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Phenylketonuria: optimizing care

Demirdas, S.

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PHENYLKETONURIA:

OPTIMIZING CARE

Serwet Demirdas

Phenylketonuria: optimizing care

Thesis, University of Amsterdam, Amsterdam, The Netherlands

© S. Demirdas, M.D.

All rights reserved. No part of this publication may be reproduced, stored or transmitted

in any form or by any means, without prior permission of the author.

Cover : Eva Bakker, Niet iedereen kent Eva, Amsterdam, The Netherlands

Layout : Lukman Özer, Amsterdam, The Netherlands

Printed by : Gildeprint Drukkerijen, Enschede, The Netherlands

ISBN/EAN : 978-94-6108-995-3

About the cover:

The cover represents the accumulation of the phenylalanine molecule in the brain of

patients with phenylketonuria.

The printing of this thesis was financially supported by:

Nutricia Research BV, Vitaflo International Ltd, The University of Amsterdam,

Actelion Pharmaceuticals Nederland B.V. and Genzyme BeNeLux

PHENYLKETONURIA:

OPTIMIZING CARE

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het College voor Promoties ingestelde

commissie, in het openbaar te verdedigen in de Agnietenkapel

op vrijdag 11 september 2015, te 14:00 uur

door

Servet Demirdas

geboren te Tunceli, Turkije

PROMOTIECOMMISSIE

Promotor: Prof. Dr. F.A. Wijburg Universiteit van Amsterdam

Copromotor: Dr. A.M. Bosch Universiteit van Amsterdam

Overige leden: Prof. Dr. P. Burgard University of Heidelberg

Prof. Dr. M.A. Grootenhuis Universiteit van Amsterdam

Prof. Dr. H.S.A. Heymans Universiteit van Amsterdam

Prof. Dr. B.T. Poll-The Universiteit van Amsterdam

Prof. Dr. F.J. van Spronsen Rijksuniversiteit Groningen

Prof. Dr. R.J.A. Wanders Universiteit van Amsterdam

Faculteit: Faculteit der Geneeskunde

This thesis is dedicated to my parents:

You have thought me to always be the best of who I am.

THESIS CONTENT

Chapter 1 General introduction 8

Chapter 2 Clinical pathways for inborn errors of metabolism: warranted and feasible

Orphanet J Rare Dis 2013, 8: 37

34

Chapter 3 Evaluation of quality of life in PKU before and after introducing tetrahydrobiopterin (BH4); a prospective multi-center cohort study

Mol Genet Metab 2013, 110 Suppl: S49-S56.

42

Chapter 4 The time consuming nature of phenylketonuria: a cross-sectional study investigating time burden and costs of phenylketonuria in the Netherlands

Mol Genet Metab 2013, 109: 237-242

72

Chapter 5 Bone health in phenylketonuria: a systematic review and meta-analysis


90

Chapter 6 Micronutrients, fatty acids and bone health in phenylketonuria

Submitted

140

Chapter 7 Discussion and summary 170

Chapter 8 Future perspectives 188

Chapter 9 Dutch summary 194

Addendum List of publications

Co-authors and affiliations

List of abbreviations

Portfolio

Acknowledgements

About the author

208

General introduction

Chapter 1

~ 10 ~

General Introduction

~ 11 ~

GENERAL INTRODUCTION

In 1950, the Nobel and Pulitzer prize winning author Pearl S. Buck published a personal

memoir about the life of her daughter with severe cognitive impairment who was born

in 1921, with the title: The child who never grew [1]. During the majority of her

daughter’s life, the cause of her impaired mental functions remained unclear. She never

received disease-specific medical treatment and spent most of her life institutionalized

at the Vineland Training School in New Jersey [2]. In those days, having a child with

cognitive impairment, especially as an accomplished upper class citizen, was never

spoken about because the concept of eugenics, which was en vogue at that time, taught

that ‘feeblemindedness’ was linked to the genetic pool of those less fortunate in the

population [3]. It took Ms. Buck almost 30 years before she dared to come forward and

write about her own daughter, intending to inform other parents of cognitively impaired

children on how to deal with their ‘burden.’ She writes on the first two pages: “The final

reason for setting down this story is that I want my child’s life to be of use in her

generation. She is one who has never grown mentally beyond her early childhood,

therefore she is forever a child, although in years she is old enough now to have been

married and to have children of her own...”[1]. It was later discovered that PKU was the

cause of her daughter’s severe cognitive impairment.

Phenylketonuria (PKU, ORPHA79254, MIM 261600) is an autosomal recessive

inherited disorder of metabolism that arises due to mutations in the gene coding for the

hepatic enzyme phenylalanine hydroxylase (PAH; EC 1.14.16.1). Because of this

mutation, the essential amino acid phenylalanine (Phe) cannot be converted to tyrosine

and accumulates in the blood and in the brain, causing severe cognitive impairment [4].

The incidence of PKU in European and Oriental Asian populations is approximately

1:10.000 [4,5]. PKU is therefore one of the most prevalent inborn errors of metabolism

found in Europeans [6,7].

The book by Ms. Buck on the life of her daughter is an emotional tale of the difficulties

of growing up with severe cognitive challenges; fortunately, the picture of a young,

untreated patient with PKU is currently rare in developed countries. In 1934, Asbjörn

Fölling, a Norwegian physician and biochemist, discovered that a disorder in the

Chapter 1

~ 12 ~

biochemical pathway involved in the degradation of Phe was associated with severe

cognitive impairment in several patients based on the identification of high levels of

phenylpyruvic acid in the urine [8-11]. The disease was initially named ‘imbecillitas

phenylpyruvica’ by Fölling in 1934. The name was later adapted to oligophrenia

phenylpyruvica by Jervis in 1937 and it was finally changed to PKU in that same year by

Penrose and Quastel in their work concerning one of the first Phe loading tests in

untreated patients [4,9,11-13]. In 1951, treatment became available when the German

physician Horst Bickel developed the first Phe free amino acid mixture to supplement all

essential amino acids except for Phe in patients placed on a protein-restricted diet. This

strategy has been corner stone of the treatment of PKU up to now [8]. The discovery of a

screening test in dried blood spots by the American physician Robert Guthrie in the

1960’s enabled the early introduction of dietary treatment in developed countries,

resulting in near normalization of the cognitive outcomes for patients with PKU [14].

The enormous success of treatment has, however, led to new thresholds to overcome in

the life-long treatment of PKU, and the road to optimizing care in PKU is open [14-21].


~ 13 ~

BIOCHEMISTRY

In patients with PKU, PAH is deficient and Phe catabolism is subsequently hampered,

which causes increased levels of Phe throughout the body, including, most importantly,

the brain. PAH is expressed in the liver [22], and it is a non-heme iron enzyme that

catalyzes the hydroxylation of Phe into tyrosine using iron, molecular oxygen and

tetrahydrobiopterin (BH4) as cofactors [23] (Figure 1). Phe is an essential amino acid

and therefore must be ingested through dietary protein intake. In addition, protein

catabolism contributes to the body’s free Phe pool.

The impaired degradation of Phe leads to an increase in the excretion of its metabolites

in urine: phenylacetate, phenylpyruvate, and phenylethylamine. Of these metabolites

phenylpyruvate is the most prominent and contains a ketone group, which is why the

disease is called ‘phenylketonuria’ (Figure 1).

Chapter 1

~ 14 ~

Figure 1. Degradation of phenylalanine


~ 15 ~

GENETIC SUBSTRATE

The PAH gene is located on the long arm of chromosome 12 (bands q22-q24). Almost

800 different mutations causing PKU have been identified in the PAH gene (the Human

Gene Mutation Database; www.hgmd.org), with most patients being compound

heterozygotes. The most frequently encountered allele is c.1222C4T, and the most

frequent genotype is c.[1066-11G4A;[1066-11G4A][23,24]. Mutations in the catalytic

domain, splice site variants and missense mutations (most prevalent) may be present,

resulting in a full lack of enzyme activity, a truncated and non-functional protein or

misfolded enzyme with low to absent activity, respectively [23,25]. Genotype based

prediction of the phenotype is complex. However, several steps towards predicting

phenotypic severity based on genetic changes have been taken [6,23,26,27]. Predicting

clinical severity based on mutation analysis, in combination with the blood Phe

concentration at diagnosis, may assist in selecting an optimal treatment approach for

individual patients. Certain genotypes can predict responsivity to treatment with BH4

[28-30]. Personalized medicine based on known genotype-phenotype correlations is

therefore rapidly becoming an option. Studies concerning the prediction of PAH activity

and BH4 sensitivity based on genotype are ongoing [22], and several databases (such as

the Human Gene Mutation Database) are used to collect new mutations of the PAH

gene.

Chapter 1

~ 16 ~

PATHOPHYSIOLOGY

The exact pathophysiology of the cognitive impairment in PKU is not fully known, and

several hypotheses have been postulated. The following mechanisms are thought to play

a role in causing brain damage in patients with PKU and will be discussed below: Phe

toxicity, low concentrations in the brain of large neutral amino acids (LNAA) other than

Phe, specific properties of the blood-brain barrier (BBB), impaired neuronal synthesis of

neurotransmitters and impaired myelination [31-33].

Phenylalanine Toxicity

Higher levels of Phe in the blood are related to more extensive CNS damage than lower

levels [34-36]. The most widely supported theory is that direct toxicity of Phe causes

irreversible damage to brain cells and synapsis, leading to cognitive impairment [37,38].

Waisbren et al. performed a meta-analysis and found that for children up to the age of

12 years, an increase of mean lifetime blood Phe levels of 100 mol/L (levels between

423 and 750 mol/L) caused a decrease of 1.3 to 3.1 IQ points. [39]. Furthermore Jahja

et al. showed in a preliminary study that executive functioning was better in pediatric

patients with mean lifetime Phe levels below 240 mol/L than in those with higher

levels [40]. The effect of high Phe concentrations on the brain is age dependent, and Phe

toxicity is more pronounced in the developing brain in utero and up to the age of 12

years than later in life [41-44].

Large Neutral Amino Acids and the Blood-Brain Barrier

Decreased LNAA concentrations in the brain may also play a role in the cerebral

dysfunction in patients with PKU [32,45,46], but the precise role remains to be

elucidated. Several studies have demonstrated that Phe competes with eight other LNAA

(tyrosine, tryptophan, valine, isoleucine, leucine, threonine, methionine, and histidine)

when crossing the BBB because they all use the same L-type amino acid transporter

(LAT1, SLC7A5) [36,45,47-50]. Because the LAT1 transporter has a greater affinity to

Phe than to the other LNAA, high levels of Phe in the blood lead to preferential


~ 17 ~

transport of Phe over the BBB, resulting in elevated Phe and diminished LNAA

concentrations in the brain [48,51]. Furthermore, LNAA are deficient intra-cerebrally

because high Phe concentrations lead to decreased protein synthesis. An indication that

LNAA other than Phe play a role in brain pathology in patients with PKU is

demonstrated by several authors describing improvement of symptoms and increased

LNAA brain concentrations after supplementation with specific LNAA [45,46,52-54].

Neurotransmitter Synthesis

Neurotransmitter synthesis is decreased in patients with PKU [55], and this may

consequently play a role in the pathophysiology of cerebral dysfunction in patients.

Tyrosine is the precursor of dopamine and it is a degradation product of Phe [50,56].

Due to PAH deficiency, tyrosine is not properly formed, and it is hypothesized that PKU

may lead to low levels of both tyrosine and dopamine. Small studies with patients on a

natural protein-restricted diet reported that influx and efflux of tyrosine across the BBB

is impaired [50,57-62]. Tryptophan, another LNAA in competition with Phe at the BBB,

is a precursor of serotonin, and both have been found to be decreased in the brains of

patients on dietary treatment [63,64]. The enzymes involved in the degradation of

tyrosine and tryptophan (tyrosine- and tryptophan hydroxylase respectively) may be

reduced in patients, not only leading to lowered neurotransmitters but also to decreased

synaptic plasticity and decreased axonal growth. However, there is no proof of the role

of deficiencies of these neurotransmitters and their precursors in the pathogenesis of

CNS disease in PKU [32].

Myelination

White matter abnormalities have been observed in patients with PKU despite dietary

treatment [65,65-71]. Two explanations have been suggested. First, decreased protein

synthesis might lead to improperly formed myelin. Secondly, it is thought that white

matter lesions occur due to intermyelenic edema [65]. Both hypothesis need to be

further investigated.

Chapter 1

~ 18 ~

DIAGNOSIS

PKU is suspected when Phe is found to be elevated in dried bloodspots obtained through

newborn screening in the first week of life. The diagnosis is subsequently confirmed by

the detection of high plasma Phe levels and the exclusion of other genetic conditions

causing hyperphenylalaninemia (Figure 2). Measuring PAH activity in patient blood is

not effective because it is only detectible in hepatic tissue. Based on the level of Phe at

diagnosis, disease severity is classified as either classic PKU (≥ 1200 mol/L), moderate

PKU (900–1200 mol/L), mild PKU (600–900 mol/L) or mild hyperphenylalanine-

mia (mHPA; (360–600 mol/L) [16]. A Phe level below 120 mol/L is normal, and a

level between 120–360 mol/L is classified as mHPA not in need of treatment. Another

classification of PKU patients is based on their tolerance for natural protein or, more

specifically, for Phe intake. According to this classification, patients with a daily

tolerance of less than 500 mg Phe (which equals 10 grams of protein) are classified as

severe patients, and those with a daily tolerance of 500 mg or more of Phe are classified

as having a mild to moderate form of the disease [72,73].

Hyperphenylalaninemia may be found in disorders other than PKU. When investigating

the cause of hyperphenylalaninemia, a disorder in BH4 synthesis should be ruled out as

the cause of the elevated Phe levels [74,75]. The incidence of BH4 deficiencies is 1 to 2%

of all patients with high Phe levels at birth [76]. For this reason, a blood or urine pterin

profile (analysis of biopterin, neopterin and primapterin) should be obtained, and the

activity of dihydropteridine reductase must be measured specifically in blood (Figure 2).

If the results are abnormal, the patient should be tested for deficiencies of 1,6-pyruvoyl-

tetrahydrobiopterin synthase, dihydropteridine reductase, pterin carbinolamine-4α-

dehydratase or GTP-cyclohydrolase [6,77]. In patients with 1,6-pyruvoyl-

tetrahydrobiopterin synthase deficiency, urine biopterin is low with a decreased

biopterin/neopterin ratio. In patients with GTP-cyclohydrolase deficiency, the urine

pterin profile shows decreased levels of both biopterin and neopterin, and patients with

pterin carbinolamine-4α-dehydratase deficiency have primapterin (a specific pterin) in

the urine. Because dihydropteridine reductase does not show a specific pterin profile in

urine, the enzyme is measured in blood. The diagnosis is completed by evaluation of


~ 19 ~

neurotransmitters, pterin and folate levels in the cerebrospinal fluid, which show

distinctive alterations based on the underlying genetic disorder [76,77].

Figure 2. Tetrahydrobiopterin (BH4) synthesis

Chapter 1

~ 20 ~

THERAPY

The aim of dietary protein-restricted treatment is to ensure that patients have a safe

level of Phe, as measured in plasma or bloodspots [78]. For children up to 12 years of

age levels below 360 mol/L have the best outcomes [39,79-81], and for adults the need

for metabolic control is still under debate. However, a study by Okano et al. found

evidence that Phe levels below 500 mol/L are safest because below this value, the least

disturbance in oxidative stress status and nitric oxide metabolism are found [82].

Reference values for patients under treatment vary worldwide [16], but in the

Netherlands the consensus is as follows (based on the Dutch clinical pathway for PKU;

see chapter 2 of this thesis): 0–12 years: 120–360 mol/L, ≥ 12 years: 120–600 mol/L,

and during pregnancy: 120–240 mol/L.

Diet

The corner stone in the treatment of PKU consists of a strict natural protein-restricted

diet [6,14-16,83]. The main natural food sources restricted in the PKU diet are protein

rich foods, such as meat, fish, dairy products, bread and pasta. Patients are allowed a

limited intake of natural protein based on their individual Phe tolerance. To provide a

safe and sufficient intake of protein, patients are supplemented with special fabricated

amino acid supplements containing most amino acids except for Phe and variable

amounts of micronutrients and essential fatty acids. Patients may also use modified low

protein food products as an alternative to some natural high protein products, such as

cheese, milk, pasta and bread. Without the fortified amino acid supplements, patients

are prone to deficiencies [17,84,85]. For example, deficiencies of selenium [86,87], zinc

[88,89], folate [90], vitamin B6, vitamin B12 (in patients not adherent to amino acid

mixture intake) [91-96], carnitine [97,98], tryptophan, tyrosine [63], transferrin, ferritin

[99,100] and specific essential poly-unsaturated fatty acids (eicosapentaenoic acid and

docosahexaenoic acid) have been described in patients with PKU undergoing treatment

[101,102]. To regularly assess the nutrient status of the patients, regular visits to a

physician and dietician are important, with more frequent visits during childhood.


~ 21 ~

BH4

A novel treatment of PKU (available in the Netherlands since 2009) consists of oral

supplementation with high doses of BH4, the co-factor of PAH, which may enhance the

function of the PAH enzyme in responsive patients. Approximately 25–50% of patients

are responsive to BH4 treatment [103-106]. Mostly mild patients benefit from treatment

with BH4 because these patients more often have some residual PAH activity. Those

responsive to treatment have an increased Phe tolerance and may somewhat relax their

diet, with a minority of patients being able to go off diet entirely [6,107].

LNAA

LNAA have been studied as a treatment option for patients with PKU based on their

ability to block the uptake of Phe from the intestine and at the BBB due to competition

at the L-type amino acid transporter [45,46,54,64]. Studies that demonstrate a clear

benefit are scarce, and the effectiveness of LNAA as a single treatment is debatable.

Because LNAA do not lower Phe values satisfactorily, their use should be avoided in

pregnant women.

PEG PAL

The most recent advance in the pharmacological therapy of PKU is that of polyethylene

glycol-conjugated phenylalanine ammonia lyase (PEG-PAL) [108-110]. PEG-PAL is a

bacterial enzyme that catalyzes the degradation of Phe to trans-cinnamic acid, which is

cleared by the kidney and secreted in the urine. Trials to evaluate the efficacy of

subcutaneously administered PEG PAL injections are ongoing. Because PEG-PAL

degrades Phe by means of an enzyme other than PAH, it is applicable in all patients

regardless of their genotype.

Chapter 1

~ 22 ~

Pregnancy

Pregnant women with PKU provide a number of special challenges for dietary treatment

because moderately elevated levels of Phe may lead to adverse outcomes in the fetus

[111]. Koch et al. investigated the effect of maternal blood Phe levels on neonatal

outcomes and found that the optimum range for blood Phe values in pregnancy lies

between 120 and 360 µmol/L. It is advised to achieve this strict dietary control by 8

weeks before pregnancy to minimize the risk for fetal complications [112,113]. Elevated

levels of blood Phe may lead to maternal PKU syndrome with mental retardation,

microcephaly and congenital heart defects in the offspring [41,111,113-117]. The exact

mechanisms causing the teratogenic effects of the heart are unknown [116].


~ 23 ~

COMPLICATIONS IN PKU

Patients with untreated PKU show severe intellectual impairment, motor problems,

behavioral disturbances, epilepsy, eczema and relatively fair skin and fair hair due to

melatonin deficiency [13,118]. Paine et al. in 1957 described in detail the variability in

manifestations of untreated patients before early treatment was widely introduced and

before details about the genotype/phenotype correlations and the role of the amount of

residual enzyme activity became known [119].

Since the 1950’s, dietary treatment for patients with PKU has been available, and many

advances in the care for patients have been made. Patients diagnosed trough newborn

screening and treated early and continuously have very good outcomes. Despite the

significant achievements, treatment has also led to some new challenges in the care for

patients with PKU. Although some of these new challenges are due to dietary

restrictions, others are caused by the disease itself. These new challenges are: nutrient

deficiencies [17,120], bone health impairment [102,121-124] and hypothesized

deficiencies in neurotransmitters (serotonin and dopamine) [32,125-127]. Furthermore,

hidden disabilities [128] are postulated to exist in patients with PKU, caused by

executive functioning defects [40,129-134], social problems [130,135], possible impaired

quality of life [53,79,104,135,136] and emotional difficulties. Finally, the dietary

treatment of PKU is assumed to place a significant burden on the patient because the

diet is demanding [137], and further attention to patients’ needs is required [138].

Chapter 1

~ 24 ~

THESIS OUTLINE

Optimizing care in patients with PKU requires fine-tuning of the treatment itself as well

as evaluation and management of the adverse outcomes of the treatment. This thesis

focuses on several topics, and the outline is as follows:

Chapter 2 discusses the importance of establishing pathways to provide optimal care

for patients with inborn errors of metabolism, within the line of national consensus.

There is broad diversity in the management of care for patients with PKU (both

nationally and internationally). To standardize and optimize care, clinicians need to

reach consensus on what the best care is for their patients.

Chapter 3 presents the results of a study performed to assess the health-related quality

of life (HRQoL) in PKU patients and the effect of the novel treatment with BH4 on this

outcome. HRQoL is the ultimate outcome of provided care, and standardized

questionnaires are available to assess the subjective wellbeing of patients in relation to

pre-specified domains of life (such as work, school, and emotions).

Chapter 4 reports on a cross-sectional study investigating the burden of the time and

costs of living with PKU for adult patients and caretakers of pediatric patients in the

Netherlands. The strict dietary treatment places a significant burden on the patient.

Therefore, the burden associated with disease and treatment management needs to be

assessed to better comprehend how the patient experiences overall wellbeing.

Chapter 5 provides a systematic review and meta-analysis on bone health in patients

with PKU. It has been postulated in several publications that bone mineral density in

patients with PKU is diminished, possibly due to differences in nutrient intake or

through direct effects of the disease itself. We have pooled available patient data and we

have reviewed several topics known to affect bones in order to evaluate bone health in

patients with PKU.

Finally, Chapter 6 reports the results of a multi-center study on the nutrient status and

bone health in patients with PKU. Changes in nutrient status and a diminished bone

condition are considered important complications of a natural protein-restricted diet.


~ 25 ~

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Chapter 1

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Clinical Pathways for Inborn

Errors of Metabolism:

Warranted and Feasible


Serwet Demirdas1, Imke N van Kessel1, Marjolein J Korndewal, Carla EM Hollak, Hanka

Meutgeert, Anja Klaren, Margreet van Rijn, Francjan J van Spronsen, Annet M Bosch

and The Dutch working Group*

1 Equal contributors

Chapter 2

~ 36 ~

ABSTRACT

Inborn errors of metabolism (IEM) are known for their low prevalence and multidisciplinary

care mostly founded on expert opinion. Clinical pathways are multidisciplinary tools to organize

care, which provide a clear route to the best care and improve communication. In 2010 the

Dutch Society for Children and Adults with an Inborn Error of Metabolism (VKS) initiated

development of clinical pathways for inborn errors of metabolism. In this letter to the editor we

describe why it is warranted to develop clinical pathways for IEM and shortly discuss the

process of development for these pathways in the Netherlands.

*The Dutch working Group on clinical pathways for inborn errors of metabolism.

Folkert. W. Asselbergs1, Christiaan Blank2, Terry G.J. Derks8, Eugène F. Diekman2, Monique E. Dijsselhof6, Marc

Engelen7, Peter M. van Hasselt2, Nienke M. ter Horst6, Dorine A.M. van den Hurk3, Mirian C.H. Janssen9, Francois

P.J. Karstens11, Elles van der Louw12, Eva Morava10, Joost Nicolai14, Ludo van de Pol2, Bwee Tien Poll-The5, M Estela

Rubio-Gozalbo15, G. Peter A. Smit8, Jessica de Ruijter5, Corrie Timmer3, Catharina M.L. Touw8, Gepke Visser2, Harold

W. de Valk4, Frits A. Wijburg5, Monique Williams13.

Departments of Cardiology1, Pediatrics2, Dietetics3 and Internal Medicine4, University Medical Center, Utrecht, The

Netherlands. Department of Pediatrics5, Dietetics6, Neurology7, Emma Children’s Hospital, Academic Medical Center,

Amsterdam, The Netherlands. Section of metabolic diseases8, Beatrix Children’s Hospital, University Medical Center

Groningen, University of Groningen, Groningen, The Netherlands. Department of Internal Medicine9and

Pediatrics10, Unitversity Medical Center st Radboud, Nijmegen. Department of Internal Medicine11, Dietetics12 and

Pediatrics13, Erasmus MC, Rotterdam, The Netherlands. Department of Neurology14 and Pediatrics15, University

Hospital Maastricht, Maastricht, The Netherlands.

Clinical Pathways

~ 37 ~

INTRODUCTION

Inborn errors of metabolism (IEM) are known for their low prevalence and chronic need

of medical care. Care provided is multidisciplinary and often based on expert opinion. In

recent years, excellent guidelines on metabolic disorders have been developed and

published [1-5]. In 2010 the Dutch Society for Children and Adults with an Inborn Error

of Metabolism (VKS) initiated development of clinical pathways for 20 IEM, with

separate versions for professionals and patients. This letter discusses why clinical

pathways for IEM are warranted and feasible.

Background

Clinical pathways are a tool for multidisciplinary decision making and organization of

care processes for well-defined groups of patients [6]. Often they are based on

guidelines [7-9]. Pathways optimize clinical outcomes whilst maximizing clinical

efficiency [10]. For example, they describe which actions should be taken, when, and by

whom [11]. It has been demonstrated that use of pathways decreases the duration of

inpatient care, increases interdisciplinary communication, enhances patient knowledge

and self-awareness, leads to significant better coordination of care and reduces costs

[7,9,12-14].

Clinical pathways can be valuable for patients with IEM. Firstly, low prevalence of IEM

leads to limited knowledge about best practice. In the absence of robust evidence, expert

opinion and outcomes of clinical studies can support the establishment of a clinical

pathway [12]. When frequently updated, it presents a reference to latest state of art in

care [15,16] and provides guidance for further research. Secondly, a multidisciplinary

approach is of great importance. The complexity of multidisciplinary care may lead to

delay of care, overuse of diagnostics or therapy and miscommunication between

caregivers [7]. Multidisciplinary cooperation using a clinical pathway will improve

communication and provide a clear route to best care, based on consensus. Thirdly,

clinical pathways may improve care for patients when used in local hospitals, while the

physicians in academic referral centers can serve as consultants. Finally, clinical

pathways become more important as transition to adult care increases [17], leading to

more active participation of patients in their treatment.

Chapter 2

~ 38 ~

THE DESIGN OF CLINICAL PATHWAYS FOR IEM

Design and consensus

The initiative for development of clinical pathways was taken by the patient society

(VKS). Dutch expert pediatricians, internists and dieticians for each specific disorder in

cooperation with the VKS created the pathways. The final version was discussed in a

national consensus meeting. Separate versions were made for professionals and for

patients, presenting the Dutch consensus. All advice is substantiated by a level of

evidence [18], according to the scoring system of the Dutch Institute for Healthcare

Improvement CBO. Level 1: one systematic review or two independent high quality

randomized controlled trials (RCTs); level 2: two independent moderate RCTs or

comparative trials; Level 3: one RCT, comparative or non-comparative trial; Level 4:

expert opinion [19].

Clinical pathway for professionals

The first section of the version for professionals comprises a general introduction and

concise strategy for diagnostics and treatment. The second and third sections contain

more specific guidance for treatment and follow up in childhood and adulthood.

The pathways include responsibilities for each professional, advised frequency for

outpatient visits and laboratory studies, and recommendations on follow up of known

complications of the disorder. In the pediatric pathway one chapter is dedicated to

transition from pediatric to adult care.

In the pathways all advice is substantiated by a level of evidence. Evidence levels 3 and 4

were common. Level 1 was rarely available and mostly resulted from trials evaluating a

novel pharmaceutical agent. Most advice was therefore founded on expert opinion and

trials of moderate quality.

Clinical Pathways

~ 39 ~

Clinical pathway for patients

The first section of the patient version contains general information on the disorder and

its treatment. The second and third sections address treatment and follow up in

childhood and adulthood. The purpose of the pathway for patients is to provide insight

into current consensus of best practice and an overview of all professionals involved. It

provides clarity on responsibilities, including that of the patient/parents who take a

prominent place in the treatment team.

For active patient participation, patients must be provided evidence based information

in an appropriate and comprehensible form [20]. The fact that the patient versions are

based on the professional pathway ensures that they are in accordance with available

evidence, and comprehensibility is secured by cooperation with the VKS.

We demonstrated that development of clinical pathways for IEM is feasible and we were

able to reach national consensus. At this time, Dutch pathways are publically available

for 20 diseases including urea cycle defects, organic acidurias, mitochondrial fatty acid

oxidation disorders, galactosemia, phenylketonuria, tyrosinemia, glycogen storage

disorders, congenital disorder of glycosylation type 1a, and Niemann Pick type c [21].

Chapter 2

~ 40 ~

REFERENCES

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Campbell JK, Salehi V, Chamberlain JM:

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emergency treatment of patients with inborn

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2. Kolker S, Christensen E, Leonard JV,

Greenberg CR, Boneh A, Burlina AB, et al:

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3. Haeberle J, Boddaert N, Burlina A,

Chakrapani A, Dixon M, Huemer M, et al:

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4. Haute Autorité de Santé; Phénylcétonurie;

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u_web.pdf.

5. The National Society for Phenylketonuria

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and management of children, adolescents and

adults with phenylketonuria. http://www.nspku

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nt %20of %20PKU.pdf 2004.

6. European Pathway Association; Clinical/care

pathways. http://www.e-p-a.org/clinical---care-

pathways/index.html 2013.

7. Panella M, Marchisio S, Di Stanislao F:

Reducing clinical variations with clinical

pathways: do pathways work? Int J Qual Health

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8. Every NR, Hochman J, Becker R, Kopecky S,

Cannon CP: Critical pathways: a review.

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Clinical Cardiology, American Heart Association.

Circulation 2000, 101:461–465.

9. Hauck LD, Adler LM, Mulla ZD: Clinical

pathway care improves outcomes among

patients hospitalized for community-acquired

pneumonia. Ann Epidemiol 2004, 14:669–675.

