorphism, additional nonallelic mutations, and environmental influences represent the differences between individuals. Inconsistent metabolic and clinical phenotypes in individuals carrying identical PAH genotypes suggest other factors might influence metabolic and clinical phenotypes.  For example, studies of siblings show that the discrepancy between individuals carrying identical genotypes were attributed to: differences in the disposal of excess phenylalanine by transamination (93), protein stability, and activity of chaperones and proteolytic enzymes (94) . For some PAH gene mutations, it was hypothesized that the degradation of the mutant protein (PAH enzyme) in vivo determines the PAH activity (95).   Finally, there could be other genetic disease modifiers, such as single nucleotide polymorphism, in the genes related to hypephenylalaninemia. For example: PTS, GCH1, QDPR, 18  PCBD1 genes, responsible for BH4 deficiencies, and the relatively recently described DNAJC12 gene (74) (83) (96) .   In summary, the continuum of metabolic phenotypes adds to the inconsistencies in the clinical nomenclature as clinicians and researchers attempt to place the metabolic spectrum into defined categories.  Every algorithm of classifications of PKU severity has advantages and disadvantages. With regard to severity diagnosis in the clinical setting, variation has been reported in Europe (83) with no North American surveys to date.  1.6 Treatment of phenylketonuria Treatment strategies for PKU aim to lower Phe concentration in the bloodstream. In Canada, the most common forms of treatment for pediatric patients include protein-restricted diet, supplemented with medical formulas; and sapropterin dihydrochloride (Kuvan®). These strategies are discussed in detail, and other emerging treatment options are discussed briefly below. Comments on the recommended duration of treatment and struggles with treatment adherence close this section. 1.6.1 Dietary treatment  Discovered over half a century ago, Dr. Bickel applied the first successful trial of a Phe-free formula treatment to a 17-month old child with PKU who had presented with developmental delay, severe neurological symptoms, eczema, and deviant self-injurious behavior (4). The formula was administered and withdrawn repeatedly while all other sources of protein were eliminated from the child’s diet. The first application successfully showed dramatic improvement of the child’s behavior, mood, and motor development milestones over a period of several months. Treatment withdrawal reversed these improvements. Thus, the need for eliminating most 19  natural protein from the diet and adding a Phe-free form of protein was recognized (4). This milestone achievement has rendered immense success in the prevention of severe mental retardation associated with the accumulation of Phe. The dietary regimen has since improved tremendously with the availability of a wide assortment of commercial Phe-free medical formulas.  Today, diet remains the primary treatment of PKU and consists of a Phe-restricted (natural protein restricted) diet in combination with a medical formula (L-amino acid mixtures without Phe, usually in the form of a drink) to support protein synthesis and avoid catabolism without excess energy. This is often modified via low-protein fruits, vegetables, sugars, fats, oils, and other special low-protein foods providing essential fatty acids and other micronutrients (88) (89).  Diet starts promptly after birth with the goal of decreasing blood Phe concentrations within the first weeks of life (75) (8) (7) (97). Daily consumption of medical formula is critical to satisfy daily protein requirements and prevent macro- and micronutrient deficiencies (98). In addition to traditional medical formulas, recently the glycomacropeptide (GMP) derived from cheese whey has been used as an additional source of daily protein. Naturally low in Phe, GMP is considered safe, efficient and more palatable than formula by some (99) (100). The PKU diet aims at assuring the satiety and normal development of the individual. However, food choices are still very limited compared to a normal diet. For example, the recommended daily consumption of Phe for children from 1 to 4 years of age with severe deficiency of the PAH enzyme is around 200-320 mg/day (8).  Given that 1 gram of protein contains 47 mg of Phe, the allowance of natural protein consumption is somewhere between 4 to 6 grams of protein per day. When “translated” into natural foods, 6 grams of protein would represent just one hard-boiled egg (101). Overall, medical formula (amino acid mixtures and/or GMP) provide 80-85% of the 20  protein needs of individuals with severe PAH deficiency (102). However, subjective factors such as poor taste, smell, and undesirable texture of the medical formulas are reported by many patients which can challenge adherence (103) (98).    1.6.2 Sapropterin dihydrochloride (BH4, Kuvan®) Sapropterin dihydrochloride (Kuvan®) (hereafter sapropterin) is synthetic form of naturally occurring co-factor BH4 that increases the activity of the PAH enzyme if the residual activity of the enzyme is preserved. Not all patients equally respond to this therapy, with some studies showing a subset of 25-60% would respond to sapropterin treatment (104) (105). These patients typically have milder forms of PKU as their PAH enzyme residual activity is preserved to various degrees. In some cases, although not commonly, patients may even discontinue the low-Phe diet while on sapropterin (106). To assess the responsiveness to sapropterin, BH4 loading test is frequently used. This is another application of the BH4 loading test in addition to described above application of BH4 loading test for the differential diagnosis of HPAs. The BH4 loading test to assess the responsiveness to sapropterin (synthetic form of BH4), includes a structured trial of sapropterin with standardized measurements of blood Phe levels (with the mandatory baseline measurement) under the condition of a stable and well-documented dietary intake of natural protein (Phe) and prior accurate assessment of actual Phe tolerance. While similar to the neonatal BH4 loading test, the objective is to inform the decision on long-term sapropterin treatment. Even though there is a great variation in application of this test (107), some experts argue that BH4 loading test should be regarded as a helpful tool in assessing the responsiveness to sapropterin (108).  21  The criteria assessing the responsiveness to sapropterin treatment vary in both the research literature and clinical practice. Moreover, while attempts have been made, to date, there is no robust method of predicting an individual’s response to the treatment. Typically, 30% reduction of Phe from the baseline is used as a main criterion, however this number is arbitrary (106). Gain in Phe tolerance is also used by some but the “meaningfulness” of the change is very individual as tolerance is based on targets set between clinician / dietitian and patient (109) (110) although most recent European guideline advises 100% gain in Phe tolerance as acceptable indicator of good response to sapropterin (10).  Approved in both the US and Canada, current PKU guidelines recommend sapropterin be offered as a trial to every patient except those who have two null mutations in the trans position, which would mean that they are certain to be non-responders to sapropterin.   1.6.3 Large neutral amino acid supplementation  Despite limited evidence on their effectiveness, large neutral amino acid (LNAA) transporters are sometimes offered as a supplementary treatment to adolescents and adults who are not adherent to a typical PKU diet. At the brain-blood barrier phenylalanine shares the same transporter system with other large neutral amino acids: tyrosine, tryptophan and branched-chain amino acids.  When supplementation with LNAA showed a reduction of Phe concentration in the brain, it was hypothesized that the high concentration of blood Phe prevents the transport of other large neutral amino acids at the blood-brain barrier (111).  Other studies showed possible additional competition of Phe with other large neutral amino acids at the intestinal mucosa, potentially restricting the absorption of Phe in the gut (112). And yet, data on the safety and 22  benefits of LNAA supplementation is limited and currently it is not recommended for children or pregnant women (113) (7).   1.6.4 Emerging treatment options  Novel and experimental pharmacotherapy options include: enzyme replacement therapy (recombinant phenylalanine ammonia lyase, PAL, pegvaliase-pqpz, PALYNZIQ®), gene replacement/repair therapy, and genetically engineered probiotics.  Recombinant phenylalanine ammonia lyase represents a new type of enzyme replacement therapy where the function of the original deficient enzyme (PAH) is partially substituted by another enzyme (PAL). Unlike PAH, PAL metabolizes phenylalanine to ammonia and transcinnamate and is quite a stable protein that does not require cofactor for its activity. While initial efforts with yeast and plant extracted PAL showed minimal success, modifications increased efficiency and in May of 2018 the PEGylated form of PAL (pegvaliase)  was approved by the FDA for adults with uncontrolled blood Phe levels under the name “Palynziq” (114) (115) (116) (117) (118). While this drug is effective in Phe reduction and the possibility of diet liberalization, there is still the potential for a hypersensitive reaction. Moreover, the parenteral mode of drug delivery may prevent acceptance by all patients (119).  A gene therapy was considered an ultimate treatment option in genetic metabolic disorders caused by single-gene mutations, and while gene transfer studies show some promising results, there are many challenges with the effectiveness and stability of the gene therapy vectors that need to be addressed (120). For example, several animal studies used adeno-associated (AAV) vector and intrahepatic (via portal vein) delivery of recombinant gene. The initial normalization of blood Phe levels was not sustainable furthermore adenoviral vector triggered 23  the immune response (121)(122). Minicircle (MC)-based naked DNA vectors showed direct long-term physiologic gene expression in more recent murine model studies and could be re-administered without eliciting non-desired immune response. Overall, the results of recent studies show a good potential for the future therapeutic development of non-viral MC vectors for the treatment of genetic liver defects (123). Intensive research on gene therapy in animal models continues, yet to date there is only one active gene therapy PKU clinical trial, registered on the US-based registration platform,  clinicaltrials.gov (124), aimed at evaluating the safety and efficacy of gene therapy in adult subjects with PKU.  Genetically engineered probiotics is another treatment approach that focuses on the digestion of Phe. These probiotics degrade ingested Phe leading to the reduction of Phe in the blood. If successful, this therapy would most likely be used in conjunction with a Phe-restricted diet. Promising results have been shown in animal studies, where the reduction of  blood Phe levels by 38% was observed after the oral administration of genetically engineered Phe-degrading derivative of E. coli Nissle (125) The studies on the synthetic E.coli Nissle strain SYNB1618 have so far progressed to phase I-II clinical trials on healthy volunteers and adult patients with PKU (126).   1.6.5 Treatment duration and adherence   While it is recognized that PKU treatment begin as soon as possible after birth, the recommended duration of therapy for many years was not as clear. Early guidelines recommended strict Phe limitations up to 10-12 years, with gradual liberalization in adolescence and adulthood (127). A 1996 study concluded that a Phe-restrictive diet should be continued throughout childhood (128). Currently, PKU guidelines recommend a Phe-restricted diet for life 24  (7) (97).  Managing such a restrictive diet is not an easy task, and adherence is an everyday struggle for many patients causing them to remain at elevated risk for developing various neurocognitive and behavioural manifestations. It is quite common for children to become less adherent with age. A 2002 UK study showed the proportion of individuals with blood Phe levels above the recommended range rose from 30% in children younger than 4 years old to 80% in adolescents and young adults (129). It is a team effort to manage treatment and maintain appropriate blood Phe levels in response to metabolic changes. While treatment advances provide alternatives, the need for stringent dietary treatment, frequent clinic visits and arduous monitoring of blood Phe levels remains and provides a challenge to patients as well as their caregivers.  1.7 Outcomes 1.7.1 Intermediate treatment outcomes: determining treatment targets  Evidence collected over the years has successfully informed clinical practice on the target levels of blood Phe concentrations that optimize patients’ health outcomes. A study conducted from 1967 to 1983 in 19 metabolic treatment centres in the United States measured the neurocognitive functioning and school performance of two groups of children on strict and moderate Phe-restricted diets. At 6 years of age one group continued with the diet, while the other discontinued. Neurocognitive assessments and school performance of the children who continued on the diet were better than those who lost dietary control; moreover, the assessment results of the children whose Phe was under 600 μmol/L ranked the best compared to the groups exposed to higher concentrations.  Systematic reviews and meta-analysis studies continued to investigate the relationship between life-long and concurrent blood Phe levels and intelligence quotient (IQ) as a long-term 25  health outcome. Waisbren et al. (51) found that there is a significant inverse correlation between mean historical and concurrent mean blood Phe levels and global IQ; in children from birth to 12 years of age, every 100 μmol/L increment in mean Phe concentrations predicted a 1.3- to 3.9-point decrease in IQ over a range of 394 to 750 μmol/L. Furthermore, every 100 μmol/L increase of concurrent Phe concentrations predicted a 0.5 to 1.4-point reduction in IQ over a range of 429 to 1,664 μmol/L. Concurrent Phe was measured around the time of neuropsychological assessments (51). Moyle et al. (48) indicated that adolescents and adults who were continuously treated were more likely to have decreased cognitive functions including global IQ, attention, inhibition, processing speed, and motor control compared to healthy controls. Those with poor dietary control (Phe levels greater than 600 μmol/L after the age of 3 years) demonstrated the worst functional performance. Similarly, in a meta-analysis, Fonnesbeck et al. (130) determined that the probability of individuals with PKU to have lower IQ scores (<85) increases by 15% at blood Phe levels over 400 μmol/L (130). While Moyle et al. (48) found no difference in performance between the groups with good (120-360 μmol/L) and intermediate (360-600 μmol/L) metabolic control, other evidence suggests keeping blood Phe levels under 240 μmol/l is beneficial. One prospective cohort study found that children with PKU whose lifetime mean Phe level was lower than 240 μmol/l performed as well as, or better than, controls on cognitive tasks measuring inhibition, cognitive flexibility, and motor control compared to those with a lifetime mean Phe between 240 and 360 μmol/l (131). Another study concluded that metabolic control and neurocognitive functioning of individuals with PKU and mean blood Phe levels of 240 μmol/L or lower during the first year of life was better in the following years of childhood (132).   26  Evidence on the beneficial effect of lower blood Phe levels is limited and contradictory; there is no evidence that “normalization” of blood Phe levels is beneficial, especially considering that Phe over-restriction could potentially affect physical development and diet adherence (133). For reference, blood Phe levels in the non-PKU population are well below 100 μmol/L (134)(135). Target blood Phe levels within 120-360μmol/L has been generally accepted by the PKU research and clinical communities (7) (15). The European experts extended the upper target Phe level to 600 μmol/L in children aged ≥ 12 years given limited evidence on a harmful long-term effect of blood Phe levels that do not exceed 600 μmol/L. More evidence is needed to determine whether there should be a decrease in the currently recommended upper target level of 360 μmol/L.  1.7.2 Patient and caregiver-reported quality of life outcomes  While quality of life (QoL) is identified as one of the most important patient- and family-oriented outcomes for chronic and complex pediatric diseases (136), evidence on PKU-related QoL is limited and with mixed results. Where some authors report an overall QoL consistent with the general population (137), other studies report delayed autonomy, social relationships, and decreased emotional functioning throughout age groups in patients with PKU (138)  (139). One Swiss study reported overall QoL scores in children and adolescents to be comparable with healthy peers, but significantly lower scores in global positive emotional functioning (138). An Italian study on diet adherence and QoL in children and adults with PKU reported lower than normal global QoL scores in children, and normal overall scores in adults. In this study, adolescents reported significantly lower scores for Family Cohesion and Parental Impact-time domains compared to younger PKU patients (140). A more recent study identified significant 27  emotional impact of PKU as well as disease management-related difficulties across all age groups (141).  Caregivers of children with PKU report considerable time and financial burden associated with dietary management of PKU. A recent UK study indicated that parents spend an average of 20 hours per week managing a child with PKU, and that as many as 23% stop working to accommodate the child’s care (142). Another study reported good overall QoL, yet everyday functioning such as homework, enjoyment of school, and fear of poor grades were rated significantly lower by caregivers of children with poorer metabolic control (143). Yet in other study, parents reported stress and affected self-development, with parents of pre-school children being the most affected (144) In summary, the pressure to consistently follow the stringent diet, health-related issues, resulting chronic stress, and other factors may all directly or indirectly impact the QoL of both patients and caregivers. Moreover, caregiver stress could further affect the quality of care for children as well as family ecology.   1.8 The evolution of clinical and nutritional PKU management practices  Following Følling’s 1934 discovery, an experimental treatment using Phe-free formula was successfully administered to a PKU patient by Dr.Bickel in the 1950s (4). It became clear that simple restriction of natural protein is unsafe and that the diet should be carefully balanced to maintain adequate nutrition. It was also clear that treatment should start as soon as possible after birth (4). Dr.Guthrie enabled early diagnosing via newborn screening for HPA in the 60s, eliminating severe PKU-related complications in early and continuously treated individuals (5). With time, evidence has provided a better understanding of the biochemical and molecular basis 28  of PKU as well as the limitations of the available treatment practices. Historically, variations in PKU management practices have been reported both across and within countries. The most variation was observed in: blood Phe levels triggering treatment initiation, target Phe levels, duration and frequency of follow up, utilization of a neonatal BH4 loading test, and classification of disease severity. These differences are apparent in the variation among PKU recommendations and treatment guidelines developed by different groups in different jurisdictions (145) (146) (147)  (148) (Appendix A, Table A1).  1.9 Modern PKU management guidelines  Over recent years it became evident that PKU is more complex than initially thought. Within North America, a lack of uniformity in PKU clinical and nutritional management practices emphasized the need to further update the 2000 NIH Consensus Statements outlining standards of care. In particular, new evidence prompted the development of US-led consensus guidelines (7) and a regimen for the nutritional management of PKU (8), aiming to harmonize treatment protocols and improve patient care in North America. Both the medical and nutrition guidelines have integrated experts’ consensus with published peer reviewed evidence, addressing clinical practice areas where evidence was lacking. Both these guidelines relied upon two independent reviews conducted by experts from National Institutes of Health (NIH) and the Agency for Healthcare Research and Quality (AHRQ) (149) (79). Nutritional guidelines were developed collaboratively by Genetic Metabolic Dietitians International, the Southeast Regional Genetics Collaborative, and dietitians from the Diet Control and Management and Maternal PKU Workgroups from the National Institutes of Health Phenylketonuria Scientific Review Conference in 2012 (150).  29  Similarly to North America, the variability in practice between and within countries has prompted action in Europe, where updated PKU care recommendation guidelines were published in 2017 (151) (16). Methods of this guideline development process included extensive review, critical appraisal of available evidence, and evidence grading according to SIGN (Scottish Intercollegiate Guidelines Network). The Delphi method was used when there was no or little evidence available (15). Recommendations were published in two reports: a summary report discussing 10 key recommendations with high priority recommendations (10) and a comprehensive report consisting of 70 recommendations for PKU management (15).  While evidence was reviewed and appraised in similar ways, several inconsistencies in key PKU management recommendations between North American and European guidelines still exist (Appendix A, Table A2). 1.10 Current PKU management in Canada  Evidence on clinical and nutritional management of PKU in Canada, that is based on nation-wide studies, is limited to very few research reports. One, based on a survey of 111 treating centres conducted in 1993 in the United States and Canada (152), indicated a lack of uniformity among the various centres with variability in practices and outcomes increasing in older patients. Most healthcare providers recommended dietary Phe restriction for life, yet only one third followed patients after the age of 18 years (152).  The Canadian PKU clinics’ unpublished 2010 electronic survey, used as preliminary background preparation for the upcoming development of PKU guidelines, revealed variations in every aspect of PKU care as well as variability in management practices between treating centres in Canada (communication with Dr. John Mitchell, MD, M.Sc, FRCP, Associate Professor of Human Genetics and Pediatrics at McGill University, Montreal, Quebec, Canada, April, 2016).  30   1.11 Thesis rationale  Despite being one of the most frequently occurring inherited metabolic disorder, management of PKU is complex and some areas of uncertainty still exist. Reported practices regarding PKU management and associated patients’ outcomes vary across countries. These practices and outcomes have not been studied on a nation-wide scale in Canada. The Canadian Inherited Metabolic Diseases Research Network presented an opportunity to investigate and identify sources of important variation in PKU care in Canadian treating centres (153). The CIMDRN platform also enabled various surveys to study parent-reported outcomes in relation to clinical management. Better understanding of the variation in clinical and nutritional management practices of PKU and patient- and family-oriented outcomes will assist in optimizing management practices and creating better health and QoL outcomes for Canadian children with PKU and their families. The objective of this thesis was to analyze current Canadian practices of PKU management and associated outcomes.  1.12 Summary of objectives addressed in PhD thesis 1. To describe current nutritional and clinical management practices of PKU in Canada as reported by healthcare professionals who provide care for children with PKU.  2. To further describe current key nutritional and clinical management practices of pediatric PKU in Canadian treating centres using clinical data assembled for a large pan-Canadian pediatric cohort. 3. To explore the quality of Phe control as an intermediate outcome of PKU management in Canadian metabolic centres. 31  4. To investigate the overall impact of PKU on the wellbeing of Canadian families and whether QoL and whether the parent-reported child’s health status is associated with the quality of Phe control.  To address the research questions outlined above, four projects were carried out:  Study # 1, a nation-wide survey of metabolic dietitians, assessed current nutritional care practices for children diagnosed with PKU. Study # 2, a nation-wide survey of metabolic physicians, assessed current clinical management practices for children diagnosed with PKU. In presenting both current management practices and views on these practices, these surveys allowed individual healthcare providers the opportunity to describe their experiences and opinions on PKU management practices, including insights that could not be readily ascertained from chart data. Study #3 (CIMDRN PKU data analysis) included the analysis of clinical longitudinal data collected within the CIMDRN’s framework and had two objectives.  First, this analysis complemented survey results by incorporating “real-world” clinical data; the CIMDRN data presented an opportunity to corroborate or contrast selected results obtained from the healthcare providers’ surveys (studies #1 and 2).  Second, the CIMDRN data analysis was used to assess patients’ intermediate (surrogate) outcomes stemming from current PKU management practices. Intermediate outcomes included blood Phe levels and quality of Phe control outside and within the recommended therapeutic range of 120-360 μmol/L. Although considered a surrogate biomarker, Phe levels and proportion of Phe outside and within recommended range are immediate outcomes of treatment, thus indicating quality of treatment as well as patient’s adherence to treatment, and may be expected to influence patient-oriented endpoints (Figure 1.1.). Study #4, a quality of life survey of 32  CIMDRN participants, assessed the impact of everyday PKU management on the quality of parents’ life and child’s health from the perspective of the families who care for children with PKU. This study closes the loop between PKU management, intermediate and patient-relevant outcomes. The overall structure of this doctoral dissertation is presented in Figure 1.1.  Figure 1.1 Overall structure of the doctoral dissertation.   33  Chapter 2: Assessing the nutritional and clinical management practices of PKU1   2.1  Synopsis  Background: In 2014 the American College of Medical Genetics and Genomics (ACMG) published a clinical practice consensus guideline, accompanied by recommendations from Genetic Metabolic Dietitians International (GMDI), to guide PKU management and care (7) (8). European PKU experts also reviewed existing evidence and published comprehensive management guidelines in 2017 (10) (97). However, the landscape of practices in Canadian metabolic treating centres remains uncharted. Two country-wide surveys aimed to describe current nutritional and clinical pediatric PKU management practices in Canada.  Methods: Thirty-three dietitians and forty-five Canadian metabolic physicians, identified through online public sources, were invited to participate in two metabolic healthcare providers’ surveys. The questionnaires were designed to reflect the different roles of physicians and dietitians in PKU management.  Results: Nineteen dietitians and seventeen metabolic physicians who provided care for children with PKU completed the surveys. Both commonalities and differences in diagnosis and management practices among individual healthcare providers and between metabolic centres were revealed. To assess disease severity, 89% of dietitians and all physicians reported using pre-treatment blood Phe (Phe) levels alone or in combination with other criteria. More than half of the physicians indicated that they do not utilize neonatal BH4 tests. Those who use the test                                                  1 A version of this chapter has been published as Yuskiv N, Potter BK, Stockler S, et al. Nutritional management of phenylalanine hydroxylase (PAH) deficiency in pediatric patients in Canada: a survey of dietitians' current practices. Orphanet J Rare Dis. 2019;14(1):7. Published 2019 Jan 8. 34  administer it with a wide variety of protocols. 74% of dietitians and 59% of physicians recommend initiating a Phe-restricted diet when blood Phe levels are ≥360µmol/L. All dietitians considered 120-360µmol/L the optimal therapeutic blood Phe range for children 0-9 years of age, with less agreement for older children. More than half (65%) of physicians supported treatment targets of 120-360 µmol/L in children >2-10 years of age, but only 47% supported the same in older children. As expected, reported frequency of communication was the highest in the group of youngest patients and declined with patients’ age. 71% of responding physicians stated that they offer a trial of sapropterin to most pediatric patients at some point of care. Some dietitians (21%) reported prescribing large neutral amino acid supplementation to pediatric patients. About half of physicians recommend regular neuropsychological assessments to the majority of their patients. Finally, our surveys indicated a lack of multidisciplinary care with only 3 out of 14 centres having complete multidisciplinary teams as recommended by the ACMG.  Conclusion: Overall, Canadian PKU nutritional and clinical management practices align with current guidelines.  While Canadian children with PKU receive recommended care, variations in many aspects still exist, reflecting gaps in current PKU knowledge as well as areas where improvement is needed.       35  2.2 Introduction  New published evidence and lack of consistency in PKU management prompted updates to clinical and nutritional management guidelines. The most recent consensus-based guidelines for PKU care in North America were published by the American College of Medical Genetics and Genomics (ACMG) in 2014 (7). Companion recommendations for nutrition management of PKU were developed by Genetic Metabolic Dietitians International (GMDI) and published in the same year (8). Key recommendations and complete European guidelines for phenylketonuria diagnosis and treatment were published in 2017 (10) (97). These relatively recent developments presented an opportunity to investigate current PKU management practices in Canada based on the available guidelines, and to identify potential variations in care. Two surveys sought to understand the approaches of Canadian metabolic healthcare providers with regards to PKU nutritional and clinical management, their awareness of published PKU guidelines, and their diagnostic and treatment practices within and outside these guidelines. Providers’ views on which factors have the most influence on their treatment recommendations were also ascertained.  Identifying uncertainties in the nutritional and clinical management of pediatric PKU in Canada, from practitioners’ perspectives, is important for understanding the impact and uptake of the new guidelines, identifying areas where knowledge translation and mobilization are needed, and prioritizing questions about treatment effectiveness for future research. The dietitian and physician surveys were developed and distributed one year apart, in 2016 and 2017 respectively.  2.3 Methods  The dietitian survey was submitted to the Research Ethics Board of the University of British Columbia and received REB approval in August 8, 2015 (H15-01291). The physician 36  survey was added to existing REB application H15-01291-A007 and was approved in April 2017. 2.3.1 Development of the survey questionnaires Both surveys covered the following topics: personal and practice characteristics, self-reported awareness and use of the most recent PKU guidelines, classification of PKU severity and treatment targets.  The questionnaire for dietitians additionally covered the following topics: frequency of monitoring and target ranges for surrogate biomarkers; recommended dietary intake of key nutrients and the methods recommended for patients to self-monitor intake of these nutrients; recommended use and accessibility of medical foods; use of vitamin and mineral supplements; frequency of clinic visits and communication with patients and their families; methods of encouraging and monitoring patient adherence to therapy.  