10. Rotter T, Kinsman L, James E, Machotta A,

Willis J, Snow P, et al.: The effects of clinical

pathways on professional practice, patient

outcomes, length of stay, and hospital costs:

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Eval Health Prof 2012, 35:3–27.

11. Campbell H, Hotchkiss R, Bradshaw N,

Porteous M: Integrated care pathways. BMJ

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12. Vanhaecht K, De Witte K, Panella M,

Sermeus W: Do pathways lead to better

organized care processes? J Eval Clin Pract

2009, 15:782–788.

13. Banasiak NC, Meadows-Oliver M: Inpatient

asthma clinical pathways for the pediatric

patient: an integrative review of the literature.

Pediatr Nurs 2004, 30:447–450.

14. Neubauer MA, Hoverman JR, Kolodziej M,

Reisman L, Gruschkus SK, Hoang S, et al: Cost

effectiveness of evidence-based treatment

guidelines for the treatment of non-small-cell

lung cancer in the community setting. J Oncol

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15. de Bleser L, de Depreitere R, de Waele K,

Vanhaecht K, Vlayen J, Sermeus W: Defining

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Clinical Pathways

~ 41 ~

16. Deneckere S, Euwema M, Van Herck P,

Lodewijckx C, Panella M, Sermeus W, et al.:

Care pathways lead to better teamwork: results

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17. Van Spronsen FJ, Burgard P: The truth of



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18. Bossard N, Boissel FH, Boissel JP: Level of

evidence and therapeutic evaluation: need for

more thoughts. Fundam Clin Pharmacol 2004,

18:365–372.

19. Centraal Begeleidings Orgaan (CBO);

Evidence based guideline development.

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handleiding/A-Levels-of-evidence/ 2013.

20. Coulter A: Evidence based patient

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selingsziekten; Zorgpaden voor stofwis-

selingsziekten. http://www.stofwisselingsziekten

.nl/ziekte_informatie/zorgpaden_voor_stofwiss

elingsziekten 2012.

Evaluation of Quality of Life in PKU

Before and After Introducing

Tetrahydrobiopterin (BH4);

a Prospective Multi-Center Cohort Study


Serwet Demirdas, Heleen Maurice-Stam, Carolien CA Boelen, Floris C Hofstede,

Mirian CH Janssen, Janneke G Langendonk, Margot F Mulder, M Estela Rubio-Gozalbo,

Francjan J van Spronsen, Maaike de Vries, Martha A Grootenhuis and Annet M Bosch

Chapter 3

~ 44 ~

ABSTRACT

Background: Phenylketonuria (PKU) is a rare inborn error of metabolism caused by

phenylalanine hydroxylase enzyme (PAH) deficiency. Treatment constitutes a strict Phe

restricted diet with unpalatable amino acid supplements. Residual PAH activity enhancement

with its cofactor tetrahydrobiopterin (BH4) is a novel treatment which increases dietary

tolerance in some patients and permits dietary relaxation. Relaxation of diet may improve

health related quality of life (HRQoL). This prospective cohort study aims to evaluate HRQoL of

patients with PKU and effects of BH4 treatment on HRQoL.

Methods: Patients aged 4 years and older, diagnosed through newborn screening and early and

continuously treated, were recruited from eight metabolic centers. Patients and mothers

completed validated generic and chronic health-conditions HRQoL questionnaires (PedsQL,

TAAQOL, and DISABKIDS) twice: before and after testing BH4 responsivity. Baseline results

were compared to the general population. Data collected after BH4 testing was used to find

differences in HRQoL between BH4 unresponsive patients and BH4 responsive patients after

one year of treatment with BH4. Also a within patient comparison was performed to find

differences in HRQoL before and after treatment with BH4.

Results: 69/81 (85%) patients completed the questionnaires before BH4 responsivity testing,

and 45/69 (65%) participated again after testing. Overall PKU patients demonstrated normal

HRQoL. However, some significant differences were found when compared to the general

population. A significantly higher (thus better) score on the PedsQL was reported by children 8–

12 years on physical functioning and by children 13–17 years on total and psychosocial

functioning. Furthermore, adult patients reported significantly lower (thus worse) scores in the

TAAQOL cognitive domain. Ten patients proved to be responsive to BH4 treatment; however

improvement in their HRQoL after relaxation of diet could not be demonstrated.

Quality of Life in PKU

~ 45 ~

INTRODUCTION

Phenylketonuria (PKU; MIM 261600), caused by a deficiency of the enzyme

phenylalanine hydroxylase (PAH; EC 1.14.16.1), is an autosomal recessive disorder of

phenylalanine metabolism. The essential amino acid phenylalanine (Phe) cannot be

converted to tyrosine and therefore accumulates in the body. In the Netherlands PKU

has an incidence of 1:18,000 with approximately 12 new patients per year. Since 1974 all

newborns in the Netherlands are screened for PKU in the first week of life. With the

introduction of newborn screening and the early start of treatment, mental retardation

and neurological abnormalities due to PKU have been nearly eliminated [1–3].

The cornerstone of treatment of PKU is normalizing neurocognitive and psychological

development and functioning [4]. This is accomplished with a lifelong dietary restriction

of natural protein intake as a source of Phe and supplementation with a formula

containing all required amino acids excluding Phe. Treatment begins in the neonatal

period after confirmation of the diagnosis. A Phe restricted diet excludes many natural

high protein foods such as dairy products, meat and fish. Patients depend on low protein

food products, and the commercial Phe free amino acid supplements designed for use by

individuals with PKU are very unpalatable. The diet has been associated with adverse

feeding behaviors in young patients and compliance may be a problem [5–7].

One novel approach to the treatment of PKU is to enhance the activity of residual PAH

by treatment with pharmacological amounts of its cofactor, tetrahydrobiopterin (BH4).

BH4 is the first non-dietary treatment for patients with PKU that has been shown to be

effective in lowering blood Phe levels in randomized, double-blind trials. This can result

in increased dietary tolerance for Phe, which permits relaxation of the diet and in rare

instances a normal intake of natural protein in the diet [6,8–10]. Since June 2009 BH4

has been registered in The Netherlands for the treatment of PKU in patients from age 4

and older. Consequently thereafter, all Dutch patients with PKU aged 4 years and older

were invited to be tested for responsiveness to BH4. The patient was considered to be

responsive if Phe decreased 30% during a 48 hour test with oral BH4 in a dose of 20

mg/kg once daily [3,11]. In responsive patients BH4 treatment was started and dietary

changes were made, while unresponsive patients needed to continue their usual diet.

Chapter 3

~ 46 ~

The very strict and socially demanding diet is a severe burden on the patients and the

families [10,12,13]. A dietary relaxation may therefore positively affect the quality of life

of the patients. Several studies evaluating the health related quality of life (HRQoL) of

patients with PKU have been published. In contrast to expectations of both patients and

professionals these studies demonstrated a HRQoL in PKU which is comparable to that

of the general population [14–17], with the exception of a lower HRQoL demonstrated in

a group of Italian children [18]. A possible explanation for the unexpected normal scores

in most studies is that the presently available generic HRQoL questionnaires may not be

sensitive enough to detect the specific problems of patients with PKU. A PKU specific

HRQoL questionnaire is presently being developed.

One of the most important outcome measures of any new treatment is its effect on the

HRQoL of the patients. The introduction of BH4 in 2009 presented the unique

opportunity to study the effects of BH4 on the HRQoL of patients with PKU. This

prospective cohort study was conducted in spite of the limitations posed by the available

questionnaires. We aimed to first evaluate the HRQoL of patients with PKU compared

to the general population, and second to get an indication of the effects of the newly

introduced treatment with BH4 on HRQoL. Measurements were optimized by using

both generic HRQoL questionnaires and a questionnaire for chronically ill patients.


~ 47 ~

METHODS

Patients

The inclusion criteria for participation were: patients with PKU age 4 years and above,

who were diagnosed with PKU by newborn screening, who were early and continuously

treated with a Phe restricted diet and supplementation of amino acids, and who would

be tested for BH4 responsivity within the following 6 months. This study was approved

by the Ethical Committee of the Academic Medical Center in Amsterdam.

Procedures

Pediatricians and internists specialized in metabolic disorders of all eight Dutch

metabolic centers were asked to invite their adult patients (18 years and older) and the

mothers of pediatric patients (4–17 years) to participate in this study. Patients and

mothers were invited by a letter containing detailed information about the study, sent to

them by their treating physician and/or handed to them in person during clinic visits.

Between November 2009 and June 2010 patients (and/or mothers) who wished to

participate sent an email to the study physician expressing their informed consent. Each

participating patient (and/or mother) was sent a unique code to log in on the study

website on a secure server where the questionnaires could be completed. In the months

after completing the questionnaires all patients were tested for BH4 responsivity by

their treating metabolic physician. All patients who had participated and completed the

questionnaires in 2009 (before responsivity testing) were again invited to complete the

same questionnaires in 2011 (after responsivity testing and after the start of treatment

with BH4 if the patient turned out to be BH4 responsive) (Figure 1).

Chapter 3

~ 48 ~

Figure 1. Flowchart of the study methods

Measurement HRQoL at two time points:

- Before BH4 responsivity testing

- After BH4 responsivity testing

Pediatric patients

Self-report: 8-17

years

Proxy report: of

children 4-7 years

Adult patients

Self-report:

≥ 18 years

Generic HRQoL

questionnaire:

PedsQL

HRQoL questionnaire

chronically ill:

DISABKIDS

Generic HRQoL

questionnaire:

TAAQOL

HRQoL questionnaire

chronically ill:

modified form DISABKIDS


~ 49 ~

Measures

The patients were asked to complete both a generic HRQoL questionnaire and a HRQoL

questionnaire for the chronically ill (Figure 1). Furthermore, a number of questions were

asked about demographics, daily intake of natural protein or Phe intake, and use of

amino acid supplements. It was also asked, at both time points, if the patients had been

tested for BH4 responsivity and if they were presently treated with BH4.

Pediatric patients (age 8–17 years) and mothers of the pediatric patients (age 4–17

years) were asked to complete the Pediatric Quality of Life Inventory (PedsQL)

Measurement Model™. The PedsQL is a tool to measure generic HRQoL in children. We

used the questionnaire for generic HRQoL in four domains: physical-, emotional-,

social- and school functioning. In addition, a score for psychosocial functioning

(emotional + social + school functioning) could be calculated and a total score of all four

domains. A higher score indicates better HRQoL (on a scale of 0–100). The PedsQL has

demonstrated satisfactory reliability, validity and sensitivity for children aged 2 to 18

years old [19]. The scores of PKU patients could be compared to a Dutch norm group

[20], with the exception of PKU patients aged 4 years because the norm data of 4 year

old children were not available. The norm group was recruited from Dutch schools

stratified by geographical location (urban, suburban and rural), percentage of migrant

children and level of education. PedsQL data were available of 76 children aged 5–7

years, 47% boys (proxy parent-report); 192 children aged 8–12 years, 47% boys (child

self-report) and 148 children aged 13–17, 43% boys (child self-report) [20].

The adult patients (aged ≥18 years) were asked to complete The TNO-AZL Adult Quality

of Life questionnaire (TAAQOL). The TAAQOL is a questionnaire that measures generic

HRQoL in people aged 16 years and older, designed by the TNO Institute of Prevention

and Health and the Leiden University Hospital (TNO-AZL). The questionnaire focuses

on health problems in the past month, and, if present, the wellbeing in relation to the

health problem is assessed. The questionnaire contains 12 multi-item scales; gross and

fine motoric functioning, cognitive functioning, sleep, pain, social functioning, daily

activities, sexuality, vitality, positive emotions, depressive emotions and aggressive

emotions. The raw scale scores are converted to 0–100; higher scores indicating better

Chapter 3

~ 50 ~

HRQoL. Validity and reliability of the questionnaire is satisfactory [21]. The HRQoL in

the cohort of adults in this study was compared to norm data from the general Dutch

population aged 16 years or older gathered through a random sample of Dutch

households drawn from the national telephone registry, provided by Fekkes and

colleagues [21]. We used the age group 20–44 years (N = 2006, 40% male).

Patients (age 8 years and older) and mothers of pediatric patients (age 4–17 years) were

asked to also complete the DISABKIDS chronic generic module. This questionnaire

deals with HRQoL in children with different chronic health conditions and addresses

HRQoL aspects that pertain to chronic conditions in general. A 5-point scale of smiley

faces response scale is used for children 4–7 years old, proxy-report, resulting in a single

total score (score range 6–30). The questionnaire contains 7 multi-item scales for

patients ≥8 years old; independence (score range 6–30), emotion (7–35), social

understanding/inclusion (6–30), social stigma/exclusion (6–30), limitations (6–30),

impact treatment (6–30) and a scale representing a total score (37–185). Higher scores

indicate a better HRQoL. The DISABKIDS chronic generic module instrument has a

satisfactory validity and reliability for children aged 4 to 16 years old [22,23]. In our

study patients aged 18 years and older were asked to complete a modified form of the

DISABKIDS questionnaire; substantive changes to the questionnaire were not made,

only textual (grammatical) changes were made to fit the age of the participant.


~ 51 ~

Statistical analysis

For all analyses the Statistical Package for Social Sciences (SPSS) Windows version 19

was used. Data were recorded on online data collection forms and entered after

validation in a computer system for subsequent tabulation and statistical analysis.

Pre-analysis

First, the internal consistency of the scales was calculated. Scales with Cronbach's Alpha

lower than 0.5 were not included in the analyses. Second, chi square-tests were

conducted to test differences between the different groups with respect to gender.

Because no significant differences were found, correction for gender was not necessary

in the further analyses. In addition, Mann–Whitney U tests were conducted to test

differences between those patients who completed the questionnaires at both time

points and those who only participated at baseline; regarding age, allowed daily protein

intake and HRQoL.

Analysis used to evaluate the HRQoL of patients with PKU compared to the general

population

To compare HRQoL of PKU patients to the general population (our first aim of the

study), Mann–Whitney U tests (due to skewed distribution of the data) were performed

for the scale scores of the PedsQL and TAAQOL at first measurement, before

responsivity testing. Because the norm data of the PedsQL consisted of patients aged 5–

17 years old, patients with PKU aged 4 years old were left out of the comparison. The

DISABKIDS questionnaire is not intended for healthy children; therefore comparison

with the general population was not possible.

Chapter 3

~ 52 ~

Analysis used to get an indication of the effects of treatment with BH4 on HRQoL

The effect of BH4 treatment (our second aim of the study) was evaluated in two ways;

within-group comparisons and between-group comparisons as follows. Within the

treated BH4 responsive patients, differences between results before and after

responsivity testing regarding their scores on the PedsQL and the DISABKIDS were

tested using Wilcoxon signed rank tests. Between-group comparisons were assessed

regarding difference in the scores of the PedsQL and DISABKIDS between the treated

BH4-responsive patients and the unresponsive patients at the second measurement

(after responsivity testing) using Mann–Whitney U tests. The effect of BH4 treatment

could not be evaluated in the adult PKU patients because of too small sample size.

We displayed mean score with standard deviation for descriptive purposes. To adjust for

multiple testing, a significance level of p=0.01 was used for all tests. Significance levels

of p=0.05 are reported as trends.


~ 53 ~

RESULTS

Participants

Demographics

A total of 226 sets (patient information forms) were distributed to the physicians to

invite patients to enter in the study. We do not know how many sets were actually sent

out (the response rate therefore might be higher), but 81/226 (36%) patients consented

to participate in the study and received a personal code to enter the study website. 69/81

(85%) patients or mothers of pediatric patients completed the questionnaires before

BH4 responsivity testing. The patient group consisted of 33 males and 36 females, with

ages ranging from 4 to 44 years (mean: 18.4 years; SD: 10.2 years). 45/69 (65%)

patients or mothers completed the questionnaires again 17–24 months after being

tested for BH4 responsivity. This group consisted of 20 males and 25 females, with ages

ranging from 6 to 43 years (mean: 19.3 years; SD: 10.4 years).

The patients who completed the questionnaire before and after BH4 testing (n = 45) did

not differ from those only completing the questionnaires before BH4 testing (n = 24)

regarding age, gender, allowed daily protein intake and HRQoL.

BH4 responsivity

Of the patients who completed the questionnaires before and after being tested for BH4

responsivity, 10/45 (22%) patients were found to be BH4 responsive (mean age 13.8

years and SD: 9.7). Eight of these patients reported their intake of natural protein before

and after the start of treatment. After the start of BH4 treatment the mean intake of

natural protein increased from 14.2 (SD: 7.9) to 58.3 (SD: 27.7) grams of natural protein

per day (p = 0.012, Z = 2.52, according to the Wilcoxon signed rank test). Four patients

were able to fully relax their diet after the start of treatment. Of the 10 responsive

patients, amino acid supplementation could be stopped in three patients, reduced >60%

in three patients and a reduction of <20% was achieved in two patients. Of two patients

the amino acid supplement intake before and after start of BH4 treatment were

Chapter 3

~ 54 ~

unknown. The group of unresponsive patients did not demonstrate an increased natural

protein intake since the first report; from 10.4 (SD: 4.5) grams before responsivity

testing to 12.7 (SD: 14.8) grams of protein afterwards (p = 0.303, Z = 1.03).

Health related quality of life

HRQoL in patients with PKU compared to the general population

Children

Overall, the HRQoL of patients aged 5–12 measured with the PedsQL was comparable to

the general population. Remarkably, patients aged 13–17 reported overall a significantly

higher HRQoL score than the general population. The mothers of children aged 5–7

reported a trend towards lower HRQoL scores on the scales “school- and social

functioning”. Patients aged 8–12 reported a significantly higher HRQoL score on the

“physical functioning” scale. Patients aged 13–17 years reported a significantly higher

HRQoL score on both the “total” scale and on “psychosocial functioning” compared to

the general population. Furthermore, in patients aged 13–17 a trend towards a higher

HRQoL score was found on the scales “social functioning” and “school functioning”

(Table 1).

Adults

The total HRQoL of adult patients measured with the TAAQOL questionnaire was

comparable to the general population. However, the adults with PKU reported a

significantly lower HRQoL score on the “cognitive functioning” scale (Table 2).


~ 55 ~

Table 1. HRQoL in pediatric PKU patients before testing BH4 responsiveness: PedsQL

scores of patients with PKU versus the general population

PKU Patients General Population˙

Age Group 5-7 8-12 13-17 5-7 8-12 13-17

N 10 15 14 92 219 185

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Total 75.0 19.2 81.9 13.8 91.3* 8.0 84.2 9.0 82.1 8.9 82.2 9.1

Physical

functioning

- - 93.8* 10.0 - - - - 84.9 9.3 - -

Emotional

functioning

74.0 18.9 - - - - 75.8 13.3 - - - -

Social functioning 69.4° 21.9 - - 95.7° 6.5 86.2 12.2 - - 89.4 11.6

School functioning 71.3° 19.0 - - 84.6° 14.1 85.5 11.6 - - 74.6 13.2

Psychosocial

functioning

71.7 17.9 - - 88.8* 9.8 82.5 9.9 - - 80.2 10.2

˙ Engelen V, Haentjens MM, Detmar SB, Koopman HM, Grootenhuis MA: Health related quality of life of Dutch

children: psychometric properties of the PedsQL in the Netherlands. BMC Pediatr 2009, 9:68.

- Results of scales with Cronbach Alpha < 0.50 were not provided. HRQoL of 5-7 year old patients is assessed by proxy by their mothers HRQoL in the other age groups are self-reported. ° p < 0.05 and * p < 0.01

Chapter 3

~ 56 ~

Table 2. HRQoL in adult PKU patients before testing BH4 responsiveness: TAAQOL

scores of patients with PKU versus the general population

˙ Fekkes M, Kamphuis RP, Ottenkamp PJ: Health-related quality of life in young adults with minor congenital heart

disease. Psychology & Health 2001, 16:239-250.

¹ 1898 = complete cases, patients with scores on all scales. Number of cases varies from 1952 (Sexuality) to 2004 (Pain). * p < 0.01

PKU group General Population˙

Age in Years (mean (SD)) 28.5 (6.1) 33.4 (6.5)

N 30 1898¹

Mean SD Mean SD

Gross motoric functioning 92.9 14.2 92.5 16.3

Fine motor functioning 97.3 8.5 98.2 8.1

Cognitive functioning 67.3* 31.0 87.0 20.2

Sleep 73.8 24.0 77.1 24.3

Pain 72.3 20.5 78.2 20.8

Soc functioning 89.2 12.9 87.2 17.8

Daily activities 83.8 23.6 85.4 22.4

Sexuality 90.4 20.4 87.6 23.1

Vitality 67.5 17.1 66.5 22.1

Positive emotions 75.3 18.1 68.3 20.9

Depressive emotions 77.8 19.7 79.8 19.5

Aggressive emotions 84.8 18.3 87.5 16.5


~ 57 ~

Effects of BH4 treatment on HRQoL

Children — generic HRQoL

No difference was found in the generic HRQoL (self and mother report) of BH4

responsive patients measured with the PedsQL questionnaires before versus after the

start of BH4 treatment (Table 3). Furthermore, no significant difference was found in

the HRQoL of treated BH4 responsive patients versus unresponsive patients at the

second measurement (Table 4).

Adults — generic HRQoL

The TAAQOL data could not be used for analysis, because of the small number of

patients (N = 3).

Children and adults — questionnaire for the chronically ill

BH4 responsive children (self and proxy reports) and adults showed no significant

differences in scale scores on the DISABKIDS before treatment compared to after the

start of treatment (Tables 5 and 6). In addition, no differences were found between the

DISABKIDS scale scores of treated BH4 responsive patients and unresponsive patients

at the second measurement, after responsivity testing (Tables 7 and 8).

Chapter 3

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Table 3. HRQoL of BH4-responsive pediatric PKU patients before versus after testing

for BH4 responsiveness (and after start of BH4 treatment) measured by the PedsQL

Self-report (8-17 year old) Proxy-report (4-17 years old)

Before testing After testing Before testing After testing

N 5 5 6* 6

Mean SD Mean SD Mean SD Mean SD

Total 91.3 6.4 87.2 4.9 84.2 13.4 87.1 8.4

Physical functioning 99.4 1.4 98.8 2.8 91.1 14.2 97.7 2.6

Emotional functioning 84.0 8.2 78.0 17.9 76.4 13.5 75.8 21.2

Social functioning 90.0 14.6 93.0 5.7 84.2 21.3 85.8 16.9

School functioning 87.0 10.4 72.0 5.7 80.8 13.2 82.5 11.3

Psychosocial functioning 87.0 9.7 81.0 8.0 94.2 8.2 91.7 4.7

No significant difference on any of the scales is found. Pediatric patients that proved BH4 responsive n=7 (age 4-7 n=2; age 8-12 n=2; age 13-17 n=3). * One mother did not complete the PedsQL in 2011 and therefore could not be included in this analysis.


~ 59 ~

Table 4. HRQoL of BH4-responsive versus unresponsive pediatric PKU patients after

testing for BH4 responsiveness (and after start of BH4 treatment if responsive)

measured by the PedsQL

Self-report (8-17 year old) Proxy-report (4-17 years old)

BH4

responsive

BH4

unresponsive

BH4

responsive

BH4

unresponsive

N 5 14 6* 20


Total 87.2 4.9 85.0 14.2 87.1 8.4 81.7 16.6

Physical functioning 98.8 2.8 93.5 11.5 97.7 2.6 86.4 17.4

Emotional functioning 78.0 17.9 81.8 18.1 75.8 21.2 78.6 21.5

Social functioning 93.0 5.7 88.2 16.9 85.8 16.9 84.8 18.7

School functioning 72.0 5.7 71.4 21.0 82.5 11.3 73.5 20.0

Psychosocial functioning 81.0 8.0 80.5 16.1 91.7 4.7 79.3 17.3

No significant difference on any of the scales is found. Pediatric patients proven BH4 responsive n=7 (Age 4-7 n=2; age 8-12 n=2; age 13-17 n=3). * One mother did not complete the PedsQL in 2011 and therefore could not be included in this analysis.

Chapter 3

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Table 5. HRQoL of BH4-responsive PKU patients before versus after testing for BH4

responsiveness (and after start of BH4 treatment) measured by the DISABKIDS

questionnaire self-report (≥ 8 years)

8-17 years Adult


N 5 5 3 3


Total 159.6 10.4 160.2 16.0 152.0 20.3 163.3 16.3

Independence 26.2 2.6 25.0 1.7 25.7 2.9 27.7 2.5

Emotion 29.4 3.4 31.0 5.6 28.7 4.2 30.0 4.6

Social understanding/inclusion 25.4 2.6 26.0 2.9 24.7 1.5 25.7 2.1

Social stigma/exclusion 27.0 2.2 27.0 2.9 28.3 1.5 28.3 1.5

Limitations - - - - - - - -

Impact treatment 25.0 3.8 24.0 7.0 21.0 7.5 24.3 6.7

No significant difference on any of the scales is found. Analysis were performed for the group of patients 8-17 years old and adults as a whole. - Results of scales with Cronbach Alpha < 0.50 were not provided.


~ 61 ~

Table 6. HRQoL of BH4-responsive pediatric PKU patients before versus after testing

for BH4 responsiveness (and after start of BH4 treatment) measured by the DISABKIDS

questionnaire proxy-report (mothers of patients 4-17 year old)

4-7 years 8-17 years


N 2 2 4* 4


Total 21.0 5.7 25.0 2.8 166.2 10.2 153.0** 15.6

Independence 27.6 2.1 26.8 1.5

Emotion 30.0 4.2 31.5 5.7

Social understanding/inclusion 26.8 3.0 26.5 1.3

Social stigma/exclusion 28.8 1.3 27.3 2.2

Limitations 27.4 1.5 23.8 9.2

Impact treatment 25.6 4.6 21.7** 8.5

No significant difference on any of the scales is found. Group 4-7 years old were not statistically analysed due to the small number of patients. Pediatric patients proven BH4 responsive n=7. (Age 4-7 n=2; age 8-12 n=2; age 13-17 n=3). * One mother did not complete the DISABKIDS in 2011 and therefore could not be included in this analysis. ** n = 3 because one mother did not complete this scale of the DISABKIDS in 2011.

Chapter 3

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Table 7. HRQoL of BH4-responsive versus unresponsive PKU patients after testing for

BH4 responsiveness (and after start of BH4 treatment if responsive) measured by the

DISABKIDS questionnaire self-report (≥ 8 years)

8-17 years Adult

BH4

responsive

BH4

unresponsive

BH4

responsive

BH4

unresponsive

N 5 16 3 13


Total 160.2 16.0 153.1 19.8 163.3 16.3 153.4 26.6

Independence 25.0 1.7 25.3 4.4 27.7 2.5 25.5 4.7

Emotion 31.0 5.6 28.8 6.1 30.0 4.6 29.4 6.6

Social

understanding/inclusion

26.0 2.9 23.9 4.5 25.7 2.1 24.0 4.2

Social stigma/exclusion 27.0 2.9 27.1 3.0 28.3 1.5 26.8 4.3

Limitations - - - - - - - -

Impact treatment 24.0 7.0 23.0 4.3 24.3 6.7 23.2 6.5

No significant difference on any of the scales is found. Analyses were performed for the group of patients 8-17 years old and adults as a whole. - Results of scales with Cronbach Alpha < 0.50 were not provided.


~ 63 ~

Table 8. HRQoL of BH4-responsive versus unresponsive pediatric PKU patients after

testing for BH4 responsiveness (and after start of BH4 treatment if responsive)

measured by the DISABKIDS questionnaire proxy-report (mothers of patients 4-17 year

old)

4-7 years 8-17 years

BH4

responsive

BH4

unresponsive

BH4

responsive

BH4

unresponsive

N 2 6 4* 13***


Total 25.0 2.8 22.3 4.4 153.0** 15.6 153.3 25.9

Independence 26.8 1.5 26.1 4.0

Emotion 31.5 5.7 28.8 7.5

Social

understanding/inclusion

26.5 1.3 24.6 4.1

Social stigma/exclusion 27.3 2.2 26.5 3.9

Limitations 23.8 9.2 25.3 5.0

Impact treatment 21.7** 8.5 22.0 5.4

No significant difference on any of the scales is found. *One mother did not complete the DISABKIDS in 2011 and therefore could not be included in this analysis ** n = 3 because one mother did not complete this scale of the DISABKIDS in 2011 *** Three mothers did not complete the DISABKIDS in 2011 and therefore could not be included in this analysis.

Chapter 3

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DISCUSSION

Our study demonstrates that children and adults with PKU have a generic HRQoL

comparable to or better than the general population, as assessed by the PedsQL and

TAAQOL questionnaires. This is not an unexpected finding: most studies evaluating

HRQoL of PKU patients report normal scores [14,16,17]. It is yet unclear whether this

truly reflects HRQoL of the patients, or if this results from the use of generic

questionnaires which do not detect the possible negative consequences of “hidden

disabilities” experienced by these patients such as difficulties in planning, organizing

and reduced processing speed, which may affect treatment adherence, social

relationships, and job performance [24].

Remarkably, in the present study children between the ages 13 to 17 years reported a

significantly better HRQoL regarding total and psychosocial functioning and a trend

towards better school functioning and social functioning. This may be explained by

response shift. PKU patients grow up with the knowledge that incompliance with the

diet will be harmful to their health and abilities. Therefore, they may appreciate their

own abilities more than healthy children, possibly resulting in better HRQoL because of

this response shift [25]. In contrast, mothers reported a trend towards worse HRQoL

regarding school functioning in their 5–7 year old children. This may well be true, or

may be the result of their knowledge that patients with PKU have on average a lower IQ

than the norm [26–28], or it may be an expression of their worries about the cognitive

functioning of their child as was recently reported by Thimm et al. [17].

Adult patients reported a significantly lower HRQoL with respect to cognitive

functioning compared to the general population. This is a new finding, even though in a

previous study a group of Dutch adult patients demonstrated a trend towards a lower

score [14]. On the one hand, this lower HRQoL may result from the lower mean IQ

found in patients with PKU [29–31] or from the executive function deficits

demonstrated even in treated patients [32]. On the other hand, this particular HRQoL

scale of the TAAQOL has a strong focus on concentration. It is possible that patients

have become more aware of problems with concentration, since the negative effects of

higher Phe values on sustained attention have been extensively discussed on PKU fora


~ 65 ~

and within the Dutch PKU society [33]. Furthermore, this may be a reflection of worries

about their cognitive functioning [34] also possibly leading to response shift [25].

The latest breakthrough in the treatment of patients with PKU has been the introduction

of BH4. With the introduction of new treatments, the primary outcome in studies will

most often be the biochemical outcomes. However, the effect on quality of life of the

patients may well be considered the crucial outcome.

In our patient group the introduction of BH4 caused high expectations, followed by

strong positive emotions in responsive patients, and strong negative emotions in

unresponsive patients who lost the perspective of relaxation of the diet. From clinical

experience it is thought [35] that it takes several months for the unresponsive patients

to overcome their disappointment and for the responsive patients to adapt to the more

relaxed diet. To avoid effects of these emotions on our measurements [35] and to

measure in a stable situation only, we chose to evaluate the HRQoL of patients before

and at least one year after the BH4 responsivity test; which in practice was 17–24

months after the test (and start of BH4 treatment if responsive).

To evaluate the effects of BH4 on the quality of life of the patients several comparisons

were made. No differences between HRQoL scores before and after the start of BH4

treatment were found within the group of BH4 responsive patients. Also, no differences

were found between the treated BH4 responsive patients and the unresponsive patients

at the second measurement. This confirms the findings of Ziesch et al. [35] who also

evaluated the effects of BH4 treatment in patients with PKU and found no significant

effects. However, in that study HRQoL was measured on time points more closely to

responsivity testing: the first day of the preparation for the BH4 responsivity test and 42

and 90 days after responsivity testing.

We had expected to find effects of treatment with BH4 on the HRQoL of the patients.

Patients in our clinics could increase their daily protein intake 4 fold and during

outpatient clinic visits spontaneously report a major increase of freedom and

spontaneity in their lives. However, even on item base in the “limitations” section of the

DISABKIDS questionnaire, no significant changes are seen. The fact that we did not

demonstrate an effect may have several reasons. There may be a “ceiling effect”: the

Chapter 3

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reported HRQoL of the patients was already excellent, even before the responsivity

testing, and there may be no room for further improvement.

There are a number of limitations to this study as well. First, the study design, using the

available generic HRQoL questionnaires because of lack of a PKU specific HRQoL

questionnaire, was not optimal for studying the effect of BH4 treatment. However, the

introduction of BH4 in 2009 necessitated this timing of our study. Second, selection

bias and lost to follow-up could have been a threat to the generalizability and validity of

the results. Although we found no differences regarding age, gender, allowed protein

intake and HRQoL between those who completed the questionnaires before and after

BH4 testing versus those who were lost to follow-up, no information is available about

patients who did not participate at all. Also, from the patients lost to follow-up, we do

not know how many would have turned out to be BH4 responsive. Third, the statistical

power was limited due to the small sample size, which is a common problem in studies

with patients with inborn errors of metabolism. Based on this study, it is not possible to

conclude that BH4 use does not improve the HRQoL of patients. We may not have had

the power to detect a true difference between treated BH4 responsive patients, either

before and after treatment or versus unresponsive patients, even if such a difference

existed. Fourth, it must also be pointed out that not all outcome measures could be used

in the analyses because of unsatisfactory internal consistency of several HRQoL scales in

this relatively small cohort (Cronbach Alpha b0.50). A final limitation may be that we

did not collect the blood Phe values of the patients at the time of answering the

questionnaires, yet these might have been valuable due to the proven effect on mood

[36]. Despite the good HRQoL in our population, it may still be important to monitor

patients' quality of life during clinical practice and to be attentive to changes as a result

of development and treatment. More studies, using a PKU specific HRQoL

questionnaire and a meta-analysis are needed and could provide a definitive conclusion

regarding the effect of BH4 use on HRQoL.

In conclusion, this cohort of Dutch patients with PKU overall demonstrated a normal to

better HRQoL when compared to the general population. However, the adult patients

showed significantly lower scores on the domain of cognitive functioning. With the

currently available questionnaires and given the limitations of the study, an


~ 67 ~

improvement in HRQoL of patients with PKU could not be demonstrated after at least

17 months of treatment with BH4.

Chapter 3

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The time consuming nature of

phenylketonuria: A cross-sectional study

investigating time burden and costs of

phenylketonuria in the Netherlands

Mol Genet Metab 2013, 109: 237-242

Serwet Demirdas1, Indra Eijgelshoven1, T Alexander Smith, Jeanni MT van Loon,

Sabine Latour and Annet M Bosch 1 Equal contributors

Chapter 4

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ABSTRACT

Background: Phenylketonuria (PKU) is a rare inborn error of metabolism that affects the

ability of patients to metabolise phenylalanine (Phe). Lifelong management of blood Phe levels

is required in order to avoid the complications associated with PKU. This constitutes a severely

protein restricted diet, and regular monitoring of Phe levels. Management of PKU may be costly

and time-consuming for adult patients or caregivers of PKU-affected children. A cross-sectional

study was performed with patients or their caregivers in the Netherlands to gain insight into the

personal time burden and cost of living with PKU.

Methods: A systematic literature review was performed to identify all aspects of PKU

management that may pose a financial or time burden on patients or caregivers. Findings were

confirmed through interviews with PKU experts and feedback from patients and caregivers, and

consolidated into a questionnaire that aimed to evaluate the impact of each of these factors.

Early and continuously treated adult patients and caregivers from seven metabolic centres were

recruited to complete the questionnaire online.

Results: 22 adult patients and 24 caregivers participated in the study. Managing a Phe-

restricted diet represented an extra time burden of 1 h and 24 min for caregivers and 30 min for

adult patients per day. Caregivers reported a significantly higher time burden than adult

patients. The median total out-of-pocket cost (OOPC) for patients was €604 annually, with 99%

of expenditure on low-protein food products. Greater disease severity was significantly

associated with increased OOPC and time burden for both adult patients and caregivers.

Conclusions: Management of PKU is associated with a considerable time burden for both

caregivers of children with PKU and adult patients. Caregivers of PKU-affected children

reported a significantly higher time burden than adult patients. The OOPC of caregivers and

patients was mainly driven by the expenditure on low protein food.

The Time Consuming Nature of PKU

~ 75 ~

INTRODUCTION

Phenylketonuria (PKU, ORPHA79254, MIM 261600) is a genetic disorder that arises

due to mutations in the gene that codes for phenylalanine hydroxylase (PAH; EC

1.14.16.1), a hepatic enzyme necessary for the metabolism of the essential amino acid

phenylalanine (Phe) to tyrosine [1]. The resulting PAH deficiency leads to chronic

increases in blood and tissue Phe concentrations, with toxic effects on the brain. PKU is

an orphan disease with a mean prevalence in Europe of 1:10,000 [2]. If left untreated, it

will lead to severe mental retardation, with additional symptoms such as autism,

epilepsy and eczema. Since the 1960s, in most European countries, these severe

complications can effectively be prevented by detection through newborn screening and

early start of treatment, usually within the first two weeks of life [3].

Treatment of PKU consists of a very strict and unpalatable diet that severely restricts

intake of natural protein, with supplementation of other amino acids in a mixture with

vitamins and minerals [2,4]. Energy is provided by food that is naturally low in protein

such as fruit and non-starchy vegetables, as well as specially formulated products such

as pastas, breads, imitation cheese and baking mixes designed for low-protein diets [5].

Some patients may benefit from treatment with BH4 (sapropterin), the cofactor of the

PAH enzyme, which increases PAH activity and consequently lowers Phe blood

concentrations in responsive PKU patients [6,7].

Disease severity can be classified by the Phe blood level at the time of diagnosis, or be

based on the tolerance for the dietary intake of Phe during treatment. Several

classifications are used in the literature; one of the common classifications describes

patients as having classical PKU (untreated Phe level > 1000 μmol/L, Phe tolerance b

500 mg/day) or mild PKU (untreated Phe level ≤ 1000 μmol/L, Phe tolerance ≥ 500

mg/day) [2,4,8,9].

Even if treatment is started early, patients may suffer from executive deficits and mood

disturbances. As these effects are strongly associated with concurrent Phe levels, a diet

for life is usually advised [10–12]. Poor executive functions may lead to “hidden

disabilities” such as difficulties in planning, organising and reduced processing speed,

thereby affecting treatment adherence, social relationships, and job performance [13].

Chapter 4

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Most studies report a normal health related quality and course of life in patients who are

treated early and continuously, even though patients often report having certain

restrictions with an evident lack of spontaneity in their lives [14–17]. This reporting of

“normal” health related quality of life (HRQoL) is probably due to the fact that the

generic questionnaires available for measuring HRQoL do not evaluate specific PKU

related problems. However, the authors are currently aware of a disease specific

questionnaire being developed for PKU and this will hopefully provide more insight into

the HRQoL of PKU patients.

The PKU diet is considered a heavy burden by both patients and professionals [18] and

the management of PKU can be time consuming for both adult patients and caregivers

of PKU-affected children. Patients and caregivers need to obtain low-protein food

products, plan the daily Phe intake, prepare the daily menu (that often involves extra

cooking), and prepare and take (or supervise the intake of) supplements. Furthermore,

Phe intake has to be closely monitored with regular blood testing of Phe levels. The

Dutch Guidelines recommend once weekly testing during the first year of life, twice

monthly from age 1, monthly after age 4, and twice weekly during pregnancy if the

mother has PKU [12].

PKU may additionally present an economic burden to patients and caregivers. This

includes direct costs of living with PKU, which relates to resource utilisation in

managing their condition, such as low-protein foods, supplements, medications,

laboratory monitoring, and healthcare visits. There may also be indirect costs such as

those arising from the loss of productivity. Costs and reimbursement of diet therapy

vary widely between different countries [19]. In the Netherlands patients with PKU and

caregivers of PKU-affected children receive tax credits and amino acid supplements are

reimbursed. Although information is available on the burden of PKU to the healthcare

system [20], there is not much information available on the personal time burden and

cost of living with PKU for patients.

To gain insight into the personal time burden and cost of living with PKU in the

Netherlands, we conducted a cross-sectional study that assessed the impact of PKU on

adult patients and caregivers of PKU-affected children. Our aim was to measure the


~ 77 ~

time spent on activities related to PKU management and also to measure the out-of-

pocket costs (OOPCs). The OOPC refers to those PKU-related expenses that are not

reimbursed by the healthcare system. Any differences in OOPC and time burden

according to different categories of patients (adult patients, caregivers, age, disease

severity and adherence to diet) were also evaluated. Medical costs of PKU to the

healthcare system are not considered in this study as the study focuses solely on the

personal burden of PKU on affected individuals.

Chapter 4

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METHODS

Study design and study population

We conducted a cross-sectional study to evaluate the costs and time burden of living

with PKU in the Netherlands from the perspectives of the patient or the caregiver.

Different aspects of the PKU lifestyle that present any potential monetary costs were

presented to participants in an internet-based questionnaire through which respondents

could indicate their OOPCs and time spent on managing a PKU lifestyle. Patients were

recruited from seven metabolic centres in the Netherlands. Early and continuously

treated adult patients and caregivers of paediatric patients (who were already

participating in another internet-based PKU study including patients age 4 years and

older) were invited to participate in this study as well.

Development of the questionnaires

A systematic literature review was performed to identify all available data on costs

associated directly with PKU and aspects of PKU that affect HRQoL. This was to gain

insight into the PKU lifestyle that would facilitate preparing a script for expert

interviews. The search was specific for costs borne by patients and their families rather

than the healthcare system. This included the costs of treating the symptoms of PKU as

well as any costs associated with managing the disease or looking after patients with

PKU. Information was categorised into disease subgroups, such as severe versus mild;

this was to enable assessment of how the burden of PKU varied for different patient

characteristics. A second literature search was performed in order to obtain guidance in

the creation of the questionnaire, including the identification of any suitable pre-

validated questionnaires that captured information on productivity loss and healthcare

resource use that could be incorporated into the survey. The findings of the literature

reviews were confirmed through interviews with six opinion leaders in the field of PKU,

three of whom were from the UK and three from the Netherlands, who detailed the

various costs associated with the management of PKU. This ensured that all potential


~ 79 ~

OOPCs and time expenditure associated with living with PKU were captured in the

study questionnaires.

Similar but separate questionnaires were created for completion by adult PKU patients

and caregivers of paediatric patients with PKU. Each questionnaire consisted of five

sections covering background information on the patient, treatment and clinical history,

general life, the effect of their health on labour, and various aspects of the Phe-restricted

diet. They assessed patients' experience over weekly, monthly and annual time frames

retrospectively. The Short Form Health & Labour Questionnaire (SF-HLQ), which is

validated in both Dutch and English, was included to gather data on productivity loss.

The questionnaires, which were created in English and then translated to Dutch, were

reviewed by four experts. They were appraised at the Dutch National PKU event by four

caregivers and one adult patient who were asked to provide feedback on their ease-of-

understanding as well as any possible suggestions for further areas that may be covered.

The questionnaires were reviewed based on feedback obtained, with further input from

two experts.

Data collection

Patients were recruited on an ongoing basis through the database of the Academic

Medical Center (AMC), Amsterdam, from mid-December 2011 to early-April 2012.

Participants were sent two reminders over a three-month period to complete the

questionnaire. Informed consent was obtained from all participants, and confidentiality

was assured. Participation was voluntary and there were no implications on treatment

for participants. Participants received a fee after completing the questionnaire, of which

they were not informed prior to filling out the questionnaire. Patient data sets were

coded and ethical approval was requested from the Ethical Committee of the AMC who

deemed that their approval was not necessary for this study.

Chapter 4

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Data analysis

In addition to the overall time burden and cost of living with PKU, demographic and

disease data were used for creating subgroups to compare the time burden and financial

cost of living for different categories of patients within each subgroup: adult patient

versus caregiver; age; severity of disease; adult patient versus caregiver in combination

with disease severity; and dietary adherence compared with dietary non-adherence.

Disease severity was defined according to the amount of natural protein (or Phe)

allowed per day for an individual patient: patients who are allowed >10 g of natural

protein (500 mg of Phe) per day were classified as ‘mild’; patients who are allowed ≤10 g

of natural protein (500 mg of Phe) per day were classified as ‘severe’. For assessing

adherence, patients were asked to provide the number of days in which they did not

adhere to the diet over the preceding 100-day period; patients were regarded as showing

good adherence if this totalled 30 days or fewer. Monetary costs for patients were

reported as OOPCs.

The analyses were performed based on the available case principle, including only those

patients for which the outcome and covariates are known. The total number included in

the analyses will differ across parameter estimations. All outcomes were continuous

outcomes (time and OOPC) and inferences were described in terms of medians, means,

interquartile ranges and ranges. To test whether differences between subgroups were

statistically significant (two sided p-value b0.05) the Wilcoxon Rank Tests for skewed

outcome distributions were used.


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RESULTS

Patient characteristics

A total of 69 participants from another web based PKU study in the Netherlands were

invited to participate in the study; of these, 22 adult PKU patients and 24 caregivers of

paediatric patients with PKU (67%) completed the survey and were included in the

analyses. Adult patients had a median age of 28 years [interquartile range (IQR) 23–53]

and paediatric patients of 11 years [IQR 9–14]. Of the adult patients, 15 reported to have

a mild disease severity and 6 reported severe. Of the paediatric patients, 9 were mild

and 12 severe. No patients were excluded from the analyses. The majority of patients

(38/46) were on a protein restricted diet. Among the patients on a protein restricted

diet, 30 patients took amino acid supplementation, 1 patient was treated with protein

restriction and sapropterin, 3 patients were treated with sapropterin and amino acid

supplementation, and 4 patients took no other treatment. Eight patients were not on a

diet, of whom 3 were treated with sapropterin only, and 2 were treated with sapropterin

and amino acid supplementation. One patient reported using amino acid supplements

without dietary restriction. In total, 26 patients reported the use of low-protein food

products (11 adults and 15 caregivers).

Time burden of PKU

The primary components of the time burden of PKU are summarized in Table 1. On

regular household tasks, not associated with PKU management, caregivers of patients

with PKU spent a median of 312 h and adult patients a median of 208 h/year. The

median total time burden associated specifically with managing PKU was 265 h [IQR

129–588]/year for the complete cohort, with a median 527 h for caregivers [IQR 213–

826] and a median 175 h [IQR 125–300]/year for adult patients (Figure 1). Dietary

management represented the greatest time burden, with 46% of the total PKU time

burden consisting of cooking and preparing meals specifically for a Phe-restricted diet,

and 11% on monitoring protein/Phe intake. The time spent on managing PKU was

significantly lower for adults than for caregivers. Furthermore, severe (median 595 h

Chapter 4

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[IQR 214–862]) PKU was associated with a significant greater time burden than mild

PKU (median 235 h [IQR 123–353]) (Figure 1). Neither adherence nor different age

groups among adults or caregivers had a significant effect on the time spent of managing

PKU.

Table 1. Time spent on PKU-related tasks, overall population

Activity Median [IQR] (mean)/h/year

Blood testing 1 [0–1] (1) Keeping Phe records 30 [0–30] (35) Cooking for Phe-restricted diet 121 [15–182] (131) Weighing foods 0 [0–12] (13) Supervising protein intake (caregivers only) 30 [8–61] (64) Baking bread 0 [0–104] (130) Food research 2 [0–39] (39) Researching PKU 0 [0–12] (20) Other PKU-related tasks 0 [0–0] (19) PKU events 0 [0–9] (17) Preparing for social events 3 [0–12] (11) Ordering amino acids 1 [0–2] (1) Ordering low-proteins 1 [0–2] (2) Ordering sapropterin 0 [0–0] (0) Total time 265 [129–588] (420)

IQR = interquartile range.

Figure 1. Time burden for different subgroups of participants


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Out of pocket costs for PKU

The primary components of OOPCs for patients with PKU are summarized in Table 2.

The median OOPC per patient was €604 [IQR €28-€1206] annually, and there was also

a median one-off OOPC of €50 [IQR €30-€250]. The majority (99%) of OOPCs were

due to expenditure on low-protein food products. Other costs consisted of the postage

cost for the Phe blood test, taking extra holiday luggage to accommodate equipment for

PKU testing or dietary management, and PKU-related events such as specialised

cookery classes. OOPCs were significantly higher for patients with severe PKU (daily

allowed intake of natural protein ≤10 g) versus mild PKU (daily allowed intake of

natural protein >10 g), €1309 [IQR €716–€1755] and €393 [IQR €3–€698]

respectively. In addition caregivers taking care of a child with severe PKU (€1309 [IQR

€1054–€1793] also had significantly more OOPCs than those who were caring for a

child with mild PKU (€5 [IQR €0–€486]) (Figure 2). Neither adherence nor different

age groups among adults or caregivers had a significant effect on OOPCs.

Table 2. Out-of-pocket costs, overall population

Expense type Median [IQR] (mean)/€/year

Amino acid supplements 0 [0–0] (75) Low-protein foods 600 [0–1200] (680) Postal cost for Phe blood test 2 [0–5] (5) Extra holiday luggage for PKU equipment 0 [0–0] (54) PKU events 0 [0–8] (34) One off-expenses (PKU equipment) 50 [30–250] (189) Annual total direct OOPCs (medical and non-medical) 604 [28–1206] (798)

IQR = interquartile range.

Chapter 4

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Figure 2. Out-of-pocket costs for different subgroups of participants

Other findings

Six adult patients and 6 caregivers reported receiving tax credits towards PKU products.

Loss of productivity was reported by 3 adult patients; additionally 3 patients indicated

that health affected their performance at work. Caregivers did not report any production

loss, although 2 caregivers reported leaving their employment in order to care for a PKU

child and 1 changed their employment because of PKU.


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DISCUSSION

This is the first study to investigate the time burden and costs resulting from having

PKU or caring for a child with PKU.

The most important outcome of our study is the considerable time burden posed on

patients and families with PKU. As presented earlier in the results, the median time

burden associated with managing PKU was 527 h/year (1 h and 24 min/day) for

caregivers and 175 h/year (30 min/day) for adult patients. Time was mostly spent on

cooking and preparing meals specifically for a Phe-restricted diet, followed by

monitoring protein intake. The significantly higher time burden for caregivers versus

adult patients suggests that less time is required for PKU management as patients enter

adulthood and begin caring for themselves. Also, this fits with the idea that many adult

patients tend to somewhat relax their diet [21]. Furthermore, the higher time burden

associated with severe versus mild PKU is to be expected as greater disease severity

necessitates more stringent dietary control and PKU monitoring.

It needs to be discussed whether the time spent on PKU management will truly pose a

burden on the lives of patients and caregivers. On regular household tasks the caregivers

reported to spend a mean of 2 h and 3 min/day (median 52 min), which is comparable

to the mean 2 h and 10 min spent on household tasks in the general population by a

Dutch parent of a family with a youngest child age 6–12 [22]. Patients and caregivers

were explicitly asked to report the extra time spent on PKU management. Our study

showed that management of PKU poses a considerable additional time burden on adults

(30 min/day) and caregivers (1 h and 24 min), which could have been spent more

valuable when not having to deal with PKU. As such, the time spent on managing PKU

could take away time from other daily activities. As few caretakers reported change of

employment or production loss as a result of having a child with PKU, the extra time

needed to manage PKU will probably be taken away from leisure time. The time burden

for adult patients is significantly less than for the caregivers; however, as “hidden

disabilities” such as difficulties in planning and organizing have been reported in

patients with PKU, this time burden may affect their daily lives as well.

Chapter 4

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The additional time spent by PKU patients for managing their disease can be considered

as a loss of valuable time that could have been spent more beneficial. When this time

loss is expressed as a monetary burden (using the gross hourly earnings of a cleaner

€13.40 [23] per hour as recommended by the Dutch Healthcare Insurance Board [24])

on society, this would amount to €7066 for caregivers of PKU-affected children and to €

2341 annually for adult PKU patients.

There is a lack of published information on the time burden for other metabolic

diseases; therefore comparison to other metabolic disorders is not feasible. However, a

US study on the burden of patients with diabetes mellitus type I, found that patients

spent on average 28.3 min a day managing their condition [25]. Additionally they spent

4.4 min/week researching the internet on diabetes and 30.8 min/week on reading for

diabetes. This would indicate a total time burden of 203 h/year for diabetes

management, compared with 265 h/year PKU management in the present study.

Although diabetes is not a metabolic disorder, the diabetes study can be used for

comparison with this study as they both involve dietary restrictions and regulation, and

close disease monitoring. However, comparison between the two studies should be

interpreted with caution as the activities that contributed to the time burden were

different in each of the two studies; furthermore, the diabetes study reported mean

values instead of median values as reported in the present study.

The median OOPC per patient (€604 annually) was comparable for both patients and

caregivers. These costs were mainly due to expenditure on low-protein food products

and for a small part on costs related to PKU testing equipment, postage of Phe blood

tests, taking extra luggage on holiday to accommodate PKU equipment and attending

PKU events. It can be easily explained that OOPCs are higher for patients with severe

PKU than for those with mild PKU by the fact that greater disease severity necessitates

more stringent protein restriction with a higher intake of low-protein food products. An

important point to consider is whether these additional PKU-related costs are offset by

the potentially cheaper natural diet imposed upon PKU patients that contains very little

or no regular bread, dairy products or meat. In comparison to the general population: a

Dutch adult on a normal diet has been demonstrated to spend a mean amount of €1200

annually on meat, cheese, milk, yoghurt and bread [26]. For patients with PKU this


~ 87 ~

expenditure is replaced by the costs of the low protein products. It may be expected that

costs between protein containing and low protein food products will balance out in

patients depending on disease severity and the need for low protein food products.

Taking this into account, it is unlikely that there will be a large burden of extra OOPCs

for families of patients with PKU.

It must be stressed, however, that the costs of the Phe free protein supplements in a

mixture with vitamins and minerals, which are an essential part of the diet of patients

with PKU, vary per country but may be as high as €30,000 annually [19]. In most

European countries these costs are reimbursed by the government, or as is the case in

the Netherlands, by health insurance. To guarantee proper dietary treatment of patients

with PKU and to avoid a disproportionate financial burden for patients and families, it is

essential that costs of the Phe free protein supplements are reimbursed in all countries.

A limitation of our study was the low sample size, especially when results were analysed

within subgroups. However, this is a consequence of the rarity of this disease, with

approximately 550 earlytreated PKU patients in the Netherlands at present, a true

orphan disease. Another limitation was the age limit used in our study. Since our

questionnaire was joined on to an existing registry, patients aged 0–4 years were

excluded. Furthermore, many participants were unwilling to reveal certain details such

as their income, so it was not possible to formally analyse the impact of the cost of living

on these patients according to their socio-economic status. Nevertheless, the main

objective of the study to gain insight into the time burden and cost of living with PKU

overall (rather than comparison between patient subgroups) was achieved.

CONCLUSIONS

With an extra daily time burden of 1 h and 24 min/day for caregivers of children with

PKU and 30 min/day for adult patients, PKU management is highly time consuming. As

was expected, the time burden was significantly higher for severe versus mild patients.

For both the caregivers and the adult patients the OOPC mainly consisted of the cost

related to low protein foods.

Chapter 4

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m 2013.

Bone health in phenylketonuria:

a systematic review and meta-analysis


Serwet Demirdas†, Katie E Coakley†, Peter H Bisschop, Carla EM Hollak, Annet M Bosch† and

Rani H Singh† † Equal contributors

Chapter 5 w

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ABSTRACT

Background: Patients with Phenylketonuria (PKU) reportedly have decreased bone mineral

density (BMD). The primary aim of this study was to perform a systematic review and meta-

analysis to determine the extent and significance of low BMD in early treated patients with PKU.

Secondary aims were to assess other bone status indicators including bone turnover markers

(BTM) and to define areas for future research.

Methods: Two research teams (Amsterdam, Netherlands and Atlanta, USA) performed

literature searches for articles reporting data on BMD, osteopenia and osteoporosis, BTM or

other bone indicators in patients with PKU. Included articles were compared between research

teams and assessed for quality and risk of bias.

Results: A total of 13 unique articles were included; 11/13 articles reported BMD including a

total of 360 patients. Ten out of 11 articles found BMD was significantly lower in patients with

PKU. Meta-analyses for total BMD (TBMD; 3 studies; n = 133), lumbar spine BMD (LBMD; 7

studies; n = 247), and femoral neck BMD (FBMD; 2 studies; n = 78) Z-scores were performed.

Overall effect sizes were: TBMD −0.45 (95% CI −0.61, −0.28); LBMD −0.70 (95% CI −0.82,

−0.57); FBMD −0.96 (95% CI −1.42, −0.49). Definitions of osteopenia and osteoporosis were

highly heterogeneous between studies and did not align with World Health Organization

standards and the International Society for Clinical Densitometry positions on BMD

measurement.

Conclusions: Despite individual study findings of low BMD indicating higher risk of

osteoporosis, pooled available data suggest reduction in BMD is not clinically important when

using standard definitions of low BMD. Results from studies evaluating BTM are inconclusive.

Phenylalanine concentration, vitamin D, PTH, and nutrient intake do not correlate with BMD or

BTM. We recommend forthcoming studies use standard definitions of low BMD to determine

clinical implications of BMD Z-scores below 0, explore cause of low BMD in the subset of

patients with low BMD for chronological age (Z-score < -2) and assess fracture risk in patients

with PKU.

Bone Health: a Systematic Review

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INTRODUCTION

Phenylketonuria (PKU, ORPHA79254, MIM 261600) is a genetic disorder caused by

mutations in the gene coding for phenylalanine hydroxylase (PAH; EC 1.14.16.1). As a

consequence, the essential amino acid phenylalanine (Phe) cannot be converted to

tyrosine and accumulates in the blood. Phe is transported across the blood–brain

barrier and high concentrations can lead to mental retardation and behavioural and

physical abnormalities. Implementation of newborn screening to detect PKU across the

world since the 1960s has enabled early diagnosis and treatment. Early dietary

treatment results in near normalization of outcomes for patients with the disorder [1].

The success of dietary treatment has, however, led to the discovery of secondary issues

in the life-long treatment of PKU [1-8]. First reported in 1962, one of the complications

seen in early and continuously treated patients is abnormal bone status [9]. Initially

examined by radiological assessment, Feinberg et al. [9] described calcified spicules of

cartilage projecting into the distal metaphyses of growing long bones in a sample of 33

patients with PKU ranging from infants to young adults. These findings were later

supported by Murdoch et al. [10] and led to further studies assessing bone status in PKU

by quantitative ultrasound (QUS) [11], peripheral quantitative computed tomography

(pQCT) [12] and dual-energy X-ray absorptiometry (DXA) [13-17]. Low bone mineral

density (BMD), an important risk factor for skeletal fractures, has since been reported

by many studies [16,17].

A recent systematic review reported spine bone mineral density (BMD) was 0.100

g/cm2lower (95% CI, −0.110, −0.090 g/cm2) in 67 subjects with PKU, compared to 161

controls collected from 3 studies [18]. This review, however, has methodological

limitations: ascertainment bias by inclusion of late diagnosed patients who may suffer

from cognitive delays and less physical activity potentially affecting the bone outcomes;

lack of literature quality appraisal and assessment of bias; and no correction for age,

gender and ethnicity on BMD data (based on g/cm2).

Most studies on bone in patients with PKU agree that bone is affected; however, there

are significant gaps in knowledge and no consensus on the degree and implications of

bone abnormalities, biological causes and riskfactors for low BMD [4,5,14,19,20], and

Chapter 5

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the identification of subgroups of patients at-risk for fractures and compromised bone

status [13,20,21]. To investigate these knowledge gaps, we combined the efforts of two

international centers to perform a systematic review on bone status in PKU. Our

primary aim was to systematically review the literature concerning bone status in early

treated patients with PKU to perform a meta-analysis on BMD, corrected for bias, age

and gender. Secondary aims were to assess other indicators of bone status including

bone turnover markers (BTM) and to define areas for future research on bone status in

PKU.