The questionnaire for physicians additionally covered the following topics: methods of diagnostic confirmation; deciding whether or not to treat with sapropterin; and assessment of responsiveness to sapropterin.  The complete questionnaires can be found in Appendix B.  2.3.2 Sample selection and survey implementation Eligible participants were metabolic dietitians and physicians who provided care to children with PKU in Canada. Dietitians’ contact information was identified through the GMDI website (154). Physicians’ contact information was identified via publicly available sources such as provincial colleges of physicians and surgeons. However, from these sources it was not 37  feasible to identify clinicians who provided care specifically for pediatric PKU patients thus the survey was mailed to the broader sample of metabolic physicians.  Thirty-three Canadian metabolic dietitians and 45 physicians in nine Canadian provinces and three territories were identified. We could not be certain, based on the available information, that these professionals specifically provided care to children with PKU; this eligibility criterion was thus incorporated into the questionnaire as a screening question.  Adapting Dillman’s tailored design method (155) up to six contacts were made to invite Canadian metabolic professionals to participate in the survey. These included (a) a pre-notification email message sent out by one of the study investigators; (b) an initial mailed invitation with a copy of the survey; (c) an initial email invitation with the link to the online survey (to dietitians); (c) a mailed reminder letter with a copy of the questionnaire to the remaining non-responders; (d) an email-reminder with the link (to dietitians); and (e) a final reminder message (email or mail), sent to remaining non-responders.  Dietitians and physicians could respond to the survey by mail, using a prepaid return envelope that was included with each of the two mailed questionnaires. Additionally, dietitians were provided with the link to the online REDCap survey, hosted on a secure BC Children’s Research Institute server with participant access through a unique identification number and password.  In accordance with existing evidence regarding monetary incentives (156), dietitians were offered a $25 iTunes gift card for a completed survey. This was mentioned in the invitation letters and subsequent reminders. A $5 coffee shop gift card was included in each of the physician’s mailed survey questionnaire package.  38  Data from both surveys were entered into a REDCap database and exported to SAS for descriptive analysis. All survey questions were categorical thus reported as proportions. Many questions used 4 or 5-point Likert-type scales and, where necessary and applicable, the categories were grouped (e.g., “all” with “most”, “excellent” with “good”, “sometimes” with “rarely”) to account for small numbers. 2.4 Results 2.4.1 Response rate and distribution of sample characteristics 2.4.1.1 Physicians’ survey sample characteristics  Twenty nine (64%) physician questionnaires were returned. From those, only seventeen questionnaires were completed by physicians (and one nurse-practitioner) who provide care for children with PKU. Hereafter respondents to the physicians’ survey will be referred as “physicians” or “healthcare providers”. We received responses from 11 centres located in 6 of the eight Canadian provinces with established regional medical and/or metabolic genetic centres. Six out of 11 centres had a single respondent and 5 centres had multiple (>1) respondents.  All respondents had at least 3 years working experience and more than a third had over 16 years of experience in metabolic services. All were employed full time with less than half of their time dedicated to PKU care. One responder reported not providing care to PKU patients at the time of the survey (Table 2.1).     Table 2.1 Sample characteristics of physicians’ survey  Characteristics of the Respondents Responded, n (%) Total number of years of practice in metabolic clinical services (n=17) Less than 1 year 0 1-2 years  1 (10%) 3-5 years 5 (30%) 39  Characteristics of the Respondents Responded, n (%) 6-10 years 2 (12%) 11-15 years 2 (12%) More than 16 years 7 (40%) Working Full- or Part time (n=17) Full time 17(100%) Part-time 0 Time dedicated to care of PKU patients (n=17) All my time 0 At least half of my time 0 Less than half  16 (94%) None of my time 1(10%)  2.4.1.2 Dietitians’ survey sample characteristics  Nineteen completed surveys were returned by dietitians, a response rate of 58% (19/33). Ten out of nineteen surveys (53%) were submitted on paper and 9/19 (47%) were completed online. Responses were provided from 14 centres located in six of the eight Canadian provinces with established regional medical and/or metabolic genetic centres; 10 centres had a single respondent and 4 centres had multiple respondents. The majority of the respondents had worked in metabolic nutritional services for more than 6 years (74%), full time (68%) and dedicated at least half of their time to the care of children with PKU (53%). The majority of centres (79%) followed more than 20 pediatric and adult (79%) patients who required regular nutritional services. Only three out of 14 centres (21%) reported having a comprehensive multidisciplinary team that included a metabolic physician, metabolic dietitian, metabolic nurse, psychologist, social worker and clinical biochemist (Table 2.2).   Table 2.2 Sample characteristics of metabolic dietitians’ survey. Characteristics Responded, n (%) Reported by individual respondent (n=19 dietitians) Total number of years of practice in nutrition  services (n=19 dietitians) < 1 year 2 (11%) 40  Characteristics Responded, n (%) 1-2 years 1 (5%) 3-5 years 2 (11%) 6-10 years 5 (26%) 11-15 years 4 (21%) > 16 years 5 (26%) Working Full- or Part timea (n=19 dietitians) Full time 13 (68%) Part-time 6 (32%) Time dedicated to care of PKU patients (n=19 dietitians) All my time 3 (16%) At least half of my time 7 (37%) Less than half 9 (47%) Reported by treating centre (n=14 centres) Number of PKU children actively followed in centre (n=14 centres) < 10 1 (7%) 10-20 2 (14%) 20-40 8 (57%) > 40 3 (22%) Number of PKU  patients newly diagnosed each year (n=14 centres) < 2 12 (86%) 3-5 2 (14%) PKU patients’ population in the centre (n=14 centres) Pediatric only (0-18 years) 3 (21%) Pediatric and adults combined 11 (79%) Centre’s health care team composition (n=14 centres) Dietitian 14 (100%) Physician 13 (93%) Metabolic nurse 9 (64%) Social worker 7 (50%)  Psychologist 6 (43%) Clinical biochemist 5 (36%) Genetic counselor 4 (29%) Nurse-practitioner 1 (7%)        Team: At least Metabolic Dietitian + Metabolic Physician 13 (93%) Units used in responder’s centre (n=14 centres) mg/dl 2 (14%) a µmol/L 12 (86%) a One of the two centres reporting using mg/dl, uses mg/dl in older patients and umol/L in younger patient  2.4.2 Reported clinical practices regarding diagnosis Almost all physicians (16/17, 95%) reported routine use of PAH gene mutation analysis for most or nearly all patients, as part of diagnosis and disease severity classification.  PAH gene 41  sequencing was the starting point of genetic analysis for the majority of responders (12/17, 71%) followed by sequencing plus del/dup as the first choice (5/17, 29%).   Almost half (8/17, 47%) of physicians reported using BH4 loading test in the newborns. Three out of eight (3/8, 37%) reported using this test as a diagnostic workup to differentiate BH4 deficiencies only. The rest (5/8, 63%) utilize this test for (a) differential diagnosis of BH4 deficiencies and (b) to assess the responsiveness to sapropterin dihydrochloride (Table 2.3). The majority of physicians who stated that they used neonatal BH4 loading tests (6/8, 75%) reported using the dose of 20mg/kg and defining a positive result as a 30% Phe reduction within 24 hours (data not shown). The duration of the protocol of BH4 response testing ranged from 8 to 72 hours, with a 24-hours protocol being most frequently reported (5/8, 63%).  The timing of Phe sampling during the response assessment also varied. Slightly more than half (53%) of physicians indicated that they typically did not perform a neonatal BH4 loading test (Table 2.3). The majority of those who reported not performing BH4 loading test (67%) reported logistical difficulties as a major obstacle. Less than half (4/9, 44%) reported that they saw no benefits of this test, and the same number (44%) expressed concerns regarding future funding of sapropterin as a major barrier to using BH4 loading tests. Additional reasons for not performing neonatal BH4 loading tests were related to a lack of evidence on the benefits, delay in starting treatment, and benefits being limited to only super responder patients.   14/17 (82%) physician respondents reported testing all newborns who screened positive for PKU via newborn screening for the disorders of BH4 synthesis and regeneration, while the remaining 3/17 (18%) indicated providing further diagnostic tests only if the follow up blood Phe levels or the results of genetic tests were not supportive of a diagnosis of PKU. Pterins in urine 42  and DHPR activity in blood spots were reported by all respondents as a method of diagnosing BH4 deficiency (Table 2.3). Table 2.3 Selected reported practices related to diagnosis ascertainment among physicians treating pediatric PKU   Response options Responded, n (%) Do you routinely perform/recommend PAH gene mutation analysis for your patients with PKU? For nearly all patients 13/17(76%) For most  patients 3/17(18%) For some  patients 1/17(6%) Rarely / Never  0 What test do you prefer to start with if there is no known mutation in the family? PAH gene sequencing 12/17(71%) PAH gene sequencing+del/dup 5/17(29%) Look for the most frequent mutations first 0 N/A 0 Do you perform the neonatal tetrahydrobiopterin (BH4) loading test? Yes, to diagnose long-term BH4 responsiveness only 0 Yes, as a part of BH4 deficiency workup only 3/17(18%) To diagnose long-term BH4 responsiveness and BH4 deficiency workup 5/17(29%) I do not perform neonatal BH4 loading test 9/17(53%) If you do not perform neonatal BH4 test, what prevents you from it? a Mostly logistic difficulties 6/9(67%) I do not see any benefit 4/9(44%) Concerns regarding future funding of sapropterin 4/9(44%) Reported time points of blood Phe sampling during the neonatal BH4 loading test Baseline – 4h -8h -24h 3/8(38%) Baseline – 4h – 8h – 24h -48h 1/8(13%) Baseline – 1h – 2h – 8h – 24h 1/8(13%) Baseline – 8h – 16h – 24h 1/8(13%) Baseline – 24h – 48h – 72h 1/8(13%) 43  Response options Responded, n (%) 4h – 8h 1/8(13%) What is (are) your initial step(s) to rule out primary BH4 deficiency in a newborn with high  blood Phe levels? a HPA gene panel 3/17(18%) Pterins in urine 17/17(100%) DHPR activity in blood spot 17/17(100%) Otherb 5/17(29%) a Multiple choice question: percentage does not add up to 100% b BH4 loading test(n=4) and PAH gene sequencing (n=1)  2.4.3 Physicians’ approaches to classification of PKU severity The physicians’ approach to defining PKU severity was based on untreated blood Phe levels (17/17, 100%) however only 3 respondents (18%) reported relying on untreated Phe as a single criterion. The majority (14/17, 82%) reported combining untreated Phe with at least one of the following criteria: Phe tolerance, PAH genotype, in-vitro residual PAH enzyme activity and/or blood Phe levels during a catabolic state. 11 out of 17 (65%) physicians reported using genotype as an aid to determine PKU severity in combination with at least one other criterion. Catabolic Phe level(s) (during concurrent illness) were reported by 4/17 (24%) in combination with other criteria, and residual in-vitro PAH activity was reported by 2/17 (12%) in combination with other criteria. The combination of “Untreated blood Phe levels and Phe tolerance at certain age and PAH genotype” was reported most frequently in defining PKU severity (6/17, 36%) (Figure 2.1).   44  Figure 2.1 Basis for classifying severity of PKU by metabolic physicians.  “Catabolic”= catabolic state. The maximum blood Phe level could be triggered by catabolic state, for example, when a patient has a fever. “Phe tol”= dietary phenylalanine tolerance.  2.4.4 Dietitians’ approaches to classification of PKU severity  While confirmation of a PKU diagnosis is most often the sole responsibility of the metabolic physician, metabolic dietitians are frequently involved in determining the severity of PKU. Dietitians closely monitor patients’ dietary intake and corresponding blood Phe levels, and they communicate with patients and families between visits. Dietitians we surveyed variably reported relying on pre-treatment Phe, Phe tolerance and PAH gene mutation analysis to classify the severity of PKU. Specifically, 9 of 19 respondents (47%) reported using only newborn pre-treatment blood Phe levels to define the severity of PKU and 8/19 (42%) reported using pre-treatment blood Phe levels in combination with either Phe tolerance, PAH genotype, or all three. One respondent indicated using Phe blood levels when the patient is catabolic (Figure 2.2).   Untreated Phe + Phe tol + Genotype + Catabolic + Residual PAH (2/17)Untreated Phe + Phe tol + Genotype + Catabolic (2/17)Untreated Phe + Phe tol + Genotype (6/17)Untreated Phe + Phe tol (3/17)Untreated Phe + Genotype (1/17)Untretead Phe  only (3/17)17%17%6%36%12%12%45  Figure 2.2 Basis for classifying severity of PKU by metabolic dietitians.  a The respondent also indicated using Phe blood levels when the patient is catabolic. The maximum blood Phe level could be triggered by catabolic state, for example, when a patient has a fever. “Phe tol”= dietary phenylalanine tolerance.  2.4.5 Nutritional management practices of PKU The majority of dietitians who participated in our survey (74%) recommend initiation of dietary treatment at blood Phe levels of   ≥360 µmol/L, although some (5/19, 26%) recommend a Phe-restricted diet at Phe concentrations between 360 and 480 µmol/L. Phenylalanine and tyrosine were reported as being routinely monitored by all dietitians, with 95% also monitoring ferritin. More than half of dietitians indicated routinely monitoring pre-albumin, albumin and vitamins. Forty-seven percent reported routinely monitoring bone density, while a small minority reported routine monitoring of essential fatty acids. Fewer dietitian respondents reported monitoring other biomarkers including: homocysteine, carnitine, full amino acid quantification, alkaline phosphatase, complete blood count, trace elements (zinc, selenium, manganese), folate, B12, and 25-hydroxyvitamin D (data not shown). Pre-treatment Phe only (9/19,47%)Pre-treatment Phe+Phe tol (5/19,26%)Pre-treatment Phe+PAH genotype (2/19,11%)Pre-treatment Phe+Phe tol+PAH genotypea(1/19,5%)Phe tol only (1/19,5%)PAH genotype only (1/19,5%)46  All dietitian respondents indicated the target range of blood Phe levels as 120-360 µmol/L for younger patients, yet opinions varied slightly for patients aged >10-18 years old: most dietitians recommended 120-360 µmol/L, while some recommended higher target blood Phe levels, up to 600 µmol/L. 58% would rarely recommend keeping blood Phe levels at the lower end of therapeutic range (e.g. maintaining a more phe-restricted diet); moreover, the majority of responding dietitians would not be comfortable with patients having blood Phe levels lower than 120 µmol/L.  Finally, 47% of dietitian respondents recommended maintaining blood Phe levels at a higher-end of the therapeutic range for some patients (Table 2.4).  Table 2.4 Selected reported PKU nutritional follow up practices among dietitians treating pediatric PKU. Response options  Responded, n (%) At which blood Phe levels do you make a decision to treat patients  with a Phe-restricted diet? (Consistently elevated blood Phe levels) ≥360 µmol/L 14 (74%) 360-420 µmol/L 1 (5%) ≥420 µmol/L 1 (5%) ≥480 µmol/L 2 (10%) ≥600 µmol/L 1 (5%) Which biomarkers do you routinely monitor in most of your PKU patients? Phenylalanine 19(100%) Tyrosine  19(100%) Ferritin 18(95%) Pre-albumin 14 (82%) Vitamins 13 (72%) Albumin 12 (63%) Bone Density 9 (47%) Essential FA 3 (18%) What is your optimal target range of blood Phe? Age groups:      0-12 months 120-360 µmol/L 19(100%) >1-2 years 120-360 µmol/L 19(100%) 47  Response options  Responded, n (%) >2-10 years 120-360 µmol/L 19(100%) >10-18 years 120-360 µmol/L 14a (78%) 120-600 µmol/L 2a (11%) 320-600 µmol/L 2a (11%) What would typically be your lowest acceptable average level of blood Phe as a long-term   treatment goal? 0-12 months 100 µmol/L 1a (6%) 120 µmol/L 17 a (94%) 150 µmol/L 0 200 µmol/L 0 >1-2 years 100 µmol/L 0 120 µmol/L 16b (94%) 150 µmol/L 1 b (6%) 200 µmol/L 0 >2-10 years 100 µmol/L 0 120 µmol/L 16b (94%) 150 µmol/L 1 b (6%) 200 µmol/L 0 >10-18 years 100 µmol/L 1 a (6%) 120 µmol/L 16b (94%) 150 µmol/L 0 200 µmol/L 1 a (6%) Would you ever be comfortable with a patient’s steady blood Phe levels being below 120 µmol/L (if “yes”, please explain)c Yes 6 (32%) Comments: “I would be comfortable with <120umol/L in maternal PKU where I was certain formula and calorie intake was optimized and the patient was careful with foods they chose to ensure good nutrition.” “If patients are experiencing rapid growth (usually in infancy).”“Only for super responders to Kuvan [sapropterin dihydrochloride] tolerating DRI total protein from regular protein foods with minimal or no PKU foods.” No 13 (68%) Comments: “If levels were consistent and testing was done weekly, I would be ok it with somewhat lower levels, perhaps as low as 80.”  “I would be more comfortable with an older child (>2years), but this rarely happens” “On Kuvan & hard to increase Phe intake; On restricted diet but growing well”  Do you recommend that patients maintain higher-end therapeutic range blood Phe levels and more liberal natural protein intake? For most/nearly all patients 5 (26%) 48  Response options  Responded, n (%) For some patients 9 (47%) Rarely / Never  5 (26%) a 1 missing response  b 2 missing responses  2.4.6 Clinical management practices More than half of physicians (10/17, 59%) reported initiating dietary treatment at blood Phe levels of ≥360 µmol/L, 4 out of 17 (24%) initiate treatment at blood Phe levels of ≥600 µmol/L, and the remaining three reported treatment initiation at various Phe levels as follows:    ≥ 240 µmol/L, ≥400 µmol/L and ≥420 µmol/L. The reported long-term follow up for male patients with mild hyperphenylalaninemia who do not require dietary treatment (non-PKU HPA) ranged from “not at all” to “life-long” follow up. For female patients with mild hypephenylalaninemia who do not require dietary treatment (non-PKU HPA) majority of respondents reported considerably longer follow up, either more than 2 years or life-long follow up (14/17, 82%).  The majority recommend life-long treatment for male and female patients with untreatable blood Phe levels from 360 to 600 µmol/L however responses were varied (Table 2.5).  Optimal target Phe levels reported by physician respondents varied for all age groups and across physicians. The guideline-recommended range of 120-360 µmol/L was reported as recommended by most physicians for children from 1 to 10 years old, however the recommended treatment target varied. Only 6 out of 17 (35%) physician respondents stated that they support a target range of 120-360 µmol/L in patients 0-12 months old, 9/17 (53%) for patients >1-2 years old, 11/17 (65%) in patients >2-10 years old and slightly less than half (8/17, 47%) in older children.  One third of respondents (6/17, 34%) identified blood Phe levels of ≤120 µmol/L as too low for most or nearly all patients (Table 2.5).  49  Table 2.5 Clinical PKU management practices (reported by physicians).  Response options  Responded, n (%) At what blood Phe level do you make a decision to initiate dietary  and/or pharmacotherapy in a newly diagnosed newborn with PKU? ≥240 µmol/L 1 (6%) ≥360 µmol/L 10 (59%) ≥400 µmol/L 1 (6%) ≥420 µmol/L 1 (6%) ≥600 µmol/L 4 (24%) For how long do you follow patients with non-PKU HPA  who do not require dietary treatment in the clinic? Male patients <1 year follow up  1(6%) 1-2 years follow up 4 (24%) >2 years follow up 8(47%) Life-long follow up  1 (6%) Not at all 1(6%) Other  0 Female patients <1 year follow up  0 1-2 years follow up 0 >2 years follow up 4(24%) Life-long follow up  10(59%) Not at all 0 Other  3(18%) For how long would you recommend treatment for a patient whose untreated blood  Phe levels are between 360 μmol/L and 600 μmol/L? (n=17) Male patients Not applicable  4(24%) Life-long 10(59%) Up to 12 years of age 3(18%) Would treat when pregnant n/a Female patients Not applicable  3(20%) Life-long 12(71%) Up to 12 years of age 2(12%) 50  Response options  Responded, n (%) Would treat when pregnant 4(24%) In your opinion, what is the optimal target range (µmol/L) of blood Phe for children with PKU in the following age groups? (n=17)  0-12 months 60-120 µmol/L  1(6%) 60-240 µmol/L 1(6%) 60-360 µmol/L 4(24%) 120-240 µmol/L 5(29%) 120-360 µmol/L 6(35%) 120-600 µmol/L 0 360-600 µmol/L 0 >1-2 years 60-120 µmol/L  0 60-240 µmol/L 2(12%) 60-360 µmol/L 4(24%) 120-240 µmol/L 2(12%) 120-360 µmol/L 9(53%) 120-600 µmol/L 0 360-600 µmol/L 0 >2-10 years 60-120 µmol/L  0 60-240 µmol/L 1(6%) 60-360 µmol/L 5(29%) 120-240 µmol/L 0 120-360 µmol/L 11(65%) 120-600 µmol/L 0 360-600 µmol/L 0 >10-18 years 60-120 µmol/L  0 60-240 µmol/L 1(6%) 60-360 µmol/L 2(12%) 120-240 µmol/L 0 120-360 µmol/L 8(47%) 120-600 µmol/L 2(12%) 360-600 µmol/L 4(24%) 51  Response options  Responded, n (%) If patient’s Phe intake is not severely restricted, to what extent would you regard  blood Phe levels in the range of ≤120 µmol/L as “low”? (n=17) For nearly all patients 3(18%) For most patients 3(18%) For some patients 6(34%) Rarely regard this as low 4(24%) Never regard this as low 1(6%) Do you refer your PKU patients for the formal neuropsychological assessment? (n=17) Yes all PKU patients, to screen 6(35%) Yes, only selected PKU patients 10(59%) I do not order formal neuropsychological 1(6%)   2.4.7 Frequency of communication and clinical follow up with dietitians Dietitians reported that clinic visits for children with PKU were the most frequent in infants 0-12 months old and declined with patients’ age. After the first year of life, the majority of dietitians indicated seeing their patients less than once per month but at least once per year. The majority of dietitians also reported that between-visit communications took place most frequently with the parents of the youngest patients. Telephone calls were most frequently used for between-visit communication with families (100%), followed by email (89%) and mail (58%).  All dietitian respondents reported team discussions of individual patients’ nutritional management, however only slightly more than half (58%) indicated discussing most of their patients on a regular basis (data not shown).  Only one third (36%) of dietitians reported that they considered multidisciplinary healthcare team communication at their centre as “highly effective”, while the majority of dietitian respondents (68%) reported that they found the within-team communication as “somewhat effective” (Table 2.6).  52  Table 2.6 Reported frequency of clinic visits and provider-family communications. Patients’ age At least once            per week Less than once per week but at least once per month Less than once per month but at least once per year Less than   once per year Other b,c Clinic visits frequency, n (%) < 1 year 3a (17%) 6a (33%) 8a (44%) 0 1a (6%) b 1-2 years 0 4a (22%) 14a (78%) 0 0 3-10 years 0 2a (11%) 16a (89%) 0 0 11-18 years 0 2a (11%) 16a (89%) 0 0 Frequency of communication with patient/family/caregivers (via phone, email, fax, and other means of communication outside of the clinic visit), n(%)  < 1 year 15 (79%) 3 (16%) 0 0 1 (5%)b 1-2 years 4a (22%) 13a (72%) 0 0 1a (6%) c 3-10 years 0 13 (68%) 5 (26%) 0 1 (5%) c 11-18 years 0 9 (47%) 8 (42%) 0 1 (5%) c a  1 missing answer (n=18). b “Each month until 3 months old then every 3 months” (n=1). c Other: “Based on how frequently family/patients monitor Phe levels (n=2); “Some (monitor) weekly, some every 2 weeks, some monthly (n=1)”.  2.4.8 Physicians’ approaches to the utilization of neuropsychological assessments Less than half of physician respondents (6/17, 35%) reported referring all PKU patients to a psychologist for the neuropsychological assessments, even if their patients do not show/experience any symptoms. If psychological problem was suspected in a patient, more than half of respondents (10/17, 59%) would consider referring a patient to a psychologist. The reported barriers to referring to a psychologist were: limited accessibility of psychological services (n=5), perceiving the assessment as “not beneficial” in assessing non-symptomatic patients (n=2). The following comments were provided by physician respondents: “psychology services are not easily accessible for a patient” (n=3); “I do not see any benefit of screening non-symptomatic PKU patients for potential neuropsychological deficits” (n=1); “psychologists indicate the repeat assessment is worthless. Also it is discriminatory vs well controlled normal PKU child” (n=1); “I 53  would refer all PKU patients but am limited with access to the psychologist” (n=1) and “Not able to do formal testing as no access” (n=1). 2.4.9 Clinical practices of pharmacological treatment with sapropterin among physicians All physician respondents reported offering a trial of sapropterin to their patients at any point of their follow up. The majority of physician respondents (12/17, 71%) offering a trial of sapropterin to “nearly all and most of their pediatric patients” while the rest (5/17, 19%) reported offering sapropterin to “some patients and rarely”. The majority of physicians (14/17, 82%) reported that their main criterion in defining a good response to sapropterin treatment is the reduction (at least 30%) in blood Phe levels from the baseline.  The majority also reported determining long-term response to treatment over a 6-month period. Along with reduction in blood Phe levels, Phe tolerance was reported to be used by 15/17 (88%) physicians to assess response to sapropterin treatment. However, there was no consensus among survey respondents on what constituted a meaningful gain in Phe tolerance, with a “good response” to sapropterin reported across a range of >10% to a 100% increase from baseline.  Regarding the monitoring of sapropterin treatment outcomes, a baseline (pre-treatment) neuropsychological assessment was reported to be used by slightly fewer than half of physician respondents (8/17, 47%), with the majority of those reporting the use of a follow up assessment, typically after a year (5/8, 63%). Parent- and patient-reported feedback was reported as routinely used in assessing sapropterin treatment outcomes by 14 out of 17 respondents (82%) with parent-reported feedback as the most reliable tool (11/14, 79%) followed by patient-reported feedback (9/14, 64%). Standardized QoL questionnaires reported to be used by 3 out of 14 respondents 54  (21%) who responded “yes” to the routine use of parent and/or patient-reported feedback when assessing the outcome of treatment with sapropterin (Table 2.7).    Table 2.7 Reported practices on treatment with sapropterin dihydrochloride (reported by physicians).   Response options  Responded, n (%) For pediatric patients with PKU, do you offer a trial of sapropterin treatment? (n=17) For nearly all 6 (35%) For most  6 (35%) For some  4(24%) Rarely 1(6%) Never  0 Before starting treatment with sapropterin, do you determine a patient’s  upper limit of Phe tolerance? (n=17) For nearly all 11 (65%) For most  6 (35%) For some  0 Rarely 0 Never  0 How do you determine reduction in Phe levels from baseline in response to  sapropterin treatment?a (n=17) (multiple choice) Mean baseline mean Phe vs mean treatment Phe 16 (94%) General Phe level trend observation 5(29%) Last Phe before treatment vs lowest Phe on treatment 4(24%) Otherb 1(6%) What increase in Phe tolerance (%) do you consider sufficient to determine a good response?(n=14) c (multiple choice) >10 %  1(7%) 20% 1(7%) >25% 1(7%) 30% 7(50%) 50% 1(7%) 100% 1(7%) 55  Response options  Responded, n (%) Other c 3(21%) From your clinical experience with PKU, what was the longest time period in determining responsiveness to sapropterin (approximately)?d (n=17) up to 7 days 0 1- 4 weeks 6(35%) 1-6 months 10(59%) 6 months – 1 year 0 Otherd 1(6%) What would you consider the single best indicator of sapropterin effectiveness? (n=17) Stable reduction of blood Phe levels 11(65%) Stable increase in Phe tolerance 4(24%)   Reported QoL improvement  2(12%) Neuropsychological scores improved 0 Do you perform a baseline neuropsychological assessment test for children  treated with sapropterin?a,e (n=17) For nearly all or most patients 6(35%) For some patients 2(12%) Rarely 4(24%) Never 5(29%) In what time interval, after starting sapropterin, do you typically order a follow up  neuropsychological assessment test? (n=17) In 3 months 0 In 6 months 1(6%) In 1 year 5(29%) Not applicableg 10(59%) Otherh 1(6%) If you use patient-reported outcomes in assessment of sapropterin treatment (n=14), what tools do you use to assess patient-reported outcomes? a (multiple choice) Parent-reported feedback 11(79%) Patient-reported feedback 9(64%) History taking only 4(29%) Standardized QoL questionnaires 3(21%) Non-standardized QoL questionnaires 1(7%) Structured interviews 1(7%) a This is multiple choice question: the percentage does not add up to 100%  b“[Compare] one month level with weekly levels” cOther:n=3 do not regard Phe tolerance as an indicator of response to sapropterin treatment. 56  Comments: “Must be clinically meaningful”; “Dietitian decides - any increase in phe tolerance is good”; “this has more to do with what it practically means: another piece or two of bread? Foods previously not tolerated? Drop in formula needed?” d “From 7days to 4weeks for the first stage; from 1month to 6months for the second stage, (to assess) phe tolerance (change)”. eComments: “psychology services are not easily accessible for a patient“ (n=3); “ I do not see any benefit of screening non-symptomatic PKU patients for potential neuropsychological deficits” (n=1). fComments: “psychologists indicate the repeat assessment is worthless. Also it is discriminatory vs well controlled normal PKU child”; “I would refer all PKU patients but am limited with access to the psychologist”. gNot applicable: I do not routinely order a follow up neuropsychological assessment test to evaluate the treatment outcome in children with PKU treated with sapropterin. h Other: “Not able to do formal testing as no access”.    2.4.10 Medical formulas, foods and supplements from dietitians’ perspectives  The discontinuation of the medical formula was reported to be never considered by 8/19 (42%) of dietitians, while the remaining dietitian respondents stated that they would consider discontinuing formula in certain patients with mild PKU and good responders to sapropterin. The most important factors that dietitians reported as influencing their decisions regarding prescribing medical formulas were nutritional composition of formula, patient’s age, preferences of the patient or family and availability of the product, reported by 95%, 89%, 89% and 79% of dietitians, respectively. One third (32%) reported that their choice of formula is limited by the hospital “formula contract”. The most frequently used medical formulas and factors influencing prescription of one or another medical formula are presented in Appendix B, Table B.3.1. Dietitians from 4 centres reported full provincial coverage of the cost of low protein foods, while the remainder reported partial coverage. Good and excellent accessibility to low protein foods was reported by almost all dietitian respondents (89%). Unexpectedly, with regards to other treatment options, 4/19 (21%) dietitians reported prescribing large neutral amino acid supplements (LNAA).   57  2.4.11 Monitoring of dietary Phe intake A majority of dietitians (89%) reported 3-day diet records as the most frequently used tool for monitoring the adequacy of their patients’ nutritional intake. The most commonly recommended method for self-monitoring of Phe intake, according to dietitian respondents, was counting grams of dietary natural protein (89%), followed by regular bloodwork (74%), counting milligrams of dietary Phe (63%), counting Phe exchanges (47%) and use of computer applications for PKU (53%) (dietitians could endorse multiple responses). Those who reported using computer applications indicated “How much Phe?” as the most frequently reported application (67%), followed by “Accugo” (25%) and “Metabolic Diet App” (25%) (dietitians could endorse multiple responses). Those dietitians who reported using Phe exchanges (9/19,47%) stated that they calculated 1 exchange as 15 mg of Phe.  Home sample collection was most frequently reported as a method of routine Phe monitoring, followed by a “local lab or hospital close to patient's house” and “metabolic clinic” (Table 2.8).   