~ 95 ~

MATERIALS AND METHODS

Research question

Two centers for metabolic diseases in Atlanta, Georgia (USA) and Amsterdam

(Netherlands) performed separate searches for literature concerning bone health in

patients with PKU according to the Preferred Reporting Items for Systematic Reviews

and Meta-Analyses (PRISMA) [22]. Both centers included similar research questions,

review strategies and proposed outcomes, thus efforts were combined. Whereas the

Atlanta research group focused on assessing the effects of nutrient intake, blood Phe

concentration, and adjunctive therapy on bone status indicators (search 1); the group in

Amsterdam focused on a comprehensive meta-analysis of BMD and an assessment of

BTM in early diagnosed patients with PKU (search 2). The protocol for the Atlanta

systematic review is registered with the ‘International prospective register of systematic

reviews’ (PROSPERO) as systematic review number CRD42014009176 [23].

Inclusion criteria for search 1 were primary research or review articles, human research

including subjects with PKU or hyperphenylalaninemia, and written in English only.

Exclusion criteria were articles unrelated to PKU, animal studies, studies including in

vitro results only, and studies that did not include BMD, bone mineral content (BMC),

bone turnover, or measures of bone metabolism.

Inclusion criteria for search 2 were original studies (randomized controlled trial, cohort

or case–control studies) of early diagnosed and treated patients with PKU studying

either BMD or BTM with a quality rating of acceptable or better according to quality

appraisal. Exclusion criteria were reviews (however reference lists were viewed for

relevant articles), studies that include pregnant patients, articles published in a

language other than English or Dutch and articles not meeting the inclusion criteria.

Chapter 5

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Methodology

Databases

Literature eligible for inclusion in search 1 was retrieved from PubMed and EMBASE

databases through a computerized search with assistance from a trained Emory

University librarian. The initial search was performed in 2013, with an updated search

completed in May 2014 to ensure the inclusion of recently published articles. As an

example, we provide here the MEDLINE® search: (pku[All Fields] OR

(“phenylketonurias” [MeSH Terms] OR “phenylketonurias” [All Fields] OR

“phenylketonuria” [All Fields])) AND (“bone and bones” [MeSH Terms] OR (“bone” [All

Fields] AND “bones” [All Fields]) OR “bone and bones”[All Fields] OR “bone” [All

Fields]). All articles and abstracts retrieved through PubMed and EMBASE searches

were downloaded in PDF format through Emory University open access or requested

through Illiad, a document-delivery service, if full text was not available.

A computerized search with the help of a trained University of Amsterdam librarian in

MEDLINE®, EMBASE and The Cochrane Library [24] was performed for search 2. The

databases were searched initially in October 2013 and last in June 2014. No limits were

used in the searches. As an example, we provide here the search used in MEDLINE®:

(“Phenylketonurias” [mh] OR phenylketon* [tiab] OR “PKU” [tiab] OR

hyperphenylalaninaemia [tiab] OR hyperphenylalaninemia [tiab]) AND ((minerals[mh]

OR mineral*[tiab] OR “Bone Diseases, Metabolic” [Mesh] OR “Osteoporosis” [Mesh]

OR osteoporosis [tiab] OR “Bone Density” [Mesh] OR “Bone Demineralization,

Pathologic” [Mesh] OR “Bone Resorption” [Mesh] OR “Bone Development” [Mesh]) OR

“Bone Remodelling” [Mesh] OR osteolysis [tiab] OR decalcification [tiab] OR bone

[tiab] OR bones [tiab]).

MEDLINE® contains references of articles published since 1966, the majority of which

are published in the USA. EMBASE also contains articles published in Europe, with

references dating back to 1976. The Cochrane library contains over 250,000 records of

Cochrane Controlled Trials [24].


~ 97 ~

Screening literature

Retrieved titles and abstracts were screened for inclusion eligibility and applicability by

one researcher for search 1 and two separate researchers for search 2. Articles not

related to the research question or not meeting inclusion criteria were discarded and the

reason for exclusion was noted. Remaining articles were screened as full text and

included in the final analysis if they met inclusion criteria. Abstracts concerning

conference meetings were included in the search to prevent publication bias; however

abstracts not containing adequate information related to research questions were

discarded. Bibliographies of all included articles and of review articles that were

excluded from the meta-analysis were reviewed for missed relevant articles.

Data extraction

Two investigators extracted data from all included articles for search 1 (author KEC) and

for search 2 (author SD) using validated abstraction forms [Genetic Metabolic Dietitian

International (GMDI)/Southeast Regional Collaborative (SERC) Evidence Abstract

Worksheet (search 1) [25], Cochrane Renal Group protocol guidelines appendix 4

(search 2) [26]]. Data extracted included characteristics of study populations and

control groups, study design, outcome measures, results, and limitations. Outcome

measures of bone status were BMD [total body (TBMD), lumbar spine (LBMD) and/or

femoral bone (FBMD)]; BTM; BMC; incidence or prevalence of osteopenia,

osteoporosis, low BMD, or fractures; vitamin D and/or parathyroid hormone (PTH)

status; and other indicators. DXA is the preferred and most commonly reported method

to measure BMD in both children and adults [27-29]. Studies of bone in patients with

PKU primarily report DXA estimates of BMD; however, other techniques such as pQCT,

QUS and several X-ray methods used to measure BMD are available and are compared

elsewhere [30,31]. We included studies measuring BMD using any recognized method.

Chapter 5

~ 98 ~

Quality appraisal search 1

The Academy of Nutrition and Dietetics Evidence Analysis Process (AND EA Process

[32]) was adapted by the GMDI/SERC effort to create nutrition management guidelines

[25] for inborn disorders of metabolism and applied as the foundation for search 1. The

AND Evidence Analysis Process provides a method to abstract data and assign a quality

grade to primary and review articles retrieved through systematic searches. All included

articles were reviewed, graded, and abstracted by author KEC, trained in the Evidence

Analysis Process through participation in the development of PKU guidelines [33].

Quality criteria checklists (QCCs) were completed for all studies included in search 1.

Each QCC included four relevance questions addressing the purpose and applicability of

the study and 10 validity questions with a varying number of sub-questions (Tables 1

and 2). Answers to validity questions were used to assign a quality score of positive,

negative or neutral to each article. For a positive quality rating, specific validity

questions including an unbiased selection of patients, comparable study groups (i.e.

matched controls for age, height and weight), sufficient description of study

intervention and procedures, and clearly defined outcomes were required. Articles that

did not meet these validity criteria, but did include other strengths were assigned a

neutral quality rating. Articles that did not contain most of the validity components (6

out of 10 or more) received a negative quality rating.


~ 99 ~

Table 1. Quality criteria checklist used in search 1— Primary research

Relevance Questions

1. Would implementing the studied intervention procedures (if found successful) result in improved outcomes for the patients/clients/ population group? (N/A for some Epidemiological studies)

2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about?

3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dietetics practice?

4. Is the intervention or procedure feasible? (N/A for some Epidemiological studies)

Validity questions

1. Was the research question clearly stated? 1.1 Was the specific intervention(s) or procedure (independent variable(s)) identified? 1.2 Was the outcome(s) (dependent variable(s)) clearly indicated? 1.3 Were the target population and setting specified? 2. Was the selection of study subjects/patients free from bias? 2.1 Were inclusion/exclusion criteria specified (e.g. risk, point in disease progression, diagnostic or

prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? 2.2 Were criteria applied equally to all study groups and/or all subjects? 2.3 Were health, demographics, and other characteristics of subjects described? 2.4 Were the subjects/patients a representative sample of the relevant population? 3. Were study groups comparable? 3.1 Was the method of assigning subjects/patients to groups described and unbiased? (Method of

randomization identified if Randomized Controlled Trial (RCT)) 3.2 Was the distribution of disease status, prognostic factors, and other factors (e.g. demographics)

at baseline similar across study groups (original or created post hoc)? 3.3 Were concurrent controls used? (Concurrent preferred over historical controls.) 3.4 If cohort study or cross-sectional study, were groups comparable on important confounding

factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? (Criterion may not be applicable in some cross-sectional studies.)

3.5 If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.)

3.6 If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")?

4. Was method of handling withdrawals described? 4.1 Were follow-up methods described and the same for all groups and/or all subjects? 4.2 Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate),

and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%)

4.3 Were all enrolled subjects/patients (in the original sample) accounted for? 4.4 Were reasons for withdrawals similar across groups? 4.5 If diagnostic test, was decision to perform reference test not dependent on results of test under

study? 5. Was blinding used to prevent introduction of bias? 5.1 In intervention study, were subjects, clinicians/practitioners, and investigators blinded to

treatment group, as appropriate?

Chapter 5

~ 100 ~

5.2 Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.)

5.3 In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded?

5.4 In case control study, was case definition explicit and case ascertainment not influenced by exposure status?

5.5 In diagnostic study, were test results blinded to patient history and other test results? 6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s)

described in detail? Were intervening factors described? 6.1 In RCT or other intervention trial, were protocols described for all regiments studied? 6.2 In observational study, were interventions, study settings, and clinicians/provider described? 6.3 Was the intensity and duration of the intervention or exposure factor sufficient to produce a

meaningful effect? 6.4 Was the amount of exposure and, if relevant, subject/patient compliance measured? 6.5 Were co-interventions (e.g., ancillary treatments, other therapies) described? 6.6 Were extra or unplanned treatments described? 6.7 Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? 6.8 In diagnostic study, were details of test administration and replication sufficient? 7. Were outcomes clearly defined and the measurements valid and reliable? 7.1 Were primary and secondary endpoints described and relevant to the question? 7.2 Were nutrition measures appropriate to question and outcomes of concern? 7.3 Was the period of follow-up long enough for important outcome(s) to occur? 7.4 Were the observations and measurements based on standard, valid, and reliable data collection

instruments/tests/procedures? 7.5 Was the measurement of effect at an appropriate level of precision? 7.6 Were other factors accounted for (measured) that could affect outcomes? 7.7 Were the measurements conducted consistently across groups? 8. Was the statistical analysis appropriate for the study design and type of outcome indicators? 8.1 Were statistical analyses adequately described the results reported appropriately? 8.2 Were correct statistical tests used and assumptions of test not violated? 8.3 Were statistics reported with levels of significance and/or confidence intervals? 8.4 Was “intent to treat” analysis of outcomes done (and as appropriate, was there an analysis of

outcomes for those maximally exposed or a dose-response analysis)? 8.5 Were adequate adjustments made for effects of confounding factors that might have affected the

outcomes (e.g., multivariate analyses)? 8.6 Was clinical significance as well as statistical significance reported? 8.7 If negative findings, was a power calculation reported to address type 2 error? 9. Are conclusions supported by results with biases and limitations taken into consideration? 9.1 Is there a discussion of findings? 9.2 Are biases and study limitations identified and discussed? 10. Is bias due to study’s funding or sponsorship unlikely? 10.1 Were sources of funding and investigators’ affiliations described? 10.2 Was there no apparent conflict of interest?


~ 101 ~

Table 2. Quality criteria checklist used in search 1 — Reviews

Relevance Questions

1. Will the findings of the review, if true, have a direct bearing on the health of patients? 2. Is the outcome or topic something that patients/clients/population groups would care about? 3. Is the problem addressed in the review one that is relevant to dietetics practice? 4. Will the information, if true, require a change in practice?

Validity questions

1. Was the research question clearly focused and appropriate? 2. Was the search strategy used to locate relevant studies comprehensive? Were the databases

searched and the search terms use described? 3. Were explicit methods used to select studies to include in the review? Were inclusion/exclusion

criteria specified and appropriate? Were selection methods unbiased? 4. Was there an appraisal of the quality and validity of studies included in the review? 5. Were specific treatments/interventions/exposures described? Were treatments similar enough to

be combined? 6. Was the outcome of interest clearly indicated? Were other potential harms and benefits

considered? 7. Were processes for data abstraction, synthesis, and analysis described? Were they applied

consistently across studies and groups? Was there appropriate use of qualitative and/or quantitative synthesis? Was variation in findings among studies analyzed? Were heterogeneity issues considered? If data from studies were aggregated for meta-analysis, was the procedure described?

8. Are the results clearly presented in narrative and/or quantitative terms? If summary statistics are used, are levels of significance and/or confidence intervals included?

9. Are conclusions supported by results with biases and limitations taken into consideration? Are limitations of the review identified and discussed?

10. Was bias due to the review’s funding or sponsorship unlikely?

Chapter 5

~ 102 ~

Assessment of bias search 1

QCC-derived quality ratings reflected the likelihood of bias in each study. Those rated

positive were unlikely to contain significant bias, while those with neutral quality ratings

included some elements likely to produce bias. Negative quality ratings indicated that

bias in the study was very likely and these articles were excluded from the review.

Quality appraisal search 2

Quality appraisal and assessment of bias were performed for search 2 on all assessed full

text articles by two separate researchers (SD and AMB) and outcomes were discussed.

The ‘Scottish Intercollegiate Guidelines Network’ (SIGN) checklists were used [34] to

assess quality based on the study design (RCT, cohort or case–control study). SIGN

checklists are based on the Grading of Recommendations Assessment, Development and

Evaluation (GRADE) [35] approach. Articles were appraised as of low, acceptable or

high quality and those assessed as low quality were excluded from the review.

Assessment of bias search 2

Quality ratings reflected the likelihood of bias in each study. Those rated high quality

were unlikely to contain significant bias, while those with acceptable quality ratings

included or did not include some elements likely to produce bias. Low quality ratings

indicated bias in the study was likely and these studies were not included in the review.


~ 103 ~


Z-scores and T-scores for BMD are calculated to clinically assess an individual’s bone

status. T-scores describe the number of standard deviations (SD) by which a patient’s

BMD differs from the expected mean value in a healthy young adult. The World Health

Organization

(WHO) defines osteopenia in adults as a T-score between -1 and -2.5, and osteoporosis

as a T-score below -2.5 [29]. Z-scores describe the number of SDs by which the BMD in

an individual differs from the mean value expected for age and sex. For children, Z-

scores are mostly used. The International Society for Clinical Densitometry (ISCD)

states that the diagnosis of osteoporosis in children, premenopausal women and males

under 50 years of age should not be based on densitometric criteria alone. Instead, a

BMD Z-score below -2 is defined as “BMD below the expected range for age” or “low

BMD for chronological age” and cannot be defined as osteoporosis unless coupled with a

significant fracture history [28]. The ISCD does however stress that Z-scores above -2 do

not preclude the possibility of skeletal fragility. Most recent studies of bone health in

PKU report BMD Z-scores only [14,36], because the patient population is relatively

young and pediatric patients are assessed together with adult patients. BMD can be

measured at a variety of locations and we included studies that measured BMD in total

body, spine and femur. Spinal BMD reflects BMD in trabecular bone, and femoral BMD

is significant for cortical bone [37].

Qualitative and quantitative analyses were performed to assess BMD in patients with

PKU. Qualitative analysis was performed to review evidence on bone health and assess

the prevalence of low BMD in patients with PKU. Quantitative analysis was performed

in the form of a meta-analyses to analyse whether if BMD Z-scores are different in

patients with PKU than reference values (deviant from 0 SD of reference). If the full text

of an article did not contain BMD or BMD Z-scores, the authors were contacted to

obtain data. The meta-analysis was performed in Review Manager [38]; a fixed effects

model or random effects model was used to pool the patient-based data. The choice for

selecting a fixed effects model or a random effects model was based on heterogeneity of

the data per meta-analysis. Low heterogeneity between studies led to the use of a fixed

Chapter 5

~ 104 ~

effects model, and high heterogeneity to the use of a random effects model to pool data

[39,40]. Heterogeneity was tested by calculating I2(heterogeneity is low when I2 is

25%, moderate if 25 50% and high if 75%) [39]. The presence of publication bias was

visually assessed by means of a funnel plot and calculation of Egger’s test with statistical

software (IBM SPSS statistics 20) [40]. By using outcomes from a specific population

(early diagnosed and treated patients with PKU), it should be noted that the effect

cannot be extrapolated to other patients with PKU.

To assess BMD Z-scores considered low for chronological age (below -2), we used a

normal distribution curve to estimate prevalence in a healthy population and developed

a PKU-specific normally distributed curve based on estimated effect size for LBMD.

Since these estimates are hypothetical, we also calculated prevalence in a normal

population using the 2007–2008 National Health and Nutrition Examination Survey

(NHANES) [41] data. We limited the analysis to participants 8–45 years of age with

lumbar spine and femoral neck BMD measurements since NHANES does not collect

data in children under eight years of age, and included studies in this review do not

report on patients over the age of 45 years (older patients are often late diagnosed). Z-

scores for age and sex were calculated using the Centers for Disease Control and

Prevention’s (CDC) references [42] for lumbar spine and femoral neck BMD. We used

the computer program SAS-Callable SUDAAN 11.0.1 to calculate weighted population

prevalence of low BMD for chronological age (Z-score <-2), taking into account primary

sampling units and strata. To calculate final prevalence, we limited analyses to a

subpopulation of non-Hispanic Caucasian participants only.

All other outcomes including BTM, BMC, and blood vitamin D and PTH status were

evaluated qualitatively. Overview tables of results were generated to summarize results

and draw conclusions. Factors examined in association with bone-related outcomes

were included in overview tables with statistical methods, direction of association, and

statistical and clinical significance. If analyses controlled for variables, these were also

noted. Factors significantly associated with bone-related outcomes in multiple studies

were noted to identify potential underlying causes of low BMD. Finally, all data collected

and summarized through quality appraisal and overview tables were used to identify

gaps in the bone health evidence base in PKU.


~ 105 ~

RESULTS

Study selection

Twenty-three articles were included in this review as a result of study selection criteria

for search 1, resulting from 437 initially identified records from both EMBASE and

MEDLINE® (Figure 1). A total of 13 articles were included for search 2 after selection

from 418 initially identified records from EMBASE, The Cochrane Library and

MEDLINE® (Figure 2).

Included studies

Articles included in search 1 and search 2 were combined and 23 unique articles were

identified. We found that both study teams included 13 records (57%), listed in Table 3

with their study characteristics. Studies were published between 1994 and 2013, with

the majority published since 2000 (69%). Twelve were cohort studies whereas one was a

case–control study. Search 1 included 10 articles that were not included in search 2.

Most of these articles were published before 2000 (80%) and identified due to

differences in search terms (n = 5) or discrepancy in quality ratings (n = 5). Of these 10

articles, 5 were discarded when appraised as low quality in search 2 and the remaining 5

articles did not address BMD and were therefore excluded in search 2. We limited our

analyses to articles included in both searches only (n = 13).

Chapter 5

~ 106 ~

Figure 1. Inclusion process flow diagram for search 1 (PRISMA 2009 [22])


~ 107 ~

Figure 2. Inclusion process flow diagram for search 2 (PRISMA 2009 [22])

Chapter 5

~ 108 ~

Ta

ble

3.

art

icle

s in

clu

ded

aft

er s

tud

y s

elec

tio

n i

n b

oth

sea

rch

1 a

nd

2 (

n=

13)

Re

fere

nce

St

ud

y ai

m

In-

and

exc

lusi

on

cr

ite

ria

Stu

dy

typ

e

Stu

dy

po

pu

lati

on

O

utc

om

e

me

asu

res

Co

akle

y K

E, D

ou

glas

TD

, Sin

gh R

H. U

sin

g p

red

icti

ve m

od

elin

g to

est

imat

e b

on

e m

iner

al

de

nsi

ty in

ch

ildre

n a

nd

ad

ult

s w

ith

p

he

nyl

keto

nu

ria.

Jo

urn

al o

f In

he

rite

d M

eta

bo

lic

Dis

ea

se. 2

01

3;3

6(2

):S1

26

. R

efe

ren

ce [

46

]

To u

se c

linic

al

par

amet

ers

colle

cted

in

PK

U p

atie

nts

to

pre

dic

t to

tal b

od

y B

MD

.

Incl

ud

ed:

Pat

ien

ts a

ge 4

yea

rs

and

old

er, e

arly

tr

eate

d P

KU

pat

ien

ts.

No

exc

lusi

on

cri

teri

a.

Co

ho

rt s

tud

y (c

on

fere

nce

ab

stra

ct)

N =

57

♂

34

♀

24

M

ean

age

1

7.3

± 1

1 y

- B

MD

mea

sure

d

by

DX

A: Z

-sco

re

TBM

D

De

Gro

ot

MJ,

Ho

eks

ma

M, V

an

Rijn

M, S

lart

RH

JA,

Van

Sp

ron

sen

FJ.

Re

lati

on

ship

s b

etw

ee

n lu

mb

ar

bo

ne

min

era

l den

sity

an

d b

ioch

em

ical

p

aram

ete

rs in

ph

en

ylke

ton

uri

a p

atie

nts

. M

ole

cula

r G

en

eti

cs a

nd

Me

tab

olis

m.

20

12

;10

5(4

):5

66

-57

0.

Re

fere

nce

[1

4]

To in

vest

igat

e th

e re

lati

on

ship

s b

etw

een

Z-

sco

res

of

lum

bar

BM

D

and

age

, clin

ical

se

veri

ty

of

PK

U, m

ean

P

he

con

cen

trat

ion

an

d

Ph

e va

riat

ion

of

the

year

pri

or

to D

XA

sc

ann

ing,

as

we

ll as

blo

od

co

nce

ntr

atio

ns

of

vita

min

s, m

iner

als,

an

d

alka

line

ph

osp

hat

ase.

Incl

ud

ed:

Pat

ien

ts w

ith

PK

U

dia

gno

sed

by

new

bo

rn s

cree

nin

g,

earl

y an

d

con

tin

uo

usl

y tr

eate

d,

free

of

con

com

itan

t d

isea

se.

No

exc

lusi

on

cri

teri

a.

Co

ho

rt s

tud

y (f

ull

arti

cle

)

N =

53

♂

25

♀

28

M

edia

n a

ge

16

y

Age

ran

ge

2 –

35

y

- B

MD

mea

sure

d

by

DX

A: Z

-sco

re

LBM

D

- b

loo

d v

itam

in D

, al

kalin

e p

ho

sph

atas

e

Me

nd

es

AB

, Mar

tin

s FF

, Cru

z W

MS,

Da

Silv

a LE

, A

bad

ess

o C

BM

, Bo

ave

ntu

ra G

T. B

on

e

de

velo

pm

en

t in

ch

ildre

n a

nd

ad

ole

sce

nts

wit

h

PK

U. J

ou

rnal

of

Inh

eri

ted

Met

abo

lic D

ise

ase

. 2

01

2;3

5(3

):4

25

-43

0.

Re

fere

nce

[2

0]

To d

escr

ibe

the

imp

act

of

die

tary

fac

tors

an

d

con

tro

l of

pla

sma

Ph

e le

vels

on

bo

ne

age

and

B

MD

.

Incl

ud

ed:

Pat

ien

ts w

ith

PK

U

dia

gno

sed

by

new

bo

rn s

cree

nin

g.

No

exc

lusi

on

cri

teri

a.

Co

ho

rt s

tud

y (f

ull

arti

cle

)

N =

13

♂

4

♀ 9

A

ge r

ange

8

-16

y

- B

MD

mea

sure

d

by

DX

A: Z

-sco

re

LBM

D

- X

-ray

of

han

d

and

fis

t: b

on

e ag

e


~ 109 ~

Ad

amcz

yk P

, Mo

raw

iec-

Kn

ysak

A, P

lud

ow

ski P

, B

anas

zak

B, K

arp

e J

, Plu

skie

wic

z W

. Bo

ne

m

eta

bo

lism

an

d t

he

mu

scle

-bo

ne

re

lati

on

ship

in

child

ren

, ad

ole

sce

nts

an

d y

ou

ng

adu

lts

wit

h

ph

en

ylke

ton

uri

a. J

ou

rnal

of

Bo

ne

an

d M

iner

al

Me

tab

olis

m. 2

01

1;2

9(2

):2

36

-24

4. R

efe

ren

ce [

13

]

To a

sse

ss b

on

e m

etab

olis

m in

yo

un

g su

bje

cts

wit

h P

KU

usi

ng

rou

tin

e D

XA

par

amet

ers,

fu

nct

ion

al m

usc

le-b

on

e an

alys

is e

stim

ated

on

th

e b

asis

of

DX

A

mea

sure

men

ts a

nd

se

vera

l lab

ora

tory

va

riab

les.

Incl

ud

ed:

Pat

ien

ts w

ith

PK

U

dia

gno

sed

by

new

bo

rn

scre

enin

g w

ho

sta

rted

tr

eatm

ent

wit

hin

th

e fi

rst

mo

nth

of

life.

N

o e

xclu

sio

n c

rite

ria.

Co

ho

rt

stu

dy

(fu

ll ar

ticl

e)

N =

45

♂

25

♀

20

M

ean

age

1

3.8

±5

.2 y

- B

on

e tu

rno

ver

mar

kers

: IC

TP,

bA

LP, o

steo

calc

in

- B

MD

mea

sure

d

by

DX

A: Z

-sco

re

LBM

D a

nd

TB

MD

-

BM

C

- O

ther

: p

arat

ho

rmo

ne,

ca

lcit

on

in, t

ota

l an

d io

niz

ed

calc

ium

N

agas

aka

H, T

suka

har

a H

, Ta

kata

ni T

, et

al.

Cro

ss-

sect

ion

al s

tud

y o

f b

on

e m

eta

bo

lism

wit

h n

utr

itio

n

in a

du

lt c

lass

ica

l ph

en

ylke

ton

uri

c p

atie

nts

d

iagn

ose

d b

y n

eo

nat

al s

cre

en

ing.

Jo

urn

al o

f B

on

e

and

Min

era

l Me

tab

olis

m. 2

01

1;2

9(6

):7

37

-74

3.

Re

fere

nce

[2

1]

To o

bta

in f

un

dam

enta

l d

ata

for

esta

blis

hin

g an

o

pti

mal

tre

atm

ent

stra

tegy

fo

r b

on

e d

isea

se in

PK

U.

Incl

ud

ed:

Ad

ult

pat

ien

ts w

ith

P

KU

dia

gno

sed

by

new

bo

rn s

cree

nin

g an

d w

ho

wer

e ea

rly

trea

ted

. N

o e

xclu

sio

n c

rite

ria.

Co

ho

rt

stu

dy

(fu

ll ar

ticl

e)

Pat

ien

ts

N =

34

♂

13

♀

21

A

ge r

ange

2

0 -

35

y

Co

ntr

ols

N

= 3

6

♂ 1

4

♀ 2

2

Age

ran

ge

19

- 4

0 y

- B

on

e tu

rno

ver

mar

kers

: bA

LP,

OC

, pro

colla

gen

ty

pe

1

carb

oxy

term

inal

p

rop

epti

de

(PIC

P),

-

Oth

er:

vita

min

D, P

TH

Chapter 5

~ 110 ~

Lage

S, B

ue

no

M, A

nd

rad

e F

, et

al.

Fatt

y ac

id

pro

file

in p

atie

nts

wit

h p

he

nyl

keto

nu

ria

and

its

rela

tio

nsh

ip w

ith

bo

ne

min

era

l de

nsi

ty.

Jou

rnal

of

Inh

eri

ted

Me

tab

olic

Dis

ea

se.

20

10

:1-9

. R

efe

ren

ce [

15

]

To a

sse

ss t

he

infl

uen

ce

of

fatt

y ac

id p

rofi

le o

n

BM

D in

pat

ien

ts w

ith

P

KU

.

Incl

ud

ed: g

enet

ical

ly

pro

ven

PK

U, n

o

neu

rolo

gica

l dam

age

and

ind

epen

den

tly

fun

ctio

nin

g in

dai

ly

acti

viti

es.

N

o e

xclu

sio

n c

rite

ria.

Co

ho

rt

stu

dy

(fu

ll ar

ticl

e)

Pat

ien

ts

N =

47

♂

30

♀

17

A

ge r

ange

6

– 4

2 y

C

on

tro

ls

N =

77

♂

65

♀

12

A

ge r

ange

6

-4

5 y

- B

MD

mea

sure

d

by

DX

A: Z

-sco

re

LBM

D a

nd

FB

MD

-

Oth

er:

Fatt

y ac

id p

rofi

le,

ph

enyl

alan

ine,

ca

lciu

m, 2

5-

hyd

roxy

vit

amin

D

, die

tary

co

ntr

ol

and

cal

ciu

m

inta

ke

Po

rta

F, R

oat

o I,

Mu

ssa

A, e

t al

. In

cre

ase

d

spo

nta

ne

ou

s o

ste

ocl

asto

gen

esi

s fr

om

pe

rip

he

ral

blo

od

mo

no

nu

cle

ar c

ells

in p

he

nyl

keto

nu

ria.

Jo

urn

al o

f In

he

rite

d M

eta

bo

lic D

ise

ase

. 20

08

:1-4

. [R

efe

ren

ce 4

8]

To in

vest

igat

e sp

on

tan

eou

s o

steo

clas

toge

nes

is in

p

atie

nts

wit

h P

KU

co

mp

ared

wit

h t

hat

in

hea

lth

y co

ntr

ols

.

Incl

ud

ed: P

atie

nts

w

ith

PK

U d

iagn

ose

d

by

new

bo

rn s

cree

nin

g w

ho

sta

rted

tre

atm

ent

wit

hin

th

e fi

rst

mo

nth

o

f lif

e.

Excl

ud

ed: P

atie

nts

w

ith

su

bo

pti

mal

n

utr

itio

n, s

ho

rt

stat

ure

, his

tory

of

imm

ob

ility

, dru

gs

infl

uen

cin

g b

on

e an

d/o

r co

nco

mit

ant

dis

ease

wit

h b

on

e o

r b

loo

d in

volv

em

ent.

Co

ho

rt

stu

dy

(fu

ll ar

ticl

e)

Pat

ien

ts

N =

20

♂

8

♀ 1

2

Mea

n a

ge

14

± 7

.1 y

C

on

tro

ls

N =

20

‘A

ge-

and

se

x-m

atch

ed’

- O

steo

clas

t n

um

ber

an

d s

ize


~ 111 ~

Mo

dan

-Mo

ses

D, V

ere

d I,

Sch

war

tz G

, et

al. P

eak

b

on

e m

ass

in p

atie

nts

wit

h p

he

nyl

keto

nu

ria.

Jo

urn

al o

f In

he

rite

d M

eta

bo

lic D

ise

ase

. 2

00

7;3

0(2

):2

02

-20

8.

Re

fere

nce

[4

5]

To e

valu

ate

pea

k b

on

e m

ass

in a

du

lt P

KU

p

atie

nts

an

d t

o r

elat

e B

MD

to

nu

trit

ion

al

par

amet

ers.

Incl

ud

ed:

Pat

ien

ts w

ith

cla

ssic

al

PK

U d

iagn

ose

d b

y n

ewb

orn

scr

een

ing

wh

o s

tart

ed t

reat

men

t w

ith

in t

he

firs

t m

on

th

of

life.

N

o e

xclu

sio

n c

rite

ria.

Co

ho

rt

stu

dy

(fu

ll ar

ticl

e)

N =

31

♂

13

♀

18

M

ean

age

2

5 ±

5.3

y

- B

MD

mea

sure

d

by

DX

A: Z

-sco

re

LBM

D a

nd

FB

MD

-

Oth

er:

vita

min

D

, PTH

, alk

alin

e p

ho

sph

atas

e,

calc

ium

B

arat

P, B

arth

e N

, Re

do

nn

et-

Ve

rnh

et

I, P

arro

t F.

Th

e im

pac

t o

f th

e c

on

tro

l of

seru

m p

he

nyl

alan

ine

le

vels

on

ost

eo

pe

nia

in p

atie

nts

wit

h

ph

en

ylke

ton

uri

a. E

uro

pe

an J

ou

rnal

of

Pe

dia

tric

s.

20

02

;16

1(1

2):

68

7-6

88

. R

efe

ren

ce [

41

]

To d

eter

min

e th

e ro

le o

f th

e co

ntr

ol o

f p

hen

ylal

anin

e b

loo

d

leve

ls in

th

e p

ath

oge

nes

is o

f o

steo

pen

ia.

Incl

ud

ed:

Pat

ien

ts w

ith

PK

U

dia

gno

sed

by

new

bo

rn

scre

enin

g w

ho

wer

e ea

rly

and

co

nti

nu

ou

sly

trea

ted

. N

o e

xclu

sio

n c

rite

ria.

Cas

e-

Co

ntr

ol

stu

dy

(fu

ll ar

ticl

e)

N =

13

♂

7

♀ 5

M

ean

age

n

ot

des

crib

ed,

stu

dy

con

cern

ing

ped

iatr

ic

pat

ien

ts

- B

MD

mea

sure

d

by

DX

A: Z

-sco

re

LBM

D

Zem

an J

, Bay

er

M, S

tep

an J

. Bo

ne

min

era

l den

sity

in

pat

ien

ts w

ith

ph

en

ylke

ton

uri

a. A

cta

Pae

dia

tric

a, In

tern

atio

nal

Jo

urn

al o

f P

aed

iatr

ics.

1

99

9;8

8(1

2):

13

48

-13

51

. R

efe

ren

ce [

16

]

To a

sse

ss t

ota

l bo

dy

BM

D a

nd

lum

bar

BM

D in

p

atie

nts

wit

h P

KU

in

rela

tio

n t

o n

utr

itio

nal

in

take

an

d p

rote

ins

fro

m

nat

ura

l fo

od

s an

d

arti

fici

al s

ou

rce

s.

Incl

ud

ed:

Pat

ien

ts w

ith

cla

ssic

al

PK

U d

iagn

ose

d b

y n

ewb

orn

scr

een

ing

wh

o s

tart

ed t

reat

men

t w

ith

in t

he

firs

t m

on

th

of

life.

N

o e

xclu

sio

n c

rite

ria.

Co

ho

rt

stu

dy

(fu

ll ar

ticl

e)

N =

44

♂

19

♀

25

M

ean

age

1

6.1

y

Age

ran

ge

6 -

29

y

- B

MD

mea

sure

d

by

DX

A: Z

-sco

re

TBM

D a

nd

LB

MD

Chapter 5

~ 112 ~

Sch

wah

n B

, Mo

kov

E, S

che

idh

aue

r K

, Le

ttge

n

B, S

cho

nau

E. D

ecr

eas

ed

tra

be

cula

r b

on

e

min

era

l de

nsi

ty in

pat

ien

ts w

ith

p

he

nyl

keto

nu

ria

me

asu

red

by

pe

rip

he

ral

qu

anti

tati

ve c

om

pu

ted

to

mo

grap

hy.

Act

a P

aed

iatr

ica,

Inte

rnat

ion

al J

ou

rnal

of

Pae

dia

tric

s. 1

99

8;8

7(1

):6

1-6

3.

Re

fere

nce

[1

2]

To m

easu

re B

MD

in

pat

ien

ts w

ith

PK

U

usi

ng

per

iph

eral

Q

CT

to d

eter

min

e if

o

steo

pen

ia o

ccu

rs.

Incl

ud

ed:

Pat

ien

ts w

ith

PK

U

dia

gno

sed

by

new

bo

rn

scre

enin

g, t

reat

ed

acco

rdin

g to

ac

cep

ted

re

com

men

dat

ion

s.

No

exc

lusi

on

cr

iter

ia.

Co

ho

rt

stu

dy

(fu

ll ar

ticl

e)

Pat

ien

ts

N =

14

♂

8

♀ 6

A

ge r

ange

5

-28

y

Co

ntr

ols

N

= 1

4

♂ 8

♀

6

Age

ran

ge

5-2

8 y

- B

MD

mea

sure

d

by

per

iph

eral

Q

CT:

to

tal b

on

e B

MD

an

d s

po

ngy

b

on

e B

MD

of

the

rad

ius

- p

lasm

a P

he.

Hill

man

L, S

chlo

tzh

aue

r C

, Le

e D

, et

al.

De

cre

ase

d b

on

e m

iner

aliz

atio

n in

ch

ildre

n

wit

h p

he

nyl

keto

nu

ria

un

de

r tr

eat

me

nt.

Eu

r J

Pe

dia

tr. J

ul 1

99

6;1

55

Su

pp

l 1:S

14

8-1

52

. R

efe

ren

ce [

37

]

To a

sse

ss t

he

eff

ect

of

die

t an

d/o

r d

isea

se o

n B

MD

in

pat

ien

ts w

ith

PK

U

by

com

par

ing

child

ren

wit

h P

KU

to

no

n-P

KU

ch

ildre

n

for

par

amet

ers

of

bo

ne

min

eral

izat

ion

an

d h

om

eost

asis

.

Incl

ud

ed:

Ped

iatr

ic p

atie

nts

w

ith

PK

U.

No

exc

lusi

on

cr

iter

ia.

Co

ho

rt

stu

dy

(fu

ll ar

ticl

e)

Pat

ien

ts

N =

11

♂

5

♀ 6

M

ean

age

1

0.9

±4

.2 y

C

on

tro

ls

N =

64

♂

32

♀

32

M

ean

age

1

1.4

±4

.2 y

- B

on

e tu

rno

ver

mar

kers

: C

arb

oxy

term

inal

te

lop

epti

de

of

typ

e I c

olla

gen

(I

CTP

), (

bA

LP),

o

steo

calc

in i

n

seru

m. T

artr

ate

resi

stan

t ac

id

ph

osp

hat

ase,

ca

lciu

m/c

reat

inin

e ra

tio

in u

rin

e.

- B

MD

mea

sure

d

by

DX

A: Z

-sco

re

LBM

D a

nd

FB

MD

-

Oth

er: c

alci

um

, m

agn

esi

um

, zin

c,

ph

osp

ho

rus,

al

bu

min

, cr

eati

nin

e,

25

-hyd

roxy

vi

tam

in D

, 1.2

5

dih

ydro

xy v

itam

in

D, P

TH


~ 113 ~

Alle

n J

R, H

um

ph

ries

IRJ,

Wat

ers

DL,

et

al.

De

crea

sed

bo

ne

min

eral

den

sity

in

child

ren

wit

h p

hen

ylke

ton

uri

a. A

mer

ican

Jo

urn

al o

f C

linic

al N

utr

itio

n.

199

4;5

9(2

):4

19-4

22.

Re

fere

nce

[1

7]

To e

valu

ate

TBM

D

and

SB

MD

an

d

nu

trie

nt

inta

ke in

a

gro

up

of

child

ren

w

ith

PK

U o

n

die

tary

th

erap

y in

co

mp

aris

on

wit

h a

gr

ou

p o

f ag

e-

mat

ched

co

ntr

ol

sub

ject

s

Incl

ud

ed:

Pre

-pu

ber

tal

pat

ien

ts w

ith

P

KU

dia

gno

sed

b

y n

ew

bo

rn

scre

en

ing,

ear

ly

and

co

nti

nu

ou

sly

tre

ate

d.

Co

ho

rt

stu

dy

(fu

ll ar

ticl

e)

Pat

ien

ts

N =

32

♂

20

♀

12

M

ean

age

7

.7 ±

2.3

y

Co

ntr

ols

N

= 9

5

♂ 5

7

♀ 3

8

Mea

n a

ge

8.1

± 2

.1 y

- B

MD

m

easu

red

by

DX

A: Z

-sco

re

LBM

D a

nd

TB

MD

♂ in

clu

ded

mal

e su

bje

cts,

♀ in

clu

ded

fem

ale

sub

ject

s.

Chapter 5

~ 114 ~

Quality appraisal

Of the 13 included studies, seven (54%) were graded neutral quality and six (46%) were

graded positive quality according to the AND Evidence Analysis Process applied during

search 1 (Figure 3). As defined by SIGN checklists, applied during search 2, the majority

of papers were graded acceptable quality (n = 11) and two [12,21] were high quality

(Figures 4 and 5).

While quality was determined on separate scales by each search team, scores of eight

articles (62%) corresponded between AND and SIGN quality scores. Seven of the eight

were assessed as neutral quality by AND criteria in search 1 and a corresponding

acceptable quality by SIGN checklists in search 2. One article was graded positive

quality by AND criteria and a corresponding high quality by SIGN checklist. The

remaining five of the 13 (38%) included studies with quality ratings that did not agree

between AND and SIGN scales. All five were scored with the highest quality rating

(positive) by AND criteria in search 1, but rated acceptable quality by SIGN checklists in

search 2. Most of these papers did not fully describe the number of patients recruited for

the study versus the number of actual study, a requirement for a high quality rating on

the SIGN checklist. Two articles that were scored positive based on AND criteria also

scored very well on the SIGN checklists, but they were retrospective studies,

automatically disqualifying them for a rating above acceptable quality [14,43].


~ 115 ~

Figure 3. Risk of bias summary table search 1

Figure 4 and 5. Risk of bias summary tables search 2

Chapter 5

~ 116 ~

BMD in early treated patients with PKU

A total of 11 articles studied BMD in early treated patients with PKU; 10 cohort studies

and one case–control study. Combined, a total of 360 patients (range 11 – 57 per study)

were included. Five studies included pediatric patients only, one study selectively

included adult patients [44] and 5 studies included pediatric and adult patients. Ten of

the 11 studies found that BMD was significantly lower in patients with PKU compared to

a reference group or controls. A single study, including children and adolescents, did not

find altered BMD in patients with PKU [20].

BMD in pediatric patients with PKU

All 5 studies that included pediatric patients used DXA to measure BMD. Four reported

a reduced BMD in patients and one study did not find a significantly altered LBMD

when comparing 8 pediatric patients with PKU to a control population [20]. In this

study, however, two of the eight (25%) patients had a LBMD Z-score below -2, meeting

the criteria for low BMD for pediatric patients defined by the ISCD [28,45]. Both are

described as adolescent patients not adherent to diet.

Of the 4 articles reporting lower BMD in patients with PKU, Adamczyk et al. [13]

described a group of 45 children (mean age 13.8 ± 5.2 years) and concluded that skeletal

status is impaired in patients with PKU (mean Z-score LBMD -0.572 ± 1.270 and TBMD

-0.117 ± 1.347). They also found that in patients who were sexually mature, those who

were non-adherent to diet had a significantly lower BMD than those who adhered to

diet.

Furthermore, Barat et al. [43] investigated a group of 13 pediatric patients with PKU,

reporting a mean LBMD Z-score of -1.36 ± 1.586.

Similarly, a study by Hillman et al. [37] established that BMD at multiple sites was

significantly lower in a group of 11 pediatric patients with PKU compared to age-

matched controls [LBMD 0.61 ± 1.5 g/cm2vs 0.72 ± 0.24 g/cm2and FBMD 1.56 ± 0.30

g/cm2vs 1.87 ± 0.56 g/cm2].


~ 117 ~

Finally, Allen et al. [17] investigated 32 pre-pubertal patients (mean age 7.7 ± 2.3 years)

and found significantly lower BMD compared to age-matched, non-PKU controls

(TBMD 0.770 ± 0.085 g/cm2vs 0.814 ± 0.075 g/cm2 and LBMD 0.619 ± 0.100 g/cm2vs

0.701 ± 0.097 g/cm2). TBMD of patients with PKU was 97.1% of predicted BMD for

children of the same gender and age while LBMD was 92% of predicted BMD. Clinical

fracture risk was not directly evaluated by any of the studies.

BMD in pediatric and adult patients with PKU

Four of the 5 studies that described a mixed group of pediatric and adult patients used

DXA to assess BMD and 1 study [12] used pQCT to assess BMD in the radius. All 5

studies reported altered BMD in patients with PKU.

A conference abstract by Coakley et al. [46] reported TBMD in a population of 57

patients over 4 years of age. The authors found that 16 patients (28%) had a TBMD Z-

score between -1 and -2.5 and three patients (5%) had a Z-score below -2.5. TBMD was

positively correlated with age (controlling for BMI, sex, metabolic control, and medical

food intake) in their population with a mean age of 17.5 years. Similar results were

obtained by three other studies. de Groot et al. [14] reported a mean LBMD Z-score of -

0.78 ± 1.1 in a group of 53 patients with PKU and low BMD (LBMD Z-score below -2) in

10 patients (19%). A subgroup analysis showed that younger patients had a higher

prevalence of low BMD though no significant correlations were established between

BMD and age.

Lage et al. [15] investigated BMD in 47 patients with PKU and found a mean Z-score

significantly below 0 (mean FBMD Z-score -1.2 ± 1.0; LBMD Z-score -0.4 ± 0.8). A Z-

score between -1 and -2.5 was found in 13 patients (28%) and a Z-score below -2.5 in 6

patients (13%) of at least one site. The authors found a negative correlation between age

and LBMD in patients 6–10 years of age and a positive correlation between age and

FBMD in patients 11–18 years of age.

Chapter 5

~ 118 ~

Zeman et al. [16] studied 44 patients with PKU and described that 14 (32%) had a

TBMD Z-score below -1 and 20 patients (45%) had a LBMD Z-score below -1, of whom 6

had a Z-score below -2.5. No correlation between age and LBMD or TBMD was evident.

A final study by Schwahn et al. [12] used pQCT in 14 patients with PKU ages 5–28 years

to assess BMD of both spongy and total bone of the non-dominant distal radius. They

found that spongy bone BMD was significantly lower in patients with PKU compared to

14 age, gender, weight and height-matched controls [139.7 ± 23.5 mg/cm3 vs 169.3 ±

31.5 mg/cm3]. Mean total bone BMD of the radius in patients with PKU was slightly

lower than controls, but not significant. Within the group of PKU patients, TBMD and

LBMD were lower in adolescents ages 13–16 years compared to younger children and

adults. The authors hypothesized that patients with PKU have altered trabecular bone

architecture indicated by low spongy bone BMD and/or altered mineralization, but

show minor changes of cortical bone. They emphasize this hypothesis by describing the

case of an untreated severely retarded female patient who showed lower BMD,

especially of trabecular bone, at 10 years of age, which could not be explained by a

history of malnutrition or immobilization.

BMD in adult patients with PKU

The only study included in this review examining exclusively adult patients is by Modan-

Moses et al. [44]. In a group of 31 patients, 42% had compromised BMD (Z-score <-1).

Mean TBMD Z-scores (-0.474 ± 0.719) and FBMD Z-scores (-0.727 ± 0.66) were

significantly lower than expected for individuals of the same sex and age without PKU (p

= 0.002 and p < 0.001, respectively). Mean LBMD was also lower than expected, but not

statistically significant.


~ 119 ~

Prevalence of compromised bone status

Five studies examined the prevalence of low BMD. In cohort studies, prevalence of

osteopenia (defined in all papers as a Z-score between -1 and -2.5) ranged from 28–46%

[15,16,20,44,46]. A single study estimated the prevalence of osteopenia retrospectively,

finding 62% of children with PKU had a Z-score between -1 and -2.5 at age 12 [43]. The

prevalence of osteoporosis (defined in each study as a BMD Z-score below -2.5) ranged

from 5–14% [15,16,20,44,46]. A single study defined low BMD as a Z-score below -2,

consistent with ISCD recommendations, and reported a prevalence of 19% in children

and adults [14]. Seven studies included in this review did not report the prevalence of

low BMD [12,13,17,19-21,37], and none of the 13 studies reported BMD T-scores.

In known literature databases, we found no reports on low BMD prevalence (Z-score <-

2) in a reference population of adolescents or young adults for comparison. A self-

performed pilot analysis of weighted NHANES data [41], however, suggests Z-scores

between -1 and -2.5 are found in 14.9% (95% CI 12.6–17.4%) at the proximal femur and

14.3% (95% CI 12.1–17.0%) at the lumbar spine in non-Hispanic Caucasians ages 8–45

years. Z-scores below -2.5 were found in an additional 0.13% (95% CI 0.03–0.53%) at

the proximal femur and 0.53% (95% CI 0.13–2.10%) at the lumbar spine. The

prevalence of low BMD for chronological age, defined by ISCD criteria as a Z-score

below -2, was 1.8% (95% CI 1.0–3.3%) at the lumbar spine and 1.6% (0.8–3.0%) at the

proximal femur in NHANES data. These findings confirm a normal distribution of BMD

in the general population, in which a 2.3% of the population would be expected to have a

score below -2 SD.

Height-corrected bone mineral density

Three studies included a height-correction of DXA measured BMD to correct for height

bias.

Adamczyk et al. [13] reported SD-scores on DXA results with correction for patient

height and gender. TBMD and LBMD SD scores were significantly lower in adolescent

patients who were not compliant with diet compared to compliant patients. Total body

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and lumbar spine BMC SD scores were also significantly lower in non-compliant versus

compliant patients.

Allen et al. [17] reported lower LBMD in children with PKU compared to controls,

adjusting for height and weight. There was no difference in mean age and SD height and

weight scores between the PKU and control children. Based on predictions for LBMD

derived from control data, LBMD of the children with PKU was 92% of what was

expected.

De Groot et al. [14] report a positive correlation between BMD and height in children

with PKU under age 18, but not in adults. They conclude Z-scores of BMD found in their

whole study population (n = 53; mean age 16.7 ± 9.1) are not significantly correlated to

height and weight.

Blood Phe levels and BMD

Nine studies investigated the correlation between Phe blood levels and BMD [3,13-

17,37,44,46], seven of which found no correlation [14-17,37,44].

de Groot et al. [14] found no significant correlations between BMD and frequency or

proportion of Phe blood concentrations below the recommended threshold, or the mean

cumulative variation of blood Phe concentrations.

Two studies, however, did find a negative correlation between Phe levels and BMD

[13,43]. First, Barat et al. [43] describe that although mean Phe concentration did not

correlate with BMD outcomes, patients with BMD Z-scores below -1 had a significantly

higher mean cumulative Phe variation than controls [3.1 ± 0.4 mg/dl versus 2.5 ± 0.4

mg/dl (p-value = 0.006)]. Based on their findings, the researchers suggest variations of

Phe concentrations may contribute to lower BMD in children with PKU. Second,

Adamczyk et al. [13] report that among pediatric patients who had reached sexual

maturity, those who were compliant to diet had significantly higher BMD Z-scores, and

lower plasma Phe levels than non-compliant patients. A regression analysis also showed


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serum Phe concentration had the most negative influence on BMD values of all variables

examined including demographics such as age, sex, and body mass index (BMI).

Dietary intake and BMD

Seven cohort studies examined the impact of total protein and/or medical food protein

intake on BMD [13,15-17,37,44,46]. Evidence is consistent for total protein intake with 4

studies reporting no correlation with BMD [17,37,44,46]. Studies assessing medical food

protein intake and BMD, however, are inconsistent. Coakley et al. [46] found a positive

correlation between medical food prescription (grams of protein per day) and actual

medical food intake and TBMD. Zeman et al. [16] reported no correlation between daily

intake of Phe-free amino acid mixture per kilogram body weight and TBMD or LBMD Z-

scores.

Meta-analysis of BMD in early treated patients with PKU

A meta-analysis was performed on mean BMD Z-scores in the spine (7 studies), whole

body (3 studies) and femur (2 studies). All studies used DXA to measure BMD. Pooling

of data was performed by using available BMD Z-scores provided by the authors, either

in the article [14,20,44,46] or through added information on request per e-mail

[13,15,43,46]. A fixed or random effects model of generic inversed variance was used to

examine the mean difference between patients with PKU and normal values for healthy

age and sex-matched controls (BMD Z-score = 0).

Exploration of heterogeneity

Seven papers measured LBMD for a total of 247 patients [13-15,20,43,44,46]. Mean Z-

scores ranged from -1.363 to -0.4 (Figure 6). A moderate heterogeneity was observed

(I2= 59%), justifying the pooling of results and the use of a fixed effects model [39,47].

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Three papers measured TBMD for a total of 133 patients [13,44,46]. Mean Z-scores

ranged from -0.55 to -0.12 (Figure 7). A moderate heterogeneity was seen (I2= 42%),

justifying the pooling of results by the use of a fixed effects model [39,47].

Two papers were available providing FBMD for a total of 78 patients [15,44]. Mean Z-

scores ranged from-1.2 to-0.727 (Figure 8). High heterogeneity between the two studies

was observed (I2= 84%), probably due to the low amount of included studies, therefore

a random effects model was used to pool patients-based results [39,47].

Assessment of publication bias

There was no evidence of publication bias for LBMD as visual assessment of the funnel

plot (Figure 9) shows a symmetrically distributed inversed funnel and Egger’s test was

not significant (p = 0.407). Evaluation of publication bias by funnel plot or Egger’s test

for TBMD and FBMD was not reliable due to the limited number of studies included.

Pooled patient-based BMD

In 247 pooled patients with PKU included in 7 studies, mean LBMD Z-score was -0.70

(95% CI-0.82,-0.57). The overall effect is significantly different (P < 0.00001) from the

norm and none of the individual studies crossed the line of no effect (Figure 6).

In 133 pooled patients with PKU included in 3 studies, mean TBMD Z-score was -0.45

(95% CI-0.61,-0.28). The overall effect is significantly (P < 0.00001) different from the

norm (Figure 7). One of the individual studies crossed the line of no effect [13].

In 78 pooled patients with PKU included in 2 articles, mean FBMD Z-score was -0.96

(95% CI-1.42,-0.49). The overall effect is significantly different (P < 0.0001) from the

norm and both included studies do not cross the line of no effect (Figure 8).


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Bone turnover markers in early treated patients with PKU

Four cohort studies examined BTM in patients with PKU. Combined, a total of 110

patients (range 11 – 45 per study) were included. Two studies included only pediatric

patients [13,37], one study included only adult patients [21] and one study included both

pediatric and adult patients [48]. All included studies found significant alterations in

one or more BTM.

Adamczyk et al. [13] measured 3 bone formation markers including carboxyterminal

telopeptide of type I collagen (P1NP), bone-specific alkaline phosphatase (bALP) and

osteocalcin in serum of 45 pediatric patients with PKU. They compared BTM by

subgroups of patients based on sexual maturity and compliance to diet, but did not

provide mean values for the group as a whole. Among those compliant with diet,

sexually immature patients (Tanner stage below 5) had higher P1NP (10.33 ± 2.97 lg/l vs

6.62 ± 2.10 lg/l) and bALP (75.67 ± 49.60 U/l vs 30.67 ± 37.05 U/l) compared to

sexually mature patients.

On the other hand, among sexually mature patients, differences were found between

non-compliant and compliant patients including higher bALP (63.0 ± 46.43 U/l vs

30.67 ± 37.05 U/l) and higher osteocalcin (48.87 ± 23.0 ng/ml vs 33.15 ± 11.88 ng/ml).

These findings are in line with physiological concentrations of BTM, which are increased

during active periods of bone remodeling including growth in childhood and pre-

pubertal adolescence [49,50].

Hillman et al. [37] assessed BTM in 11 children with PKU in comparison to 11 age-

matched controls. Bone formation markers bALP (6.1 ± 6.3 U/l vs 13.1 ± 2.0 U/l) and

osteocalcin (72 ± 30 U/l vs 126 ± 43 U/l) were significantly lower in patients with PKU

compared to controls, whereas P1NP was lower (290 ± 174 U/l vs 400 ± 159 U/l) but not

significant. Bone resorption markers including urinary tartrate resistant acid

phosphatase and calcium creatinine ratio did not differ between subjects and controls.

Nagasaka et al. [21] reported BTM in adult patients (n = 34) compared to age-matched

controls (n = 36). The bone resorption markers blood pyridinoline cross-linked

telopeptide domain of type I collagen, urinary deoxypyridinoline, and urinary N-

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telopeptide of type I collagen were significantly higher in patients with PKU than in the

control group. Blood osteoprotegerin, an inhibitor of bone resorption, was also

significantly lower in individuals with PKU. No differences were found in the bone

formation markers bALP and osteocalcin between the PKU and control groups.

Porta et al. [48] examined spontaneous osteoclastogenesis, the differentiation of mature

osteoclasts from precursors to initiate the process of bone resorption, in pediatric and

adult patients with PKU compared to 20 age and sex-matched controls. Their results

show that osteoclasts, generated through spontaneous osteoclastogenesis from

peripheral blood monocytes, were larger and nearly double in number compared to

those of control subjects.


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Figure 6. Forest plot of LBMD (Z-score) in patients with Phenylketonuria

Figure 7. Forest plot of TBMD (Z-score) in patients with Phenylketonuria

Figure 8. Forest plot of FBMD (Z-score) in patients with Phenylketonuria

(SE = standard error, IV = Inversed Variance, CI = confidence interval)

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Figure 9. Funnel plot LBMD (Z-score) in patients with PKU.

SE = standard error; MD = mean difference (Egger's test: p = 0.407)

Blood Phe levels and BTM

Four studies investigated correlations between blood Phe concentrations and individual

BTM [13,21,37,48]. Bone formation markers including bALP and osteocalcin were

reported as higher in patients with Phe above recommended levels compared to patients

with recommended Phe levels [13]. Moreover, mean serum Phe over a period of one year

was significantly correlated with the number of osteoclasts, indicators of active bone

resorption, in patients with PKU (r = 0.576; p-value = 0.010) [48]. Other studies report

no correlation between serum Phe concentrations and BTM [21,37].


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Other indicators of bone status in early treated patients with PKU

Bone mineral content

BMC was examined in a single study [13] by Adamczyk et al. (2011). The authors

reported higher total body BMC and spine BMC in mature patients with concurrent

recommended threshold Phe levels at time of measurement compared to mature

patients with Phe levels above recommendations. Moreover, in non-compliant patients,

the total body BMC to lean body mass (LBM) ratio was reduced, an indicator of

increased risk for fragility fractures. In compliant patients, however, the BMC/LBM

ratio was not different than expected for age and height.

Vitamin D status in patients with PKU

Six included studies measured blood vitamin D status, all are cohort studies

[14,15,21,37,44,46]. Among the cohort studies, findings varied by age group. One study

of 31 adults with PKU showed that all patients had normal 25-hydroxyvitamin D

concentrations, the primary indicator of vitamin D status [44]. Two studies report

associations between vitamin D and indicator of bone status in children and adults with

PKU [14,46], but do not mention the prevalence of vitamin D deficiency or insufficiency.

A case–control study suggests 25-hydroxyvitamin-D and 1,25-dihydroxyvitamin-D

concentrations in children with PKU do not differ from controls matched on sex and age

[37]. In male and female adults with PKU on the other hand, 1,25-dihydroxyvitamin-D

was reported as significantly higher than in controls and 25-hydroxyvitamin-D was

significantly lower than controls [21]. Coakley ea. report a significant positive

association between TBMD and 1,25-dihyroxyvitamin D [46]. All other studies report no

correlation between plasma 25-hydroxyvitamin-D and BMD at any site [14,15,44].

Parathyroid Hormone (PTH) in patients with PKU

Four of the 13 included studies measured PTH, all are cohort studies [13,21,37,44].

Overall, children with PKU have similar PTH concentrations to healthy controls [37],

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but differences are reported in subgroups. PTH appears to be significantly higher in

non-compliant children and adolescents compared to those with recommended Phe

levels [13]. PTH is also reported to be higher in female and male adults with PKU

compared to controls, but the difference is not statistically significant in males [21]. PTH

above the normal reference range was reported in two of 31 (6%) adults with PKU

examined in one study [21].

Other indicators of bone status

Fracture history was examined in a single study. Modan-Moses et al. [44] reported that

4 patients (13%) included in their study had a significant fracture history, though all

were the result of physical trauma. Two patients had normal BMD, one had a LBMD Z-

score of -1.9, and one had a FBMD Z-score of -2.4. Greeves et al. [51] provided the first

investigation of fractures in patients with PKU, reporting the risk of fracture is 2.6 times

greater in patients with PKU over 8 years of age compared to controls. Though the study

did not meet inclusion criteria for this review, Greeves ea. provides the only estimate of

fracture risk in known literature on patients with PKU.

Concentrations of vitamins and minerals related to bone metabolism, including calcium,

phosphorus and magnesium, were also measured in several studies.

Calcium was measured in six of the 13 included studies [13-15,21,37,44]. Serum calcium

was reported as significantly lower in children with PKU compared to controls [37],

although no difference in total calcium was found between compliant and non-

compliant subgroups of children and adolescents with PKU [13]. In adults with PKU,

urinary calcium excretion was significantly higher than in controls [21], though all

patients’ blood calcium concentrations fell within normal range [44]. A negative

correlation between blood calcium and BMD Z-score in individuals with PKU of all ages

was reported by de Groot et al. [14], but no correlation between plasma calcium and

BMD Z-score was found by Lage et al. [15].