Table 2.8    Monitoring dietary Phe intake (reported by dietitians). Response options  Responded, n (%) How often do you ask your PKU patients to track and submit any diet intake information for clinic analysis? (n=19) Depends on the needs of the patient 15 (79%) With every blood Phe 2 (11%) Only at clinic visits 2 (11%) Other 0 If you request diet records from your patients, what diet records  do you request most often? (n=19) 3 days diet record 17 (89%) 2 days diet record 2 (11%) 1 day diet record 0 7 days diet record 0 58  Response options  Responded, n (%) Other 0 What methods do you recommend for patients with PKU and their caregivers to self-monitor intake of Phe? (n=19)a (multiple choice) Counting grams of dietary natural protein 17 (89%) Regular blood work 14 (74%) Counting mg of dietary Phe 12 (63%) Computer apps for PKU 10 (53%) Counting Phe exchanges 9 (47%) Where do your patients collect blood samples for routine monitoring of Phe? (n=19)  (multiple choice) Home  18 (95%) Local laboratory 13 (68%) Metabolic clinic 12 (63%) aMultiple choice question   2.4.12 Monitoring adherence to the medical formula and low protein foods Dietitians most often reported relying on the verbal report of the parent and/or caregiver to assess patients’ adherence to formula intake (89%), followed by monitoring blood Phe levels (84%), monitoring weight and height (79%) and checking how much formula was released by the dispensing authority (63%). Among biochemical biomarkers used for assessing adherence to the diet, phenylalanine, tyrosine, pre-albumin, ferritin and B12 were monitored most frequently. A majority of respondents consider high blood Phe levels as the most reliable indicator of patients’ non-adherence to the diet and/or drug therapy (reported by 10/19, 53%). Nutrients, phenylalanine, protein, calories, minerals (any) and vitamins are routinely monitored for most patients, as reported by the majority of dietitians. All participants reported performing anthropometric measurements at every clinical visit, while nutrition analysis and nutrition education were reported as always included in routine visits by half of the respondents (58%).  Individualized PKU nutritional counseling was recommended as a strategy to improve patients’ 59  diet adherence by all respondents (19/19, 100%), along with motivational interview techniques and reporting results of blood dots to patients (each 14/19,74%) Individualized nutritional counseling was considered the most successful in improving diet adherence by more than half of respondents (11/19,58%) while “motivational interviewing counseling techniques” and “reporting results of blood dots to patients” reported as less successful (each 4/19, 21%). Regular reminders of Phe dots were not considered successful in improving diet adherence (0/19). “Other” means to improve adherence (3/19, 16%) shared were: “involving other team members (i.e. clinic nurse, social worker), “more blood Phe control”, and “reinforcement of (the) importance of diet adherence by other team members (especially physician) to a patient” (Table 2.9).   Table 2.9 Reported practices on monitoring adherence to the diet (reported by dietitians). Distribution of the responses to survey questions on monitoring adherence, n (%) Which nutrients do you routinely monitor, based on diet records?a (n=19) (multiple choice)  For most patients For some patients Rarely or never Dietary Phe intake 19 (100%) 0 0 Protein intake 18 (95%) 1 (5%) 0 Calorie intake 14 (74%) 5 (26%) 0 Mineral intake (any) 12b (67%) 6a (33%) 0 Vitamin intake (any) 12 (63%) 7 (37%) 0 Fat intake 5c (29%) 8c (47%) 2c (12%) Other 0 1 (5%) 0 How often does your routine clinical visit assessment typically include a: (n=19) (multiple choice)  Always/Often Sometimes Rarely  Anthropometric measurements  19 (100%) 0 0 Diet education 17 (90%) 2 (10%) 0 Dietary analysis 17 (90%) 1 (5%) 1 (5%) How do you assess your patients’ adherence to formula intake? (n=19) (multiple choice) By patient’s/caregiver’s verbal report 17 (89%) 60  Distribution of the responses to survey questions on monitoring adherence, n (%) Monitoring weight and height 15 (79%) By laboratory tests:  Phenylalanine 16 (84%) Pre-albumin 13 (68%) Tyrosine 13 (68%) Iron 12 (63%) B12 12 (63%) Albumin 5 (26%) Checking how much formula was released by the dispensing authority 12 (63%) By analyzing written dietary questionnaires 10 (53%) Which technique do you think is most successful in improving adherence to diet?(n=19) Individualized PKU nutritional counseling 11 (58%) Motivational interview techniques 4 (21%) Reporting results of blood dots to patients 4 (21%) Regular reminders for Phe blood dots 0  a Multiple choice question  bOne missing response (n=18) b Two missing responses (n=17)  2.4.13 Use of published PKU management guidelines  All respondents, physicians and dietitians, reported awareness of published PKU guidelines, with the majority referencing the ACMG PKU consensus guideline (7) and the companion recommendations for the nutrition management of PKU (8). Additionally, other guidelines reported by dietitians were “SERC-GMDI PKU Nutrition Management Guidelines” (157), “NIH Consensus Guideline for Management of PKU” (158), “European Guidelines (not specified)”, and “Publications by Anita Macdonald (not specified)”.  More than one third (38%) of physicians reported using the European guidelines in their practice (10), and 25% and 19% referred to NIH (historic) guidelines (158) and UK PKU guidelines respectively. Seven physicians (41%) reported using a locally developed “sick day management protocol”. It was suggested that more consensus was needed concerning neonatal 61  BH4 loading tests, follow up of benign non-PKU male patients, and individualization of the Phe therapeutic range for adult patients based on their functional status.   2.5 Discussion  These surveys revealed several points of variability in key nutritional and clinical practices in relation to existing guidelines. Variation was found in diagnostic and follow up practices such as classification of PKU severity, neonatal BH4 loading test performance, neuropsychological follow up, treatment targets, frequency of communication between healthcare providers and patients/caregivers, and organization of care in metabolic centres. For example, reported criteria for defining the severity of PKU varied considerably between treating centres and individual providers. It is worth noting that approaches to defining PKU severity also differed between disciplines: 76% of physicians reported using Phe tolerance in combination with other criteria, compared to only 37% of dietitians.  Nearly half (47%) of physician respondents reporting using a neonatal BH4 loading test, and among those using this test, both test objectives and testing protocols varied. This variability could partially stem from the very purpose of the test: while the shorter test is consistent with differential diagnosis in distinguishing PKU from BH4 deficiencies, a longer duration of the test is more beneficial in assessing responsiveness to sapropterin dihydrochloride. Reports that describe BH4 loading test practices in European treating centres also point to variability in practices (159).  Use of regular neuropsychological assessments for patients with suspected deficiencies was reported by slightly more than half of physician respondents, while some respondents indicated they would find it beneficial to refer all PKU patients to psychology services.  Both 62  perspectives could be justifiable. While the literature suggests there is an increased prevalence/incidence of neurocognitive and psychosocial symptoms in PKU patients (42) (46)(47) neuropsychological monitoring might be limited in some centres  due to limited access to neuropsychological services, as outlined in physicians’ comments. The most recent North American guidelines recommend keeping blood Phe levels within the 120-360 µmol/L range during the life time.  While most Canadian metabolic providers follow these guidelines, some reported a relaxation of the diet up to 600 μmol/L starting at age 10. There is emerging evidence suggesting that mean levels within 120-240 μmol/L might be more beneficial for patients in the long-term (132).    Nearly half of the dietitian survey respondents reported communicating with older patients less frequently than is recommended. Decreased frequency of contact with older children is likely due to decreased frequency of home blood Phe monitoring, especially as patients learn to become independent in managing their daily diets and home blood draws. However, other factors may also offer explanation: staff shortages in the metabolic clinic and subsequent time limitations; disappointment with non-adherent patients; or other social, psychological, economical and human resource related barriers (129) (142) (160). Decline in the frequency of communication might potentially contribute to non-adherence with treatment in adolescents.  Our survey of dietitians indicated a lack of multidisciplinary care with dietitians at only 3 out of 14 centres reporting a complete multidisciplinary team as recommended by the ACMG guideline. Moreover, dietitians at only two centres reported having dietitians whose time is fully dedicated to the care of patients with PKU. This lack of staffing might impact quality of care, leading to suboptimal outcomes, as corroborated by the results of another survey (161). While 63  evidence with respect to the impact of a coordinated team approach on improved outcomes in the treatment of PKU is very scarce, one Canadian retrospective study reported that a multidisciplinary centralized approach results in better outcomes in terms of adherence to the diet, control of blood Phe, and fewer patients lost to follow up (162).  In conclusion, variation in reported nutritional and clinical practices of PKU in Canada was observed in the surveys of metabolic healthcare providers. The question remained whether the variation in practices is indeed observed in real-world PKU care. Thus, the findings of these surveys informed the direction for the analysis of the longitudinal clinical PKU data, which portrayed observed key clinical and nutritional practices abstracted from the clinical charts of patients with PKU. The surveys provided invaluable information on current nutritional and clinical practices, as well as opinions on these practices, from the perspectives of individual healthcare providers. These results benefitted from being compared and contrasted with “real-world” clinical data abstracted from medical charts provided by the Canadian Inherited Metabolic Disease Network. The analysis of clinical data is presented in Chapter 3.      64  Chapter 3: Observed PKU management practices and intermediate outcomes  3.1 Synopsis  Background:  The survey results have served two purposes. First, they revealed that in some areas of PKU management lack of consensus and variation in care still exists. Secondly, they informed and narrowed the direction of the analysis of comprehensive longitudinal clinical PKU data collected from the Canadian Inherited Metabolic Diseases Research Network (CIMDRN) platform. This secondary data analysis of an existing cohort aims to: (1) describe key practice-based nutritional and clinical management of pediatric PKU in Canadian treating centres selected from the survey results and, (2) assess intermediate outcomes resulting from these practices. Methods: The CIMDRN cohort gathered chart-derived clinical information of children born between 2006 and 2015 who received care in one of 13 participating Canadian metabolic treatment centres. Study participants were followed until March 31, 2017. The following variables were descriptively analyzed: participants’ demographic characteristics, selected practices regarding diagnosis ascertainment, clinical and nutritional monitoring, individuals’ blood Phe levels during the entire follow up period, and frequency of Phe monitoring and communications with metabolic healthcare providers. Results: The study population included 215 children born from 2006 to 2015, diagnosed with PKU with complete diagnostic data recruited across 12 participating metabolic centres. Mean blood Phe levels were within the guideline-recommended range of 120-360 μmol/L across age groups with a tendency to increase with age from 241±63 μmol/L in children 1-12 months old to 331±151 μmol/L in children older than 7 years. Analysis of the proportion of Phe levels 65  within the desired therapeutic range reflected rather slow achievement of guideline-recommended blood Phe levels by infants with classic PKU during the first month of life. By the fourth week of life, only one third of infants with classic PKU had more than 60% of their blood Phe levels within recommended therapeutic range. 65% of children classified as having mild PKU and 83% of children classified as having mild HPA achieved good metabolic control, defined as having more than 60% of their blood Phe levels within 120-360 μmol/L during the first month of life. However, the majority of “out-of-range” blood Phe levels were below 120 μmol/L rather than above 360 μmol/L in the neonatal period. Beyond the first month of life overall Phe control was still less than ideal across age groups. In particular, only 28% of 8-9 year olds had more than 60% of their levels within recommended range, with the majority of “out-of-range” levels being above 360μmol/L. Discrepancies in disease severity classification were also observed: 43% of participants were considered to have classic PKU despite 30% of this group having never had a recorded peak blood Phe measurement greater than 1,200µmol/L. On the contrary, 4% of the participants who experienced at least one peak Phe level corresponding to classic PKU (>1,200 µmol/L) were assigned to milder forms of PKU. It is of note that peak Phe levels was reported as the main criterion used for classification of PKU severity in both surveys. Contrary to the survey results, the data showed communication between healthcare providers and patients with classic PKU is aligned with recently published nutritional guidelines in the majority of treating centres although there was variation between centres. Participants with milder forms of PKU had considerably fewer Phe tests and clinical visits compare to individuals diagnosed with classic PKU.  Conclusion: This is the first comprehensive description of a nation-wide pediatric PKU cohort in Canada, providing an understanding of the current nutritional and clinical practices as well as 66  immediate treatment outcomes (blood Phe levels) that result from these practices. Complementing the findings from the surveys of healthcare providers, the results of the clinical data analysis also recognized variation in key nutritional and clinical treatment practices across treating centres. Moreover, while mean Phe levels beyond the first month of life remained within the recommended therapeutic range of 120-360 μmol/L, we found the quality of Phe control (presented by the proportions of blood Phe levels outside the recommended therapeutic range) in children with PKU to be suboptimal; this decline in diet adherence with age might contribute to the development of suboptimal health outcomes. This study provides critical information to support the optimization of care with the goal of subsequent improvement in health outcomes.     3.2 Background  Evidence shows that elevated blood Phe levels contribute to suboptimal health outcomes in a dose-dependent manner among children with PKU; lower mean blood Phe levels correlate with better neurocognitive performance (51) (50) (132). It is possible that different approaches to PKU management result in variable treatment outcomes in PKU patients, and in some cases, these outcomes could be less than ideal.  Evidence points towards discrepancies in modern PKU management across treating centres and countries (16) (161). This evidence is corroborated by the survey results described in Chapter 2, indicating variation in key nutritional and clinical practices of PKU management across Canadian treating centres (163). Using the results of the surveys as direction, analysis of clinical chart-abstracted data was employed to further describe and complement information on key practices of PKU management. The intermediate treatment outcomes including mean Phe levels and proportions of Phe levels within and outside therapeutic 67  range (120-360μmol/L) resulting from these practices were also described. These goals were met by analyzing the longitudinal clinical PKU data available from a previous cohort study conducted by the Canadian Inherited Diseases Research Network (CIMDRN). The CIMDRN initiative, a Canadian research network of 12 hereditary metabolic disease treatment centres, was established in 2012 upon receiving a Canadian Institutes of Health Research Emerging Team grant (153). The network is designed to develop the evidence needed to improve health care and outcomes for children with inherited metabolic disorders (IMD) and includes over 40 investigators in the fields of pediatric care for IMD patients, epidemiology, health services and policy research. This comprehensive analysis of longitudinal CIMDRN PKU data is the first Canadian study exploring long-term metabolic control in a pediatric PKU cohort where blood Phe levels are analyzed in the context of clinical and nutritional management practices in metabolic treating centres. The objectives of this study are twofold: (1) to complement the results of the survey, describing key nutritional and clinical management practices of pediatric PKU in Canadian treating centres based on the analysis of chart-derived clinical data, and (2) to assess patients’ blood Phe levels resulting from these practices. Phe levels were described through both mean blood Phe levels, as well as the proportions of blood Phe levels within and outside the guideline-recommended therapeutic range.  3.3 Methods 3.3.1 CIMDRN data compilation  The CIMDRN cohort study collected clinical data from medical charts of children diagnosed with one of 31 eligible inherited metabolic diseases, including PKU. These children were born between 2006 and 2015, enrolled with parental consent at 12 participating treatment 68  centres, and followed until the end of March 2017 (164).  Some children began receiving care at a participating centre partway through the data collection window and others were discharged from a participating centre before the end of the follow-up period. Thus, follow-up time among participating children varied according to birth year and these other factors. Enrollment and data collection for the CIMDRN cohort study was previously approved by research ethics boards (REB) at all participating centres as well as the University of British Columbia (CW14-0023 / H13-03285). PKU, along with several other inherited metabolic disorders, was selected as one of several conditions for which more in-depth longitudinal data were collected.  The baseline PKU data cover diagnostic information (clinical, biochemical, molecular) and basic patient demographic characteristics. The PKU longitudinal data include biochemical markers of disease monitoring and interventions and services received or prescribed, including dietary interventions and pharmacotherapy prescriptions. This information was entered into the CIMDRN central database (housed on the secure server Children’s Hospital of Eastern Ontario’s Research Institute) retrospectively from the child’s medical chart. Data were entered by research coordinators based at each site through a secure web-based data entry system (REDCap) (165).  All data underwent a verification process to ensure that they were complete and to identify potential errors.  The data entry was finalized in all centres as of June 2019. The detailed quality assurance practices applied in CIMDRN database were described in detail elsewhere (153).  3.3.2 Key PKU management practices selected for the analysis of clinical data  The surveys provided understanding of the current nutritional and clinical practices from the perspectives of individual healthcare providers. CIMDRN clinical data provided an opportunity to corroborate or contrast these practices based on the data abstracted from the 69  medical charts. The selection of the key PKU nutritional and clinical management practices was based on the results of the surveys of healthcare providers described in Chapter 2, and on the availability of chart data reflecting these areas of practice. The selection of practices was also supported by ACMG/GMDI PKU practice guidelines  (7) (8). Table 3.1 provides the list of key practices selected for the analysis in CIMDRN database and the availability of relevant data in CIMDRN database.  Table 3.1 Key practices identified by surveys and further complemented by the analysis of CIMDRN clinical PKU data Practice area related to: Practices assessed by surveys Practices selected for the analysis in CIMDRN database based on chart-abstracted data  Diagnosis  Approaches to classification of PKU severity  Assessing peak Phe levels and corresponding chart-derived severity of PKU  Diagnosis  Experience and opinions on the performance of the neonatal BH4 loading test  Chart-documented performance of the neonatal BH4 loading test  Follow up  Reported treatment targets Patients’ mean blood Phe levels  Follow up  Experiences and opinions on neuropsychological assessments  and its applicability at local centres Data were not available in the medical charts thus virtually absent in the CIMDRN database Follow up  Reported frequency of Phe tests, communications with patients / families Frequency of communication as assessed by frequency of Phe tests and clinic visits  Treatment  Experience and opinion on sapropterin trial  Documented sapropterin trial at any time of the follow up Treatment  Assessment of the responsiveness to sapropterin  Not assessed / information is not available   70  Practice area related to: Practices assessed by surveys Practices selected for the analysis in CIMDRN database based on chart-abstracted data  Treatment  Dietary protein intake / Phe tolerance   Not assessed / information is not available Treatment  Blood Phe levels at treatment initiation  Not assessed / information is not available Treatment  Age at the treatment onset Not assessed / information is not available    3.3.3 Definition of PKU severity  Traditionally, the clinical classification of metabolic phenotype is based on the highest (peak) blood Phe concentration occurring before the initiation of treatment (“untreated” Phe), and is frequently reported as four phenotypic categories (37):  Classic PKU (Phe >1,200 μmol/L), Moderate PKU (900-1,200 μmol/L), Mild PKU (600-900 μmol/L) and Mild HPA (120-600 μmol/L). For the purpose of preserving sufficient sample size, the moderate and mild PKU were combined into “Mild PKU” category thus the above classification was modified to three phenotypic categories:   Classic PKU: (Phe >1,200 μmol/L)   Mild PKU: (>600-1,200 μmol/L)   Mild Hyperphenylalaninemia (HPA): (120-600 μmol/L)  Further, where necessary, to account for small numbers, the mild PKU and mild HPA categories were combined into one category: “Milder PKU”. For the primary analysis, I relied on 71  the chart-reported classification. The highest Phe level during the first month of life was used as an indicator of severity in cases where it was not specified in the medical chart. 3.3.4 Assessment of PKU severity from the perspective of peak Phe levels  Assignment of PKU severity was assessed by describing the range of blood Phe levels associated with each severity classification as indicated in the patient’s chart. The median of individuals’ maximum Phe levels during the first month of life was calculated and presented as the range for each PKU severity category. The modified classification of severity described in the paragraph above and corresponding Phe ranges were used as benchmark: Classic PKU (Phe >1,200 μmol/L), Milder PKU (600-1,200 μmol/L) and Mild HPA (120-600 μmol/L) (37).  The observed peak Phe level was defined as the highest value of blood Phe concentration during the first month of life or the highest Phe level observed at any point during the entire follow up time.   3.3.5 Frequency of Phe monitoring and communication  As discussed in Chapter 2, respondents to the dietitians’ survey reported a potentially declining frequency of communications between healthcare providers and patients/caregivers as children became older.  Thus, the communication patterns between patients / caregivers and metabolic healthcare providers were further assessed by the analysis of the CIMDRN clinical database. Physical clinic visits and telehealth sessions were combined into the category “frequency of communication” as both provide an opportunity for health care team members to meet with the patient and/or parents/caregivers. The frequency of communication between providers and patients and/or patients/caregivers during specified patients’ age groups was assessed during the following age periods: 1st month of life, 1st year of life, 1 to 7 years, and in 72  participants older than 7 years of age. The age categories and corresponding recommended frequency of communication were adapted from ACMG/GMDI recommendations on PKU nutrition management as described in the “Age” paragraph below (8). The following variables were derived from the CIMDRN clinical database:  Phe level monitoring frequency (frequency of any Phe tests: dried blood spot or plasma Phe), based on blood sample collection dates associated with Phe test results as documented in the patient chart;   Frequency of communications with a healthcare provider (either clinic visits or telehealth sessions) based on dates associated with these events.  3.3.6 Assessment of Phe levels and quality of Phe control While the most recent blood Phe levels are crucial for guiding the optimal nutritional management of an individual with PKU, there is much less certainty in the clinical utilization and methods of assessment of long-term exposure to elevated levels of phenylalanine in blood.  In research, the assessment of long-term blood Phe levels is traditionally expressed by central tendency measures during defined periods of time. For example, mean and/or median blood Phe levels are broadly used in research as an indicator of the quality of metabolic (Phe) control and adherence to the diet (106) (131).  Phe fluctuation is also described by various methods. For example: standard deviation (50), standard error of the estimate of the regression of Phe concentration (166), and a proportion of deviation episodes within and outside of the recommended Phe therapeutic range (50) (167). In attempt to identify the most optimal measurement of the quality of Phe control, Hood et al compared different methods to assess “lifetime” Phe concentrations and their association with neurocognitive outcomes (66). The 73  authors assessed Phe exposure with connection to inhibitory control, working memory, and strategic processing in school-age children with early and continuously treated PKU. The indices representing average blood Phe levels and variation of Phe levels included: mean blood Phe levels, the index of dietary control (IDC: the mean of the yearly medians of blood Phe levels), standard deviation (SD), standard error of estimate, % of Phe spikes above the accepted upper limit and a slope reflecting change in Phe as a function of age. The authors reported a correlation between long-term Phe fluctuations and suboptimal neurocognitive outcomes however there was no clear evidence to determine which method of fluctuation assessment is the most optimal as indices of Phe variability were highly correlated with each other. The authors concluded that the use of mean lifetime Phe and SD alone would be sufficient for analyses.   3.3.7 Mean Phe levels and quality of Phe control as intermediate outcomes of PKU treatment  Blood Phe levels remains a key element in the monitoring of PKU treatment outcomes. In this thesis, the term “intermediate outcomes” is used in relation to two indicators related to Phe levels: (a) mean blood Phe levels and (b) quality of Phe control, defined as the proportion of Phe levels within and outside of recommended therapeutic range of 120-360 μmol/L. The terminology “intermediate” is used for these surrogate endpoints because blood Phe levels have been demonstrated to be associated with patient-oriented endpoints including neuropsychological and quality of life outcomes, as described in detail in the Introduction (130) (144) (51).  We looked at blood Phe levels from three different perspectives: (a) “lifetime” mean blood Phe levels by age; (b) blood Phe levels during the first month of life and (c) proportion of 74  Phe levels within and outside of recommended therapeutic range of 120-360 μmol/L defined as “quality of Phe control”.  3.3.7.1 Mean blood Phe levels   Mean “lifetime” blood Phe levels and standard deviations were calculated during the individual’s entire follow up period and where applicable, by age and PKU severity. To account for different frequencies of Phe tests at different age periods and/or severity of PKU, the sample means (SDs) were calculated as mean of individual participants’ mean Phe levels during a specified time period (age group) so as to give each participant contributing to an analysis an equal weight (given that some participants had more individual Phe values in the dataset than others). These mean of mean Phe levels were compared with the recommended therapeutic range of 120-360 μmol/L (7) which was used as a benchmark.   Blood Phe levels during the first month of life were excluded from the lifetime mean Phe analyses due to the significantly higher observed Phe levels compared to the rest of the follow up period (167). Blood Phe levels during the first month of life were analysed separately.   3.3.7.2 Quality of Phe control  The proportion of Phe levels within and outside the recommended therapeutic range (using various thresholds) defines the quality of Phe control in several observational studies. For example, some authors use a 70% benchmark in defining “good” Phe control in individual with PKU (i.e., 70% of Phe values inside the treatment range) (168), (169). The longitudinal retrospective cohort study conducted at the University of British Columbia determined the quality of Phe control as “good” when at least 60% of an individual’s blood Phe levels were 75  within therapeutic range (Appendix C, Table C1), and this approach was applied to the current analysis. Adapting the categorization of Hartnett et al. (167) Phe control was defined dichotomously as follows:   Good Phe control was defined as more than 60% of blood Phe levels within therapeutic range (120-360 μmol/L), among all available blood Phe levels for an individual within the period of time of interest;   Limited Phe control was defined as fewer than or equal to 60% of blood Phe levels within therapeutic range.  A limitation of this categorization of the quality of Phe control is that blood Phe levels that are outside the therapeutic range could be either higher than 360 µmol/L or lower than 120 µmol/L. Depending on the prevalence of “low” or “high” Phe values, individuals with different patterns of “out of range” values could be quite different with respect to diet adherence. An abundance of elevated blood Phe levels indicates a potentially more liberal protein intake (less diet adherence), or other reasons such as acute illness (especially in younger child). On the contrary, lower blood Phe levels could indicate over-restriction of natural protein (or Phe) intake. To address the potential difference between “high Phe” and “low Phe” groups an additional sensitivity analysis was performed to further describe “limited control” group. The main analysis included two mutually exclusive categories (as defined above):   Good control group: >60% of Phe levels are within the 120-360 µmol/L range;   Limited control: <=60% of blood Phe levels are within the 120-360 µmol/L.   Finally, the “limited control” group was further divided by three sub-groups: Limited control group: proportion of individuals with <=60% of blood Phe levels are within the 120-360 µmol/L range and out of those: 76  o Limited control, high Phe: individuals with >60% of their out of range blood Phe levels being >360 µmol/L. This group of individuals potentially has a tendency to be less adherent to the diet; o Limited control, low Phe: individuals with >60% of their out of range blood Phe levels being <120 µmol/L. This group of individuals potentially has a tendency to over-restrict natural protein; o Limited control, variable Phe: individuals with <=60% of out range levels that are above 360 µmol/L or below 120 µmol/L. This group of individuals has a combination of high and low blood Phe levels and thus have variable control.  3.3.8 Age  Age categories were adapted from the current GMDI guidelines on nutritional management of PAH deficiency where age groups were defined as follows: 0-1 year; 1-7 years; 8-18 years  (8). To better address the research questions of this study, the original categories were modified:  0-1 year was separated to 0-1 month and >1-12 months, the category of >1-7 years remained the same and the category 8-18 years was transformed to >7 years given that children in the CIMDRN cohort were followed to a maximum age of 11 years.  