Phosphorus was measured in four of the 13 included studies [14,21,37,44]. Children and

adults with PKU were reported to have serum phosphorus concentrations within normal


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ranges and comparable to healthy reference groups [37,44]. While no difference in

phosphorus concentration was found between adults with PKU and controls [21],

children with PKU had lower phosphorus excretion and higher phosphorus reabsorption

compared to controls [37]. Children and adults with low BMD Z-scores were described

to have higher blood phosphorus concentrations compared to those with normal BMD,

but the correlation between blood phosphorus and BMD Z-score was not significant

[14].

Two studies examined serum magnesium and found lower concentrations in children

with PKU compared to controls [14,37]. Children and adults with PKU with low BMD

also had lower, though not significant, magnesium than those with normal BMD [14].

Magnesium did not correlate significantly with BMD Z-score [14] or any measure of

bone status [37] in either study.

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DISCUSSION

BMD in early treated patients with PKU

The results of our qualitative and quantitative review suggest that mean BMD is lower in

PKU patients compared to reference groups but within the normal range in most

patients, thus the clinical relevance of this finding is questionable.

The meta-analysis of pooled data from 247 patients with PKU shows an overall effect

size for LBMD Z-score of -0.70 (95% CI-0.82,-0.57). The overall effect size for TBMD Z-

score in 133 patients is also below zero [-0.45 (95% CI-0.61,-0.28)]; however one of the

studies [13] shows a large range and crosses the no effect line (Z-score = 0). Because

heterogeneity is moderate, it can be assumed the overall effect size is reliable and that

TBMD Z-score in patients with PKU is indeed below 0. Our meta-analysis for FBMD

shows a similar effect, although heterogeneity of the populations and outcomes in these

studies hamper a firm conclusion.

In our qualitative analysis of BMD Z-scores in patients with PKU, we found study-

defined prevalence of osteopenia and osteoporosis [15,16,20,44,46] to be higher than

prevalence of comparable Z-score categories in a reference population of adolescents

and young adults; however, our meta-analysis of pooled BMD Z-scores reported in

patients with PKU challenges this hypothesis. Overall effect sizes of Z-scores for LBMD,

TBMD and FBMD calculated in our meta-analyses are categorized as normal by ISCD

standards. The 95% confidence intervals of effect size for separate studies do, however,

show a number of patients with LBMD Z-scores below -2. Thus, a subset of patients with

PKU reported in articles included in this study might have a higher risk for skeletal

fragility and fractures. Based on the assumptions that our data are normally distributed

and the overall effect size for LBMD Z-score is -0.70, approximately 10% of early treated

patients with PKU may have a LBMD below -2 SD. This means 90% of early treated

patients with PKU are not at risk for low BMD, a much better outcome than expected

from single studies from the literature. The projected 10% of patients with a Z-score

below -2 may be at risk for osteoporosis and may benefit from the same preventative

and treatment strategies defined for healthy individuals [52].


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A large limitation to these findings is the lack of standardization between individual

study’s definitions of osteopenia and osteoporosis and clinical diagnostic criteria. In

pediatric patients, fracture history must be assessed alongside BMD Z-score before

diagnosis can be made. In adult patients, WHO guidelines require T- scores to diagnose

osteopenia or osteoporosis. Currently, most studies in patients with PKU report Z-

scores, regardless of age groups studied, and only 2 studies report fracture history, but

do not mention its relevance to clinical diagnoses. Thus, studies reporting prevalence of

osteopenia and osteoporosis in patients with PKU are missing essential information

necessary to qualify patients for these diagnoses.

We also examined the impact of Phe status and dietary compliance on BMD. Most

studies researching correlations between Phe values and BMD did not find a correlation.

Dietary compliance and dietary intake assessed as reported medical food intake [16,46],

total protein [17,37,44] or Phe intake [16,17] were not correlated to BMD or BTM. The

impact of overall protein status, including biological value of intact versus medical food

protein and percent of total protein derived from medical food, on bone were not

considered by any studies.

Age in relation to BMD was examined, but outcomes are very heterogeneous with

associations varying across age groups. We are not able to draw conclusions about BMD

in different age categories based on the included studies; however children under the

age of 10 years and those from 13–16 years of age may have a higher prevalence of low

BMD than other age groups [12,14].

Bone turnover in early treated patients with PKU

Results on bone turnover in PKU were ambiguous, though the 4 studies examining BTM

in children and/or adults with PKU found significant alterations in one or more marker.

Investigated markers were heterogeneous and populations studied were not similar in

age and thus cannot be reliably compared. Examining correlations between Phe

concentration and individual BTM provides mixed results. Differences in findings could

be due to differences in methods to measure and report Phe and diversity in reported

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markers. Consensus on the clinical utility of BTM including reliable methods of

measurement and reference ranges, and the establishment of markers suitable for

(various age groups of) patients with PKU must be established in future investigations.

Other factors related to bone status in PKU

Other indicators of bone status that were investigated in patients with PKU were BMC,

vitamin D, PTH, calcium, phosphorus and magnesium concentrations. Most outcomes

were reported by a small number of studies with heterogeneous groups of patients and,

sometimes, contradictory outcomes. BMC may be reduced in non-compliant individuals

with PKU, but the clinical implications of low BMC are unknown. Vitamin D and PTH

status do not seem to influence BMD based on found results. Calcium seems to be lower

in children with PKU, but the impact on bone is ambiguous. Phosphorus and

magnesium blood levels do not seem to affect bone status. At this time, it is not possible

to draw conclusions on these indicators of bone status without additional evidence from

high-quality studies in large groups of patients with consistent measurements. Although

the results are inconclusive, including additional bone status indicators in future studies

could add to the standard evaluation of bone health in patients with PKU of all ages.

Summary of evidence

We examined the strongest current evidence on bone health in patients with PKU. All

studies were of adequate to high quality, with low to no risk of bias and included only

patients who were early diagnosed and treated. Our results suggest that patients with

PKU have lower BMD as shown by the mean effect sizes in our meta-analyses. Clinical

significance of these outcomes is debatable as the mean effect size Z-scores are within

the range for normal BMD according to ISCD recommendations. Though prevalence of

low BMD for chronological age is higher in patients with PKU than in the normal

population (estimated 10% vs 2.3%, assuming a normal distribution), definitions used

for osteopenia and osteoporosis are highly heterogeneous between studies and ISCD

positions and WHO standards are rarely followed. Even though several studies


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reporting on limited cohorts of patients report osteopenia and hypothesize poor bone

health in patients with PKU, our review and metaanalyses of all available data suggests

bone is not clinically compromised in most early treated patients with PKU.

With the data at hand, we do not have sufficient evidence to establish conclusions on

BTM and other indicators of bone status we examined, nor define relationships between

Phe or nutrient intake and bone. Further research with more consistent measurements

in larger studies is necessary to provide better insight.

Clinical implications

Mean total body, lumbar spine, and femoral hip BMD Z-scores in patients with PKU are

lower than in their healthy peers, but well within the reference range for normal. The

clinical relevance of a slightly lower BMD Z-score is unclear. A projected 10% of patients

have a BMD Z-score below -2; however 90% of early treated patients with PKU are not

at risk for low BMD. Fracture risk must be established before developing final

conclusions on bone health in patients with PKU.

In order to evaluate the risk for skeletal fragility or fractures in individual patients, a

single assessment of BMD by DXA scan in all adolescent patients with PKU could be

considered [33]. Patients with a BMD Z-score above -2 may not require additional

follow-up; however patients with low BMD for chronological age (Z-score below -2)

and/or a significant fracture history may need follow-up.

For prevention and treatment of low BMD, factors related to bone health in healthy

individuals may be applied to prevent low BMD in patients with PKU. We suggest

following recommendations for the general population outlined in the ‘National

Osteoporosis Foundation’s Clinician’s Guide to Prevention and Treatment of

Osteoporosis’ to preserve bone strength [53]. In particular, an adequate intake of

calcium and vitamin D, lifelong participation in regular weight-bearing and

musclestrengthening exercise, cessation of tobacco and excess alcohol use if applicable,

and treatment of risk factors for falling are also appropriate recommendations for

patients with PKU.

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Future studies

Forthcoming studies will need to establish whether slightly lower BMD from an early

age increases the risk for osteoporosis or fractures acutely or long-term. Furthermore,

for patients with low BMD, preventative and treatment strategies to improve BMD in

PKU should be defined. To harmonize evidence, where to measure BMD; valid markers

of bone turnover; and definitions of osteopenia/osteoporosis, metabolic control, dietary

compliance and protein intake must be concretely defined and standardized and related

to fracture risk. Finally, studies are needed of factors impacting BMD that may not be

related to PKU such as physical activity.

Strengths and limitations

One strength of this review is the inclusion of only early-diagnosed and treated patients.

By excluding studies on patients who were late diagnosed and at-risk for nutrient

deficiencies and potentially impairments in physical activity, which are known to have a

negative impact on bone status, we excluded significant potential ascertainment bias.

Another strength is that two metabolic centers from two continents performing

independent searches reached the same conclusion before combining efforts. Finally, we

were able to include a large group of patients in our meta-analysis by contacting authors

personally to request data, resulting in meta-analyses of Z-scores from multiple BMD

sites.

Our study also has some limitations. There were differences in methodology between

study team 1 and study team 2 during individual literature searches, quality and risk of

bias assessment, and data extraction, though standardized tools were used by each study

team. After comparing data extraction, overview tables were compared and essentially

identical. Therefore data extraction was easily and justifiably combined.

Neither search included “fractures” as a clinical outcome in search terms; however,

search 1 was broad and captured all literature related to bone in PKU including articles

related to fractures.


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A small number of the included articles report a correlation between BMD and height of

the patient. This is a known restriction of BMD measured by DXA [28]; patients with

lower height for age may be falsely diagnosed with a low BMD . Based on the data

published however, we were not able to provide height adjusted BMD Z-scores for our

pooled data.

Finally, all included studies reported osteopenia and osteoporosis based on Z-scores,

contrary to ISCD positions which do not recommend these diagnoses in pediatric

patients. Instead, the ISCD recommends the term “low BMD for chronological age”

when Z-scores are less than or equal to -2, and does not recommend the diagnosis of

osteoporosis without a clinically significant fracture history [28]. These are important

caveats to the current literature in patients with PKU and important evidence that

criteria for low BMD, osteopenia, and osteoporosis must be concretely defined.

CONCLUSIONS

BMD in early diagnosed and treated patients with PKU is below healthy population

average but within normal range. These findings are important to provide preliminary

evidence that bone does not appear to be compromised to the extent previously

hypothesized. However, while the overall effect size of BMD Z-scores are between -0.4

and -1 in patients with PKU, there is lack of data on a corresponding higher risk of

fracture in these patients.

Other indicators of bone status in early treated patients with PKU are inconclusive due

to the small number of studies and the heterogeneity in groups examined and

measurement methods. Though we now conclude that low BMD does not seem to be an

exaggerated concern in patients with PKU, research is needed on the effect of the PKU

diet on bone, the reliability of bone turnover markers in bone assessment, and a

concrete estimate of fracture risk in patients with PKU.

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Micronutrients, fatty acids and

bone health in Phenylketonuria

Submitted

Serwet Demirdas, Carla EM Hollak, Johanna H van der Lee, Francjan J van Spronsen, M Estela

Rubio-Gozalbo, Peter H Bisschop, Fred M Vaz, Nienke M ter Horst, Frits A Wijburg and Annet

M Bosch

Chapter 6

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Abstract

Introduction: In phenylketonuria the natural protein restricted diet, supplemented with

micronutrient fortified amino acid mixtures, prevents severe cognitive impairment due to high

phenylalanine levels. Nutrient deficiencies may occur as a result of the dietary restrictions. We

aimed to evaluate intake and blood levels of micronutrients and essential fatty acids (FA), bone

mineral density (BMD), bone turnover markers (BTM) and fracture history.

Methods: Sixty early diagnosed phenylketonuria patients (aged ≥1 year) were included in a

multi-center cross sectional study. We assessed micronutrients, FA and BTM in blood, dietary

intake and fracture history using a questionnaire, and BMD retrospectively..

Results: Dietary intake and serum levels of selenium (14 and 46% of patients) and 25-OH

vitamin D2+3 (20 and 14% of patients) were inadequate . Zinc serum levels were below normal

in 14% of patients despite adequate intake. Folic acid serum and intake levels were above

normal. Despite safe total protein and fat intake, arginine plasma levels and erythrocyte

eicospentaenoic acid were below reference values in 19% and 6% of patients respectively. Low

BMD (Z-score <-2) was slightly more prevalent in patients but lifetime fracture prevalence was

comparable to the general population. Both resorption and formation BTM were elevated.

Conclusions: In general, patients with phenylketonuria have a normal nutrient status.

However, the risk of low intake and blood levels of zinc, selenium, 25-OH vitamin D2+3,

arginine, eicosapentaenoic acid, as well as observed high levels of folic acid, need attention.

Fracture prevalence is normal but a slightly more prevalent low BMD and elevated BTM

warrant further investigation.

Micronutrients, fatty acids and bone health

~ 143 ~

INTRODUCTION

Phenylketonuria (PKU; MIM 261600) is an autosomal recessive disorder of

phenylalanine (Phe) metabolism caused by a deficiency of the enzyme phenylalanine

hydroxylase (PAH; EC 1.14.16.1), leading to severe cognitive impairment due to

accumulation of Phe in the brain. With the introduction of newborn screening and the

early institution of dietary treatment, cognitive impairment caused by PKU has nearly

been eliminated in developed countries [1]. Based on the level of blood Phe at diagnosis,

disease severity is classified as either classic or severe PKU (≥ 1200 μmol/L), mild to

moderate PKU (600–1200 μmol/L), or mild hyperphenylalaninemia (mHPA; (360–600

μmol/L). Treatment consists of dietary Phe intake restriction (an essential amino acid

(AA)) through a low protein diet in order to achieve safe Phe blood levels. Severely

affected patients tolerate <500 mg of Phe, which is <10 grams of natural protein per

day [2,3]. In order to guarantee a sufficient intake of daily protein, patients use

designated amino acid mixtures (AAM) containing most amino acids (not Phe), vitamins

and other micronutrients. Many different AAM are available with a highly variable

composition of nutrients. In some AAM, calculation of the amount of added nutrients is

based on needed daily calories and in others on advised intakes of protein per kilogram

bodyweight [4]. Few studies evaluated intake and deficiencies of nutrients in patients

with PKU and both normal and reduced intakes and blood levels of nutrients have been

reported [5,6]. A minority of patients is responsive to treatment with

tetrahydrobiopterin (BH4), a cofactor of PAH, which increases dietary Phe tolerance

and thus permits relaxation of the diet [7]. Because BH4 has only recently (2009)

become available for PKU treatment in the Netherlands, responsive patients on a

relaxed diet find it difficult to make healthy food choices after years on a strict diet and

deficiencies have been reported [6].

Furthermore, a decreased BMD in PKU has been frequently reported, although a recent

systematic review showed that the prevalence of osteoporosis according to

internationally accepted standards is low [8]. The relationship between bone health and

(micro)nutrient deficiencies is insufficiently studied. In order to optimize treatment,

Chapter 6

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and to prevent deficiencies or potential toxic concentrations of micronutrients, there is a

need for more insight into the nutrient intake and blood levels of patients with PKU [4].

The main objective of this study was to evaluate the intake of micronutrients and

essential FA from natural protein containing food and AAM in patients with PKU, and

to investigate the association between intake and blood levels of these micronutrients

and essential FA. The secondary objectives were to investigate BMD, bone turnover

markers (BTM) and fracture history in patients with PKU, and their associations with

blood levels and intake of micronutrients and essential FA.


~ 145 ~

METHODS

Study Design

This cross-sectional multicenter study was performed in three Dutch metabolic centers

(Amsterdam, Groningen and Maastricht) between May 2013 and May 2014. Inclusion

criteria were: PKU diagnosed through newborn screening; age ≥1 year; continuous

treatment with either a protein restricted diet with the use of an AAM, a protein

restricted diet with AAM in combination with BH4 treatment or BH4 treatment without

dietary protein restriction. Exclusion criteria were: changes in AAM in the month before

inclusion and (planned) pregnancy.

Micronutrients, essential FA and BTM were assessed in blood after at least three hours

fasting. An investigator-designed-questionnaire was used to evaluate daily dietary

intakes, fracture history and amount of physical activity (sports, walking and cycling in

the past year). Medication (including BH4) and dietary intake of AAM, natural protein

containing food sources and supplementary vitamins/minerals were assessed. Patient

reported intake was compared to the most recent dietary prescription from their

treating dietician[9]. Patient records were studied to obtain Phe levels from dried blood

spots over the last 12 months, and BMD Z-scores from dual-energy X-ray

absorptiometry scans (DXA) performed between two years before to 6 months after

inclusion. The study protocol was approved by the Ethics Committee of the AMC and

patients/parents provided informed consent before participation.

Laboratory measurements

Results from collected blood specimens were all obtained from centralised laboratories,

except for plasma amino acids which were assessed at the medical centre where the

patient was under treatment. However, all three centres are certified by the

ERNDIMQA, quality control for amino acid measurements

(http://cms.erndimqa.nl/Control-Materials.aspx), and for this reason assessments are

http://cms.erndimqa.nl/Control-Materials.aspx

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easily comparable. Chemical analyses were performed at the Clinical Chemical

Laboratory of the AMC; Fatty acid (FA) assessments were performed at the Laboratory

Genetic Metabolic Disease of the AMC; Bone turnover markers (BTM) were provided by

the Vrije Universiteit medical centre (VUmc) endocrinology laboratory.

Analysis of micronutrients

Analysis of micronutrients were performed after serum centrifugation at 2000 x g and

being stored at -20°C until analysis. Roche diagnostics equipment was used to analyse

albumin and calcium (the spectophometry method), sodium and potassium (the

ion selective electrode assay indirect method ), magnesium (the colorimetric method),

phosphate (the molybdate reduction method), transferrin (the immunotubidimetry

method) and folic acid and vitamin B12 (the electrochemiluminescence

immunoassay method). Atomic absorption spectroscopy using Beun de Rionde B.V.

equipment was used to analyse zinc, and selenium measurements were performed

using the Zeeman atomaire absorptionspectofometry. 25OH vitamin D2 + D3 was

assessed with Liaison Diasorin equipment using the competitive chemiluminescence

immunoassay. Finally, full blood vitamin B1 and B6 were evaluated with HPLC

Gynkotek Dionex equipment using reverse phase HPLC fluorimetric detection.

Analysis of total Fatty acids

We measured erythrocyte FA. Erythrocytes of venous EDTA blood were washed three

times in isotonic saline, counted by routine hemocytometric analysis and frozen

overnight in a BHT (2,6-di-tert-butyl-4-methylphenol)-coated eppendorf cup. Fifty

microliters of the resulting hemolysate –for plasma also 50 μl– was transmethylated in 1

ml 3 M HCl by incubating for 4 hours at 90°C in the presence of 10 nmol internal

standard; the methyl ester of 18-methylnonadecanoic acid. After cooling, the aqueous


~ 147 ~

layer was extracted in 2 ml hexane, and this extract was taken to dryness under nitrogen

flow and resuspended in 80 μl of hexane. One microliter of this solution was injected

into a Hewlett Packard GC 5890 equipped with an Agilent J&W HP-FFAP, 25m,

0.20mm, 0.33µm GC Column and eluting fatty acid methylesters were detected by flame

ionization detection. Fatty acid concentration were calculated using the known amount

of internal standard and expressed as pmol/106 cells for erythrocytes and in μM for

plasma.

Analysis of plasma amino acids

To assess amino acids plasma was prepared from heparinized blood by centrifugation at

1000 x g and stored at -20°C until analysis. Plasma amino acids were analysed using the

JEOL AminoTac amino acid analyser JLC-500/V according to the manufacturer’s

instructions.

Bone Mass Density measurements

Hologic Discovery imaging equipment was used with reference data from the general

Dutch population to compare patient outcomes to [10]. According to the Society for

Clinical Densitometry (ISCD) a diagnosis of osteoporosis in children, men and

premenopausal women is based on a BMD Z-score below -2 coupled with a significant

fracture history (two or more long bone fractures by age ten years and/or three or more

long bone fractures at any age up to nineteen years, or at least one vertebral

compression fractures in the absence of trauma) [11].

Dietary intake and growth parameters

Weight and height were collected, and age and gender appropriate Z-scores were

calculated based on Dutch population references [12]. From the questionnaire the daily

intake of micronutrients, protein and fat was calculated. Results were compared to the

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recommended daily dietary allowance provided by the Federal Public Service Health of

Belgium (basing its recommendations on previously performed research in the Dutch

population and on recommendations of the USA [13]), and the safe advised range (SAR)

of intake as recommended by the European Food Safety Authority (EFA) [14]. The

recommended dietary allowance is based on +2 SD of the required dietary intake and

thus provides sufficient intakes for 97.5% of the general population [15].


For all analyses the Statistical Package for Social Sciences (SPSS) Windows version 19

was used. Descriptive statistics were used to assess intakes and blood levels of

micronutrients, AA, FA, BMD Z-scores and BTM. Concerning laboratory and dietary

intake levels, we decided that levels outside of the reference range are of interest to

study (remarkable). The reference range provides mean ± 2 SD for a healthy age

matched population, hence remarkable results represent levels below the P5 or above

P95 of normal. Results are reported as medians and displayed in scatterplots in which

reference ranges are indicated; data are shown separated in age groups equal to

available age groups used to indicate reference ranges. In addition, Mann–Whitney U

(not normally distributed continues variables) or Chi square tests (nominal data) were

conducted to test differences between patients based on disease severity, BH4 and

supplement (not AAM) use.

Multivariable linear regression analyses was intended to investigate associations

between blood levels, BMD, BTM and intake.


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RESULTS

Participants

Sixty out of 102 eligible patients (58.8 %) agreed to participate in the study (43/51

Amsterdam, 10/32 Groningen and 7/19 Maastricht). The main reason for refusal was

time constraints of patients. One enrolled patient was not included in the dietary

analyses, because answers given in the questionnaire compared to the prescribed intake

were unequal. None of the participants had restricted mobility, or used medication

affecting bone status. Patient characteristics are displayed in Table 1.

Protein intake, phenylalanine levels and BH4 use

Detailed information on protein intake and Phe levels is shown in table 1. All patients

had a total protein intake above minimal safe recommendations. Twenty-four percent of

patients had a Phe tolerance of <500 mg per day (severe phenotype). BH4 was used by

14 patients; 10/14 additionally treated with natural protein restriction and AAM, 4/14

were off diet. Based on coincidence and taste preferences twenty-five different AAM

were used, most frequently Milupa PKU-2-prima (n=11), Milupa PKU-2-mix (n=10),

Vitaflo PKU cooler (n=9) and Milupa PKU-3-advanta (n=6).

The median percentage of Phe measurements above recommended range in the year

before inclusion varied from 33-60%, increasing with age (Table 1); comparable to

findings in other cohorts of PKU patients [16,17].

Laboratory results

Laboratory results of two patients were incomplete: in one AA were not evaluated and in

the other only vitamins and FA were measured. The laboratories assessing AA made

different choices on AA measured in routine PKU follow up, and therefore the total

Chapter 6

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number of assessed samples differs. We report on remarkable differences when

compared to reference ranges that might have clinical implications.

Table 1. Patient characteristics

Population

Age

(years)

Frequency

N (%)

Age in years

Median (IQR)

Male

N (%)

BMI

SD for age and sex

(SD)

All 60 (100) 13.0 (6 – 17) 25 (41.7) 0.45 (-0.18 - 1.24)

1 - 11 25 (41.7) 6.0 (4.5-9) 10 (40) 0.45 (0.08 - 0.96)

12 - 17 20 (33.3) 15 (13.3-15.8) 10 (50) 0.42 (-0.49 - 1.06)

18 – 39 15 (25) 29 (20.8-35.8) 5 (33.3) 0.49 (-0.40 - 2.65)

Protein intake in g/day

Age

(years)

n Total protein

intake

median (IQR)

Prot. natural

sources

median (IQR)

n Prot. amino-acid

supplement

median (IQR)

BH4 use

n (%)

All 59 64.8 (43.1-76.6) 14.6 (10.1 – 26.4) 55 45.00 (25.2 – 62.2) 14 (24)

1 - 11 24 39.7 (34.5-58.1) 10.8 (7.9-25.4) 23 25.2 (15.2-39.0) 7 (28)

12 - 17 20 74.7 (63.9-83.5) 16.3 (12.0-27.3) 19 60.0 (40.0-63.0) 5 (25)

18 - 39 15 81.3 (72.5-86.1) 19.0 (12.5-28.2) 13 60 (50.5-71.7) 2 (14)

Phenylalanine

Age

(years)

n Plasma Phe values

(µmol/L) median (IQR)

% of bloodspot Phe values above

range per patient median (range)

1 - 11 24 302 (193 – 342) 33 (0 – 67.5)

12 - 17 20 611 (401 – 778) 57 (0 – 100)

18 - 39 15 804 (522 – 978). 60 (0 – 100)


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Micronutrients

Remarkable dietary results are reported in Figure 1 and 2, blood levels in Figure 3 and 4.

The intake of vitamin D was below the advised minimum intake of 5 µg/day [18] in

12/59 patients. Vitamin D supplements were used in 12/60 patients. The 25-OH vitamin

D2+3 serum level was below the reference range of 50 nmol/L in 8/59 patients and

below 25 nmol/L in 2 of them. The lowest serum levels were found in one patient using

AAM and in four patients treated with BH4 off diet. No significant differences in serum

levels were found between supplemented and un-supplemented patients for vitamin D

(72 vs 70 nmol/L).

Daily selenium intakes varied from 5-125 μg/day, and 27 patients had serum levels

below the reference range (7/27 using BH4 with AAM). Selenium supplements were

used by 6/60 patients. No significant differences in serum levels were found between

supplemented and un-supplemented patients for selenium (0.87 vs 0.80 μmol/L).

Despite dietary zinc intake being well above the norm (48/59 patients), serum levels

below the reference range were found in 8/59 patients (none using BH4). Severe

patients showed significantly higher median zinc levels than mild patients: 12.5 vs 10.6

μmol/L.

Intakes of folic acid, magnesium, vitamin B6 and B12 were above the advised range in

our patients. Folic acid intake was above SAR in 5 patients and 26/56 patients showed

serum levels above reference range. Patients used the following supplements:

magnesium (n=1), multivitamin tablets (n=4), vitamin B complex (n=1). Blood levels

above reference were also found for magnesium (9/59 ), vitamin B6 (52/60) and

vitamin B12 (11/58). Patients off diet treated with BH4 did not show elevated

magnesium and folic acid levels (n=5). Severe patients showed significantly higher

levels of vitamin B12 (median 600 vs 482 pmol/L). No significant differences were

found between supplemented and un-supplemented patients for the micronutrients

folic acid, magnesium, vitamin B12 and B6.

Chapter 6

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Figure 1. Dietary intake vitamin D, selenium, zinc and folic acid

Median, minimum and maximum recommended daily dietary allowance

N = patients m = male f = female Safe Advised Range (SAR)

SAR for vitamin D = 100 µg/day

SAR for selenium ages ≥14 years = 250 µg/day


~ 153 ~

Figure 2. Dietary intake magnesium, vitamin B6, vitamin B12 and fat

Median, minimum and maximum recommended daily dietary allowance

N = patients m = male f = female Safe Advised Range (SAR)

SAR for vitamin B6 ≥4 years = 7 mg/day

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Figure 3. Blood levels of 25-OH vitamin D2+3, selenium, zinc and folic acid

Median, minimum and maximum Reference range

N = patients


~ 155 ~

Figure 4. Blood levels of vitamin B6, vitamin B12 and magnesium


N = patients

Chapter 6

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Amino acids

Plasma levels were below reference ranges for asparagine (22/59), 2-aminobutyric acid

(10/50), tyrosine(13/59) and arginine (33/58). Hydroxyproline plasma levels were

elevated (11/40) and ornithine levels normal to high (16/59) (Figure 5). Phe levels were

above reference range in all ages. Low asparagine was significantly more often found in

patients not using BH4 (2/15 patients using BH4 versus 20/44 not using BH4).

Erythrocyte fatty acids

In 57/59 patients the daily total fat intake of patients was below 40% of the total caloric

intake (SAR) and for 34 patients below the minimal recommended 20% (Figure 1). Of 55

patients treated with AAM 35 used essential FA containing AAM and none used

additional FA supplements.

Patients had total erythrocyte FA levels within reference range. Levels of the essential

FA linoleic (C18:2ω6, LA) and α-linolenic acid (C18:3ω3, ALA) were unremarkable. The

following metabolite levels of these FA were remarkable: 10/60 patients showed

lowered levels for eicosapentaenoic acid (C20:5ω3, EPA), and elevated levels were found

in 11/60 in homo-γ-linolenic acid (C20:3ω6), 23/60 in docosatetraenoic acid

(C22:4ω6), 26/60 in docosapentaenoic acid (C22:5ω6), and 10/60 in docosahexaenoic

acid (DHA; c22:6ω3) (Figure 6).

Median DHA and EPA levels were higher both in patients using FA supplemented AAM

and in patients without restriction of natural protein, when compared to patients using

AAM without FA supplementation (DHA 23 and 22 vs 16.5 pmol/10E6 cells; EPA 2.7

and 2.4 vs 2.1 pmol/10E6 cells).


~ 157 ~

Figure 5. Plasma amino acids

Median, minimum and maximum Reference range N = patients

Tyrosine data points:

patients that fasted overnight patients that did not fast overnight

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Figure 6. Essential fatty acids: a-linolenic acid and metabolites


N = patients FA = fatty acids AAM = amino acid mixture


~ 159 ~

Bone Mass Density and Bone Turnover Markers

BTM were above reference range for resorption marker CTx (21/56; 20/43 children and

1/13 adults) and formation marker PINP (42/56; all 13 adults and 28/43 children)

(Figure 7). Mean Z-scores for lumbar, femoral and hip BMD were overall normal with Z-

scores below -2 in 4.9% (n=2/41), 7.4% (n=2/27) and 5.9% (n=2/34) of patients

respectively (Figure 8). No differences in BMD or BTM were found based on BH4 use or

severity of disease.

Median physical exercise for adults was 205 min/week, for children 12-17 years 325

min/week and those 1-11 years 180 min/week. A total of 25 patients (41,7%) have

suffered a fracture. Fifteen patients suffered a single fracture episode, 9/25 two and 1/25

three. All fractures were caused by compatible trauma and healed without

complications. One patient had a positive fracture history as defined by the ISCD but

BMD was within normal range.

Multivariable linear regression

One of our aims was to use multivariable linear regression to investigate associations

between blood levels, BMD, BTM and intake. However, because assumptions needed to

justify such a model were not met (the data of the dependent variable was not

distributed in a straight-line), we do not report results concerning associations between

intake and serum levels of micronutrients, nor between serum levels of micronutrients

and bone health.

Chapter 6

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Figure 7. Bone turnover markers


N = patients

PINP = resorption marker procollagen type I N-terminal propeptide

CTX = formation marker carboxy-terminal collagen crosslinks


~ 161 ~

Figure 8. Bone mineral density (BMD)


N = patients

Chapter 6

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DISCUSSION

We evaluated dietary intake and deficiencies of micronutrients and essential FA, BMD,

BTM, physical activity and fracture history in a large group of patients with PKU. In

spite of the complex diet with many natural food products replaced by designated AAM

and low protein foods, most blood levels of micronutrients are in the normal range.

Some exceptions need to be addressed specifically.

Micronutrients

Vitamin D

Serum 25-OH vitamin D2+3 levels were below reference range in 14% of patients, fully

comparable to individuals in the general population in whom concentrations below

reference range are also frequently observed [18]. However, 2 patients showed levels in

the range associated with clinical symptoms (<25 nmol/L [18]). For this reason, despite

near normal bone health outcomes (BMD, fracture risk and BTM), it may be advisable

to yearly evaluate intake and determine blood levels, and to supplement patients when

levels are <50 nmol/L [18,19].

Zinc

Zinc serum levels were below reference range in 14% of patients, despite an intake above

SAR in 52% of patients. This finding has been reported before [4,6]. As a low intake of

animal protein in a vegetarian diet may decrease absorption of zinc [20], it seems

probable that the severe restriction of natural protein in the PKU diet decreases zinc

absorption. Zinc deficiency is associated with abnormalities in growth, sexual

maturation, wound healing, hair loss, visual dark adaptation and anorexia [21]. Hair

loss has been described at serum levels below 11 µmol/L (72 µg/dL) [22] and skin

lesions below 9.2 µmol/L (60 µg/dL) [23]. The clinical relevance of our findings is

debatable and further studies need to be done on how to effectively increase zinc uptake

in PKU patients. Especially considering the fact that a large proportion of our patients

already have intakes exceeding SAR, and that to achieve an increase of 6% of serum

levels intake needs to be doubled [24].


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Selenium

Low plasma selenium serum levels have previously been described in PKU [6]. In our

cohort dietary intake was below reference in 41% and serum levels in 46% of patients.

Selenium is best absorbed when ingested as an organic form while inorganic forms, such

as sodium selenite used in AAM, are less well absorbed. Selenium deficiency may lead to

cardiomyopathy, depressive symptoms or osteoarthropathy [25-27]. Mood disorders

and depressive symptoms have previously been described in PKU [28].An increased risk

for depressive symptoms has been reported at serum levels of <1.04 µmol/L (82 µg/L)

[26], which is above the lowest reference range of normal (0.8 µmol/L) in the present

study. Because intake in many patients is low it seems advisable to annually evaluate

intake and blood levels and consider supplementation if levels are below advised

reference ranges (supplementing up to 400 μg/day is considered to be safe in adults

[29]). However, no large studies are available concerning the clinical relevance of

lowered selenium levels. Therefore, larger studies are indicated to further assess the

clinical relevance of our findings.

Folic acid

Folic acid intake and blood levels were remarkably high in patients using AAM. Five

patients showed intake levels above SAR (Figure 1). Because all patients off diet showed

serum levels in the normal range, elevated serum levels appear to be due to the fortified

AAM [30]. As there is discussion on the safety of high levels [31] it deserves due

consideration to lower folic acid amounts in AAM. Our findings are confirmed by the

study by Stolen et al reporting similar results [32].

Magnesium, vitamin B6 and B12 are elevated in dietary intake and blood levels.

However as intakes are within SAR and these micronutrients are not known to be toxic

adaptation of intake may not be warranted [13].

Amino acids

Plasma arginine (a conditionally essential amino acid) was below reference range in 19%

of patients . This decrease cannot be assigned to hemolysis (when arginase converts

arginine to ornithine) as ornithine levels were normal. Arginine is important for many

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cellular functions, including nitric oxide production and urea cycle function [33,34].

Glutamine levels were fully normal demonstrating normal function of the urea cycle.

Further research is necessary to determine whether it may be advisable to increase

supplementation of arginine in AAM.

Plasma tyrosine was low in our patients, especially in children and adolescents. Patients

who fasted overnight had lower median tyrosine levels (40 umol/L) than those fasting

3-6 hours (54 umol/L), confirming previous reports of low fasting tyrosine values in

PKU [35]. AAM use has been demonstrated to cause large fluctuations in plasma

tyrosine over the day and effects of extra tyrosine supplementation on outcome are yet

unclear [36]. Further investigations on effects of intake on plasma levels are warranted.

Asparagine (a non-essential amino acid ) levels were low in 1/3 of patients, but not

much is known about implications of deficiency. Asparagine is not supplemented in

AAM. Further examination is needed to objectify if asparagine deficiency is clinically

relevant, and if it should be added to AAM.

2-Aminobutyric acid is formed from methionine and threonine[37], and was low in our

patients. Low plasma levels have been reported before in a small patient sample (n=4)

[37]. It is unclear if decreased levels have a clinical effect.

Hydroxyproline (a bone resorption marker) is solely elevated in adolescents,

representing increased protein turnover in bone during growth[38].

Erythrocyte fatty acids

Essential FA are precursors of thromboxanes, leukotrienes and prostaglandins [39].

DHA and EPA are known to have cardio-protective effects [40] and deficiencies may

lead to CNS disease (specifically DHA). Deficiencies of EPA and AA may affect the

immune system [39,39].

Our patients had normal to elevated levels of LA and it’s metabolites. This can be

explained as both LA and arachidonic acid are supplemented in AAM.

Of the ALA metabolites EPA and DHA are frequently supplemented in AAM. Perhaps

EPA is insufficiently supplemented as it is below reference levels in 6% of our patients,

and for this reason it may be considered to increase EPA supplements in AAM. DHA is


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more often and higher supplemented in AAM, explaining why levels are high compared

to the reference range in our patients. Significantly lower levels of DHA and EPA are

found in patients using AAM without FA versus those using FA supplemented AAM. For

this reason it may be advisable to prescribe FA containing AAM or warrant sufficient

intake with other means of supplementation.

Bone health

In our population 4.9% and 7.4% of patients had a lumbar and femoral BMD Z-score <-

2 respectively. This finding is in line with data reported in a previously published meta-

analysis by our study group [8]. None of our patients had osteoporosis as defined by the

ISCD and the lifetime fracture prevalence of patients with PKU seems comparable to the

age-standardized lifetime fracture prevalence of the general population in England

(41,7% versus 38.2%) [41].

Physical exercise in adults met the WHO recommended 150 min/week. Of children aged

12-17 years 80% did not meet the recommended 60 min/day of exercise, which is

comparable to the general population [42]. We have no reason to believe that

insufficient physical activity in this patient group has a negative effect on bone health

other than it would in the general population.

BTM are remarkably elevated, formation more than the resorption marker. Implications

of these findings are yet unclear and outcomes of other studies have shown

contradictory results with elevated, decreased or even normal BTM in patients [8]. We

hypothesize that this disbalance of bone turnover in our population may have effect on

bone health when patients are over the age of 50 years. However, in the light of the

near normal BMD and fracture prevalence in this patient group the clinical implications

of these findings are yet unclear and need further study.

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Multivariable linear regression

Unfortunately we were not able to properly investigate associations between blood and

intake levels of assessed nutrients, nor between outcomes of researched nutrients and

BMD, BTM. However, because these relations are of great interest to achieve further

knowledge on the etiology of nutrient deficiencies and bone health, larger cohort or

case-control studies are indicated.

CONCLUSIONS

In conclusion, we detected lower levels of several micronutrients and FA in this sample

of patients with PKU. However, specific complications that may be related to these

alterations, other than BMD, were not assessed in this study. Those micronutrients that

have been studied in large cohorts as potentially leading to risk (e.g. vitamin D and

selenium) could be considered to be supplemented. At this time there is no convincing

evidence for supplementation of the other nutrients.

Furthermore, although fracture prevalence is normal, a slightly more prevalent low

BMD and elevated BTM are found. The clinical implications may be limited as none of

the patients have osteoporosis as defined by the ISCD. However, the meaning of these

outcomes is yet unclear and follow up into older age is warranted.


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15. The Health Council of the Netherlands. Dietary refrence intakes: energy, proteins, fats, and digestible carbohydrates. [2001/19]. 2015. The Hague, The Hague: Health Council of the Netherlands. http://www.gezondheidsraad.nl/ sites/default/files /0119 ER.PDF

16. Ten Hoedt AE, Hollak CE, Boelen CC, van der Herberg-van de Wetering NA, Ter Horst NM, Jonkers CF et al.: "MY PKU": increasing self-management in patients with phenylketonuria. A randomized controlled trial. Orphanet J Rare Dis 2011, 6: 48.

17. Bekhof J, van Rijn M, Sauer PJ, Ten Vergert EM, Reijngoud DJ, van Spronsen FJ: Plasma phenylalanine in patients with phenylketonuria self-managing their diet. Arch Dis Child 2005, 90: 163-164.

18. van Schoor NM, Lips P: Worldwide vitamin D status. Best Pract Res Clin Endocrinol Metab 2011, 25: 671-680.

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23. Kumar P, Lal NR, Mondal AK, Mondal A, Gharami RC, Maiti A: Zinc and skin: a brief summary. Dermatol Online J 2012, 18: 1.

24. Lowe NM, Medina MW, Stammers AL, Patel S, Souverein OW, Dullemeijer C et al.: The relationship between zinc intake and serum/plasma zinc concentration in adults: a systematic review and dose-response meta-analysis by the EURRECA Network. Br J Nutr 2012, 108: 1962-1971.

25. Loscalzo J: Keshan disease, selenium deficiency, and the selenoproteome. N Engl J Med 2014, 370: 1756-1760.

26. Conner TS, Richardson AC, Miller JC: Optimal serum selenium concentrations are associated with lower depressive symptoms and negative mood among young adults. J Nutr 2015, 145: 59-65.

27. Rayman MP: The importance of selenium to human health. Lancet 2000, 356: 233-241.

28. Brumm VL, Bilder D, Waisbren SE: Psychiatric symptoms and disorders in phenylketonuria. Mol Genet Metab 2010, 99 Suppl 1: S59-S63.

29. Kieliszek M, Blazejak S: Selenium: Significance, and outlook for supplementation. Nutrition 2013, 29: 713-718.

30. Wiig I, Motzfeldt K, Loken EB, Kase BF: Nutritional Consequences of Adhering to a Low Phenylalanine Diet for Late-Treated Adults with PKU : Low Phe Diet for Adults with PKU. JIMD Rep 2013, 7: 109-116.

31. Choi JH, Yates Z, Veysey M, Heo YR, Lucock M: Contemporary issues surrounding folic Acid fortification initiatives. Prev Nutr Food Sci 2014, 19: 247-260.

32. Stolen LH, Lilje R, Jorgensen JV, Bliksrud YT, Almaas R: High dietary folic Acid and high plasma folate in children and adults with phenylketonuria. JIMD Rep 2014, 13: 83-90.


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33. Coman D, Yaplito-Lee J, Boneh A: New indications and controversies in arginine therapy. Clin Nutr 2008, 27: 489-496.

34. Tapiero H, Mathe G, Couvreur P, Tew KD: I. Arginine. Biomed Pharmacother 2002, 56: 439-445.

35. van Spronsen FJ, van DT, Smit GP, van Rijn M, Reijngoud DJ, Berger R et al.: Large daily fluctuations in plasma tyrosine in treated patients with phenylketonuria. Am J Clin Nutr 1996, 64: 916-921.

36. Webster D, Wildgoose J: Tyrosine supplementation for phenylketonuria. Cochrane Database Syst Rev 2013, 6: CD001507.

37. Yudkoff M, Blazer-Yost B, Cohn R, Segal S: On the clinical significance of the plasma alpha-amino-n-butyric acid:leucine ratio. Am J Clin Nutr 1979, 32: 282-285.

38. Szulc P, Seeman E, Delmas PD: Biochemical measurements of bone turnover in children and adolescents. Osteoporos Int 2000, 11: 281-294.

39. Tapiero H, Ba GN, Couvreur P, Tew KD: Polyunsaturated fatty acids (PUFA) and eicosanoids in human health and pathologies. Biomed Pharmacother 2002, 56: 215-222.

40. Morris MC: Dietary fats and blood pressure. J Cardiovasc Risk 1994, 1: 21-30.

41. Donaldson LJ, Reckless IP, Scholes S, Mindell JS, Shelton N.J.: The epidemiology of fractures in England. J Epidemiol Community Health 2008, 62: 174-180.

42. WHO Global Health Observatory. WHO. Global Health Observatory: prevalence of insufficient activity, situation and trends. http://www.who.int/gho/ncd/risk_ factors/physical_activity_text/en/ 2015.

Discussion and Summary

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GENERAL DISCUSSION and SUMMARY

The treatment of phenylketonuria (PKU) has been very successful since the introduction

of newborn screening in the 1960’s, Newborn screening has enabled doctors to identify

patients and start dietary natural protein restriction within the first weeks of life. Early

and continuous treatment has led to prevention of severe cognitive impairment and

near normalization of outcomes. Despite these great achievements the treatment of PKU

has also led to some new challenges in patient care. Some of these newly encountered

issues may be due to the dietary restrictions, while others may be caused by the disease

itself as early treated patients reach adulthood providing new challenges. Optimizing

care in patients with PKU needs fine-tuning of the treatment itself and evaluation and

management of adverse outcomes of the treatment. It is therefore of importance to

provide continuous and specific care for newly emerging issues as patients reach

adulthood. This thesis presents several studies focussing on the optimisation of care in

PKU, which will be summarised and discussed in this chapter: multidisciplinary

consensus on optimal care; the time burden, out-of-pocket-costs (OOPC) and health

related quality of life (HRQoL) of patients with PKU and their parents; bone health in

PKU; dietary intake and blood levels of micronutrients and essential fatty acids (FA)

(chapter 1).

Consensus on optimal care

Summary

To provide the best possible care for patients with PKU one of the challenges to

overcome are the differences observed in treatment between the different metabolic

centers (chapter 2). To harmonize care provided in the Netherlands, the Dutch patient

society for inborn errors of metabolism (the VKS) initiated the development of clinical

pathways for 20 different inborn errors of metabolism (IEM), including PKU, in

cooperation with pediatricians, internists and dieticians specialized in metabolic

disorders. The aim was to achieve national consensus about the best provided care for

these diseases using clinical pathways. Clinical pathways are of use to accomplish such

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national consensus as they are a tool for multidisciplinary decision making and

organization of care processes for well-defined groups of patients [1]. It has been

demonstrated that the use of pathways increases interdisciplinary communication,

enhances patient knowledge and self-awareness and leads to significant better

coordination of care [2,3]. When frequently updated, it also presents a reference to the

latest advancements in care [4] and provides direction for further research. We believe

that multidisciplinary cooperation using a clinical pathway will improve communication

and provide a clear route to best care, based on national consensus. For each IEM

clinical pathways (one version for professionals and one for patients) were made freely

available online through the VKS website [5].

Implications

The development of national clinical pathways for IEM has been successfully executed

and the present best available multidisciplinary approach to care in PKU has been

established. National consensus is of great importance in optimizing care for the patient

with PKU. The pathways help to clarify to both patient and caregivers what the route in

the care of individual patients broadly will be, and they are known to decrease duration

of inpatient care, increase interdisciplinary communication, enhance patient knowledge

and self-awareness, lead to significant better coordination of care and reduce costs [3].

The patient’s perspective and involvement is very important in this context as patients

wish, and need, to be more and more involved in their own treatment. Patients are

asking for more unified and consistent care trajectories [6]. Increasing patient

involvement and self-management may lead to better outcomes and clinician-patient

relationships [6,7]. To guarantee up-to-date information about the care trajectories

stated in clinical pathways it is necessary for clinicians to remain in dialogue about the

content, and all clinical pathways need to be regularly updated.

In chapter 2 we discuss the implementation and need for clinical guidelines nationally.

In the care for PKU international diversity in treatment also exists [8-10]. For example,

there is much debate on which blood Phe ranges are safest to target [6,8-10].

Furthermore, there are differences between countries and/or medical centers when it


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comes to the prescribed total dietary protein intake and care providers also define the

food produce which patients are allowed to eat without restriction differently [8,9,11].

Transition to adult care [12-15], acceptable Phe blood concentrations during pregnancy

[9,12] and the use of breastfeeding in newly diagnosed neonates with PKU [8] are also a

matter of attention as these subjects are approached unalike internationally. It is

important to acknowledge such dissimilarities in care and to strive to overcome them

using multidisciplinary consensus on the best available treatment options. Open

communication between providers of care for PKU (and patients) leads to uniformity in

care, a better understanding of lacunas in knowledge/evidence based medicine and

provides insight in areas that need further research. A European guideline involving

physicians, dietitians and a patient society is currently being developed [16].

Health related quality of life, out-of-pocket costs and time burden

The impact of the severe dietary restrictions on patients is thought to be considerable in

PKU. For this reason we hypothesized that HRQoL may be impaired. Furthermore, we

expected that disease management could cause a considerable time burden for the

patient and that it could lead to additional OOPC. To objectify the impact of disease

management we used online questionnaires to investigate HRQoL and to assess OOPC

and time burden concerning disease management.

Summary

Health related quality of life

We hypothesized that HRQoL in patients with PKU is impaired. Patients frequently

express psychosocial issues resulting from disease and dietary management, and this

possibly affects treatment adherence, social relationships, and job performance [14,17].

Therefore, we evaluated HRQoL of patients with PKU (chapter 3). First, we aimed to

obtain knowledge of the patients’ HRQoL during the period that only dietary treatment

was available. Second, we intended to gain insight into the effects of the newly

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introduced treatment with tetrahydrobiopterin (BH4) on HRQoL. We used online

questionnaires aimed at measuring generic HRQoL and questionnaires assessing

HRQoL in patients with chronic disease. We asked both patients and parents

(depending on the age of the patient) to fill out the questionnaires at two time-points.

The first time patients and parents answered our questionnaire was well before BH4

was introduced in the Netherlands as a treatment option in PKU. The second time was

at least one year after BH4 responsive patients had started treatment with the new

medication. In this manner we were able to evaluate baseline HRQoL of our patients,

and we were also able to compare differences between the first and second

measurements in patients who had started using BH4. In contrast to our hypothesis,

outcomes of our study demonstrated that patients with PKU overall have a HRQoL

comparable to or better than the general population (baseline measurement). Results

also did not show any changes in HRQoL scores measured before and after the start of

BH4 treatment within the group of BH4 responsive patients, nor did we find any

differences between the treated BH4 responsive and unresponsive patients at the

measurement well after BH4 implementation. Comparable outcomes on HRQoL have

been published [18,19] and it is yet unclear whether found results truly reflect the

HRQoL of patients, or if this results from the use of generic questionnaires which are

not disease specific and therefore do not detect the possible negative consequences

experienced by our patients. Ideally a PKU specific HRQoL questionnaire would have

been used, but the introduction of BH4 in 2009 necessitated the timing of our study

because one of our aims was to measure HRQoL before and after implementation of

BH4. Furthermore, the fact that we did not find differences in HRQoL at the first and

second measurement may be explained by the fact that the reported HRQoL of patients

at baseline was already excellent, leaving no room for further improvement (‘ceiling

effect’) when patients were able to relax their diet as a result of BH4 use. Based on this

study, it is not possible to conclude that the use of BH4 improves the HRQoL of

patients.


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Time burden and costs

We hypothesized that the disease management of PKU would impose a considerable

time burden on patients and families with PKU (chapter 4). In our study we showed

that the median time burden associated with managing PKU was 1 h and 24 min/day for

caregivers and 30 min/day for adult patients. Time was mostly spent on cooking and

preparing meals specifically for a Phe-restricted diet, followed by monitoring protein

intake. The significantly higher time burden for caregivers versus adult patients suggests

that less time is required for PKU management as patients enter adulthood and begin

caring for themselves. This fits with the idea that many adult patients tend to somewhat

relax their diet. The outcome of time burden placed on caregivers of pediatric patients

with PKU is considerable, especially because we measured disease specific time burden

which is therefor cumulative to time spent on daily household tasks. As such, the time

spent on managing PKU could take away time from other daily activities.

The median OOPC per patient was around € 604 annually. As amino acid supplements

are reimbursed by the health insurances in the Netherlands, this amount was mainly

spent on low-protein food products. It needs to be discussed if these costs are a true

burden, because an average Dutch adult on a normal diet has been demonstrated to

spend a mean amount of € 1200 annually on meat, cheese, milk, yoghurt and bread

[20], products not or little consumed by the patient with PKU. For patients with PKU

this expenditure is replaced by the costs of the low protein products. Taking this into

account, it is unlikely that there will be a large burden of extra OOPC for families of

patients with PKU. However, it must be stressed that to guarantee proper dietary

treatment of patients with PKU and to avoid a disproportionate financial burden for

patients and families, it is essential that costs of the Phe free protein supplements

remain reimbursed by the Dutch health insurances. Costs of these supplements are of

such a scale (over €30.000 per year per patient [21]) that not reimbursing them could

be harmful for patient care outcomes and treatment.

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Implications

HRQoL is an important outcome of treatment and disease management using patient

(and/or parent) reported outcomes [22]. Decreased HRQoL in both parents and

patients with IEM have been reported [23-25] and it has been an area of great interest

for researchers in the PKU field [14,18-20,26]. It seems that available questionnaires are

too generic to pinpoint quality of life related problems in patients with PKU. In order to

assess HRQoL adequately it is of importance that the used questionnaire is evaluative

and discriminative for the researched group of patients [22]. Our results did not show a

change in HRQoL in patients when dietary restrictions were relaxed in BH4 responsive

patients, but since our study others have been able to show an improvement using a self-

designed PKU specific HRQoL questionnaire before and after the introduction of BH4

[27]. This specific questionnaire however is yet not validated and as such not widely

applicable. For this reason it is important to design an internationally validated HRQoL

questionnaire specifically for patients with PKU to further study the impact of PKU and

of treatment on HRQoL. Very recently the validation process of an internationally

developed PKU specific HRQoL questionnaire for all ages was published[28]. This type

of questionnaire may be used in patient follow up as a tool to monitor problems and

changes in HRQoL on a frequent basis, to intercept psychosocial problems early and

intervene when needed, as well as to evaluate effects of new treatment options [29,30].

Our study on time burden and OOPC in patients with PKU shows that patients do not

have a high burden of costs as a result of their disease management. This is partly

because patients do not use food sources rich in protein. However, the most important

reason is that patients in the Netherlands are reimbursed by the healthcare insurances

for the expensive amino acid substitutes that they depend on for their dietary intake

[21]. In contrast, we showed that time burden is considerable especially for caregivers of

pediatric patients and imposes a significant burden on patients and families. It is of

importance for clinicians to realize this impact of imposed dietary treatment on patients

in order to improve the physician-patient relationship and to perhaps better understand

problems in adherence.


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Bone health

Many studies have postulated that bone mineral density (BMD) is affected in patients

with PKU. Hypotheses have been stated that the cause of such an impairment in BMD

may either be caused by high or fluctuating blood phenylalanine (Phe) values [31], or by

nutritional deficiencies as a result of the natural protein restricted diet of patients [32].

In order to assess the BMD of patients and the clinical implications of a possibly

impaired BMD, we systematically reviewed the literature on this subject and performed

a meta-analysis on pooled patient data from available studies. Furthermore, to objectify

the bone health of our Dutch early and continuously treated patients, we conducted a

clinical multi-center study in which we researched BMD measured with dual-energy X-

ray absorptiometry scans (DXA) and we looked at bone turnover markers (BTM) in

blood.

Summary

Meta-analysis

The systematic review and meta-analysis (chapter 5) that we conducted showed that

BMD Z-scores in early and continuously treated patients with PKU were lower in some

patients when compared to the general population, but within the normal range in most

patients. We found that the overall effect sizes of BMD Z-scores calculated from pooled

data of 247 patients, retracted from 11 studies, were: total body BMD −0.45 (95% CI

−0.61, −0.28); lumbar spine BMD −0.70 (95% CI −0.82, −0.57); femoral BMD −0.96

(95% CI −1.42, −0.49). These outcomes of Z-scores for BMD are categorized as normal

by recommendations of the Society for Clinical Densitometry (ISCD), stating that BMD

is low if the Z-score is below -2. Based on the assumptions that our data are normally

distributed and the overall effect sizes for BMD Z-score are as stated, approximately

10% of early treated patients with PKU may have a lumbar spine BMD Z-score below -2.

These patients with low BMD may benefit from care aimed at preventing osteoporosis.

However, 90% of early treated patients with PKU seem not to be at risk for low BMD,

which is a much better outcome than expected from earlier literature. It was not

possible to assess fracture risk of patients with PKU, or effect of BTM, dietary outcomes

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and Phe blood values based on the systematic review because evidence from the

included studies was limited and heterogeneous.

Clinical study

The results on bone health from the clinical multi-center study (chapter 6), which we

performed in collaboration with three metabolic centers in the Netherlands, are in

accordance with findings from our review. We showed that BMD in our population is

overall normal, although Z-scores below -2 were found in 4.9% of our patients for

lumbar BMD and 7.4% for femoral BMD. None of our individual patients had

osteoporosis as defined by the ISCD and the lifetime fracture prevalence of patients with

PKU seemed comparable to the age-standardized lifetime fracture prevalence of the

general population in England.

Both the measured bone formation and resorption marker, the first more than the latter,

were elevated. With these abnormalities in BTM, we hypothesize that bone turnover in

our population may be affected. Even though most patients have a BMD within the

normal range, this could possibly lead to adverse outcomes after the age of fifty years.

Implications

Over the last few decades an impaired BMD has been often suggested to occur in

patients with PKU. For this reason we performed a systematic review to determine the

extent and significance of low BMD in early treated patients with PKU. To objectify

BMD results in patients with PKU as reported in literature we used ISCD

recommendations which state that the diagnosis of osteoporosis (and thus an increased

fracture risk) can be made when the patient has a BMD Z-score below -2 and a

significant fracture history (two or more long bone fractures by age ten years and/or

three or more long bone fractures at any age up to nineteen years, or at least one

vertebral compression fractures in the absence of trauma) [33]. Our meta-analysis

showed that BMD Z-scores were within the normal range in most patients. However,


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based on the assumption that the pooled data was normally distributed, 10% of early

and continuously treated patients with PKU have a BMD Z-score below -2. Fracture risk

could unfortunately not be assessed because data was not available about fracture

history from the included studies. We found consistent results on BMD Z-scores in our

clinical multi-center study, which showed that around 7% of Dutch patients with PKU

had a Z-score below -2. This is a slightly higher prevalence of low BMD than the 2.3%

found in the general population. Furthermore, in our study we saw that BTM were

elevated indicating that there is an imbalance in bone formation and resorption. The

combination of these findings may have effect on bone health when patients become

older. To objectify these possible effects, further research is indicated. The oldest patient

in our study was 39 years old as a result of newborn screening only being implemented

in the Netherlands in 1974. Female patients turning fifty years old will be a particular

important group to be studied in the future. Especially because women of that age in the

general population already have a higher prevalence of osteoporosis related fractures,

and early and continuously treated women with PKU might have an even greater risk

based on our findings.

Nutrient status

We evaluated dietary intake and deficiencies of micronutrients, amino acids and fatty

acids (FA) in one of the largest studied cohorts of patients with PKU (chapter 6),

because the natural protein restricted diet has been reported to lead to nutrient

deficiencies [34,35].

Summary

Micronutrients

Our results showed that the dietary intakes of micronutrients of patients with PKU were

overall normal. However, the intake of vitamin D was inadequate in 20% of patients and

serum 25-OH vitamin D2+3 levels were below reference range in 14% of patients.

Despite near normal bone health outcomes (BMD, fracture risk and BTM), it seems

advisable to yearly evaluate intake and determine blood levels of vitamin D, and to

supplement patients when serum levels are <50 nmol/L. Furthermore, in our cohort

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dietary intake of selenium was also below the advised range in 41% of patients and

serum levels were below reference range in 46% of patients. Clinical symptoms linked to

selenium deficiency such as cardiomyopathy and depression have been reported in the

general population. Because the dietary intake of selenium in many of our patients is

below the advised range, it seems advisable to annually evaluate intake and blood levels

and consider supplementation of selenium. Especially if levels are also below the

advised reference ranges.

Additionally, zinc serum levels were below reference range in 14% of patients, despite an

intake above the safe advised range in 52% of patients. Zinc deficiency has been

reported to cause several symptoms, one of which is impaired wound healing. The

clinical relevance of our findings is debatable and further studies need to be done on

how to effectively increase zinc uptake in PKU patients. Especially considering the fact

that a large proportion of our patients already have intakes exceeding the safe advised

range.

In contrast, folic acid was found to be high both in the dietary intake as in the serum

levels of patients. As there is discussion on the safety of high levels [36,37] it deserves

due consideration to lower folic acid amounts in amino acid mixtures.

Because serum levels below reference range of 25-OH vitamin D2+D3, zinc and

selenium might also have clinical implications, it may be advisable to annually check-up

on intake and blood levels of these micronutrients.

Amino acids

Protein intake in our patients was well above the minimally required daily intake.

Investigating plasma amino acids we found that arginine, amongst others, was below

the reference range. Further research is indicated to determine whether it is warranted

to increase supplementation of arginine as it plays a main role in nitric oxide formation

and in removing ammonia from the body. Other amino acids that were below the

reference range were asparagine, 2-aminobutyric acid and tyrosine. Clinical

implications of these findings is however unclear.