The same participants could contribute data to multiple age categories as the data were longitudinal, with repeated measurements in the same children over time.     3.4 Statistical Analysis Descriptive statistics were used to describe sample characteristics. The central tendency and variability measurements, mean, mean of means, and standard deviation or median and range 77  were used to describe exposure to Phe and the fluctuation of blood Phe levels. Frequency of communication(s) was calculated as a rate of communication(s) per year as participants contributed various years of follow up in different age categories. Average frequency of communication was calculated using the formula: Rate (frequency) of communication (per year) within specified age group equals a total count of events (e.g. Phe tests) within the age category divided by sum of individually contributed years of follow up. Mean levels of phenylalanine by age were presented graphically using boxplots. If an individual had more than one Phe measurement recorded during the defined time period, individual’s mean Phe level was calculated with subsequent computation of mean of individual means; these were plotted in the boxplots. The horizontal line within each box represented the median of the individual means and the diamond shape within each box represented the mean of means.  In reporting descriptive results, characteristics that summed to fewer than 5 participants had to be combined or suppressed due to confidentiality issues. Due to small numbers, only centres with 20 or more PKU participants were reported separately, where applicable.   3.5 Results 3.5.1 Sample characteristics Complete diagnostic data were available for 215 participants who were diagnosed with PKU. Of this group, 109 (51%) were males and 106 (49%) were females. Due to the longitudinal nature of the collected data, the same participants contributed various total numbers of follow up years. Out of 12 treating centres, three reported at least 20 participants with PKU: Hospital for Sick Children (66, 31%), BC Children’s Hospital (36, 17%) and McMaster Children's Hospital - Hamilton Health Sciences (20, 9%). The majority of CIMDRN PKU participants were diagnosed 78  with classic PKU (92, 43%), followed by mild hyperphenylalaninemia (85, 40%) and mild to moderate PKU (25, 12%), based on the notation present in the child’s medical chart. PKU severity was not specified in the medical charts in 13 (6%) cases. The vast majority of cases were identified via PKU newborn screening. Seven PKU cases identified outside of NBS were either diagnosed directly because of older siblings with PKU or born outside of Canada. Slightly more than one third of newborns with PKU (35%) were administered BH4 loading tests during the first three weeks of life. Less than one third (26%) were documented to have received sapropterin trial at any point of the follow up and genetic testing results were available in the chart for half of the participants (Table 3.2).   Table 3.2 Characteristics of PKU cohort with complete minimum data (n=215).  n % Sex   Male 109 51 Female 106 49 Year of birth   2006 22 10 2007 21 10 2008 14 7 2009 20 9 2010 18 8 2011 18 8 2012 26 12 2013 22 10 2014 27 13 2015  27 13 Treatment centre at time of consenta   BC Children's Hospital (Vancouver) 36 17 Alberta Children's Hospital (Calgary) 11 5 Stollery Children's Hospital (Edmonton) 12 6 Children's Hospital - Health Science Centre Winnipeg 11 5 Children's Hospital - London Health Sciences Centre 16 7 McMaster Children's Hospital - Hamilton Health Sciences 20 9 79   n % Hospital for Sick Children (Toronto) 66 31 Children's Hospital of Eastern Ontario (Ottawa) 14 7 L'Hopital de Montreal pour enfants du Centre universitaire de sante McGill 11 5 Izaak Walton Killam Health Centre (Halifax) 9 4 Chart-reported disease classification   Classic PKU 92 43 Mild PKU 25 12 Mild Hyperphenylalaninemia (HPA) 85 40 Not specifiedb  13 6 Identified by newborn screening (NBS) (n=214)c 207 97 BH4 loading test reported during the first 3 weeks of life 74 35 Molecular genetic testing results in chart during follow up  107 51 Sapropterin treatment and/or trial documented in chart at any point during follow up  56 26 a Centres with reported <5 cases: Kingston General Hospital and Le Centre hospitalier universitaire Sherbrooke. bSeverity of PKU was not recorded in the patient’s chart.  c Ascertainment missing for one participant as the diagnosis was ascertained at a non-CIMDRN centre.     3.5.2 Diagnosis of metabolic phenotype in treating centres  Almost one third (30%) of children who were assigned, according to the chart, as having  classic PKU never reached the diagnostic Phe level of classic PKU set at 1,200 µmol/L (37) at any point of the entire follow up (Table 3.3).  Table 3.3 Maximum (peak) blood Phe levels at any point in time among children diagnosed with classic PKU as reported in their medical chart (n=92). Treatment centre at time of consent Children diagnosed with classic PKU as reported in their medical charts Children diagnosed with classic PKU and a “lifetime” maximum Phenylalanine level < 1,200 µmol/La All centres 92/215 (43%) 28/92 (30%) Centre-specific resultsb  Sick Kids 22/66 (33%) 6/22 (27%) BCCH 19/36 (53%) 6/19 (32%) McMaster 9/20 (45%) <5 80  a NBS phenylalanine levels are included in the analysis bOnly includes centres with >=20 participants diagnosed with classic PKU   Assignment of PKU severity was further assessed by describing the range of blood Phe levels associated with each severity classification as indicated in the patient’s chart (Table 3.4). The median of individuals’ maximum Phe levels during the first month of life among those assigned as having classic PKU was 1,620 µmol/L, with a range from 443 µmol/L to 2,950 µmol/L. Beyond the first month, the median of individuals’ maximum blood Phe levels during the entire follow up among those classified with classic PKU decreased to 828 µmol/L.  Among participants classified as having mild PKU, the median of individuals’ maximum blood Phe levels was 705 µmol/L during the first month of life with a corresponding upper peak Phe level of 1,380 µmol/L. This declined to a median of maximums of 660 µmol/L beyond the first month of life. Among participants with mild HPA, the median of maximum Phe values was 224 µmol/L during the first month and 338 µmol/L beyond the first month of life, with a wide range among individuals. For some study participants, data extractors were not able to identify a “chart-assigned” severity classification. Among these participants, the median maximum Phe value was 812 µmol/L (upper 1,888 µmol/L) during the first month with a decrease to 638 µmol/L (upper 1,570 µmol/L) beyond the first month of life (Table 3.4).  Table 3.4 Median of individuals’ maximum (peak) blood Phe levelsa during the first month of life (28 days) and after the first of month of life by PKU severity. PKU severity as reported in the medical chart n Median (min-max) of individuals’ peak Phe level during the first month of life (μmol/L)  n=202b n  Median (min-max) of individual peak blood Phe levels during follow-up after the first month of life (μmol/L) n=214c Classic PKU  87 1,620 (443-2,950) 91  828 (378-1,944) Mild PKU  25 705 (171-1,380) 25  660 (314-1,212) 81  PKU severity as reported in the medical chart n Median (min-max) of individuals’ peak Phe level during the first month of life (μmol/L)  n=202b n  Median (min-max) of individual peak blood Phe levels during follow-up after the first month of life (μmol/L) n=214c Mild HPA  78 224 (132-1,036) 85  338 (108-1,180) Not specified  12 812 (332-1,888) 13  638 (479-1,570) aPhe levels do not include NBS Phe level. b13 cases are missing Phe in the first month of life (moving centres, information was not transferred with the charts. cOne case has incomplete longitudinal data collection.   The extracted data on criteria used to define the severity of PKU diagnoses showed a considerable variation in classification of the PKU metabolic phenotypes. The chart-derived criteria to determine the severity of PAH deficiency used in Canadian CIMDRN-participating treating centres are shown in Table C2, Appendix C.   3.5.3 Frequency of communication  As expected, the frequency of blood Phe tests and clinic visits declined with increasing age. Frequency of blood Phe tests during the first month of life aligned with the recommended frequency of Phe blood tests based on a recent nutrition guideline from GMDI (8). Frequency of Phe monitoring tests met or exceeded GMDI-recommended norms for classic PKU, but frequency of Phe monitoring tests for milder PKU was much lower.    Overall, patients with milder forms of PKU (including mild PKU, moderate PKU and mild HPA) experienced considerably fewer communications with healthcare providers when descriptively compared to patients with classic PKU.  The lower frequency of communication was consistent across all age groups of patients with milder PKU (Table 3.5).  82  Table 3.5 Average Frequency (rate)a of PKU monitoring and communication by the severity of PKU and age. PKU severity Frequency (rate) of Phe tests per month  Frequency (rate) of clinic visits per monthb  0-1 month >1-12 months >1-7 years >7 years 0-1 month >1-12 months >1-7 years >7 years GMDI guidelinec 4-8 per month 4-8 per month 1-4 per month 1-4 per month 1-4 per month 1-4 per month 0.16-1 per month 0.08-0.16 per month Overall 5.81 (n=185) 2.79 (n=185) 1.34 (n=181) 0.80 (n=59) 1.19 (n=185) 0.38 (n=185) 0.14 (n=181) 0.13 (n=59) Classic PKU 8.98 (n=82) 4.40 (n=82) 1.90  (n=82) 1.07 (n=33) 1.59 (n=82) 0.54  (n=82) 0.17 (n=82) 0.15 (n=33) Milder PKU d 3.28 (n=103) 1.47 (n=103) 0.77  (n=99) 0.32 (n=26) 0.88 (n=103) 0.26 (n=103) 0.11 (n=99) 0.09 (n=26) a Average frequency of communication was calculated using the formula: Rate (frequency) of communication (per year) within specified age group equals a total count of events (e.g. Phe tests) within the age category divided by sum of individually contributed years of follow up. b Clinic visits include: physical clinic visits and telehealth sessions (diagnosis acquisition visits were excluded from the analysis)  c GMDI (Genetic Metabolic Dietitians International) -recommended frequencies of monitoring and communication were adapted from Singh et al, 2014 (8) d“Milder” forms of PKU include: mild and moderate PKU and mild HPA (as derived from medical charts)  There was considerable variation in the observed frequency of communications between treating centres. For example, frequency of Phe monitoring tests for children with classic PKU ranged from 5 times per month to 16 times per month in newborns. Variation was also observed in the frequency of clinic visits, ranging from roughly once a month to once a week in newborns.  Between-centre variation in both frequency of monitoring Phe and clinical visits were observed in all age groups. The majority of the results for children older than 7 years could not be reported by centre due to small numbers. The same tendency of between-centre variation in monitoring and visit frequency was observed in children with milder forms of PKU (Appendix C, Tables C3 and C4).   83  3.5.4 Intermediate treatment outcomes 3.5.4.1 Phenylalanine levels beyond neonatal period As demonstrated by Figures 3.1 - 3.3, levels of blood Phe and/or Phe fluctuation were considerably higher during the first month for all PKU types. Therefore, they were analyzed separately. The boxplots show the progression of mean blood Phe levels throughout five age periods. After the first month of life where the observed blood Phe levels were the highest, mean blood Phe levels overall remained within recommended therapeutic range for the vast majority of patients with classic PKU. However, variation in mean blood Phe levels among different children was substantial throughout childhood. While within the recommended therapeutic range, a slight increase of mean blood Phe levels was observed in the group older than 7 years (Figure 3.1). In patients diagnosed with mild PKU, the mean of individual mean blood Phe levels during the first month of life was lower compared to patients with severe PKU, although slightly above the upper level of recommended treatment range. After the first month, mean blood Phe levels declined so that mean levels for all children were within the recommended range of 120-360 µmol/L and subsequently remained within recommended range for nearly all groups throughout early childhood, slightly rising in the group of older children. The greatest variation of blood Phe among this group was observed during the first month of life and in children >7 years old (Figure 3.2). In the cohort of patients with mild hyperphenylalaninemia (HPA), initial mean blood Phe levels were within therapeutic range and remained within the recommended norms throughout early childhood. Phe levels among participants with mild HPA showed low variability (Figure 3.3). 84    Figure 3.1 Distribution of mean blood Phe levels in classic PKU by age.          Figure 3.2 Distribution of mean blood Phe levels in mild PKU by age. 85   Figure 3.3 Distribution of mean blood Phe levels in mild HPA by age.    During the first month of life the highest mean blood Phe levels (mean of individual means, hereafter “mean”) were observed in both classic and milder forms of PKU, though less so in the mild HPA group. Beyond the first month of life, observed mean blood Phe levels were within the guideline-recommended therapeutic range of 120-360 µmol/L in all age groups and 86  PKU severities. A slight increase in mean blood Phe levels and higher variation in mean Phe across the sample was observed in children older than 7 years, indicating some relaxation of the diet (Table 3.6).  Table 3.6 Meana (SD) of age-specific blood Phe levels by PKU severity..  Age group Meana (SD) blood Phe levels (µmol/L)b, n Classic PKUc Mild PKU/Mild HPAc 0-1 month 763 (393), n=88 272 (120), n=109 >1-12 month 241 (63), n=84 217 (53), n=101 >1-7 years 266 (90), n=90 242 (72), n=102 >7 years 331 (151), n=37 260 (112), n=24 a Mean of individual participants’ mean Phe levels, bNBS Phe levels were included in this analysis; c Diagnosis of severity is based on chart-derived PKU severity classification   When analyzed by treating centres, the overall “lifetime” (until a maximum age of 11 years in this cohort) mean of individuals’ blood Phe means excluding the first month of life were within recommended therapeutic range across centres and PKU severity diagnoses. The lifetime mean blood Phe levels in children with classic PKU ranged from 221 µmol/L in the treating Centre C to 332 µmol/L in Centre D. In milder forms of PKU, the mean blood Phe levels ranged from 189 µmol/L at Centre B to 284 µmol/L at Centre I. The observed standard deviations of the lifetime Phe means was ranging from 26 to 98 for classic PKU and from 32 to 81 for milder forms of PKU (Table 3.7).  Table 3.7 Lifetime meana (SD) of blood Phe levelsb by severity of PKU and by treating centrec, Treating centre at time of consent Meana (SD), Phe (µmol/L) Classic PKU Mild PKU/ Mild HPA  Overall 262 (73), n=91 228 (61), n=110 Centre A 254 (98), n=22 220 (65), n=44 Centre B 235 (34), n=19 189 (32), n=10 Centre C 221 (37), n=5 267 (40), n=6 87  Treating centre at time of consent Meana (SD), Phe (µmol/L) Classic PKU Mild PKU/ Mild HPA  Centre D 332 (93), n=8 <5 Centre E 261 (31), n=7 235 (50), n=9 Centre F 245 (48), n=9 201 (41), n=11 Centre G 243 (26), n=7 247 (75), n=7 Centre I <5 284 (81), n=8 Centre H <5 245 (54), n=7 a Mean of individuals’ mean Phe levels. bPhe levels during the first month of life were excluded from the analysis). cOnly reported cells with 5 or more observations.   3.5.4.2 Quality of Phe control during the first month of life Blood Phe levels during the first month of life were given special consideration. This is the time of diagnosis, diet adjustment, and one of the crucial periods in childhood development. Overall, the first month of life was characterized by rather poorer quality of Phe control in newborn children with classic PKU, as limited proportion of Phe levels were within recommended range, as observed in the majority of newborns during this period (Table 3.8; Figure 3.4). During the first week of life, only one quarter of children with classic PKU had good Phe control, defined as having >60% of their blood Phe levels within therapeutic range. This proportion decreased to 10% during the second week of life, slowly improving towards the end of the first month. At the end of the first month about one third of all newborn children with classic PKU attained “good” Phe control.  A similar trend of decreasing quality of Phe control during the second week of life was observed in newborns with mild PKU. Slightly more than one third had the majority of their blood Phe levels within therapeutic range at the first week of life. Phe control subsequently worsened at the second week and improved at the end of the first month of life with 65% of all newborns achieving “good” Phe control. A slightly different trend 88  was observed in the “mild HPA” group. Almost all newborns with mild HPA had the majority of their blood Phe levels within therapeutic range at “week one” with a slight decline in quality of Phe control during subsequent weeks. At the end of the month fewer newborns had “good” Phe control compare to the first week (83% vs 94% respectively) (Table 3.8); the data are graphically presented in Figure 3.4.   Table 3.8 Proportion of newborns with more than 60% of their Phe levels within therapeutic range 120-360μmol/L during the first four weeks of life. PKU severity Proportion of infants n (%) attaining therapeutic Phe rangea  Week 1 (age 0-6 days) Week 2 (age 7-13 days) Week 3 (age 14-20 days) Week 4 (age 21-27 days) Classic PKU  21/84 (25%) 8/82 (10%) 15/75 (20%) 22/69 (32%) Mild PKU  8/25 (32%) 6/22 (27%) 7/18 (39%) 13/20 (65%) Mild HPA 77/84 (92%) 49/58 (84%) 25/36 (69%) 30/36 (83%) a Therapeutic range is 120-360μmol/L (7); newborn screening blood Phe levels included in the analysis.   Figure 3.4 Proportion (%) of newborns with more than 60% of their Phe levels within treatment range 120-360μmol/L during the first four weeks of life by PKU severity.   89  3.5.4.3 Quality of Phe control beyond neonatal period Beyond the first month of life, at most ages, more than half (54%) of children with classic PKU had “limited” Phe control, including either higher or lower than acceptable levels of Phe (Table 3.9 and Figure 3.5); note that in this longitudinal dataset, many children contributed data to multiple age categories as they aged through the follow-up period). Between the first and sixth month of life, 46% of participants with classic PKU had more than 60% of their blood Phe levels in the acceptable range. From 6-12-months of age, the quality of Phe control somewhat declined among children with classic PKU, with 36% considered to have good control.  After one year of age, the proportion of children with more than 60% of their Phe test results within treatment range steadily increased to approximately 5 years of age. Around 5 years, the proportion of children with more than 60% of their blood Phe levels within treatment range started to steadily decline from 55% at 4-5 years old to 28% at 8-9 years old (Table 3.9).  The results are graphically presented in Figure 3.5.   Not surprisingly, infants with milder forms of PKU, including mild PKU and mild HPA, experienced more than 60% of their Phe levels within the recommended therapeutic range throughout childhood. However, there was a drop after 7 years of age (Table 3.9; Figure 3.5). In participants at 6-7 years old, 76% of children had more than 60% of their Phe tests within therapeutic range while at 7-8 years old only slightly more than half (56%) had 60% or more of their blood Phe levels within recommended therapeutic range. There were too few children with data at ages >8 years with milder forms of PKU to report on these age groups.  90  Table 3.9 Proportion (%) of children with “good” and “limited” quality of Phe control in different age groups by PKU severitya.     Age Classic PKU Milder forms of PKUb Good Phe controlc  n(%) Limited Phe controld n(%) Good Phe controlc  n(%) Limited Phe  controld  n(%) >1-6 months 39/84 (46%) 45/84 (54%) 84/97 (87%) 13/97 (13%) >6-12 months 30/84 (36%) 54/84 (64%) 69/85 (81%) 16/85 (19%) >1-2 years 35/86 (41%) 51/86 (59%) 75/95 (79%) 20/95 (21%) >2-3 years 37/80 (46%) 43/80 (54%) 52/72 (72%) 20/72 (28%) >3-4 years 31/67 (46%) 36/67 (54%) 43/59 (73%) 16/59 (27%) >4-5 years 34/62 (55%) 28/62 (45%) 36/49 (73%) 13/49 (27%) >5-6 years 23/52 (44%) 29/52 (56%) 21/29 (72%) 8/29 (28%) >6-7 years 14/47 (30%) 33/47 (70%) 19/25 (76%) 6/25 (24%) >7-8 years 13/36 (36%) 23/36 (64%) 10/18 (56%) 8/18 (44%) >8-9 years 8/29 (28%) 21/29 (72%) - <5 >9 years 9/24 (38%) 15/24 (62%) - <5 a Diagnosis of severity is based on chart-derived PKU severity classification; bMild PKU and Mild HPA were combined into “Milder forms of PKU”. cGood Phe control is defined as having Phe levels within 120-360 µmol/L in > 60% of all of a child’s test results. d Limited Phe control is defined as having Phe levels within 120-360 µmol/L in ≤60% of all of a child’s test results.  Figure 3.5 Proportion (%) of children with “good” Phe control a beyond the first month of life by PKU severity and age category.  a Good Phe control is defined as having Phe levels within 120-360 µmol/L in > 60% of all of a child’s test results.  91  3.5.5 Sensitivity analysis of the quality of Phe control utilizing a different definition of the quality of Phe control among children with classic PKU.  As previously discussed in the “methods”, the definition of good metabolic control, while established in the literature, may not be ideal. Good metabolic control was defined as having greater than 60% of all blood Phe levels within therapeutic range of 120-360 µmol/L. Among those considered to have “limited control”, this could reflect blood Phe levels either above or below the recommended therapeutic range.  Patients who tend to have lower blood Phe levels and those with a tendency to have higher blood Phe levels may be different with regard to diet adherence or other diet-related or health factors. Thus, a sensitivity analysis was conducted to investigate these groups separately.   Within groups of children with classic PKU who were considered to have “limited” control the higher proportion of children having blood Phe levels below therapeutic range (<120 μmol/L) was at a younger age, then the proportion of children with blood Phe levels above therapeutic range (>360μmol/L) increased as early as at 2-3 years of age group and was steadily increasing with increasing age (Table 3.10 and Figure 3.6). The milder PKU group did not have a sufficient sample size to analyze the trend of limited Phe quality as the majority of children had good Phe control (data not shown).  Table 3.10 The proportion of children with classic PKU having various quality of Phe control given as percent of Phe levels within and outside therapeutic range (120-360 μmol/L) by age.   Age Proportion of children n(%) with “good” Phe controla   Proportion of children with “limited high” Phe control  Proportion of children n (%) with “limited low” Phe control Proportion of children n (%)  with “limited varying” Phe control >1-6 months 39 (46%) 12 (14%) 14(17%) 19 (23%) >6-12 months 30 (36%) 14 (17%) 27 (32%) 13 (15%) 92   Age Proportion of children n(%) with “good” Phe controla   Proportion of children with “limited high” Phe control  Proportion of children n (%) with “limited low” Phe control Proportion of children n (%)  with “limited varying” Phe control >1-2 years 35 (41%) 12 (14%) 22 (26%) 17 (20%) >2-3 years 37 (46%) 17 (21%) 11 (14%) 15 (19%) >3-4 years 31 (46%) 21 (31%) 8c(12%) 7 (10%) >4-5 years 34 (55%) 15 (24%) - <5 >5-6 years 23 (44%) 14 (27%) 10 (19%) 5 (10%) >6-7 years 14 (30%) 19 (40%) 7 (15%) 7 (15%) >7-8 years 13 (36%) 13 (36%) - <5 >8-9 years 8 (28%) 12 (41%) - <5 >9years 9 (38%) 11 (46%) <5 <5 aPhe control defined as:  Good Phe control: >60% of levels where within the therapeutic range (120-360 μmol/L).   Limited high: <=60% of levels were within the therapeutic range, and of those levels outside of the therapeutic range, >60% of those levels were above 360 μmol/L.   Limited low: <=60% of levels were within the therapeutic range, and of those levels outside of the therapeutic range, >60% of those levels were below 120 μmol/L.   Limited varying: <=60% of levels were within the therapeutic range, and of those levels outside of the therapeutic range, <=60% of those levels were above 360 μmol/L and <=60% of those levels were below 120 μmol/L.  Figure 3.6 The distribution (%) of children with classic PKU with “limited low”a and “limited high”b quality of Phe control by age.   a Limited low: <=60% of levels were within the therapeutic range, and of those levels outside of the therapeutic range, >60% of those levels were below 120 μmol/L.  b Limited high: <=60% of levels were within the therapeutic range, and of those levels outside of the therapeutic range, >60% of those levels were above 360 μmol/L.    93  3.6 Discussion This study is the first comprehensive analysis of longitudinal clinical data for children with PKU across multiple Canadian treatment centres. The findings of this study corroborate findings of the surveys of Canadian healthcare providers described in Chapter 2 and previously conducted studies with regards to variability in key nutritional and clinical practices (161) (14). This study also shows that while the mean Phe levels of children beyond the first month of life are within the therapeutic range of 120-360 μmol/L, the quality of Phe control associated with existing practices is not ideal.  Corroborating the results of dietitians’ and physicians’ surveys described in Chapter 2, this study found variation in diagnostic and follow up practices such as classification of PKU severity, neonatal BH4 loading test performance, neuropsychological follow up, and treatment with sapropterin. For example, 47% of physicians reported perfoming neonatal BH4 loading tests and nearly same number (35%) of newborns were administered BH4 loading tests during the first three weeks of life. 35% of physicians reported referring all patients to a psychologist for a formal neuropsychological assessment while the data on neuropsycological assessments was virtually absent in the chart-abstracted database. Finally, while 35% of physicians who responded to a survey, reported offering sapropterin treatment to nearly all patients, less than one third of patients (26%) were documented to have received sapropterin trial at any point of the follow up.  In contrast to the survey findings, the frequency of communication between clinics and families was within recommended norms, particularly for classic PKU, while communication with the families of children considered to have milder PKU was less frequent.  94  While criteria for defining the severity of PKU varied considerably across centres according to survey results, we could not be sure from the analysis of CIDRN data what proportion of children have their severity “overdiagnosed” or “underdiagnosed”. Discrepancies between individual peak levels and diagnoses of severity were detected and may reflect multiple factors related to diagnostic criteria and disease management. For example, we found that one third of children with classic PKU did not reach the “diagnostic” blood Phe levels of “classic PKU”, defined as >1,200 μmol/L. While the majority of these children most likely have true classic PKU (severe PAH deficiency) with well-controlled Phe levels that never reached a high peak due to very early and continuous management, this cohort might also include children with milder forms of PKU who were “overdiagnosed” as having severe disease. Such “overdiagnosis” of severity might lead to unintentional and unnecessary over-restriction of the natural protein (84).  A neonatal BH4 loading test was performed only in one third of the cohort, and a trial of sapropterin was received at any point of care by only one fourth of the study population. There was very limited data in the medical charts on neuropsychological assessments, likely highlighting inadequate neuropsychological follow up which was also identified in the survey of metabolic physicians. In addition, inadequate charting procedures that do not always incorporate neuropsychological test results into the metabolic clinic chart were identified by CIMDRN team (153).Other practices, such as the patient’s age at the initiation of treatment, the actual Phe levels triggering the dietary treatment initiation, and Phe tolerance could not be assessed as the data describing these practices were not available in the clinical database (153). Intermediate outcomes were represented by blood Phe levels compared to the guideline-recommended therapeutic range of 120-360 μmol/L and the proportion of the Phe levels within 95  and outside of the therapeutic range. While mean Phe levels beyond the first month of life remained within the therapeutic range of 120-360 μmol/L, the levels increased from 241 μmol/L at 1 year of age to 331 μmol/L in children older than 7 years. Data on children 11-18 years of age were not available given the eligibility criteria for the cohort study (birth between 2006 and 2015), presenting another limitation of this study; we could not compare the data of the dietitians’ survey that reported possible relaxation of the diet in this adolescent group. The analysis revealed that in children with classic PKU, a large proportion of all blood Phe levels fall outside of therapeutic range during the neonatal period. At the end of the first month of life only one third of children with classic PKU achieve “good” metabolic control as defined by more than 60% of levels within recommended range (Table 3.7). Beyond the first month of life the quality of Phe control improves, but is not ideal and tends to decline with age although lifetime mean blood Phe levels are maintained within accepted Phe range. Similarly, a decreasing quality of Phe control with increasing age was observed in patients with milder forms of PKU. Declining adherence to the PKU diet with increasing age is well known and was reported in some Canadian centres and elsewhere (161) (103). This might contribute to the development of suboptimal neuropsychiatric outcomes (167) (170) (171). In conclusion, while Canadian children with PKU receive care aligning with recommended practices, the key nutritional and clinical practices of PKU and related quality of Phe control remain less than ideal.  This may contribute to the development of suboptimal neurocognitive outcomes as blood Phe concentration during childhood is the major determinant of these outcomes (172) (51) (48). Ideally, I would like to investigate neurocognitive outcomes as these are one of the main PKU endpoint outcomes frequently compromised in early and continuously treated patients (12) . Absence of data on neuropsychological assessments made 96  this objective impractical, as well as is a limitation of this study. However, another endpoint outcome of equal importance was assessed: the quality of life of caregivers who provide care for children with PKU and parent-reported child’s health. This was explored using the PKU-QoL questionnaire and is described in the next chapter.  97  Chapter 4: The quality of life of parents who care for children with PKU in Canada 4.1 Synopsis Background: Parents/caregivers play a central role in caring for children with PKU. Daily PKU management is challenging, and the pressure to achieve good treatment outcomes demands responsibility and discipline potentially creating a stressful environment. Evidence on health-related parental quality of life (HRQoL) for families of children with PKU is limited generally and there are no Canadian studies of HRQoL in this population.  This study explored the impact of PKU on parents’ QoL in association with a child’s metabolic control. The study had two main objectives: (1) to describe the health-related QoL of the parents/caregivers of children with PKU, and (2) to explore whether quality of metabolic control was associated with the overall impact of PKU and parent-reported overall child’s health status.  Methods: Parents of children 12 years of age or younger with PKU who were participating in a Canadian cohort study were invited to complete a 54-item parent version of the Phenylketonuria Impact and Treatment QoL Questionnaire (PKU-QoL©). The PKU-QoL questionnaire includes information on the emotional, practical, social and overall impact of PKU on parents, parental perceptions of child health status and symptoms, dietary restrictions, and administration of Phe-free medical formulas. Scores ≤25 indicated little or no impact of PKU or its management on parental well-being, >25 and ≤50 indicated a moderate impact (or moderate symptoms), >50 and ≤75 indicated major impact (or severe symptoms) and scores above 75 indicated severe impact (or very severe symptoms). The median and interquartile range were used to report domain scores.   With parental consent, we linked survey findings with existing clinical data from children in this cohort (the Canadian Inherited Metabolic Diseases Research Network, CIMDRN cohort) 98  by study ID. From the clinical data, which was abstracted longitudinally from metabolic charts at centres participating in the cohort study, the quality of metabolic control was calculated as the proportion of blood Phe levels within the guideline-recommended range and mean blood Phe levels during the two years preceding the survey. The data were analyzed descriptively, reporting proportions for categorical variables and medians with interquartile ranges (IQR) for continuous variables. We used univariable and multivariable linear regression models to investigate the relationship between the quality of Phe control and mean blood Phe levels with the overall impact of PKU on parental HRQoL and parent-reported child’s health status from the survey. Results: Out of the 127 mailed questionnaires, 87 were returned; from those, 16 indicated that the patients were not treated with diet and thus were ineligible for the survey. Thus the overall response rate was 71/111 (64 %).  With respect to child health status and symptoms, PKU-related symptoms such as tiredness, lack of concentration, irritability, and mood swings, were reported by parents as borderline severe overall (median (IQR) score 50(25-75)). Reported guilt due to poor adherence to the diet and supplements was reported as having the most severe impact on parents (median (IQR) 75 (50-100)). The impact of children’s anxiety regarding blood tests on parental well-being was severe (median (IQR) 63 (63-88)). The practical impact of dietary protein restriction was also reported as having a major impact on the everyday life of parents of children with PKU (median (IQR) 54(36-71)).  The quality of metabolic control was not associated with the overall impact of PKU on parental QoL, nor on parental assessments of overall child health status.   Conclusion: Parental guilt due to dietary non-adherence, the impact of child’s anxiety and the practical impact of dietary restrictions on Canadian parents were the most important impacts based on the results from the HRQoL survey. More family-focused strategies of care 99  should be implemented to ameliorate the chronic long-term stress on Canadian families living with children with PKU.   4.2 Introduction Health-related QoL (HRQoL) is identified as one of the most important patient- and family-oriented outcomes in chronic and complex pediatric diseases (136). In spite of this, research on HRQoL of parents/caregivers (hereafter “parents”) of children with PKU is sparse and virtually non-existent in Canada. PKU requires strict adherence to a lifelong challenging diet prescription that is very low in Phe, particularly in early childhood (75). Parenting a child with PKU thus requires constant close supervision and monitoring of a child’s nutritional intake, frequent clinical appointments, and regular blood tests all of which place significant demands on parents (173).  It is not uncommon that parents of children with PKU report significant stress related to the stringent dietary requirements and uncertainty regarding the child’s future (174). One German cross-sectional study assessed HRQoL of parents of children with PKU based on the 29-item Ulm QoL Inventory for Parents of chronically ill children, and demonstrated elevated stress and challenges with self-development (144). Another study assessed children- and parent-reported HRQoL with the Revised Children's QoL Questionnaire (KINDL-R). While the study identified normal overall scores of children’s collective HRQoL, parents were concerned about everyday functioning of the child such as completion of school homework and enjoyment of school, and they expressed fears of poor grades for schoolwork especially when Phe concentrations in their children were above the therapeutic range (143). Financial challenges due to PKU care have been also reported. For example, in one cross sectional survey conducted in the UK, parents reported spending as many as 20 hours weekly (on average) to manage a child’s 100  PKU and 23% of caregivers reported discontinuing work in order to accommodate their child’s care (142) (175).    Common limitations of existing studies include relatively small sample sizes and use of generic QoL questionnaires designed for chronic illnesses, which may not capture the entire spectrum of PKU care (141).  In studies using a recently developed PKU-specific HRQoL questionnaire for parents, authors have reported: significant emotional impacts of caregiving, guilt due to non-adherence to the recommended dietary restrictions, anxiety about high blood Phe levels, and negative impacts related to both children’s anxiety about blood tests and the practical challenges associated with dietary limitations (141) (176).   HRQoL of parents of children with PKU has not been previously studied in Canada. A retrospective cohort study of Canadian children and their families conducted by the Canadian Inherited Metabolic Research Network (the CIMDRN cohort) offered the opportunity to investigate the HRQoL of parents who care for children with PKU. Clinical data regarding metabolic control (blood Phe levels over time) that were collected in the cohort study were linkable to the QoL data collected for the present study. The objectives of this project were: (1) to describe the HRQoL of the parents/caregivers of children with PKU; and (2) to explore whether quality of metabolic control for children with PKU (based on blood Phe levels) was associated with the overall impact of PKU and overall child’s health status as reported by parents.   4.3 Methods We used a mailed survey to implement the parent version of the PKU-specific HRQoL questionnaire (PKU-QoL©) (177). Eligible participants were parents of children with PKU 101  enrolled in the CIMDRN cohort who had agreed to be contacted to participate in survey research. Children in the PKU cohort were born between 2006 and 2015, had a confirmed diagnosis of PKU, and had received care at one of 13 participating treatment centres.   Adapting Dillman’s tailored approach to survey implementation (155), up to 5 contacts were made by mail to invite eligible parents to participate in the study: a pre-notification letter, an invitation mailed 2 days later with a copy of the questionnaire and a token incentive ($2 coffee shop gift card) (156), a first reminder to non-responders sent 2 weeks later with a replacement questionnaire, and then a second and final reminder 2 weeks later with a replacement questionnaire. Parents could opt to complete the questionnaire in English or French. Return of the questionnaire by mail (using envelopes with pre-paid postage provided with each mail-out) was considered consent to participate. Parents were invited to participate between December 2016 and April 2018, depending on the timing of site-specific Research Ethics Board approvals, which were obtained at all participating centres.   4.3.1 Health-related QoL measures: Parent health-related quality of life (HRQoL) was assessed using the 54-item parent version of the Phenylketonuria Impact and Treatment Quality of Life Questionnaire (PKU-QOL)  (177). The impact of PKU was assessed across 30 domains compiled into four modules: (1) child’s PKU-related symptoms (reported by parent), comprised of 11 single-item domains including parent’s reports of their child’s health status, headaches, stomach aches, tiredness, lack of concentration, slow thinking, irritability, aggressiveness, moodiness, sadness, anxiety (2) the “PKU in general” module consists of 9 “impact” domains that measure the impact of managing the child’s PKU on parental well-being, including the practical, social, emotional, financial and 102  overall impact of PKU. This module also measures parents’ perceptions of the child’s anxiety due to blood tests, parental anxiety due to potential high blood Phe levels, and associated impacts on parents; (3) the “Supplement administration” module includes 5 domains related to adherence to Phe-free protein supplements (medical foods), guilt due to poor adherence to Phe-free protein supplements, and the impact of Phe-free protein supplements on daily life and family; and (4)  the “Dietary protein restriction” module includes 5 domains related to adherence to dietary protein restrictions, overall difficulty following dietary restrictions, guilt if the diet is not followed, social impact of the diet, practical impact of the diet, and parental perceptions of the child’s food enjoyment. For each domain, the percentage of items completed within the domain was calculated and the domains with scores of at least 70% were analyzed (178). The Parent PKU-QoL questionnaire was finalized and validated in a European multicenter, prospective, non-interventional, observational study including 253 parents (177). The concurrent validity of the Parent PKU-QoL was assessed by the tool’s authors by evaluating the correlations between Parent PKU-QoL scores and Child Health Questionnaire–Parent Form 28 (CHQ-PF28) (179) using Spearman correlation coefficients(177) with the highest correlation coefficients observed between scores assessing  close concepts. Internal consistency was estimated by the tool developers using Cronbach’s alpha and was good for most multi-item domain scores in their sample (emotional impact, α=.70; practical impact, α=.78; social impact, α=.78; overall impact, α=.83; anxiety, blood test, α=.83; practical impact of dietary protein restriction, α=.87), although reliability for some single item scores was not fully satisfactory. For example, Cronbach’s alpha was lower for impact of anxiety-blood test, α=.59; practical impact of supplements, α=.52; and management of dietary protein restriction, α=.35 (177).  103   Individual responses to each item were assessed with 5-point Likert scales. When specified, the recall period was 7 days.  Based on the response of the survey participant, an item score ranging from 0 to 4 was obtained for each item. Based on the guidelines provided by the tool’s developers (178), domain scores were calculated by summing the item scores within a domain, dividing by the number of non-missing item scores within the domain, and multiplying by 25. Scores for each domain were thus converted to have a range from 0 to 100.  For symptom scores, a lower score is associated with a lower severity of PKU symptoms (better health). For adherence scores, a lower score is associated with a better adherence. For other scores, a lower score is associated with a lower impact of PKU. An entire domain score was set to missing if there were fewer than 70% of items completed within that domain. Scores ≤25 indicated little or no impact of PKU or its management on parental well-being, >25 and ≤50 indicated a moderate impact (or moderate symptoms), >50 and ≤75 indicated major impact (or severe symptoms) and scores above 75 indicated severe impact (or very severe symptoms). The median and interquartile range were used to report domain scores.    Because the PKU-QoL questionnaire is suitable only for parents of children whose child’s PKU is managed with diet, we included a screening question on prescribed diet in the invitation letter for the study and those who were ineligible on this basis were asked to return a blank questionnaire together with the answer to the screening question. The questionnaire also collected some demographic information, including the parental role (father/mother) of the person completing a survey.    104  4.3.2 Measures of metabolic control and mean blood Phe levels The QoL survey data was linked to the CIMDRN PKU clinical longitudinal dataset by the participants’ CIMDRN study ID. Thus the classification of PKU severity and blood Phe levels for each QoL survey participant were derived from the clinical dataset, which was abstracted from children’s medical charts at clinical metabolic centres participating in the cohort study. Mean blood Phe levels were calculated in the 2 years prior to PKU-QoL survey. Similarly, the quality of Phe control was calculated during the two years preceding PKU-QoL survey. “Good” Phe control was defined as more than 60% of blood Phe levels within therapeutic range (120-360 μmol/L) and limited Phe control was defined as less than or equal to 60% of blood Phe levels within therapeutic range (167). “Limited” Phe control was set as the reference category in the regression analyses.   4.3.3 Statistical analysis The health-related quality of life data, including parent-reported child’s health status and symptoms, were analyzed descriptively, reporting proportions for categorical variables and medians with interquartile ranges (IQR) for continuous variables. We also compared HRQoL variables (parent-reported child health status, parental impact of child PKU overall and specific to social, emotional, and practical aspects) in children with severe and milder PKU.  We used univariable and multivariable linear regression models to investigate the relationship between the quality of Phe control and mean blood Phe levels during the two years preceding the distribution of the QoL questionnaire with two outcomes: (1) the overall impact of PKU on parental HRQoL from the survey, and (2) parent-reported child’s health status from the 105  survey. Covariates were patient age at the time of the survey and chart-derived PKU severity classification. The child’s age at the time of the survey was treated as a continuous variable. Because the measure of “overall impact of PKU” on parental HRQoL was derived from 15 questions which represent multiple aspects of PKU care, it was considered the primary outcome and was reported on a continuous scale. There was only one question that created the variable “child’s health status” thus the linear regression was performed on an ordinal dependent variable with five categories for this secondary outcome. Regression analyses were performed using SAS version 9.4 “proc glm” procedure.   The extent to which the data have met the regression assumptions was verified. The relationships between the individual continuous explanatory variables: mean Phe levels, age in years, and the overall impact of PKU and parent-reported child’s overall health status were checked for linearity, normal distribution of errors (normality) and constant error variance (homoscedasticity). The data also were investigated for cases of collinearity among explanatory variables as well as outlying observations. Due to potential collinearity between mean blood Phe levels and quality of Phe control and the difficulty in interpreting one of these variables adjusted for the other, two separate multivariable regression analyses were performed with either mean Phe level or the quality of Phe control as the main predictor variable.    4.4 Results 4.4.1 Sample characteristics  Of the 127 families approached for this study, 87 parents returned completed PKU-QoL surveys from which 16 indicated that they did not use PKU diet, therefore returned blank surveys (response rate of 71/111, 64 %). The majority of children (69%) were considered to have classic 106  PKU, based on the chart-reported disease severity classification, and slightly more than half (52%) were considered to have good Phe control in the two years leading up to the survey. The majority (87%) had mean blood Phe level at 360 μmol/L or less and few cases (8,13%) had mean blood Phe levels over 360 μmol/L in the two years leading up to the survey (Table 4.1).   Table 4.1 PKU-QoL sample characteristics.   n % Parent completed survey (n=71 respondents)       Mother  58 82     Father  13 18 Age of the child at the time of the survey (n=71)       0-2 years  11 15     3-4 years 26 37     5-12 years 34 48 Quality of Phe control (n=62)a       Good Phe control 32 52     Limited  Phe control 30 48 Mean Phe level in the two years leading up to the survey (n=62) a       360 μmol/L or less 54 87    >360 μmol/L 8 13 Chart-reported disease classification (n=62) a       Classic PKU 43 69     Mild PKU 12 19     Mild hyperphenylalanemia (HPA) 7 11  a 9 cases with unspecified severity and/or non-sufficient number of blood Phe levels were removed from the initially eligible QoL sample (n=71)  4.4.2 Parent-reported child’s health status and symptoms  Parent-reported overall child health status scores indicated little to no health concerns, with a median score of 25 and interquartile range (IQR) of 0-50. Tiredness, lack of concentration, irritability and mood swings were reported as symptoms with moderate severity, with median scores of 50 for these single-item domains. The remaining reported symptoms such 107  as slow thinking, sadness, aggressiveness, anxiety and stomach ache were reported as low severity with median scores of 25 (Table 4.2).    Table 4.2 Child’s health status and symptoms as reported by parents.  Parent-reported child’s health status and symptoms Median (IQR) Child health status 25 (0-25) Headaches 0 (0-25) Stomach aches 25 (0-50) Tiredness 50 (25-50) Lack of concentration 50 (25-63) Slow thinking 25 (0-50)    Irritability 50 (25-50) Aggressiveness 25 (0-50) Moodiness 50 (25-50) Sadness 25 (0-50) Anxiety  25 (0-50)  When analyzed by PKU severity, parents of both severe and milder PKU groups reported fairly good overall health status for their children.  There was no statistically significant difference in reported child symptoms although descriptively parents of children with classic PKU reported more profound slow thinking and sadness in their children relative to parents of children with milder PKU, 25(0-50) vs 0(0-25) and 25(0-50) vs 13(0-50) respectively (Table 4.3). Table 4.3 Parent-reported health status and symptoms in children with severe and milder PKU    Parent-reported child’s health status and symptoms Median (IQR) p-valueb Classic PKU Milder types of PKUa Child health status 25(0-50) 25(0-50) .95 Headaches 0(0-25) 0(0-0) .55 Stomach aches 25(0-50) 25(0-50) 1.00 Tiredness 50(25-50) 50(25-50) .57 Lack of concentration 50(25-75) 50(0-50) .80 Slow thinking 25(0-50) 0(0-25) .57 108  Parent-reported child’s health status and symptoms Median (IQR) p-valueb Classic PKU Milder types of PKUa Irritability 50(25-50) 50(25-75) .68 Aggressiveness 25(0-50) 25(0-50) .62 Moodiness 50(25-50) 50(25-50) .49 Sadness 25(0-50) 13(0-50) .22 Anxiety  25(0-50) 25(0-50) .94 a Milder PKU types include combined Mild PKU and Mild HPA b P-values calculated using a normal approximation. Exact p -values could not be calculated due to a lack of required computing power.  4.4.3 Impact of child’s PKU on parental health-related QoL Among the domains measuring impact of PKU on parental HRQoL, the highest median scores were for parental guilt related to poor adherence to dietary restrictions, median (IQR) 75 (50-100); and parental guilt related to poor adherence Phe-free amino acid supplementation, 75 (50-100). Parents also reported relatively high impact for them of the child’s anxiety during blood tests, median (IQR) 63 (63-88) and a high practical impact of dietary protein restriction, 54 (36-71). Parents’ perceptions of their children’s anxiety related to having blood (Phe) tests was moderately high, with a median severity score of 50 (25-63); and the emotional impact of PKU for the parents was also rated as moderate on average, with a median score of 44 (31-63). Finally, the financial, practical and social impact domains demonstrated the lowest reported impact on parent well-being, with medians of 25 (0-25), 21 (8-46) and 15 (10-30) respectively (Table 4.4).   Table 4.4 PKU-QoL domain scores in the study sample. Domain scores N of items  in the domain  Median (IQR) PKU in general   Emotional impact of PKU 4 44 (31-63) Practical impact of PKU 6 21 (8-46) Social impact of PKU 5 15 (10-30) Overall impact of PKU 15 28 (18-38) Child anxiety-blood test 2 50 (25-63) Impact of child anxiety-blood test 2 63 (63-88) 109  Domain scores N of items  in the domain  Median (IQR) Child anxiety-Phe levels 1 38 (25-75) Financial impact of PKU 1 25 (0-50) Information on PKU 1 25 (25-50) Supplement administration   Adherence to supplements 1 0 (0-25) Guilt if poor adherence to supplements 1 75 (50-100) Impact of supplements on family 1 25 (0-25) Practical impact of supplements 3 33 (17-50) Management of supplements 1 0 (0-25) Dietary Protein Restriction   Adherence to dietary protein restriction  1 0 (0-0) Management of dietary protein restriction  6 33 (17-42) Practical impact of dietary protein restriction  7 54 (36-71) Guilt if dietary protein restriction not followed 1 75 (50-100) Child food enjoyment  1 25 (0-25)  4.4.4 Reported PKU-QoL in parents of children with severe and milder PKU    There were also no statistically significant differences in the scores related to the impact of PKU among parents of children with classic PKU relative to those with milder PKU across all domains (Table 4.5). Descriptively, in keeping with the overall results, parents of children with both classic and milder PKU reported feeling severe parental guilt if dietary protein restriction not followed, with reported median scores (IQRs) of 75(25-100) and 75(50-100). Also descriptively, parents of children with classic PKU reported more impactful feelings of guilt due to poor adherence to supplements, 75(75-100) vs 63(25-100).  Descriptively, parents of children with classic PKU also reported being more impacted by their children’s anxiety compare to parents of children with milder forms of PKU 75(56-94) vs 63(63-75).  However, children with milder forms of PKU were reported by their parent as being more anxious about taking blood tests compared to their peers with classic PKU 63(13-63) vs 50(25-75). Parents of children with classic PKU also descriptively reported feeling more worried 110  that their child’s blood Phe levels might be high 50(25-75) vs 25(0-50). The practical impact of maintaining everyday diet requirements was descriptively greater in the classic PKU group compare to the milder PKU group: 57(39-71) vs 48(21-61). The practical impact of supplements was also descriptively higher yet moderate for parents of children with classic PKU 33(17-54) vs 25(8-42) (Table 4.5).    Table 4.5 Reported parental PKU-QoL scores by severity of PKU.   PKU-QoL Domains Median (IQR) p-valueb Classic PKU Milder PKUa PKU in general  Emotional impact of PKU 44(38-69) 38(25-50) .15 Practical impact of PKU 25(8-46) 21(8-46) .51 Social impact of PKU 15(10-40) 15(10-20) .82 Overall impact of PKU 28(22-42) 28(17-38) .92b Child anxiety-blood test 50(25-75) 63(13-63) .76 Impact of child anxiety-blood test 75(56-94) 63(63-75) .48 Child anxiety-Phe levels 50(25-75) 25(0-50) .06 Financial impact of PKU 25(0-50) 25(0-50) .73 Information on PKU 25(25-50) 25(25-50) .65 Supplement administration  Adherence to supplements 0(0-25) 0(0-25) .57 Guilt if poor adherence to supplements 75(75-100) 63(25-100) .22 Impact of supplements on family 0(0-25) 0(0-25) .95 Practical impact of supplements 33(17-54) 25(8-42) .51 Management of supplements 0(0-50) 0(0-25) 1.00 Dietary protein restriction  Adherence to dietary protein restriction  0(0-0) 0(0-0) 1.00 Management of dietary protein restriction  33(17-50) 29(17-38) .88 b Practical impact of dietary protein restriction  57(39-71) 48(21-61) .29 b Guilt if dietary protein restriction not followed 75(50-100) 75(25-100) .54 Child food enjoyment  25(0-50) 25(25-25) .76 a Milder PKU types include combined Mild PKU and Mild HPA b P-values calculated using a normal approximation. Exact p-values could not be calculated due to a lack of required computing power.  111  4.4.5 Child’s health status as predicted by the quality of Phe control.  Children’s overall health status as assessed by parents yielded a relatively low median score, indicating good health status, across all potential predictors (Table 4.6). Health status had a slightly higher score, indicating worse health, in the group of children with relatively elevated mean blood Phe levels although there were only 8 cases in that group, median (IQR) 38 (13-50) (Table 4.6).   Table 4.6 Child health status by PKU severity, age, mean Phe levels and quality of Phe control (n=61) a.  Child’s health status  n(%) median IQR Chart-reported disease classification        Classic PKU 42(69) 25 0-50     Mild PKU 12(20) 0 0-50     Mild hyperphenylalaninemia (HPA) 7(11) 25 25-50 Patients’ age at the time of the survey (years)        0-2 yearsb 10(16) 13 0-50     3-4 years 24(39) 25 0-50     5-12 years 27(44) 25 25-50 Mean Phe level in the two years leading up to the survey        360 μmol/L or less 53(87) 25 0-50    >360 μmol/L 8(13) 38 13-50 Quality of Phe control        Good Phe control 29(48) 25 25-50     Limited  Phe control 32(52) 25 0-50 a9 cases with unspecified severity and/or non-sufficient number of blood Phe levels and 1 case with a missing score for child health status were removed from the initially eligible QoL sample (n=71). bThe age group of 0-1 y.o. had less than 5 cases and therefore could not be reported separately The simple (univariable) linear regression analysis did not identify significant relationships between any individually chosen predictor (mean Phe level, quality of Phe control, age and severity of PKU) and overall child’s health status as reported by the parent (Table 4.7).  112  Table 4.7 Univariable associations between mean Phe levels, quality of Phe control, age, PKU severity and child health status (n=61)a.   ß 95% CI R2 p-value Mean Phe level in the two years leading up to the survey  0.03 -0.02, 0.08 .03 .21 Mean Phe level in the two years leading up to the survey (10 unit change) 0.31 -0.17, 0.79 .03 .21 Quality of Phe control -6.98 -19.38, 5.42 .02 .26 Patients’ age in years at the time of the survey 1.06 -1.16, 3.28 .01 .34 Chart-reported disease classification   .03 .43     Classic PKU 12.50 -7.35, 32.35  .21     Mild PKU 13.39 -9.73, 36.52  .25     Mild HPA (reference category) - -   a 9 cases with unspecified severity and/or non-sufficient number of blood Phe levels and 1 case with a missing score for child health status were removed from the initially eligible QoL sample (n=71). The results of the multiple linear regression model also did not identify a strong association between any of the predictors and child’s health status. The strength of relationship between the parent-reported overall child’s health status, mean Phe, age, and PKU severity was very weak with our predictor variables explaining only 1% of variance in the data (R2= .01) and p=0.51. Due to collinearity issues described in “Methods”, two separate multiple regression models were analyzed; one with mean blood Phe levels and another with quality of Phe control as the main predictors of parent-reported child’s health status (Table 4.8). When the analysis was performed using the model containing the quality of Phe control (replacing mean Phe), the results did not identify any significant relationship between quality of Phe control, age at the survey, and severity of PKU and parent-reported child’s health status. The same weak relationship was observed (R2= .01, p=0.53) (Table 4.8)   113  Table 4.8 The association between mean Phe levels/quality of Phe control and parent-reported child’s health status, adjusted for age and severity of PKU. The adjusted association between mean Phe level and child health status  (n=61a, adjusted R2= -0.01, p = .51)  β 95% CI p-value Mean Phe level in the two years leading up to the survey (10 unit change) 0.03 -0.02, 0.08 .29 Patients’ age in years at the time of the survey 0.40 -2.04, 2.83 .76 Chart-reported disease classification        Classic PKU 10.34 -10.16, 30.85 .32     Mild PKU 13.97 -9.57, 37.51 .24     Mild HPA (reference category) - - -  The adjusted association between quality of phenylalanine control and child health status  (n=61 a, adjusted R2= -0.01, p = .53)  β 95% CI p -value Quality of phenylalanine control -6.36 -19.01, 6.28 .32 Patients’ age in years at the time of the survey 0.68 -1.64, 3.01 .56 Chart-reported disease classification        Classic PKU 10.63 -9.89, 31.14 .30     Mild PKU 12.68 -10.72, 36.07 .28     Mild HPA (reference category) - - - a 9 cases with unspecified severity and/or non-sufficient number of blood Phe levels and 1 case with a missing score for child health status were removed from the initially eligible QoL sample (n=71).  4.4.6 Overall impact of PKU on parental QoL as predicted by the quality of Phe control Parents reported relatively low scores regarding the overall impact of PKU on parental quality of life, indicating little or no impact, across all potential predictors (Table 4.9). An exception was a moderate overall impact of PKU as reported by parents of children with relatively elevated mean blood Phe levels, although there were only 8 cases in that group, median (IQR) 37, (22-63) (Table 4.9).   114  Table 4.9 Overall impact of PKU by PKU severity, age, mean Phe levels and quality of Phe control (n=62)a.  Overall impact of PKU  n median Q1-Q3 Chart-reported disease classification        Classic PKU 43 30 20-42     Mild PKU 12 38 18-43     Mild hyperphenylalaninemia (HPA) 7 25 7-28 Patients’ age at the time of the survey (years)        0-2 years 10 22 12-30     3-4 years 25 32 27-43     5-12 years 27 32 17-42 Mean phenylalanine level in the two years leading up to the survey     360 umol/L or less 54 29 18-38    >360 umol/L 8 37 22-63 Quality of phenylalanine control        Good Phe control 32 29 18-37     Limited  Phe control 30 31 25-42 a9 cases with unspecified severity and/or non-sufficient number of blood Phe levels were removed from the initially eligible QoL sample (n=71).  The univariable linear regression analysis did not identify significant relationships between any individual chosen predictor (mean Phe level, quality of Phe control, age and severity of PKU) and overall impact of PKU on parental QoL. The strongest association, although statistically non-significant, was observed between the severity of PKU and the outcome, where the severity of diagnosis was explaining 6% of the variance in the data (compare to 1% for the remaining predictors) (Table 4.10).    Table 4.10 Univariable associations between mean Phe levels, quality of Phe control, age, PKU severity and the overall impact of PKU on QoL (n=62)a.   ß 95% CI R2 p -value Mean Phe level in the two years leading up to the survey 0.01 -0.02, 0.05 .01 .35 Mean Phe level in the two years leading up to the survey (10 unit change) 0.15 -0.17, 0.47 .01 .35 Quality of Phe control -3.23 -11.34, 4.87 .01 .43 115   ß 95% CI R2 p -value Patients’ age in years at the time of the survey 0.69 -0.76, 2.15 .01 .34 Chart-reported disease classification   .06 .17     Classic PKU 12.09 -0.71, 24.88  .06     Mild PKU 11.71 -3.22, 26.64  .12     Mild HPA (reference category) - -   a9 cases with unspecified severity and/or non-sufficient number of blood Phe levels were removed from the initially eligible QoL sample (n=71).  Due to collinearity issues described in “Methods”, two separate multiple regression models were analyzed; one with mean blood Phe levels and another with quality of Phe control as the main predictors of the overall impact of PKU with results presented in Table 4.11. The results of the multiple linear regression analysis including the mean blood Phe levels did not identify a strong association between overall impact of PKU and mean Phe, age, and PKU severity. In fact, the strength of the relationship between the predictors and outcome was very weak, with the predictor variables explaining only 1% of variance in the data (R2= .01) and  p =0.37. Similarly, when analysis was performed using the model containing the quality of Phe control (replacing mean Phe), the results did not identify any significant relationship between predictor variables (quality of Phe control, age at the survey, and severity of PKU) and overall impact of PKU. Thus, the same weak relationship was observed (R2= .01, p=0.37) where only 1% of variance in the data could be explained by the chosen predictors (Table 4.11).   Table 4.11 The association between mean Phe levels/quality of Phe control and the overall impact of PKU on parental QoL, adjusted for age and severity of PKU.  The adjusted association between mean Phe level and the overall impact of PKU on QoL  (n=62a, adjusted R2= .01, p = .37)  β 95% CI p -value Mean Phe level in the two years leading up to the survey (10 unit change) 0.12 -0.23, 0.46 .48 Patients’ age in years at the time of the survey 0.25 -1.34, 1.83 .76 116  Chart-reported disease classification        Classic PKU 11.07 -2.23, 24.37 .10     Mild PKU 11.85 -3.46, 27.16 .13     Mild HPA (reference category) - - - The adjusted association between quality of Phe control and the overall impact of PKU on QoL (n=62a, adjusted R2= .01, p = .37)  β 95% CI p -value Quality of Phe control -2.72 -10.86, 5.42 .51 Patients’ age in years at the time of the survey 0.37 -1.13, 1.88 .62 Chart-reported disease classification        Classic PKU 11.10 -2.20, 24.40 .10     Mild PKU 11.31 -3.89, 26.51 .14     Mild HPA (reference category) - - - a9 cases with unspecified severity and/or non-sufficient number of blood Phe levels were removed from the initially eligible QoL sample (n=71).  4.5 Discussion   QoL is one of the most important outcomes for children with chronic illness and their parents. This has been increasingly recognized in clinical research in recent years. Evidence regarding the QoL of parents of children with rare metabolic diseases, including PKU, is sparse. This is the first Canadian study investigating the HRQoL of parents of pediatric patients with PKU while using a validated PKU-specific health-related QoL questionnaire (177). We found that the areas where parents reported the greatest impact of their children’s PKU on their QoL was parental guilt due to poor adherence to the dietary treatment, 75(50-100) and guilt to due poor adherence to supplements (medical foods), 75 (50-100). Parents also reported an important impact on their well-being of their perceptions of their child’s anxiety during blood tests, 63 (63-88) and an important practical impact for them related to their child’s dietary protein restriction, 54 (36-71). As expected, parents who have children diagnosed with classic PKU reported more severe impacts on QoL with regard to practical aspects of dietary restriction relative to parents of children with milder PKU, although none of these differences was statistically significant. Symptoms that included tiredness, lack of concentration, irritability and mood swings were 117  reported as having moderate impact regardless of diagnosis severity (all with median score of 50). In our study, neither the overall impact of PKU on parental QoL nor overall child’s health status correlated with mean blood Phe levels in the two years preceding the survey or with the quality of Phe control.   The findings of this study echo the results of a recently published Australian survey of 18 mothers of children with PKU that used the same PKU-specific QoL questionnaire (176). Similar to Canadian parents, the parents in Australia reported guilt associated with poor adherence and anxiety related to the blood tests as the major implications of PKU. Contrary to our findings, this study found an association between overall impact of PKU on parents and the child’s lifetime mean blood Phe levels. Another study using the PKU-specific parental questionnaire was conducted in Europe, and reported similar results with some exceptions (141). In contrast to our findings, parental perceptions of the child’s blood test-related anxiety had little or no impact on European parents. On the other hand, anxiety regarding blood Phe levels was reported to have an important impact in the European sample, with little impact in the Canadian sample.  In conclusion, the findings of this study indicate that parents of children with all severities of PKU experience a considerable level of chronic stress associated with the disease. It is suggested that parents may need support to work with their feelings of guilt and to alleviate both theirs and their children’s distress regarding blood tests.        118  Chapter 5: Discussion  This chapter discusses key findings derived from the four studies conducted within this PhD project, including: (a) two surveys of Canadian metabolic healthcare providers, describing physicians’ and dietitians’ perspectives on clinical and nutritional management practices of PKU in Canada; (b) the analysis of CIMDRN chart-abstracted longitudinal PKU data, directed by the results of both surveys, and further describing PKU management practices, and associated intermediate outcomes and, (c) the quality of life of caregivers and parent-reported child’s overall health status in association with the quality of Phe control.  Overall, this PhD project describes the observed variation in PKU care in Canadian treating centres and discusses its potential implications on health outcomes. This work identified less than ideal intermediate outcomes in children with PKU, potentially resulting from the observed variation in care; and some evidence of reduced parental quality of life in particular domains. Finally, while there was no association between Phe control and parental quality of life or child’s health status, considerable parental guilt due to dietary non-adherence, impact of child’s anxiety and practical impact of dietary restrictions due to PKU were identified. As a result, it is suggested that strategies focused on improving parental wellbeing might in turn improve suboptimal outcomes in children with PKU.       5.1 Variation in care and its impact on health outcomes  Both survey results and chart-abstracted data showed that, overall, PKU management in Canada is consistent with the published guidelines. However, it was also clear that variation in key clinical and nutritional practices in Canadian metabolic treatment centres still exists. Some observed variation could be attributed to gaps in current knowledge and associated with 119  uncertainties in guideline recommendations. For example, variation in PKU classification stems from the absence of a standard method to diagnose PKU severity. The observed variations in PKU care and potential implications on patients’ outcomes are expanded in detail below.   5.2 Observed variation in PKU diagnostic practices  5.2.1 Variation in classification of PKU severity PKU is a highly heterogeneous condition and presents with a spectrum of metabolic phenotypes. The diagnosis of PKU is determined by consistently elevated concentrations of Phe in blood, after the primary deficiencies in the biosynthesis or recycling of the cofactor BH4 diagnoses have been excluded. Traditionally, severity is based on the highest Phe level reached before treatment intervention (37). Seemingly straightforward, classification based on untreated blood Phe levels is not always accurate. Prompt diagnosis and treatment initiation may not allow blood Phe levels to reach maximum concentrations (37), resulting in the proposal of alternative classifications. In some cases classification requires prolonged monitoring of blood Phe levels, especially in milder forms of PKU, and considers a combination of criteria including untreated blood Phe levels, dietary Phe tolerance, and genotype (37) (163).  Uncertain classification methods prevent guidelines from offering robust standard definitions of PKU severity. For example, ACMG guidelines defined two phenotypic categories based on untreated Phe: “Classical PKU” with untreated blood Phe levels typically higher than 1,200µmol/L, and “Hyperphenylalaninemia” with untreated blood Phe levels less than 1,200 µmol/L (National Institutes of Health (NIH) Consensus Development Conference Statement, 2000 (7).   European guidelines proposed categories of PKU severity as (a) not requiring treatment, and (b) requiring diet, BH4, or both. The recommendation was graded as level “D” as it was largely based on 120  expert opinion  (15). Given this uncertainty, it was not surprising that this study’s surveys and accompanying analysis of the CIMDRN PKU clinical data confirmed limited consensus among Canadian metabolic healthcare providers on the definition of PKU severity. As identified by the surveys, approaches to define the severity of PKU phenotypes varied considerably across Canadian treatment centres and individual professionals. According to the physicians’ survey, maximum untreated Phe levels alone, or in combination with Phe tolerance or genotype or both, were utilized by all healthcare providers in determining severity. Neither Phe tolerance nor genotype was used as a single criterion to define PKU severity.  In line with the survey results, CIMDRN clinical data analysis indicated considerable variation in the definitions of PKU severity. Chart-abstracted data showed that nearly half of participating children (43%) were assigned classic phenylketonuria (PKU), despite 30% of them never having had a recorded peak blood Phe measurement greater than 1,200 µmol/L. It is possible that these may be true classic PKU cases with good metabolic control, or it could indicate an inaccurate severity diagnosis. On the contrary, 4% of patients who reached peak blood Phe levels corresponding to classic PKU (>1,200 µmol/L) were in fact diagnosed with milder forms of PKU, potentially representing “under-diagnosed” cases or cases that had perhaps initially been considered classic PKU but were eventually resolved as milder.  Finally, a considerable number of cases with non-identified severity in the chart-derived data accounted for as much as 7% of all PKU cases. To date, there are no universal criteria that can precisely characterize a spectrum of metabolic severity of PAH deficiency. Measuring direct PAH enzyme activity in hepatocytes is a highly precise method, but it is not ethical outside of specific research contexts due to its invasive nature (180) (117). Several existing classification schemes, described in detail in the 121  Introduction of this dissertation, are based on biochemical phenotype including untreated blood Phe levels and Phe tolerance (81)(181) others used high throughput PAH phenotyping in response to Phe exposure (88). These existing classification schemes have limitations. As mentioned above, blood Phe might not reach its highest levels before treatment initiation, potentially misleading the diagnosis of metabolic PKU severity (67). With regard to Phe tolerance, the age-specific trajectory of actual Phe intake supporting acceptable blood Phe levels is not well studied. Limited evidence shows that Phe tolerance becomes a fairly reliable biomarker of phenotypic severity around 2 years of age (82). While a good predictor of metabolic severity, Phe tolerance is challenging to accurately evaluate in clinical settings (84). Thus, current classification of PKU severity based on Phe tolerance is fairly impracticable where the diagnosis of PKU, including severity, is typically established soon after birth.  In summary, a lack of reliable criteria for defining PKU severity in clinical practice creates potential for misinterpretation. While determining PKU severity is not a requirement for optimal treatment, misinterpretation of the severity of PKU could potentially impact several important aspects of care, including kinds of treatment and the quality of life of patients and their parents. These are discussed further below.  5.3 Potential implications for patient care 5.3.1 Over-restriction of natural protein consumption The clinical data analysis identified a potential over-diagnosing of PKU severity, possibly resulting in unnecessary additional dietary restrictions (although as described, this could also reflect children with truly severe disease treated from a very early age who never reached a peak untreated Phe level). Recent evidence shows that the over-restriction of natural protein in clinical 122  practice commonly occurs, partially supporting this suggestion. Thus, Scala and colleagues (182) re-assessed Phe tolerance in 46 adolescents with PKU. Slightly less than a half (43.5%) were able to tolerate larger than prescribed amounts of natural protein while keeping their blood Phe levels within recommended range.  Another retrospective review study (84) found that as many as 65% of observed adolescents’ natural protein was over-restricted. A PKU diet is already extremely restrictive; unjustified over-restriction of natural protein would only introduce unnecessary practical and emotional burden to patients and their caregivers.  5.3.2 Averting trial of sapropterin  Current ACMG guidelines recommend that every patient, except those with two null mutations “in trans” (obligatory BH4-non-responder), should be offered a sapropterin trial to assess BH4 responsiveness (7). According to the published evidence, patients with classic PKU are less likely to be identified as good responders to sapropterin (183). Given the high cost and limited access to sapropterin, the diagnosis of “classic PKU” could, in some cases, prevent healthcare providers from offering a trial of medication due to a presumptive non-responsiveness to therapy (184)(185) (186).   5.3.3 Potential impact of over-diagnosis of PKU severity on caregivers’ quality of life The PKU-QoL survey identified significant emotional impact on parents of children with PKU. Guilt over non-adherence to the diet or supplements, the impact of child’s anxiety, and the practical impact of dietary restrictions affected parental QoL. Not surprisingly, parents of children with classic PKU were potentially (but not statistically significantly) more affected compared to those whose children had milder PKU as this cohort of patients require more care.  Over-diagnosis of PKU severity imposes an additional and unjustified challenge on parents.  123  5.3.4 Impact of uncertainty in PKU classification on PKU research While diagnosing PKU severity may not be crucial for clinical management (considering treatment is based on observed blood Phe levels) PKU classification is essential in research as it is commonly used in various studies.  Yet, as discussed classification approaches vary (83) (182) (105), using different case definitions can cause a misinterpretation of findings, as well as limited or biased comparability of results across studies. This might impede the utilization of systematic literature reviews and meta-analyses, consequently preventing the generation of quality research evidence.   5.4 The use of the neonatal BH4 loading test in Canadian treating centres The objectives and methods of BH4 loading test were described in more detail in the Introduction. To recap, BH4 loading test is especially helpful in two ways. Firstly, in the newborn, the test helps to distinguish between PAH enzyme deficiency and BH4 cofactor deficiencies, as these conditions present with hyperphenylalaninemia. Secondly, it assesses responsiveness to treatment with sapropterin. While differentiation between PAH deficiency and BH4 deficiencies must be made in the neonatal period in order to promptly initiate appropriate disease-specific treatment, other, more precise laboratory methods exist to do so, although there are circumstances when BH4 loading test could be helpful. On the other hand, sapropterin is considered an adjuvant therapy to the Phe-restrictive diet and there is no urgency to assess the responsiveness in the neonatal period as the treatment could be initiated later (149). Thus these differences create a degree of uncertainty in applications of BH4 loading test in clinical practice. The survey of Canadian metabolic healthcare providers described in Chapter 2 revealed considerable variation between treating centres in performing the neonatal BH4 loading test. A 124  subsequent analysis of the CIMDRN PKU data supported this finding. Almost half physician respondents (9/17, 53%) reported not using the neonatal BH4 loading test in their practice, while the other half of respondents (8/17, 47%) utilize the test for both differential diagnoses of BH4 deficiency and assessing long-term BH4-responsiveness (5/8, 63%). The minority of BH4 test users (3/8, 38%) use the test for differential diagnosis of BH4 deficiencies only. Reported BH4 doses and schedules of blood sampling patterns also varied. For example, a 20mg/kg dose was typical, while some prescribed 10mg/kg. Logistical difficulties, potential delays in initiation of treatment, and limited benefit for patients who are not “super responders” to BH4 were the most commonly reported barriers to performing the BH4 loading test in newborns. The Canadian experience is not unique; similar variations in BH4 loading test practices across countries was also described in a recent publication, where the most prominent barriers were lacking evidence on test effectiveness and absence of standard guidelines regarding test administration and response evaluation (108). The ACMG guidelines did not provide specific recommendations, and European guidelines (in cases of delayed results of pterins and DPHR analyses) recommend using the 24-hr BH4 loading test as an auxiliary test in the differential diagnosis of BH4 deficiencies. As per current standards of care, measurement of pterins in blood, urine, and DHPR activity in DBS are the most common screening methods for inborn errors of BH4 metabolism. Some experts suggest the importance of molecular investigation as mandatory testing in the current scheme of differential diagnosis of HPAs as genetic tests become more available (7) (40). This will most likely be the case when the turnaround time of gene sequencing is reduced to several days; currently the average turnaround time of PAH gene sequencing is approximately one month, not including the time it takes for parental consenting, sample collection, and shipping. Because accurate diagnosis and subsequent early initiation of disease-specific 125  treatment affects long-term morbidity, prompt diagnosis is crucial.  While experts do not discard the neonatal BH4 loading test from the list of diagnostic tests of HPAs, the added benefit of the test is uncertain, especially when access to the more sensitive diagnostic tests are satisfactory.   With regard to the use of BH4 loading test to assess the responsiveness to sapropterin, according to one European survey, the test is typically performed in patients of at least four years of age and lasts for at least 48 hours (159). The current ACMG guideline advises that a BH4 trial should be performed to determine responsiveness to sapropterin. Along with baseline Phe, ACMG recommends measuring subsequent blood Phe levels at regular intervals, usually at 24 h, 1 week, 2 weeks, and in some cases, 3 or 4 weeks with the administered dose of 20mg/kg of sapropterin.  As per the Canadian physicians’ survey, almost two thirds of respondents reported offering a trial to the majority of their patients during their pediatric follow up. They would also assess responsiveness to the drug before the trial.  In the clinical database the information on BH4 loading tests to assess the responsiveness was inconsistent. To some degree, this reflected the uncertainty in utilization of the test. On the other hand, the data from the clinical database are from 2006-2017. If we had a later-born cohort and more recent data, we may see more trials of sapropterin. Overall, the lack of robust evidence and subsequent uncertainty and variation in management practices on BH4 loading tests might have certain implications on patient care.  5.4.1 Potential implications on patient care Limited evidence shows that BH4 loading tests might delay appropriate dietary management for as long as 24 hours in children who are not responsive to BH4 (187), thus unnecessarily prolonging some newborns to elevated blood Phe levels. Negative results of the neonatal BH4 loading test are not always predictive of the long-term non-responsiveness to BH4, 126  however the evidence is limited (159). Yet, it is likely that a patient diagnosed as a non-responder to BH4 in the neonatal period will not be considered for the trial of sapropterin later in life.  Presently, there is no published consensus on the optimal blood-sampling schedule. A 24 hour test duration is recommended by some authors with pre-dose blood sampling frequency as follows: 2, 4, 6, 8, 12, and 24 hours (15), (185). With regard to dosing, some authors suggest 20 mg/kg (189).  In conclusion, the benefits of practical application of neonatal BH4 loading tests in PKU care is not well supported by the robust evidence, hence direct recommendations are not conclusive. While a BH4 loading test might be a good diagnostic tool if applied consistently, presently there is large variation in application of the test in treating centres. It is perhaps the time to address the utilization and standardization of the neonatal BH4 loading test protocol in clinical practice in Canadian metabolic treating centres, as well as to explore other, more efficient alternatives to assess for BH4 responsiveness in the neonatal period and in older children.   5.5 Variation in follow up practices 5.5.1 Frequency of Phe monitoring and clinic visits  As expected, the most frequent Phe tests and clinic visits were during the first months of life and these gradually declined with increasing age. When comparing the frequency of Phe tests and clinic visits with the current PKU guideline recommendations, the observed age-specific monitoring frequency was comparable for classic PKU. Unsurprisingly, patients with 127  milder forms of PKU had considerably fewer Phe tests and clinical visits compared to individuals diagnosed with classic PKU.  Outside of clinic visits, the frequency of communications between healthcare providers and patient/caregivers is driven by the results of Phe blood tests. Limited evidence suggests that the reduced frequency of tests is associated with patient’s non-adherence to the diet (173). According to personal communications with metabolic dietitians, it appears that current approaches to Phe monitoring practice is feedback-based; the dietitian communicates test results to a patient, adjusting the diet according to the Phe level and patient-reported Phe intake. Thus, less frequent monitoring of Phe will result in fewer communications with the clinical team, and in turn, this may be associated with a potential decline in adherence to the dietary treatment (161) (190). In contrast to the survey findings, the results of the CIMDRN data showed that frequency of communication was within recommended norms for classic PKU. While sufficient communication should promote better quality of Phe control, as described later in this chapter, the observed quality of Phe control is not ideal, declining with increasing age. This might be due to factors other than communication.   The observed communication frequency for milder forms of PKU was almost half that of classic PKU. There is no doubt that frequent monitoring and communication is especially beneficial for patients with severe PAH deficiency; perhaps it is time to re-evaluate the need and importance of communication with patients with milder forms of PKU, considering that quality of Phe control is also not ideal in this group. The data shows that, for both classic and mild PKU, the quality of Phe monitoring and, possibly, adherence to the diet, diminishes with age. Not much data on milder cases was available due to non-frequent monitoring and clinic visits. The data showed that the proportion of children with limited Phe control increases with age, however 128  due to small sample sizes in the older age categories it was not possible to determine whether the limited control trended towards “low” or “high” levels of Phe.    In summary, frequency of communication is in accordance with the proposed recommendations for classic PKU. However, decreasing quality of Phe control in spite of frequent communication suggests possible other causes.   5.6 Neuropsychological assessments  Healthy neurocognitive development is one of the core priorities in PKU care. This statement is supported by research evidence, and is identified as the highest priority outcome by PKU patients and their metabolic healthcare providers (191), (51), (58), (62), (50) (47). The ACMG guideline recommends monitoring the neurocognitive development of PKU patients on a regular basis (7). While analyzing CIMDRN data, a virtual absence of neuropsychological assessments data was identified. This absence of data in the chart-abstracted database reflects the fact that such data was also missing in the metabolic charts across many treating centres. It is also possible that neurocognitive assessments might not be performed on a regular basis in Canadian PKU treating centres. This finding is supported by the survey results described in Chapter 2, where very few providers reported performing regular neuropsychological assessments of their PKU patients.  The physicians’ survey also revealed several barriers to performing neuropsychological assessments in the PKU population, with limited access to a psychologist being one of the main obstacles, as outlined in the comments. The survey also identified that the majority of Canadian PKU metabolic clinics do not include a psychologist as a member of metabolic team. Some respondents expressed the opinion that existing 129  neuropsychological questionnaires and scales are not sensitive to detect subtle changes or deviations in neuropsychological development in the PKU pediatric population.   Developing a simple screening tool could be a good solution to integrate neuropsychological monitoring into standard care. Such an instrument should be user-friendly, brief, simple to administer, conventional to practitioners, patient and caregivers, and sensitive enough to effectively identify the most common neuropsychological symptoms across a range of ages. The evidence on the application of screening tools in clinical settings is emerging. According to one study, 22% of regular patients with PKU younger than 12 years screened positive for executive function impairment and 19% screened positive for psychiatric distress. The study used the Pediatric Symptom Checklist (PSC), Youth Report or the Brief Symptom Inventory, and the Behavior Rating Inventory of Executive Function screening tool which could only take 15-20 minutes to complete (47). This study showed that a simple standardized screening for psychiatric distress of patients with phenylketonuria could be implemented in the existing metabolic clinic routine. Furthermore, with easy access to mobile technologies, various online applications could be used to screen for neuropsychological problems in children with PKU during the regular clinic visit.  Further studies are needed to address other areas of uncertainty, for example: whether   neuropsychological screening is an effective approach in monitoring the individual outcomes of PKU care; whether the available neuropsychological testing tools are specific enough to detect subtle initial deficits and subsequent changes; and whether neuropsychological screening in PKU patients with no apparent clinical need is cost-effective. With the aim of moving towards the optimization of outcomes for PKU patients, effort should be made to both incorporate screening 130  of neuropsychological development as a standard of care, and to secure optimal access to psychological services for all patients with PKU.     5.7 Quality of Phe control in Canadian pediatric population  As described earlier in the Introduction, bloodstream Phe levels serve as the most important biomarker reflecting the quality of dietary treatment, as well as patient adherence to treatment regimens. The main goal of PKU treatment is to maintain the patient’s blood Phe at a level that supports optimal physical and neuropsychological development while providing a nutritionally complete diet. The association between elevated blood Phe levels and suboptimal outcomes was described in the Introduction in more detail.  Based on current evidence, the ACMG guideline sets the target blood Phe levels within a 120-360 µmol/L range across the life span (7).  The ACMG guideline also recommends reaching therapeutic Phe levels “as quickly as possible” within the first two weeks of life. CIMDRN data analysis showed that despite early diagnosis and treatment, newborns with severe PAH deficiency might be exposed to elevated and unstable blood Phe levels during the first month of life. Beyond the first month, the means of participants’ mean blood Phe levels are within the recommended range, although there is a tendency for them to increase with age. In older children the means of mean blood Phe levels were close to the upper acceptable limit, accompanied with greater Phe fluctuation, in patients with both severe and milder forms of PKU. These results are corroborated with survey results where some dietitians and physicians reported acceptance of a relaxation of the diet in older children, up to Phe levels of 600µmol/L.  The results of our data analysis agree with other reports that describe an overall tendency of blood Phe levels to increase with age. One retrospective study reported an overall increase of 131  blood Phe concentrations with age, from a median of 175 µmol/L in infants to 465 µmol/L in 11-16 years old children in western European countries (170). The signs of weakening metabolic control with age are also described in other countries (171). Many different factors could contribute to the worsening of metabolic control. As was indicated by the healthcare providers’ surveys, some treating centres have limited human and/or time resources to provide consistent feedback to the patients and families, and/or deliver continuing education and counselling.   The current analysis revealed that overall Phe control in Canadian PKU patients of all age groups is less than ideal. Fewer than half of patients with classic PKU across all pediatric age groups (except 4-5 years old) have more than 60% of their Phe test results within the recommended therapeutic range. Moreover, it appears that there is a trend in the quality of Phe control that might potentially be explained by physiological and social milestones; the first decline in the quality of Phe control in patients with classic PKU occurs around 6-12 month of age. This, perhaps, could reflect the introduction of solid foods to a child’s diet, reflecting the adjustment to the new dietary regimens. After the period of relative increase in the quality of Phe control, the proportion of “PKU-normal” Phe levels starts to decline around 5-7 years and remains lower in older children. At the same time, the proportion of blood Phe levels above the recommended range increases in older children. The second decline in quality of Phe control coincides with the time of entering elementary school. This decline could potentially reflect the new social environment that perhaps might not always be fully supportive of children with PKU needs.  Similar patterns of decline in dietary adherence were described in other study (161). Other factors that could affect Phe control, including Phe fluctuation include: poor adherence, relaxed recommendations, and residual physiological diurnal fluctuations of Phe or spikes of blood Phe levels due to illness and catabolic state (172)(161)(65). Even if the adherence to the 132  diet is ideal, perhaps some residual or occasional elevations of blood Phe levels should be expected.   5.8 The quality of life of a parent who provides care for child with PKU One of the objectives of this PhD project was to explore the quality of life of the parents who care for children with PKU. Decreased parental wellbeing can affect their ability to provide good care for their children, thus contributing to the development of less than ideal health outcomes. The continuous supervision of appropriate intake of nutrients, frequent blood monitoring, clinic appointments, and communication with healthcare providers puts strains on caregivers. Domestic care provided by a parent is at least as important and complementary to care provided by metabolic health professionals. While parental quality of life is an outcome on its own, with appropriate support it also could become a powerful tool to improve children’s health outcomes.  While the analysis revealed no association between “overall impact of PKU” (including emotional, social and practical aspects of PKU) and blood Phe levels or quality of Phe control, there were considerable emotional challenges reported by parents. The highest observed median scores, indicating moderate to severe impact on caregivers, were observed for feeling guilty due to non-adherence to either the Phe-restrictive diet or medical foods. PKU dietary treatment is difficult; it demands a great degree of supervision and discipline from a parent to ensure a child is adherent to the treatment regimen. Some parents indicate that they feel guilt even if their child only occasionally skips the diet due to the uncertainty over whether or not elevated Phe will result in brain damage (192). Interestingly, the reported impact of anxiety related to a child’s blood test on parents was descriptively greater than parent reports of their child’s anxiety of 133  having blood tests. Moderate anxiety regarding high blood Phe levels was also reported. The emotional challenges and anxiety possibly indicate higher levels of stress in families with PKU. According to one recent study, mothers who reported a higher impact of various aspects of PKU on their daily lives also had higher levels of parental stress (176) however more research is needed to explore the predictors of parent-reported PKU-QoL in Canadian culture.   The practical impact of dietary restriction was also identified as somewhat challenging for parents, especially in cases of severe PKU. Scrupulous daily dietary management, frequent blood tests for monitoring blood Phe levels, and regular visits to a metabolic treating centre make the management of PKU quite time consuming, requiring considerable effort from the parents (144).  The results of our QoL study identified quite a striking difference with regards to the impact that different aspects of everyday PKU care have on quality of life. On the one hand, little or no impact was reported on practical impact of PKU related to financial burden, missing work hours, time spent for administrative tasks related to PKU, and burden of physicians visits. On the other hand, median scores indicated moderate to severe impact were reported on “practical impact of dietary restriction” including items on the taste of low protein food, lack of spontaneity/freedom due to PKU dietary protein restriction, difficulty eating out due to PKU dietary protein restriction, planning meals in advance, time consuming aspects of dietary protein restriction, the complexity of cooking, and difficulty travelling and transporting PKU food for special event situations (Mapi Group, 2015). Similar to the findings of this study, others have reported similar results with regards to difficulties with traveling and dining out, and the taste of the medical food being reported as a barrier to good diet adherence by some patients (193).  This parental PKU-QoL study is one amongst a limited number of others that used a PKU-specific QoL questionnaire. It is the first one, to date, in Canada. The findings of this 134  survey are in line with a recent study conducted in Australia which uses a Parent PKU-QoL questionnaire. Similar to our study, guilt due to non-adherence and impact of child’s anxiety were the domains where the higher impact on parents was observed. The authors also did not find any association between lifetime metabolic Phe levels and QoL module scores, except Dietary Protein Impact scores (176).  Another survey conducted in seven European countries showed similar result in the domains of guilt related to non-adherence to the dietary restrictions (141) The reproducible results increase the confidence in the results of our survey (Table 5.1). Table 5.1 Comparison of Canadian, European (141) and Australian PKU-QOL surveys (176).  Domain scores Canada Europe Australia median (IQR) median (IQR) median (IQR) Parent-reported child’s health status and child’s PKU-related symptoms Child health status 25(0-25) 25(0-25) 25(0-50) Headaches 0(0-25) 0(0-25) 0(0-50) Stomach aches 25(0-50) 0(0-25) 25(0-32) Tiredness 50(25-50) 25(0-50) 25(0-56) Lack of concentration 50(25-63) 25(0-50) 25(0-50) Slow thinking 25(0-50) 0(0-50) 0(0-25) Irritability 50(25-50) 25(25-50) 38(25-50) Aggressiveness 25(0-50) 0(0-25) 13(0-25) Moodiness 50(25-50) 25(0-50) 25(19-31) Sadness 25(0-50) 25(0-25) 13(0-31) Anxiety  25(0-50) 0(0-25) 13(0-50) PKU in general (parent QoL) Emotional impact of PKU 44(31-63) 38(25-63) 27(16-37) Practical impact of PKU 21(8-46) 13(0-25) 8(0-17) Social impact of PKU 15(10-30) 13(5-25) 15(5-32) Overall impact of PKU 28(18-38) 21(12-35) 22(8-33) Child anxiety-blood test 50(25-63) 13(0-38) 25(0-56) Impact of child anxiety-blood test 63(63-88) 13(0-38) 63(44-88) Child anxiety-Phe levels 38(25-75) 50(25-75) 38(25-56) Financial impact of PKU 25(0-50) 25(0-50) 25(0-50) Information on PKU 25(25-50) 25(0-50) 25(19-50) Supplement administration Adherence to supplements 0(0-25) 0(0-25) 25(17-42) Guilt if poor adherence to supplements 75(50-100) 50(25-75) 75(50-82) Impact of supplements on family 25(0-25) 0(0-25) 0(0-44) Practical impact of supplements 33(17-50) 8(0-33) 25(8-33) Dietary protein restriction Management of supplements 0(0-25) 0(0-25) 0(0-25) 135  Domain scores Canada Europe Australia median (IQR) median (IQR) median (IQR) Adherence to dietary protein restriction  0(0-0) 0(0-25) 29(26-42) Management of dietary protein restriction  33(17-42) 21(5-38) 21(15-31) Practical impact of dietary protein restriction  54(36-71) 29(14-46) 43(29-57) Guilt if dietary protein restriction not followed 75(50-100) 50(25-75) 75(44-81) Child food enjoyment  25(0-25) 25(0-25) 25(0-31)  The results of this study highlight the importance of acknowledging the emotional challenges, anxiety, and practical burden of dietary restrictions on parents who provide care for children with PKU in Canada. PKU requires multifaceted care, a large component of that being domestic. It is possible that parental health, emotional wellbeing, and coping mechanisms might also influence family ecology and the quality of care that children with PKU receive at home.   In conclusion, improving parental wellbeing through better supports is one of the ways to potentially improve suboptimal outcomes in their children.  It appears that challenges faced by parents who care for children with PKU are similar across countries, with some variance. More research is needed to elucidate the causes of increased parental stress in Canadian parents of children with PKU, as well as what groups of parents are at greater risk. Engaging parents / caregivers in participatory research can provide great insight into the needs of this population. These results should also encourage the development of care, support strategies, and interventions that focus on a family-oriented approach to PKU care.   In spite of the aforementioned challenges, Canadian children with PKU overall receive state-of-the-art care. Healthcare providers do their best to achieve the most optimal outcomes. Despite the advances in PKU care, current treatment modalities do not provide a cure for this condition. Perhaps advances in gene therapy will provide a cure, but until then, a Phe-restricted diet can only modify the natural course of the disease and some residual morbidity should be 136  expected. Thus, dietary treatment might have reached its therapeutic plateau leaving room for other strategies to be considered in the pursuit of improved health outcomes. Where addressing gaps in PKU knowledge and promoting better adherence to the guidelines might be employed, here we suggest that the improvement of wellbeing of caregivers may be an additional venue through which we can optimize health outcomes in Canadian children with PKU.  137  Chapter 6: Conclusion  6.1 Summary of findings and contributions to the practice-based evidence  The purpose of this thesis was to draw a comprehensive picture of the current state of PKU management in Canadian metabolic treating centres. Specifically, the associated outcomes of Phe control in children with PKU and quality of life of parents who provide care at home were focused on. Overall, Canadian metabolic healthcare providers follow published guidelines and provide state-of-the-art care to children with PKU. However, healthcare providers’ surveys demonstrated areas of variation and different approaches to care delivery. The surveys also revealed that variation in management practices was likely due to a lack of good quality empirical evidence. Directed by survey results, the analysis of longitudinal chart-abstracted clinical data supported the findings on variation in care. Yet, striking differences were found in both diagnostic and follow-up practices of PKU management, including defining PKU severity (PAH deficiency phenotype), performing neonatal BH4 loading test, setting treatment targets, neuropsychological assessments, provider-patient communication and organization of care in metabolic treating centres. These practices have the potential to directly or indirectly impact individual health outcomes, though most immediately, some of them impact patient’s blood Phe levels (or Phe control).   Furthermore, the analysis of intermediate outcomes revealed that while the blood Phe levels of most children with PKU stay within the currently recommended therapeutic range, there is a substantial proportion “outside the range”. The proportion of children with “above upper limit” blood Phe levels increases with child’s age, most likely indicating diet relaxation.   138  Finally, as demonstrated by the results of the PKU-specific quality of life survey, parents of children with PKU identified significant emotional challenges associated with PKU care. This was not associated with the quality of Phe control or mean Phe levels.  Together, these findings charted a broad landscape of pediatric PKU care and associated outcomes in Canada. This knowledge provides a basis for the direction of further improvements.   6.2 Study limitations One of the limitations of this study was that each research project was focusing only and entirely on the pediatric PKU population. Another limitation was the study population, which included children with inherited disorders born from 2006 up to 2015; thus the study did not include children older than 11 years.  6.2.1 Limitations of healthcare providers’ survey study Both surveys were cross sectional studies. There were no published studies on assessment of nutritional or clinical PKU management practices in Canada prior to the distribution of the surveys or publication of ACMG/GMDI consensus guidelines. Therefore, we could not assess changes in the nutritional or clinical management practices attributable to the potential effect of the new consensus recommendations.  Both surveys focused on pediatric PKU populations only, inhibiting comment on the transition to adult care and adult clinical management. While our response rate was reasonable and similar to other surveys of healthcare providers, representing the majority of Canadian metabolic centres, the views of participants might not represent those of all metabolic healthcare providers of Canada.  139  6.2.2 Limitations of clinical data analysis  Information on natural protein (Phe) consumption, a very important component of nutritional PKU management, was not accessible at the time of thesis data analysis resulting in a limitation.  The inclusion criteria for CIMDRN study was limited to the PKU pediatric population born between 2006 and 2015, thus the data analysis was limited to a fraction of the PKU pediatric population.      There was very little information on patients with mild HPA. This is for several reasons: (1) the patients with mild HPA are usually followed up with in the clinic for only a few years, depending on the local practices; (2) patients with mild HPA might find clinic visits redundant and stop attending; (3) even if adherent to clinic follow up, usually these clinic visits are rare and this may have reduced opportunities for enrollment into the CIMDRN cohort study, which could be done remotely but was more frequently completed in person.  Finally, the linkage between practices delivered by individual healthcare providers’ and their patients’ outcomes was not feasible due to three reasons: (1) the information on HCPs delivering care to individual patients was not recorded in CIMDNR database (2) this project did not focus on assessing the quality of care provided by individual healthcare providers. 6.2.3 Limitations of the QoL study One of the major PKU-specific quality of life questionnaire limitations was the absence of data on common QoL predictors such as information on marital status, household income, level of education, ethnicity, cultural factors, and comorbidities.  Another potential limitation of this study was that we did not compare health-related quality of life in subgroups, specifically those who were treated with BH4 (sapropterin) and 140  those who were not. Comparison of health-related quality of life in subgroups was not possible due to the small sample size of those who were treated with medication. BH4 treatment would allow a more liberalized diet in patients who respond to this treatment. We did not analyze the natural protein intake, which indirectly reflects individual’s diet restriction, because the data was not available at the time of the analysis.  Also, we were not able to separately compare the quality of life of parents of children with mild hyperphenylalaninemia (with a relatively relaxed diet) to other groups of PKU severity due to a small sample size in the mild HPA group (n=7, 11%). In addition, we did not have an opportunity to explore the type of diet prescribed to mild HPA participants: we did not know if patients were prescribed the maximum protein restriction alone, or if the protein restriction was prescribed in combination with medical formulas. However, it is recognized that type of diet could potentially influence the parents’ reported quality of life.  Finally, while the recall period of the questionnaire is mainly focused on the past seven days (except “general feeling” where the recall period was “general”) the associated quality of Phe control and mean blood Phe levels were reflecting two years preceding the survey.  6.3 Recommendations for the optimization of PKU care and future research  This thesis revealed both management practices and research needs that warrant the attention of the Canadian PKU community. These diagnostic, nutritional and clinical practices may have implications on long-term PKU-related outcomes.  Firstly, there is a need to standardize procedures of diagnostic testing and severity classification of PKU. Improved understanding of the timely phenotype classification can inform expectations about PKU severity and address severity-specific nutritional and clinical care needs.  141  In addition, standard clinical PKU classification would help to generate practice-based evidence of a better quality. It is, perhaps, beneficial that existing practice on PKU classification should be considered for revision in Canadian metabolic centres. As part of improved diagnostic testing, the need and standardized procedure of neonatal BH4 loading test should also be revised. This necessitates generating more evidence on the test efficacy and recommendations on its practical applications in the clinical setting.   Secondly, there is a need to generate reliable evidence on the association between exposure to elevated and variable blood Phe levels during the first weeks of life and long-term health outcomes. It is plausible that irreversible changes in white matter might occur during this time but the evidence on timing of white matter changes in PKU is very limited (30) (31) (32). The delay in achieving good Phe control soon after birth necessitates strategies to optimize the time it takes to reduce blood Phe levels and their fluctuation, shortly after the NBS results are received.  Thirdly, there is a need to address effective and pro-active communication between healthcare providers and patient / family / caregivers. Pro-active communication is specifically important to reinforce individual treatment adherence around the time children with PKU enter elementary school.  Family-oriented programs and strategies to reduce stress and overcome emotional challenges of PKU experienced by families and patients should be encouraged. By alleviating stress family ecology improves, potentially improving intermediate outcomes in some children. Implementing more family-focused strategies of care should help ameliorate the challenges of chronic long-term stress on Canadian families living with children with PKU.  Additionally, in order to ensure a smooth transition to school and prevent decline in diet compliance, the PKU child’s care needs should be effectively communicated with school 142  personnel.  Implementing educational programs for schools will almost certainly require more dedicated metabolic personnel, as well as multidisciplinary care resources.   Fourthly, there is a need to develop simple and sensitive screening tools for neuropsychological assessment which could be administered by a metabolic healthcare provider. Regular periodic screening for neuropsychological complications would assist in timely identification, a prompt referral to a neuropsychologist for more detailed evaluation, and early potential intervention.   Finally, future research is needed to generate more high quality evidence, and/or to use available evidence more efficiently. There is a need to develop comprehensive research strategies that would pursue practice-based research, international collaborations, and active patients’ engagement to address remaining gaps in PKU evidence. 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Key practices Recommendations France, 2005 Blood Phe levels at treatment initiation  >600μmol/L (>10mg/dL) Target blood Phe levels  120-300μmol/L (2-5mg/dL) until 10 years of age   <900μmol/L (<15mg/dL) until 15-18 years old  <1200-1500μmol/L (<20-25 mg/dL) in adult Duration of the follow up   Life-long  Frequency of Phe monitoring  1/week up to 2-3 years of age  2/month up to 5 years of age  1/month 5-10 years of age   1/every 3 month up until 16 years of age  1/year in adults  Germany, 1990 Blood Phe levels at treatment initiation ≥600μmol/L (≥10 mg/dL) Target blood Phe levels  ≤ 360μmol/L (≤6 mg/dL) up to 2-3 years of age   ≤600μmol/L (≤10mg/dL) at school age   ≤1200μmol/L(≤20 mg/dL) in adults Germany, 1997 Blood Phe levels at treatment initiation ≥600μmol/L (≥10 mg/dL) Target blood Phe levels  40-240μmol/L (0.7-4 mg/dL) up until 10 years of age   40-900μmol/L (15 mg/dL) up until 10-15 years of age   40-1200μmol/L (20 mg/dL) in adults  Frequency of Phe monitoring  1/week-2/week in 0-3 years old   2/week-1/month in 4-11 years old   Every 2-3 months in ≥17 years old  167  Key practices Recommendations United Kingdom, 1993 Blood Phe levels at treatment initiation >600μmol/L (>10 mg/dL) Target blood Phe levels  120-360μmol/L (2-6 mg/dL) until 2-3 years   120-480μmol/L (2-4mg/dL) in school age    120-700μmol/L (2-11.7mg/dL) in adults  Frequency of Phe monitoring  1/week up to 4 years of age   2/months in 4-10 years of age   1/month in >10 years old  National Institute of Health, Conference Statement 2001 Blood Phe levels at treatment initiation >600μmol/L (>10 mg/dL) Target blood Phe levels 120-360μmol/L (2-6 mg/dL) until ≤12 years   120-600 μmol/L (2-10 mg/dL) after 12 years are strongly encouraged but could be liberalized up to 900μmol/L (15 mg/dL) Frequency of Phe monitoring 1/week during the 0-12 months   2/months during >1-12 years   1/month after 12 years    168  A.2 Comparison of current key recommendations of ACMG/GMDI and European PKU guidelines.  ACMG/GMDI Guidelines, 2014 European PKU Guidelines, 2017 Treatment Initiation Infants whose (untreated) blood Phe levels >600 μmol/L (10 mg/dL) require treatment All patients with untreated Phe levels >360 μmol/L (6 mg/dL) should be treated  Initiate diet preferably within first 7 days  Treatment should be started no later than 10 days of age   Treatment of infants with sustained blood Phe levels of >360 μmol/L is recommended following appropriate review of the controversy with parents.   Treatment of patients with untreated Phe levels within 360-600 μmol/L for 12 years then discontinue if blood Phe levels are stable  Treatment for infants with Phe levels between 120 and 360 μmol/L is not recommended; follow up recommended for minimum 2 years. Patients with Phe levels <360 μmol/L do not require treatment.  Target blood Phe levels Maintain target levels of 120-360 μmol/L throughout life  120-360 μmol/L up to age 12 years  Blood Phe levels of 60-120 μmol/L are acceptable especially in patients with higher Phe tolerance 120-600 μmol/L after 12 years   Life-long treatment for patients with untreated Phe levels of >600 μmol/L PKU Monitoring other: tyrosine “With Phe level measurements” “Not routine monitoring” PKU Monitoring Other: amino acids Once a year  1st year: every 1-3 months, then every clinic visit Total protein intake 0-3 months: 2.5-3 g/kg  0-3 months: 2.1-2.5 g/kg  3-6 months: 2-3 g/kg 3-6 months: 1.7-1.9 g/kg 6-12 months: 1.6-1.8 g/kg 6-12 months: 1.6-1.8 g/kg 1-4 years: 1.5-2.1 g/kg 1-4 years: 1.3-1.6 g/kg >4 years 140% of RDA  >4 years 140% of FAO/WHO/UNU (194) Large Neutral Amino Acids (LNAA) Acknowledged use in adolescent and adults; contraindicated as monotherapy in pregnancy.  Not enough evidence to include in the guideline; not recommended under12 years of age or in pregnancy. 169  Assessment of nutritional and clinical management practices of PKU B.1 Survey questionnaire to assess nutritional management practices (dietitians’ survey)      METABOLIC DIETITIANS’ SURVEY  Assessment of the Nutrition Management Practices for Phenylketonuria in Canadian PKU Pediatric Population Principal Investigator:   Nataliya Yuskiv, MD, MPH, PhD Student at UBC (Experimental Medicine) Supervisors:   Sylvia Stocker, MD, PhD (BC Children’s Hospital, University of British Columbia)  Beth Potter, PhD (University of Ottawa, Children’s Hospital of Eastern Ontario, Research Institute)  Jean Paul Collet, MD, PhD (Canadian Family Research Institute, University of British Columbia)  Pranesh Chakraborty, MD, FRCPC (Children’s Hospital of Eastern Ontario, University of Ottawa) Co-Investigators:   Keiko Ueda, RD (BCCH, Vancouver, BC)  Valerie Austin, RD (The Hospital for Sick Children (Sick Kids), Toronto, ON)  Barbara Cheng, RD (BCCH, Vancouver, BC)   Alette Giezen, RD (BCCH, Vancouver, BC)   Erica Langley, RD (CHEO, Ottawa, ON)   Amy Pender, RD (McMaster  Children’s hospital, Hamilton, ON)   Suzanne Ratko, RD (CHWO, London, ON) In collaboration with the Canadian Inherited Metabolic Diseases Research Network (CIMDRN)170   SURVEY SECTIONS I. General questions about your practice II. Patient population and classification of PKU severity III. Guideline use IV. PKU biomarkers: monitoring frequency and target levels V. Recommended dietary intake of key nutrients VI. Recommended use of medical foods VII. PKU monitoring and communication VIII. Adherence to the diet IX. Routine clinic visit X. Health care team and communication  171   1. During the past 12 months, have you worked as a dietitian in a clinical setting that provides care to children with phenylketonuria (PKU)?       Yes                     No  If No, please briefly describe your professional interest in PKU:  The rest of the questions apply only to those dietitians who work in a clinical setting that provides care for children with PKU.  If you work as an adult metabolic dietitian or responded “No” to question # 1 for other reasons,                   we would like to thank you for your interest in our survey. We would appreciate it if you would please return your questionnaire in the envelope provided. This information is important and will be used in the survey data analysis. Thank you!   2. How many total years of experience do you personally have in providing nutritional care to patients with PKU?   < 1 year   1-2 years   3-5 years   6-10 years   11-15 years   >=16 years   Not applicable (I have never provided nutritional care to patients with PKU) 3. Do you currently work:       As a full-time dietitian        As a part-time dietitian               Other (please specify; e.g., on leave, casual)  ________________________  I. GENERAL QUESTIONS ABOUT YOUR PRACTICE If you answered “Yes” to question # 1, please proceed to the rest of the questions. Thank you! 172   4. If you currently work full or part-time as a dietitian, what proportion of your work time                         is dedicated to providing nutritional care to patients with PKU?   All of my time    Not all of my time but at least half of my time    Less than half of my time         None of my time    Not applicable   5. Approximately how many children with PKU who require nutritional management                                   (protein-restricted diet and/or BH4) are actively followed in your clinical setting currently?    5 or fewer   6-10  11-20  21-30  31-40  41-50  > 50 6. On average, approximately how many children with PKU who require nutritional management in your centre are newly diagnosed each year?  2 or fewer  3-5  6-10  11-20  21-30  >30   II. PATIENT POPULATION AND CLASSIFICATION OF PKU SEVERITY 173    7. What age range are the PKU patients who receive care within your current clinical setting?       Newborns and/or children only (up to 18)         Both adults and children         Other, please specify  _______________________________________________  8. How do you classify the severity of PKU in your patients?                                                                             (please check all that apply)    Based on blood phenylalanine (Phe) levels in the newborn (before treatment / diet)   Based on Phe tolerance at a certain age    Based on genotype   Other approaches, please specify_______________________________________  9. In your practice, what units do you use to report blood Phe levels to your patients?    mg/dl      µmol/L     Other ________ 10. Using the units you list in question 8, how do you define severity of PKU based on blood Phe levels in the newborn before treatment?          Mild HPA:  ________________________                     Mild PKU:  ________________________         Moderate PKU: ____________________                    Classical PKU:  _____________________         Other, please specify  _______________          I do not define PKU severity based on blood Phe levels in the newborn 11. At what age do you typically start using Phe tolerance to define the severity of PKU?    Less than 2 years old    2 years old    More than 2 years old     Other age, please specify  ________  174     Not applicable – I do not use Phe tolerance to define the severity of PKU   12. In your clinic practice, do you recommend the highest possible Phe intake that maintains metabolic control?                Yes                 No                     Other, please specify ________ 13. How often do you re-assess Phe tolerance?  At least once a year  Less than once a year but at least every 2 years  Less than every 2 years but at least every 3 years  Other (please specify when) _______________________________   Not applicable – I do not assess Phe tolerance 14. At which blood Phe levels do you make a decision to treat patients with a Phe-restricted diet?  (Please apply the units used in your practice.) Consistently elevated blood Phe levels of:    2 mg/dl  120 µmol/L   3 mg/dl)   180 µmol/L   4 mg/dl  240 µmol/L   5 mg/dl  300 µmol/L   6 mg/dl  360 µmol/L   7 mg/dl  420 µmol/L   8 mg/dl  480 µmol/L    Other, please specify ____________________________               Not applicable – I do not use blood Phe levels to make a decision about dietary treatment     15. Are you aware of any published guidelines on the nutritional management of PKU?         Yes     No III. GUIDELINE USE 175  15a. If yes, are any of these guidelines currently used to guide care in your centre?        Yes     No 15b. If yes to 15a, please provide the guideline/s reference/s: (i)  (ii)  (iii)   IV. PKU BIOMARKERS: MONITORING FREQUENCY AND TARGET LEVELS  16. Which biomarkers do you routinely monitor in your PKU patients? (please check all that apply)  Biomarkers: Monitor for most patients Monitor for some patients Rarely or never monitor Blood levels of phenylalanine    Blood levels of tyrosine    Blood Pre-albumin    Blood Albumin    Blood Iron levels    Bone density    Blood levels of essential fatty acids    Blood vitamin levels    Other (specify):__________________     17. What is your optimal target range of blood Phe? (please choose one that corresponds to 176  your accepted units if different from “mg/dl” or “µmol/L”):   Phenylalanine, mg/dl (µmol/L) 0-12 months >1-2 years >2-10 years >10-18 years <2 mg/dl (60-120 µmol/L)     2-6 mg/dl (120-360 µmol/L)     7-10 mg/dl (360-600 µmol/L)      Other, please specify  ________     Comments:   18. What would typically be your lowest acceptable average level of blood Phe as a long-term   treatment goal?  Please use the units you specified in question # 8 (i.e., mg/dl or µmol/L or other)       < 1 year old: ________        1-2 years old:  ________        3-10 years old: ________        11-18 years old: ________        Other, please specify: ________ 19. Would you ever be comfortable with a patient’s steady Phe levels being below 2 mg/dl (< 120 µmol/L), i.e., without the patient increasing his/her dietary Phe?         Yes           No 19a. If you answered “Yes” to question 18, please explain with example clinical scenario(s):  __________________________________________________________________________ 20. Do you recommend that patients maintain higher-end therapeutic range blood Phe levels                     and more liberal natural protein intake?      For nearly all patients 177    For most patients  For some patients  Rarely  Never  Other, please specify  ________ 21. Do you usually recommend keeping lower-end therapeutic range blood Phe levels resulting  in more restricted natural protein intake?   For nearly all patients   For most patients  For some patients  Rarely  Never  Other, please specify  ________   22. How do you assess individual nutritional needs? (please specify all methods that you use)        24-hours recall   3-day diet record  Food frequency review  Others, please specify   ________________________________________________  23. What resources (tools) do you use to calculate a patient’s daily intake of protein, phenylalanine and calories? (please specify all that apply)  23a. Online resources (websites, interactive calculators, apps, etc): __________________ 23b. Textbooks: ____________________________________________________________ 23c. Other, please specify :____________________________________________________ V. RECOMMENDED DIETARY INTAKE OF KEY NUTRIENTS 178    VI. RECOMMENDED USE OF MEDICAL FOODS  24. To what extent does your province cover the cost of medical formulas for your PKU patients?        All medical formulas are covered       Some medical formulas are covered       No medical formulas are covered       Other, please specify__________  25. How important are the characteristics below to decide which formula to prescribe for your patient? (please check one response for each characteristic)  Characteristics: Very Important Somewhat important Not at all important Patient’s age    PAH severity    Nutritional composition of formula    Availability of the product    Price of the product    Preferences of the patient or family    Other (specify):_______________     26. Is your choice of formula limited by the hospital “formula contract”?  Yes     No         Other      179     27. Please list the top 3 formulas that you currently recommend for your PKU patients: Patient's Age Formula 1 2 3 < 1 year     1-2 years    3-10 years    11-18 years     Other/Comments: _____________________________________________________________________________ 28. Do you ever discontinue prescribing formula for patients with PKU?   Yes for some patients across all forms of PKU (classical, moderate, mild)  Yes but only for some mild or moderate PKU cases (never for classical PKU patients)  Yes but only for some mild PKU cases (never for classical nor moderate PKU patients)  No, never for any patients with PKU  Other/Comments _________  29. Do you prescribe Large Neutral Amino Acids (LNAA) supplements?  Yes     No 30. To what extent does your province cover the cost of special low protein foods for your PKU patients? 180   All low protein foods are covered  Some low protein foods are covered  No low protein foods are covered  31. How would you define accessibility of the low protein foods for your PKU patients, in general:  Excellent accessibility – nearly all patients can easily access  Good accessibility – most patients can easily access  Moderate accessibility – some patients have difficulty accessing  Poor accessibility – most or all patients have difficulty accessing  VII. PKU MONITORING AND COMMUNICATION 32. On average, how often do you see patients with PKU and/or their caregivers in clinic?    Patient’s Age Visit frequency At least once            per week Less than once per week but at least once per month Less than once per month but at least once per year Less than   once per year Other < 1 year      1-2 years      3-10 years      11-18 years       33. On average, how often do you communicate* with your patients (caregivers) between clinic visits (routine monitoring)?  *Communication via phone, email, fax, etc. (outside of the clinic visit). Patient’s Age Average frequency of communication* 181  At least once            per week Less than once per week but at least once per month Less than once per month but at least once per year Less than   once per year Other <1 year      1-2 years      3-10 years      11-18 years       34. By what means do you communicate with your PKU patients and caregivers between visits?   (please check all that apply)       Email       Telephone        Text messages        Skype  Secure online platform for patients and/or caregivers        Mail         Fax        Other, please specify  __________     35. How often are your PKU patients asked to track and submit any diet intake information for clinic analysis? (please check only one)     Every day      With every monitoring blood Phe level      Only at clinic visits      This depends on the specific needs of the patient      I do not ask my patients to track diet intake      Other, please specify __________   VIII. ROUTINE CLINIC VISIT 182  36. If you request diet records from your patients, what diet records do you request most often?   (please check only one)  1 day diet record   3 days diet record               7 days diet record                            Other, please specify __________      37. Which nutrients do you routinely monitor, based on diet records? Nutrient: Monitor for most patients Monitor for some patients Rarely or never monitor Dietary Phenylalanine intake    Protein intake    Calorie intake    Fat intake    Vitamin intake (any)    Mineral intake (any)    Other (specify):__________________     38. What methods do you recommend for patients with PKU and their caregivers to self-monitor intake of phenylalanine? (please check all that apply)       Counting Phe exchanges:   1 exchange = 15 mg of Phe   Other exchange amount, please specify  __________       Regular blood work        Counting milligrams of dietary phenylalanine         Counting grams of dietary natural protein       Use of computer applications for phenylketonuria        Other, please specify  __________ 39.  Which of these methods do you recommend most often for patients with PKU and their  caregivers to self-monitor intake of phenylalanine? (please check only one) 183        Counting Phe exchanges       Regular blood work        Counting milligrams of dietary phenylalanine         Counting grams of dietary natural protein       Use of computer applications for phenylketonuria        Other, please specify __________  39a.Please provide the name(s) of the computer apps, if you use these: (i)  (ii)  (iii)  40. How often does your routine clinical visit assessment typically include: Assessment: Always Often Sometimes Rarely Never Dietary analysis      Diet education      Anthropometric measures  (e.g., height and/or weight)      Other (specify):__________       IX. ADHERENCE TO THE DIET 41. Where do your patients collect blood samples for routine monitoring of phenylalanine?                (please check all that apply)   Metabolic clinic    Local laboratory, closer to patient’s house  184   Home sample collection        Other, please specify __________   42. How do you assess your patients’ adherence to formula intake? (please check all that apply)        Monitoring weight and height       Assessing PKU-related clinical signs and symptoms        By analyzing written dietary questionnaires        By laboratory tests, below:  Phenylalanine  Albumin  Pre-albumin  Iron  B12  Tyrosine  Other lab tests,   please specify ____________________      By patient’s/caregiver’s verbal report        Checking how much formula was released by the dispensing authority       Not applicable – I do not assess patients’ adherence to formula intake       Other, please specify __________  43. How do you assess your patients’ adherence to low protein foods?  (please check all that apply)       Patients’ verbal reports       Parents’/ caregivers’ verbal reports       By analyzing written dietary questionnaires       Other, please specify __________        Not applicable - I do not assess adherence to low protein food 185  44. What causes you to be concerned regarding a patient’s non-adherence to diet or drug  therapy?  (please check all that apply)       Not doing blood dots on a regular basis       Not showing up in clinic       Not pulling formula from the sources that supply formula        High Phe levels        Low tyrosine levels       Low albumin, iron, and/or B12       No regular contact with dietitian       Not applicable       Other, please specify  __________   45. In your opinion, which one factor indicates non-adherence most reliably?   (please check only one)                                                    Not doing blood dots on a regular basis       Not showing up in clinic       Not pulling formula from the sources that supply formula        High Phe levels        Low tyrosine levels       Low albumin, iron, and/or B12       No regular contact with dietitian       Not applicable       Other, please specify __________ 46. What do you usually recommend to improve a patient’s adherence to the diet?  (please check all that apply)       Motivational interview techniques       Individualized PKU nutritional counseling       Regular reminders for Phe blood dots       Reporting results of blood dots to patients 186        Not applicable       Other, please specify __________  47. Of these, which one technique do you think is most successful in improving adherence to diet?  (please check only one)       Motivational interview techniques       Individualized PKU nutritional counseling       Regular reminders for Phe blood dots       Reporting results of blood dots to patients       Not applicable       Other, please specify __________  X. HEALTH CARE TEAM AND COMMUNICATION  48. In your centre, is there at least one dietitian who sees only PKU patients?     Yes           No  49. Your PKU health care team includes the following: (please check all that apply)  Metabolic physician/s   Metabolic dietitian/s   Metabolic nurse/s  Psychologist/s  Social worker/s  Clinical biochemist/s    Other, please specify _______________  50. How often do you discuss each PKU patient’s nutritional plan and treatment goals with others  involved in a patient’s care?  On a regular basis for most patients    Sometimes but not as a routine part of my practice 187   Rarely   Never    Other, please specify  _________  51. In your opinion, how effective are the established within-team communication method(s) in    your clinic in addressing the majority of PKU-related concerns?      Highly effective            Somewhat effective       Not effective  Not applicable  52. Where do your PKU patients transition after they reach adulthood?               Adult metabolic clinic              Continue at pediatric metabolic centre       Other, please specify: ____________________________ Thank you for completing the survey  for the assessment of PKU nutrition practices in Canada!  Sincerely, the CIMDRN team       188          B.2 Survey questionnaire to assess clinical management practices (physicians’ survey)     Project Title:  “Clinical management of PAH deficiency in Canadian metabolic centres”.   In collaboration with the Canadian Inherited Metabolic Diseases Research Network (CIMDRN)  METABOLIC PHYSICIANS’ SURVEY   189  Assessment of the PAH Deficiency (PKU) Clinical Practices  in Canadian Metabolic Centres      Funded by: Canadian Institutes of Health Research   GENERAL QUESTIONS ABOUT YOUR PRACTICE 1. During the past 12 months, have you worked as a physician in a clinical setting that provides care to pediatric patients with Phenylalanine hydroxylase (PAH) deficiency (PKU) ?  Yes   No If No, please briefly describe your professional interest in PAH deficiency:   The rest of the questions apply only to physicians who work in a clinical setting that provides care for children with PAH deficiency. The current survey is designed to assess clinical management practices in pediatric PKU. If you see only adult patients, and/or responded “No” to question # 1, we would like to thank you for your interest in our survey.  We still would appreciate it if you return your (empty) questionnaire in the envelope provided. This information will help us to understand our survey population. Thank you!    If you answered “Yes” to question # 1, please proceed to the rest of the questions. Thank you.  2. How many total years of experience do you personally have in providing clinical care to  patients with inherited metabolic diseases?   < 1 year   1-2 years   3-5 years   6-10 years 190    11-15 years   >=16 years  3. Do you currently work:  Full-time  Part-time  Other (please specify – e.g., on leave)       4. What proportion of your work time is dedicated to providing clinical care to patients with  PAH deficiency?  All of my time   At least half of my time   Less than half of my time   None of my time   Not applicable  Other: please comment    PAH DEFICIENCY CLASSIFICATION AND DIET INITIATION  5. What criteria do you use in classification of the severity of PAH deficiency in your pediatric patients? (Please check all that apply)                                                                                  Untreated blood Phenylalanine (Phe) levels in the newborn  191   Phe tolerance at a certain age   Catabolic Phe levels (when a patient is sick)  Residual in-vitro PAH activity   Genotype  I do not classify the severity of PAH deficiency in my clinical practice  Not applicable   Other approaches, please specify     6. At what blood Phe level do you make a decision to initiate dietary and/or pharmacotherapy in a newly diagnosed newborn with PAH deficiency?     120 µmol/L (  2 mg/dl)   180 µmol/L ( 3 mg/dl)  240 µmol/L ( 4 mg/dl)  300 µmol/L  ( 5 mg/dl)  360 µmol/L ( 6 mg/dl)  420 µmol/L ( 7 mg/dl)  480 µmol/L    ( 8 mg/dl)  540 µmol/L    ( 9 mg/dl)  600 µmol/L    ( 10 mg/dl)    Other, please specify:   7. For how long do you follow male patients with non-PKU HPA who do not require dietary treatment in the clinic?     < 1 year  1-2 years 192   >2 years   Life-long follow up  Not at all  Other, please specify:   8. For how long do you clinically follow female patients with non-PKU HPA who do not require dietary treatment in the clinic?     < 1 year  1-2 years  >2 years  Life-long follow up  Not at all  Other, please specify:    9. For how long would you recommend treatment for a female patient whose untreated Phenylalanine levels are between 360 μmol/L and 600 μmol/L (6-10 mg/dl)?   Not applicable – I do not commence treatment in female patients whose untreated Phe         levels are ≤ 600 µmol/L (≤ 10 mg/dl)  I would recommend life-long treatment  I would recommend treatment until the age of 12 years  I do not treat female child with Phe level of ≤ 600 µmol/L, but would treat when pregnant  Other, please specify:    193  10. For how long would you recommend treatment for a male patient whose untreated Phenylalanine levels are between 360 μmol/L and 600 μmol/L (6-10 mg/dl)?   Not applicable – I do not commence treatment in male patients whose untreated Phe levels       are ≤ 600 µmol/L (≤ 10 mg/dl)  I would recommend life-long treatment  I would recommend treatment until the age of 12 years  Other, please specify:        PAH DEFICIENCY BIOMARKERS: TARGET LEVELS  11. In your opinion, what is the optimal target range of blood Phe for children with PAH deficiency in the following age groups? (Please choose one response for each age group):   Phenylalanine, µmol/L (mg/dl) 0 to 12 months >1 to 2 years >2 to 10 years >10 to 18 years 60-120 µmol/L (1-2 mg/dl)         60-240 µmol/L (1-4 mg/dl)     60-360 µmol/L (1-6 mg/dl)     120-240 µmol/L (2-4 mg/dl)     120-360 µmol/L (2-6 mg/dl)     120-600 µmol/L (2-10 mg/dl)     360-600 µmol/L (6-10 mg/dl)      194    Other, please specify:   12 If patient’s protein intake is sufficient, to what extent would you regard blood Phe levels in the range of ≤120 µmol/l (≤2 mg/dl) as “low”?   I would regard this as low for nearly all patients  I would regard this as low for most patients  I would regard this as low for some patients  I would rarely regard this as low  I would never regard this as low  Other, please specify       DIAGNOSTIC CONFIRMATION: PAH MUTATION ANALYSIS AND  BH4 LOADING TEST   13. To what extent do you routinely perform or recommend PAH gene mutation analysis for your patients with PAH deficiency?   For nearly all patients  For most patients  For some patients  Rarely  Never  Other, please specify     195  14. If you don't perform or recommend gene mutation analysis, please let us know why:   15. If you perform or recommend PAH mutation analysis in a patient with PAH deficiency, what test do you prefer to start with if the parent/sib mutation(s) is not known?  (Please check one response only)  Sequence the entire PAH gene  PAH gene sequencing plus del/dup analysis   Look for the most frequent mutations first    Other, please specify   16. Do you perform the neonatal tetrahydrobiopterin (BH4) loading test?  Yes, to diagnose long-term BH4 responsiveness only  Yes, as a part of tetrahydrobiopterin deficiency workup only  Yes for both reasons above  No, I do not perform neonatal BH4 loading test  17. If you perform neonatal loading test to diagnose long-term BH4 responsiveness, do you perform it for:                 All newborn patients with PAH deficiency   Only for selected newborn patients with PAH deficiency  Other, please specify:     18. If you do not perform neonatal BH4 test to assess responsiveness to BH4 treatment, what prevents you from it? (Please check all that apply)                                                                             196   Mostly logistic difficulties    I do not see any benefit in performing BH4 loading test in neonatal period   Concerns regarding future funding of Kuvan   Other, please specify:     19. If you routinely perform or recommend a BH4 loading test for newborn children with PAH deficiency, please specify when you typically measure Phe levels after BH4 dosing? (Please check all that apply):  Pre-dosing (baseline)  1 hour after dose  2 hours after dose  4 hours after dose  6 hours after dose  8 hours after dose  24 hours after dose  48 hours after dose  72 hours after dose  Not applicable, I don’t routinely perform or recommend a BH4 loading test for newborn       children with PAH deficiency  Other, please specify:   20. What BH4 dose do you typically use for BH4 loading tests in newborns with PAH deficiency? (Please choose one answer)   10 mg/kg   20 mg/kg  Starting from 10 mg/kg and then increasing the dose to 20 mg/kg  197   Not applicable, I do not use BH4 loading tests in newborns with PAH deficiency  Other, please specify:   21. Do you test all newborns with elevated NBS Phe for disorders of BH4 synthesis and regeneration?   Yes  No  22. If “No” to question 21, please specify, which (if any) cohort do you usually test for disorders of BH4 synthesis and regeneration:   23. What is (are) your initial step(s) to rule out primary BH4 deficiency in a newborn with high   blood Phe levels (Please check all that apply)?  Hyperphenylalaninemia gene panel  Pterins in urine  DHPR activity in blood spot  Other, please specify:   24. If a patient with PAH deficiency shows a reduction of blood Phe level upon a neonatal BH4 loading test, in most cases, do you start KUVAN right away?   I start with KUVAN right away along with Phe restricted diet  I usually add KUVAN later, when the patient has stable blood Phe levels within the treatment         range and then see whether the diet can be liberalized  I use the result of the initial BH4 test to label the patient as potentially responsive to add      KUVAN at a later stage. 198   Not applicable, I don’t use BH4 loading tests in neonates  Other, please specify:   25. How do you define a positive result with respect to responsiveness to the neonatal BH4 loading test?  At least _______% blood Phe reduction in ________ hours  Other, please specify:   DECISION TO TREAT WITH SAPROPTERIN (KUVAN®)  26. For pediatric patients with PAH deficiency, do you offer a trial of KUVAN treatment:   For nearly all patients   For most patients   For some patients   Rarely  Never  Other, please specify:   27. Before starting treatment with KUVAN, do you determine a patient’s Phenylalanine tolerance (maximum dietary Phe consumption that still maintains blood phenylalanine within therapeutic range)?   For nearly all patients   For most patients   For some patients   Rarely 199   Never  Other, please specify:    ASSESSMENT OF RESPONSIVENESS TO KUVAN  The following questions ask how you determine long-term responsiveness to KUVAN treatment in your pediatric patients.   28. Is a reduction in blood Phe levels from baseline one of the factors that you use to define long-term responsiveness to KUVAN in children?  No  Yes  29. If yes to question 28, please provide information about how you define a long-term stable reduction in blood Phe levels compared to baseline (Please check all that apply):   At least 50% Phe reduction from pretreatment Phe levels   At least 30% Phe reduction from pretreatment Phe levels   Other, please specify:   30. When evaluating responsiveness to KUVAN in children with PAH deficiency, how do you determine reduction in Phe levels from baseline? (Please check all that apply)  General Phe level trend observation   Comparing mean Phe levels at baseline and on treatment  Use the last Phe level before treatment and the lowest Phe level on treatment   Not applicable: I do not use Phe levels in evaluating KUVAN treatment  200   Other methods, please specify:   31. Do you determine long-term responsiveness to KUVAN in children at least partly based on an increase in natural protein intake (improved Phe tolerance)?      No  Yes  32. If yes to question 31, how do you determine an improvement in Phe tolerance?  Increase in mg / day Phe intake while maintaining desired Phe levels  Increase in mg / kg / day Phe intake while maintaining desired Phe levels  Increase in consumption of natural protein (g/day)  Other, please specify:   33. What increase in Phe tolerance (%) do you consider sufficient to determine a good response?   _______________(%)  I do not regard Phe tolerance as an indicator of responsiveness to KUVAN    34. From your clinical experience with PKU, what was the longest time period in determining responsiveness to KUVAN (approximately)?     Up to 7 days   From 7 days to 4 weeks    From 1 month to 6 months                From 6 months to 1 year  Other, please specify: 201   35. In general, what would you consider the single best indicator of KUVAN effectiveness? (Choose only one answer, please)  Stable reduction of blood Phe levels  Stable increase in Phe tolerance  Improvement in neuropsychological assessment even if Phe levels did not significantly      changed from the baseline   Improved reported quality of life of patient / family even if Phe levels have not significantly      changed from the baseline   Other, please specify:   36. Do you refer your PKU patients for the formal neuropsychological assessment? (Choose only one answer, please)   Yes, I refer all PKU patients, to screen for potential neuropsychological deficits even if they        do not show/experience any symptoms   Yes, but only selected PKU patients with suspected deficiencies   No, I do not order formal neuropsychological assessment for PKU patients   Other, please specify:  * By “Formal Neuropsychological Assessment” we mean either one or all of the following: referral for registered psychologist for the evaluation of developmental and/or intellectual abilities; executive functioning; behavioral / emotional assessment.     37. If “No” to question 36, what prevents you from ordering formal neuropsychological assessment for your PKU patients? (Please check all that apply)  Psychology services are not easily accessible for a patient  I do not see any benefit of screening non-symptomatic PKU patients for potential            neuropsychological deficits   202   Other, please specify:   38. To what extent do you perform a baseline (pretreatment) neuropsychological assessment test for children treated with KUVAN?  (Please check all that apply)  For nearly all patients who plan to take KUVAN   For most patients who plan to take KUVAN  For some patients who plan to take KUVAN  Rarely  Never  Not applicable  Other, please specify:   39. If applicable, in what time interval, after starting KUVAN treatment, do you typically order a follow up neuropsychological assessment test? (Please check all that apply)   In 3 months  In 6 months  In 1 year  Not applicable - I do not routinely order a follow up neuropsychological assessment test to       evaluate the treatment outcome in children with PAH deficiency treated with KUVAN  Other, please specify:   40. Do you routinely use parent and/or patient-reported feedback when assessing the outcome of treatment with KUVAN in children with PAH deficiency?  No  Yes 203   41. If yes to question 40, what tools do you use to assess patient-reported outcomes? (Please check all that apply)  Standardized quality of life questionnaires  Non-standardized quality of life questionnaires  Parent-reported feedback  Patient-reported feedback, when applicable   Structured interviews   History taking only  Other, please specify   GUIDELINE USE    42. Do you use a “sick day management” protocol (guideline) for your patients with PKU?  No  Yes  43. If “Yes” to question 42, is your PKU sick day management protocol locally developed?    Yes   No          44. If “No” to question 43, please provide a reference or description of used guideline:    45. Do you use any published PAH deficiency guideline(s)?   No 204   Yes 46. If yes to question 45 : what published PAH deficiency guidelines do you use in your practice (Please check all that apply)?   US guidelines  ACMG PAH deficiency diagnosis and management guideline (Vockley       et al, 2014)  NIH Consensus Panel Recommends Comprehensive Approach to Life       Long Care for PKU (2000)  Nutrition management of PAH deficiency (Singh et al, 2014)  European guidelines  van Spronsen et al, 2017   UK PKU guidelines, 2004  Local guidelines   Other, please specify:    47. If you use published guidelines: overall, do you agree with what is written in the guidelines that you use?   Yes, for nearly everything  Yes, for most parts  Yes, for some parts  Other, please specify:    48. In your opinion, is there anything that you would like to add to the guideline(s)?  205  Please specify:  No   Yes (please let us know):    Thank you for completing the survey  on the assessment of current PKU clinical management practices in Canada!  Sincerely, the CIMDRN team              206  B.3  Factors influencing prescription of medical formula  Distribution of responses to survey questions on use of medical formula n(%) How important are certain factors in deciding which formula to prescribe for your patient?   Very important Somewhat Important Not at all important Nutritional composition of formula  18 (95%) 1 (5%) 0 Patient’s age 17 (89%) 2 (11%) 0  Preferences of the patient or family  17 (89%) 2 (11%) 0 Availability of the product 15 (79%) 4 (21%) 0  Price of the product 4 (21%) 13 (68%) 2 (11%) PAH severity 4 (21%) 8 (42%) 7 (37%) Other:  Palatability and convenience for the patient or family  Please list the top 3 formulas that you currently recommend for your PKU patients <1 year 1-2 years 3-9 years 10-18 years Periflex Infant (19, 53%) Phenyl-free 1 (7, 37%) Periflex Junior a (1, 5%) Phenyl-free 1 (5, 26%) Periflex Junior a (5, 26%) Periflex Junior Plus b (3, 16%) Periflex Junior Plus b (4, 21%); Periflex Junior a (4, 21%); PhenylAde Essential (3, 16%) Periflex Advance (4, 21%) PhenylAde Essential (4, 21%) Phenyl-free 2 (3, 16%) a This survey captured the transition from Periflex Infant to Periflex Junior.   b This survey captured the product discontinuation of Periflex Junior changing to Periflex Junior Plus              207  Overview of the indices of Phe control and classification of PKU severity in treating centres C.1 Overview of the indicators of Phe control reported in the published literature Author(s), year of publication/journal  Main research question  Measured indices of Phe control  Duration of measuring  Phe control and age at outcomes assessment Categories of Phe control (if applicable); method of calculation of indication Study design De la Parra A, García MI, Hamilton V, Arias C, Cabello JF, Cornejo V. 2017 MGM (132) To understand the effect of a more strict metabolic control during the first 12 months of life on metabolic control later in life, psychomotor development and cognitive functioning in individuals with PKU. Average Phe: Mean Phe ± SD Variability of Phe: mean SD in different age groups Phe control (retrospective): first year of life (12 months)  Outcome (prospective): psychomotor development at 12, 24 and 30 months and neurocognitive development at 4 and 8 years (same cohort).   Phe measured during the first 12 months of life:  Group A. (Very good metabolic control): mean Phe < 240 μmol/L, and SD < 4.5.  Group B (Good metabolic control): mean Phe (≥240 to < 360 μmol/L), and SD < 4.5.  Group C (Poor metabolic control): Phe  ≥360 μmol/L or Phe ≥ 4 with a SD > 4.5.  Groups are compared during the following age periods: 0-11m 12-23m 24-35m 3y-3y 11m 4y-5y11m 6y-7y11m 8y-9y11m  Design: Comparison of cross-sectional standardized neurocognitive assessment with historic metabolic control quality.   Hood A, Grange D, Christ S, Steiner R, White D. 2014, MGM (66) Association between indices of Phe control across the lifetime and cognitive outcomes 6 indices of Phe control: (1): Average Phe: (a) mean Phe (simple lifetime mean) and (b) IDC (index of dietary control). (2) Phe variability: (a) SD Phe, (b) SEE Phe and (c) % of spikes of Phe (number of Phe levels higher then upper limit for succeeding and preceding Phe levels). Phe control (retrospective): lifetime Phe control value prior to neurocognitive assessment, including 3 developmental epochs: <5 yo; 5-9.9yo and ≥10 yo. Outcomes (IQ/executive) measured in patients of school age (6 to 18 years) N/A Design: Comparison of cross-sectional standardized neurocognitive assessment with historic metabolic control quality.  Conclusion: “Overall, our findings were most clearly reflected by single indices of average Phe (mean Phe) and variability in Phe (SD Phe) over the lifetime”.    208  Author(s), year of publication/journal  Main research question  Measured indices of Phe control  Duration of measuring  Phe control and age at outcomes assessment Categories of Phe control (if applicable); method of calculation of indication Study design Hartnett C, Salvarinova-Zivkovic R, Yap-Todos E, Cheng B, Giezen A, Horvath G, Lillquist Y, Vallance H, Stockler-Ipsiroglu S, 2012  (167) To assess how well patients with classical PKU were meeting the therapeutic goals.  (1) Indicator of Phe control: (%) of the Phe levels outside of the therapeutic range (above or below)  (2) Exposure to Phe:  mean Phe for each individual; Phe level greater than 360 μmol/L was considered as high Phe burden  (3) Indicator of (high) Phe fluctuation:  Phe standard deviations (SDs): SD > 240 µmol/L within respective age period. Three age periods:  1m-1 y, 1-6 y, 6-12 yrs. Longitudinal retrospective cohort. Blood Phe levels during the first four weeks of life were excluded.  Three groups of Phe control: (a) Poor Phe control with less than 30% of all blood Phe levels within therapeutic range; (b) Fair Phe control with 30%-60% of Phe levels within therapeutic range and (c) Good Phe control with more than 60% of Phe levels within therapeutic range. Therapeutic Phe range was defined as 120-360 μmol/L.  Retrospective review of blood Phe concentrations in 33 patients with classical PKU MacDonald A, Nanuwa K, Parkes L, Nathan M, Chauhan D, Current medical Research and opinion, 2011 (168) Association between treatment and health outcomes % of returned Phe levels within therapeutic range: denominator – total number of eligible subjects for that period  Age groups: 0-5; 6-10, 11-17 and 18+  If 70% of Phe tests were within therapeutic range - this was considered satisfactory; Weighted mean calculation (to obtain single estimate (>70%) for all years):  (%2004xn2004+%2005xn2005+%2006xn2006 etc) where n year is the number of all patients with sufficient Phe data for that year; % year is % of patients (out of n year) who have ≥ 70% of their recorded Phe concentrations within the target range for that year  (years 2004-2008)   Retrospective review of PKU longitudinal data  209  Author(s), year of publication/journal  Main research question  Measured indices of Phe control  Duration of measuring  Phe control and age at outcomes assessment Categories of Phe control (if applicable); method of calculation of indication Study design Waisbren SE, Noel K, Fahrbach K, Cella C, Frame D, Dorenbaum A, Levy H, MGM, 2007 (51) To assess the reliability of blood Phe levels as a predictive biomarker of clinical outcomes (measured by IQ) Lifetime: Mean of 6-12-months medians; Mean Phe ± SD  Phe control during (a) critical periods: from 0 to 6-12 years), (b) lifetime –lifetime IDC, (c) concurrent Phe levels.   (b) ‘‘Lifetime’’ Phe level was defined as the mean of 6- or 12-month median assessments for each patient, from birth to the last measurement in each study, and is described as a lifetime Index of Dietary Control (IDC). (c)‘‘Concurrent’’ assessments included Phe levels obtained at the time of testing of other clinical outcomes Systematic review &meta-analysis.   210  C.2  Criteria used to determine the severity of PKU as derived from the medical charts by centre where criteria were recorded (153). Consenting Centre (de-identified) Current Disease Classification & Definition Noted in Metabolic Chart Mild hyperphenylalaninemia Mild PKU Classical PKU Not specifieda Centre A Relatively Phe tolerance; untreated Phe level <600 umol/L Untreated Phe levels 200-300 umol/L  Relatively high Phe tolerance; untreated Phe level <600 umol/L Untreated Phe level of >1200 umol/L Not applicableb Centre B Not applicable.  Definition(s) not reported Phe level >1200 umol/L Not applicable. Centre C Atypical or Benign (It appears to be based on Phe numbers remaining optimal (between 120-360 umol/L) with no dietary restrictions.) Phenylketonuria atypical (Phe levels are remaining well controlled (120-360 umol/L) with a regular diet) Definition(s) not reported Atypical PKU, Benign PKU - definitions not reported Centre D Initial Phe level 120-360 umol/L Initial Phe level 361-600 umol/L Initial Phe level <600 umol/L Initial Phe level <1200 umol/L Initial Phe level 600-1200 umol/L Phe level <600 umol/L Initial Phe level <1200 umol/L Phe level >1200 umol/L Initial Phe level >1200 umol/L Benign HPA - no definition reported Centre E Phe level 120-360umol/L classifies range that does not require treatment Not applicable. Compound heterozygote (no elaboration provided) Not applicable. Centre F Phe level 120-360 umol/L Phe level 100-400 umol/L Phe level 400-600 umol/L Phe levels elevated but remain below 500 umol/L Not applicable. Phe level >1000 umol/L Not applicable. Centre G Phe level <360 umol/L Phe level <600 umol/L Phe level 600-1200 umol/L Phe level >1200 umol/L Not applicable. Centre H Phe normal range < 75mmol/L (benign) Initial Phe level 120-360 umol/L Persistent mild elevation of Phe  Initial Phe level 361-600 umol/L Initial Phe level 600-1200umol/L Phe level >1200 umol/L Initial Phe level >1200 umol/L Biopterin unresponsive Benign HPA - untreated Phe levels <600umol/L Centre I Not applicable. Not applicable. Phe level >1200 umol/L Not applicable. Centre J Not applicable. Not applicable. Definition(s) not reported Not applicable. 211  Consenting Centre (de-identified) Current Disease Classification & Definition Noted in Metabolic Chart Mild hyperphenylalaninemia Mild PKU Classical PKU Not specifieda Centre K Untreated Phe levels between 120-360 umol/L Untreated Phe levels between 120-600 umol/L Untreated Phe levels <600umol/L Untreated Phe levels between 600-1200umol/L Untreated Phe levels >1200umol/L Moderate PKU - no definition reported Centre L Definition(s) not reported Definition(s) not reported Definition(s) not reported PKU (BH4 responsive) - definition not reported Centre M Definition(s) not reported Not applicable. Elevated Phe Phe level critically high  Not applicable. aThe diagnosis of severity was not recorded in the chart. Some classifications were changed during data cleaning based on provided definitions. bNot applicable indicates that there were zero participants at that site with the specific classification.   212   C.3 PKU monitoring and communications by treating centrea: classic PKU.   Treating centre at time of consent Rate of Phe tests per month Rate of clinic visits per monthb 0-1 month >1-12 months >1-7 years >7 years 0-1 month 0-12 months >1-7 years >7 years Centre A 5.31 (n=13) 4.06 (n=13) 2.26 (n=13) <5 2.85 (n=13) 0.78 (n=13) 0.23 (n=13) <5 Centre B 16.74 (n=19) 7.11 (n=19) 2.25 (n=19) 1.84  (n=7) 1.00 (n=19) 0.25 (n=19) 0.15 (n=19) 0.15 (n=7) Centre C 6.60 (n=5) 3.20 (n=5) 1.77 (n=5) <5 3.40 (n=5) 0.42 (n=5) 0.09 (n=5) <5 Centre D 3.25 (n=8) 1.09 (n=8) 0.31 (n=8) <5 1.00 (n=8) 0.40 (n=8) 0.10 (n=8) <5 Centre E 5.00 (n=7) 3.77 (n=7) 1.53 (n=7) <5 0.71 (n=7) 0.27 (n=7) 0.11 (n=7) <5 Centre F 6.89 (n=9) 3.53 (n=9) 1.17 (n=9) <5 0.22 (n=9) 0.24 (n=9) 0.15 (n=9) <5 Centre G 9.00 (n=7) 4.00 (n=7) 3.20 (n=7) <5 4.14 (n=7) 1.79 (n=7) 0.28 (n=7) <5 Centre H <5 <5 <5 <5 <5 <5 <5 <5 Centre I <5 <5 <5 <5 <5 <5 <5 <5 a Centres with 5 or more participants in each disease group included in the table. bClinic visits include: physical clinic visits and telehealth sessions (diagnosis acquisition visits were excluded from the analysis).   213  C.4 PKU Monitoring and Communications: milder forms of PKUa   Treating centre at time of consent Rate of Phe tests per month   Rate of clinic visits per monthb  0-1 month >1-12 months >1-7 years >7 years 0-1 month 0-12 months >1-7 years >7 years Centre A 1.68 (n=40) 0.80 (n=40) 0.37 (n=36) 0.33 (n=13) 1.15 (n=40) 0.24 (n=40) 0.10 (n=36) 0.10 (n=13) Centre B 6.20 (n=10) 2.76 (n=10) 1.41 (n=10) <5 0.80 (n=10) 0.15 (n=10) 0.12 (n=10) <5 Centre C 3.17 (n=6) 2.03 (m=6) 0.80 (n=6) <5 0.83 (n=6) 0.18 (n=6) 0.07 (n=6) <5 Centre D <5 <5 <5 n=0 <5 <5 <5 n=0 Centre E 1.33 (n=9) 0.52 (n=9) 0.26 (n=9) <5 0.67 (n=9) 0.10 (n=9) 0.09 (n=9) <5 Centre F 3.73 (n=11) 1.52 (n=11) 0.72 (n=11) n=0 0.64 (n=11) 0.18 (n=11) 0.11 (n=11) n=0 Centre G 2.57 (n=7) 1.65 (n=7) 1.21 (n=7) <5 0.86 (n=7) 0.71 (n=7) 0.10 (n=7) <5 Centre H 6.86 (n=7) 2.81 (n=7) 0.98 (n=7) <5 0.57 (n=7) 0.38 (n=7) 0.11 (n=7) <5 Centre I 0.60 (n=5) 020 (n=5) 0.45 (n=5) n=0 0.20 (n=5) 0.16 (n=5) 0.14 (n=5) n=0 a Centres with 5 or more participants in each disease group included in the table. bClinic visits include: physical clinic visits and telehealth sessions (diagnosis acquisition visits were excluded from the analysis).  

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