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Essential fatty acids

Dietary fat intakes and erythrocyte bound essential FA were overall normal in our

patients. We did, however, find that the level of eicosapentaenoic acid (EPA, C20:5ω3)

in erythrocytes was lowered. Because EPA is a precursor of prostaglandins and has a

positive effect on cardiovascular disease it may be considered to increase EPA

supplements in amino acid mixtures.

Implications

The dietary treatment of PKU limits the intake of Phe by restricting the amount of

protein ingested from natural food sources. To reach recommended intakes of total

protein, a large part of the diet consists of (vitamin, mineral and sometimes FA fortified)

amino acid mixtures, not containing Phe. In some amino acid mixtures calculation of

the amount of micronutrients added is based on the required amount of calories, while

in others the amount added is based on advised intakes of protein per kilogram

bodyweight [38]. This leads to very different intakes of these nutrients per patient.

Furthermore, some mixtures are fortified with FA and others are not. Such a diet might

easily lead to altered intakes of micronutrients and FA when compared to the general

population and deficiencies have indeed been reported [34,35,38]. Remarkably, intake

and plasma levels of most micronutrients and FA were normal in the studied patients,

however, some abnormalities were detected. To prevent clinically relevant deficiencies

we advise that patients are annually checked for dietary intake and deficiencies of 25-

OH vitamin D2+3 and selenium. Lowering folic acid amounts in amino acid mixtures

should be considered as both dietary intake and serum levels of the micronutrient are

high. Further research on altered outcomes of zinc, several amino acids and EPA is

indicated. We found abnormal levels of these nutrients, but little is known about the

clinical implications of our findings.

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20. Cazzorla C, Cegolon L, Burlina AP, Celato A, Massa P, Giordano L et al.: Quality of Life (QoL) assessment in a cohort of patients with phenylketonuria. BMC Public Health 2014, 14: 1243.

21. Belanger-Quintana A, Dokoupil K, Gokmen-Ozel H, Lammardo AM, MacDonald A, Motzfeldt K et al.: Diet in phenylketonuria: a snapshot of special dietary costs and reimbursement systems in 10 international centers. Mol Genet Metab 2012, 105: 390-394.

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26. Vegni E, Fiori L, Riva E, Giovannini M, Moja EA: How individuals with phenylketonuria experience their illness: an age-related qualitative study. Child Care Health Dev 2010, 36: 539-548.

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al.: Development and psychometric validation of measures to assess the impact of phenylketonuria and its dietary treatment on patients' and parents' quality of life: the phenylketonuria - quality of life (PKU-QOL) questionnaires. Orphanet J Rare Dis 2015, 10: 59.

29. Haverman L, van Rossum MA, van Veenendaal M, van den Berg JM, Dolman KM, Swart J et al.: Effectiveness of a web-based application to monitor health-related quality of life. Pediatrics 2013, 131: e533-e543.

30. Engelen V, Detmar S, Koopman H, Maurice-Stam H, Caron H, Hoogerbrugge P et al.: Reporting health-related quality of life scores to physicians during routine follow-up visits of pediatric oncology patients: is it effective? Pediatr Blood Cancer 2012, 58: 766-774.

31. Barat P, Barthe N, Redonnet-Vernhet I, Parrot F: The impact of the control of serum phenylalanine levels on osteopenia in patients with phenylketonuria. Eur J Pediatr 2002, 161: 687-688.

32. de Groot MJ, Hoeksma M, van Rijn M, Slart RH, van Spronsen FJ: Relationships between lumbar bone mineral density and biochemical parameters in phenylketonuria patients. Mol Genet Metab 2012, 105: 566-570.

33. Schousboe JT, Shepherd JA, Bilezikian JP, Baim S: Executive summary of the 2013 International Society for Clinical Densitometry Position Development Conference on bone densitometry. J Clin Densitom 2013, 16: 455-466.

34. Robert M, Rocha JC, van RM, Ahring K, Belanger-Quintana A, MacDonald A et al.: Micronutrient status in phenylketonuria. Mol Genet Metab 2013, 110 Suppl: S6-17.

35. Lohner S, Fekete K, Decsi T: Lower n-3 long-chain polyunsaturated fatty acid values in patients with phenylketonuria: a systematic review and meta-analysis. Nutr Res 2013, 33: 513-520.

36. Choi JH, Yates Z, Veysey M, Heo YR, Lucock M: Contemporary issues surrounding folic Acid fortification initiatives. Prev Nutr Food Sci 2014, 19: 247-260.

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37. Stolen LH, Lilje R, Jorgensen JV, Bliksrud YT, Almaas R: High dietary folic Acid and high plasma folate in children and adults with phenylketonuria. JIMD Rep 2014, 13: 83-90.

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Future Perspectives

Chapter 8

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Future Perspectives

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FUTURE PERSPECTIVES

Optimizing care in phenylketonuria (PKU) remains an important goal. Since the

introduction of newborn screening and early and subsequent continuous dietary

treatment, much research has been performed on the physical and social wellbeing of

patients with PKU. This thesis has focussed on a number of these aspects and this

chapter provides an overview of future perspectives based on our results.

First, an uniform approach is warranted when treating patients with PKU. Even though

dietary treatment is the mainstay in the care for PKU, there are still differences to

overcome in how this care is implemented. To overcome such differences (inter)national

consensus procedures, resulting in clinical pathways and/or guidelines are probably the

best tools. A Dutch national consensus has been reached on how the care for PKU is best

provided. However, to guarantee up-to-date information about the care trajectories

stated in the clinical pathway for PKU it is necessary for clinicians to remain in dialogue

about the content, and the clinical pathway needs to be regularly updated. Furthermore,

a need for international consensus also exists and efforts to achieve an uniform

European PKU guideline have been made.

Second, the burden of living with PKU and disease management needs further

investigation. Health related quality of life (HRQoL) is hypothesized to be impaired. The

available questionnaires that we used in our research (both the generic HRQoL

questionnaires as the questionnaires aimed at the chronically ill) are not disease

specific. Unfortunately, for this reason we were not able to pinpoint if and which

domains of HRQoL are affected in patients with PKU. The development of a PKU

specific and internationally validated questionnaire is of great importance, because an

adequate HRQoL questionnaire is evaluative and discriminative specifically for the

researched group of patients, in this case in PKU. A disease specific questionnaire may

also be useful in detecting changes in quality of life when new treatment options are

introduced.

Chapter 8

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Third, the burden of time placed on caregivers of pediatric patients related to disease

management is significant and it takes away time from leisure. Future research on this

matter is of use in optimizing care for the patients with PKU as it may provide insight in

effects of new treatment options and it may be used as a new outcome measure in future

research. From a clinical approach it is of importance to be aware of the effect of the

imposed dietary treatment on the lives of patients with PKU. Awareness may improve

physician-patient relationship and perhaps increase understanding of dietary adherence

problems.

Fourth, a systematic review and meta-analysis showed that bone health in patients with

PKU is less impaired than was previously hypothesized, although low bone mineral

density (BMD; Z-score below -2) was slightly more prevalent than in the general

population. We found the same results for BMD Z-scores in our own Dutch PKU

population. Bone turnover markers (BTM) were found to be elevated, both formation

and resorption markers, suggesting that the bone turnover in our population is affected.

The fracture history of our patients showed that their lifetime fracture prevalence was

comparable to the general population and none of the patients had osteoporosis. These

results suggest that bone health in patients with PKU is uncomplicated. However, the

patients investigated were relatively young and it is possible that the combination of

finding a higher prevalence of low BMD and a change in BTM may lead to adverse

outcomes of bone health when patients become older. Further research is of importance

to investigate the cause of the slightly higher prevalence of low BMD and altered BTM in

blood, and to relate outcomes to the dietary treatment that patients are subjected to.

Female patients over the age of fifty years will be a particular important group to be

studied in the future as women over 50 years of age already have a higher prevalence of

osteoporosis related fractures, and women with PKU might have an even greater risk

based on our findings.

Fifth, improvements of the present dietary treatment options, need to be developed in

order to try to reduce the burden of dietary restriction on the patient and decrease risks

of nutrient deficiencies. Examples of complications of dietary treatment are alterations

in micronutrient (vitamin D, selenium, zinc, arginine) and fatty acid (eicosapentaenoic

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acid) intakes and blood levels. The effects imposed on the patient by chronic restriction

in natural protein should be carefully examined in larger cohort studies, as should the

possible complications of nutrient deficiencies.

Finally, not studied in this thesis, but of great importance, to improve care for patients

with PKU, further advances in research on new pharmacological treatment options are

warranted. PEG-PAL, gene therapy and hepatocyte transplantation are all potential

therapies that may achieve more dietary freedom, or even help patients to go off diet

completely, by increasing Phe tolerance.

Dutch Summary

Chapter 9

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Dutch Summary

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NEDERLANDSE SAMENVATTING

Phenylketonurie (PKU, ORPHA79254, MIM 261600) is een erfelijke

stofwisselingsziekte. De ziekte ontstaat doordat patiënten een enzym genaamd

fenylalanine hydroxylase (PAH; EC 1.14.16.1) onvoldoende aanmaken. Dit enzym zet in

gezonde mensen het aminozuur (een stukje van een eiwit) fenylalanine om in een ander

aminozuur genaamd tyrosine. Doordat fenylalanine niet wordt afgebroken in het

lichaam van patiënten met PKU, stapelt het op in het lichaam en in het brein. Dit leidt

onder andere tot een ernstige verstandelijke beperking. Fenylalanine krijgt men binnen

via eiwitten in het dieet. Sinds 1974 worden patiënten in Nederland via de hielprik (dag

4 of 5 na de geboorte) opgespoord en wordt er gestart met therapie. Deze therapie is het

meest effectief wanneer vlak na de geboorte wordt gestart en langdurig (in principe

levenslang, afhankelijk van de mate van ziek zijn) wordt voortgezet. Patiënten die in

Nederland zijn opgespoord met de hielprik, zijn dus allen ‘vroeg en continu’ behandelde

patiënten. De behandeling bestaat uit een strikte beperking van fenylalanine in het dieet

door de inname van natuurlijke eiwitten te beperken. Deze beperking kan zo streng zijn

dat sommige patiënten slechts 10 gram eiwit of minder per dag mogen eten. Eiwitten

worden gevonden in melkproducten, vlees, vis, brood en pasta. De meeste aminozuren,

behalve fenylalanine, worden dan ook aangevuld door middel van speciaal

geproduceerde dieetvoeding oftewel aminozuurpreparaten. Dit levenslange dieet is zeer

effectief gebleken in het voorkómen van de verstandelijke beperking en andere

symptomen van PKU. In 2009 is er een nieuw medicijn op de markt gekomen genaamd

tetrahydrobiopterine (BH4). Dit is een co-factor van het enzym PAH en het helpt als

zodanig in de omzetting van fenylalanine naar tyrosine in het lichaam. Men heeft

ondervonden dat ongeveer een derde van de patiënten met PKU responsief (gevoelig) is

voor dit medicijn. Patiënten die responsief zijn en dus BH4 gebruiken, kunnen dagelijks

meer fenylalanine (en dus natuurlijk-eiwit rijke producten) eten dan wanneer zij niet

BH4 gevoelig zijn. Bij een klein aantal patiënten is het zelfs mogelijk om het strikte dieet

in zijn geheel te stoppen. De behandeling van PKU is zeer effectief, desondanks lijken er

echter ook nadelige effecten voor patiënten mee gemoeid te gaan. De klinische

relevantie van deze nadelige effecten en de mate waarin zij de patiënt beïnvloeden zijn

echter niet geheel duidelijk. Met als doel de zorg voor patiënten met PKU te

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optimaliseren, hebben wij te verwachten nadelige effecten onderzocht om deze te

objectiveren en de klinische relevantie ervan vast te stellen. De gebieden die wij hebben

onderzocht zijn: het opstellen van nationale zorgpaden, de gezondheidsgerelateerde

kwaliteit van leven van patiënten met en zonder BH4-gebruik, de persoonsgebonden

privéuitgaven en de tijdslast gerelateerd aan de omgang met de ziekte, de botstatus en

mogelijke verstoringen in inname en bloedwaarden van voedingsstoffen ten gevolge van

het dieet. Middels dit onderzoek hebben wij beoogd om inzicht te krijgen in de meest

optimale psychosociale en nutritionele zorg voor patiënten met PKU, en daarnaast om

grond te leggen voor toekomstig onderzoek naar deze ziekte (hoofdstuk 1). Hieronder

wordt per hoofdstuk een samenvatting gegeven van de uitkomsten van de besproken

onderzoeken in dit proefschrift.

Consensus ten aanzien van optimale zorg

Om de best mogelijke zorg te kunnen bieden aan patiënten met PKU is het van belang

om nationale overeenstemming te bereiken tussen de verschillende betrokken partijen

(zorgverleners en patiënten) over de meest optimale behandeling (hoofdstuk 2). Er

bestonden in Nederland verschillen tussen de verscheidene metabole centra in de zorg

die werd aangeboden aan patiënten met stofwisselingsziekten. Om eenduidige zorg te

kunnen leveren waar nationale consensus over bestaat, heeft de vereniging voor

Volwassenen en Kinderen met Stofwisselingsziekten (VKS) de ontwikkeling van

nationale klinische zorgpaden voor 20 verschillende erfelijke stofwisselingsziekten

(waaronder PKU) geïnitialiseerd in samenwerking met kinderartsen, internisten en

diëtisten gespecialiseerd in metabole ziekten. Klinische zorgpaden zijn ideaal om een

dergelijke consensus te bereiken omdat zij een middel bieden om multidisciplinaire

besluitvorming en organisatie van zorgprocessen te bewerkstelligen in een specifieke

groep patiënten. Het is aangetoond dat het gebruik van klinische zorgpaden

interdisciplinaire communicatie verbetert, kennis en bewustzijn ten aanzien van de

ziekte bij de patiënt verhoogt en leidt tot een betere coördinatie van zorg. Mits frequent

bijgewerkt, kunnen de zorgpaden ook gebruikt worden als een referentie naar de meest

recente vooruitgang in zorg en richting bieden voor verder onderzoek. Wij zijn van

Dutch Summary

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mening dat multidisciplinaire samenwerking door middel van het gebruik van klinische

zorgpaden communicatie zal verbeteren en een duidelijke route naar de best mogelijke

zorg zal verschaffen, gebaseerd op nationale consensus. Ten behoeve van iedere erfelijke

stofwisselingsziekte waarover nationale consensus is bereikt, zijn er klinische zorgpaden

beschikbaar op de website van de VKS (één versie voor zorgverleners en één versie voor

patiënten; http://www.stofwisselingsziekten.nl/stof wis selingsziekten/zorgpaden/).

Gezondheid Gerelateerde Kwaliteit van Leven

Patiënten met PKU suggereren regelmatig psychosociale problemen te ondervinden als

gevolg van de invloed van hun ziekte en de dieetbehandeling op het gebied van

therapietrouw, sociale relaties, en prestaties op het werk. Om deze reden

hypothetiseerden wij dat de gezondheidsgerelateerde kwaliteit van leven van patiënten

met PKU verminderd is (hoofdstuk 3). Door middel van het gebruik van online

vragenlijsten onderzochten wij de gezondheidsgerelateerde kwaliteit van leven van

kinderen en volwassenen met PKU. Het doel van ons onderzoek was, ten eerste, om

kennis te vergaren over de gezondheidsgerelateerde kwaliteit van leven van patiënten

met PKU toen er enkel nog therapeutische behandeling bestond door middel van een

dieet. Ons tweede doel was om inzicht te verkrijgen in het effect van de nieuwe

farmacologische therapie met tetrahydrobiopterin (BH4) op de kwaliteit van leven. Om

onze onderzoeksvragen te beantwoorden, hebben wij patiënten en/of hun moeders

(afhankelijk van de leeftijd van de patiënt) gevraagd om online vragenlijsten in te vullen

aangaande de gezondheidsgerelateerde kwaliteit van leven. Wij hebben hierbij gebruik

gemaakt van twee typen vragenlijsten; één generieke vragenlijst om de uitkomsten te

vergelijken met de kwaliteit van leven van de algemene bevolking, en een kwaliteit van

leven vragenlijst voor chronisch zieken om de uitkomsten van patiënten met PKU te

vergelijken met de antwoorden van patiënten met andere chronische ziekten. Patiënten

en/of moeders werden gevraagd om de vragenlijsten in te vullen op twee tijdpunten. De

eerste meting vond plaats voordat patiënten in 2009 werden getest op gevoeligheid voor

behandeling met BH4 en de tweede meting vond plaats ruim een jaar hierna. De tweede

meting vond een jaar later plaats om de invloed van emotionele reacties met betrekking

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tot de BH4 gevoeligheidstest op onze resultaten te voorkómen. Op deze manier waren

wij in staat om de gezondheidsgerelateerde kwaliteit van leven van patiënten te meten in

een relatief grote groep patiënten met PKU en om het verschil te onderzoeken tussen de

twee metingen in patiënten die waren gestart met BH4-behandeling.

In tegenstelling tot onze hypothese tonen de resultaten van onze studie aan dat

patiënten met PKU over het algemeen een normale gezondheidsgerelateerde kwaliteit

van leven hebben wanneer wij deze vergelijken met de algemene Nederlandse bevolking

(eerste meting). De resultaten toonden geen veranderingen in kwaliteit van leven

gemeten voor en na de introductie van de behandeling met BH4 in de groep van

patiënten die gevoelig waren voor BH4. Ook vonden wij geen verschillen tussen

patiënten die wel en niet gevoelig zijn voor BH4 ten aanzien van de resultaten verkregen

tijdens de tweede meting (na de implementatie van BH4-behandeling).

Vergelijkbare resultaten ten aanzien van de gezondheidsgerelateerde kwaliteit van leven

werden ook door andere onderzoekers gepubliceerd [18,19] en het is nog onduidelijk of

de gevonden resultaten werkelijk de kwaliteit van leven van de patiënten weergeven, of

dat deze voortvloeien uit het gebruik van generieke vragenlijsten die niet PKU specifiek

zijn en die dus niet de mogelijke negatieve gevolgen opsporen die worden ervaren door

onze patiënten. Idealiter zou een PKU specifieke gezondheid gerelateerde kwaliteit van

leven vragenlijst zijn gebruikt, maar de introductie in 2009 van BH4 maakte de timing

van onze studie noodzakelijk omdat het één van onze doelen was om de kwaliteit van

leven te meten vóór en na implementatie van BH4. Bovendien kan het feit dat er geen

verschil in kwaliteit van leven tussen de eerste en tweede meting gevonden werd,

verklaard worden doordat de gerapporteerde kwaliteit van leven van de patiënten bij

aanvang reeds uitstekend was en er geen ruimte voor verdere verbetering

("plafondeffect") mogelijk was wanneer patiënten hun dieet konden verslappen ten

gevolg van BH4-gebruik. Op basis van dit onderzoek, is het niet mogelijk te concluderen

dat het gebruik van BH4 de kwaliteit van leven van patiënten verbetert.

Dutch Summary

~ 201 ~

De Tijdslast van het Leven met PKU en Ziektegebonden Privékosten

Onze hypothese waarop dit onderzoek gefundeerd was, was dat de omgang met de ziekte

PKU een geruime tijdslast, en mogelijk extra kosten, oplevert voor patiënten met PKU

en hun families (hoofdstuk 4). Om deze reden hebben wij volwassen patiënten en

(ouders/)verzorgers van pediatrische patiënten gevraagd om online vragenlijsten in te

vullen die de ziekte-specifieke tijdslast en persoonlijke kosten evalueerde.

Tijdslast

Door middel van de online vragenlijsten hebben wij kunnen aantonen dat de mediane

tijdslast verbonden aan de omgang met PKU 1 uur en 24 min/dag betrof voor verzorgers

en 30 min/dag voor volwassen patiënten. Tijd werd vooral besteed aan het koken en het

bereiden van maaltijden voor een fenylalanine-arm dieet en in tweede instantie door het

monitoren van eiwitinname. De significant hogere tijdslast voor verzorgenden van

pediatrische patiënten versus volwassen patiënten suggereert dat er minder tijd nodig is

voor de omgang met PKU wanneer patiënten volwassen worden en hun zorg op zichzelf

nemen. Dit sluit aan bij het idee dat veel volwassen patiënten vaak minder strikt

omgaan met hun dieet. De tijdslast die rust op verzorgenden van pediatrische patiënten

met PKU is aanzienlijk, en omdat we de specifieke tijdslast gerelateerd aan de omgang

met de ziekte hebben gemeten is dit een tijdsbelasting bovenop de dagelijkse

huishoudtaken. Als zodanig, neemt de tijd besteed aan het omgaan van de ziekte PKU

tijd weg van andere dagelijkse activiteiten.

Persoonlijke ziektegebonden uitgaven

De mediane persoonlijke ziektegebonden kosten per patiënt bleken €604 per jaar.

Aminozuursupplementen worden vergoed door de ziektekostenverzekering in

Nederland en om deze reden werd het gevonden bedrag voornamelijk besteed door

patiënten met PKU aan het kopen van eiwitarme voedingsproducten. Het is

noodzakelijk om te bediscussiëren of dit bedrag een ware last is voor onze patiënten met

Chapter 9

~ 202 ~

PKU. Een gemiddelde Nederlandse volwassene met een normaal dieet besteedt een

gemiddelde hoeveelheid van € 1.200 per jaar aan vlees, kaas, melk, yoghurt en brood.

Dit zijn producten die niet of weinig worden verbruikt door de patiënt met PKU. Voor

patiënten met PKU worden deze uitgaven vervangen door de kosten van de eiwitarme

producten die zij gebruiken, vaak als vervanging van brood, pasta of kaas. Op basis van

de genoemde uitgaven in de gemiddelde bevolking is het onwaarschijnlijk dat er een

grote extra last qua uitgaven bestaat voor (verzorgenden van) patiënten met PKU. Het

moet echter wel worden benadrukt dat, om een goede dieetbehandeling van patiënten

met PKU te garanderen en een onevenredige financiële last voor patiënten en families te

voorkomen, het van essentieel belang is dat de kosten van de fenylalanine-vrije

eiwitsupplementen worden blijven vergoed door de Nederlandse zorgverzekeraars.

Kosten van deze supplementen zijn van een dergelijke mate (meer dan € 30,000 per

jaar per patiënt) dat het niet vergoeden schadelijk voor de behandeling zou kunnen zijn.

Botstatus

Vele studies hebben gepostuleerd dat de botmineraaldichtheid (BMD) verminderd is bij

patiënten met PKU. Er bestaan een tweetal hypotheses die de oorzaak van een dergelijke

stoornis in BMD proberen te verklaren; ten eerste zou een lage BMD kunnen worden

veroorzaakt door hoge of fluctuerende bloedspiegels van fenylalaninewaarden, en ten

tweede zouden tekorten van voedingsstoffen ten gevolge van het natuurlijk-eiwitarme

dieet van patiënten een mogelijke oorzaak kunnen zijn. Om de werkelijke BMD van

patiënten met PKU en de klinische gevolgen van een eventueel verlaagde BMD te

evalueren hebben we systematisch de literatuur over dit onderwerp onderzocht en een

meta-analyse verricht op basis van patiëntgegevens uit beschikbare studies. Daarnaast

hebben we zelf een klinisch onderzoek verricht onder patiënten uit drie klinische centra

in Nederland met als doel de botstatus te onderzoeken van vroeg en continu behandelde

patiënten. In onze klinische multicenter studie hebben we de BMD beoordeeld op basis

van dual-energy X-ray absorptiometrie scan (DXA) gegevens en we hebben gekeken

naar botomzettingsmarkers in het bloed.

Dutch Summary

~ 203 ~

Meta-analyse

Het systematische literatuuronderzoek en de meta-analyse hebben aangetoond dat

BMD Z-scores lager zijn in sommige patiënten die vroeg en continu behandeld zijn,

maar voor de meeste patiënten zijn de Z-scores normaal (hoofdstuk 5). Z-scores zijn

waarden die je kunt gebruiken om data te vergelijken van patiënten van verschillende

leeftijden en geslacht. In de meta-analyse hebben we de BMD Z-scores van 247

patiënten, uit 11 verschillende studies, kunnen samenvoegen om het verschil met de

algemene bevolking te berekenen. De resultaten waren als volgt: voor het totale lichaam

-0,45 (95% BI -0,61, -0,28); voor de lumbale wervelkolom -0,70 (95% BI -0,82, -0,57)

en voor het femur (dijbeen) -0,96 (95% BI -1,42, -0,49). Deze uitkomsten van de Z-

scores voor BMD zijn te interpreteren als niet afwijkend, zoals aanbevolen door de

Vereniging voor Klinische Densitometrie (ISCD). De ISCD stelt dat de BMD laag is als

de Z-score kleiner is dan -2. Op basis van de veronderstelling dat onze data normaal

verdeeld zijn en dat de uitkomsten van de BMD Z-scores van de samengevoegde

patiënten data zijn zoals gemeld, zal ongeveer 10% van de vroeg behandelde patiënten

met PKU een lumbale wervelkolom BMD Z-score onder -2 hebben. Deze patiënten met

een lage BMD zouden gebaat zijn bij zorg gericht op de preventie van botontkalking.

Echter, 90% van de vroeg en continu behandelde patiënten met PKU lijken geen lage

BMD te hebben, wat een veel beter resultaat blijkt dan wat is beschreven in eerdere

literatuur. Het was niet mogelijk om op basis van het systematische review het risico op

botbreuken bij patiënten met PKU en het effect van botomzettingsmarkers,

voedingskundige uitkomstmaten en fenylalanine bloedwaarden op botten van patiënten

te beoordelen, omdat het bewijs van de gebruikte studies beperkt en zeer verscheiden

was.

Klinische studie

De resultaten van onze klinische multicenter studie aangaande botstatus zijn in

overeenstemming met de bevindingen van ons systematische review (hoofdstuk 6).

We toonden aan dat de BMD Z-score in onze patiënten over het algemeen normaal is.

Desondanks werden Z-scores lager dan -2 gevonden in 4,9% van onze patiënten

Chapter 9

~ 204 ~

betreffende de lumbale wervelkolom BMD en 7,4% voor de BMD van het femur

(dijbeen). Geen van onze individuele patiënten had osteoporose (botontkalking).

Osteoporose wordt gedefinieerd door de ISCD als een BMD Z-score lager dan -2 in

combinatie met een significante fractuurgeschiedenis; twee breuken van de lange

beenderen voor het tiende levensjaar of drie breuken van de lange beenderen voor het

negentiende levensjaar, dan wel een spontane breuk van een wervellichaam. De kans op

een botbreuk gedurende het leven van patiënten met PKU bleek gelijk aan die van de

algemene bevolking in Engeland.

Zowel de gemeten botvormingsmarker als de botresorptiemarker in het bloed van

patiënten met PKU bleek verhoogd. Op basis van deze afwijkingen in de

botomzettingsmarkers veronderstellen wij dat het botmetabolisme in onze patiënten

afwijkend kan zijn. Alhoewel de meeste patiënten een BMD hebben binnen het normale,

kunnen de gevonden afwijkingen mogelijk wel leiden tot negatieve resultaten na de

leeftijd van vijftig jaar. Op dit moment is onze oudste patiënt in de studie 39 jaar oud,

toekomstige studies zullen van belang zijn om de BMD te evalueren in vroeg en continu

behandelde patiënten met PKU van 50 jaar of ouder.

Nutriënten status

We evalueerden inname en bloedwaarden van micronutriënten, aminozuren en

vetzuren in één van de grootst onderzochte cohorten van patiënten met PKU

(hoofdstuk 6), omdat in eerdere literatuur wordt gemeld dat het dieet van patiënten

kan leiden tot een tekort van voedingsstoffen aan inname via het dieet en tot een tekort

in het lichaam (bloed).

Micronutriënten

Onze resultaten toonden aan dat de inname van micronutriënten van patiënten met

PKU over het algemeen normaal was. De inname van vitamine D bleek beneden de

aanbevolen hoeveelheid in 20% van de patiënten en de vitamine D waarde onder de

referentiewaarden in het bloed van 14% van de patiënten. Ondanks de relatief normale

uitkomsten ten aanzien van de botstatus (BMD, risico op botbreuken en botmarkers)

lijkt het raadzaam om de inname en de bloedspiegels van vitamine D jaarlijks te

Dutch Summary

~ 205 ~

evalueren. Bij lage inname of bloedwaarden (<50 nmol/L in serum) kan men de inname

van vitamine D aanvullen door middel van tabletten.

Daarnaast bleek in ons cohort de inname van selenium ook lager dan de aanbevolen

hoeveelheid in 41% van de patiënten en in 46% van de patiënten waren de bloedspiegels

onder de referentiewaarden. Klinische symptomen ten gevolge van een seleniumtekort,

zoals cardiomyopathie en depressie, zijn eerder beschreven in literatuur over de

algemene bevolking. Omdat de inname van selenium in veel van onze patiënten laag is,

lijkt het raadzaam om jaarlijks de inname en bloedspiegels te evalueren en tekorten aan

te vullen door middel van tabletten.

Verder bleken de bloedwaarden van zink onder de referentiewaarden bij 14% van de

patiënten, ondanks een orale inname boven de veilige aanbevolen hoeveelheid bij 52%

van de patiënten. Over een tekort aan zink is vermeld in eerdere literatuur dat

verscheidene symptomen kunnen optreden, waaronder een vertraagde wondgenezing.

De klinische relevantie van onze bevindingen ten aanzien van zink zijn discutabel omdat

er geen grote studies bekend zijn over de gevolgen van een zinktekort. Verdere studies

moeten worden verricht om te onderzoeken of zinktekorten daadwerkelijk klinisch

relevant zijn bij patiënten met PKU en om te bekijken hoe men effectief de zinkopname

uit het dieet kan verhogen. Zeker gezien het feit dat een groot deel van onze patiënten

reeds een inname van meer dan de aanbevolen veilige hoeveelheid heeft.

In contrast, werd er een te hoog foliumzuur van zowel de voedselinname als de

bloedspiegels van patiënten gevonden. Aangezien er discussie bestaat over de veiligheid

van een hoog foliumzuurgehalte in het lichaam, is het nuttig om te bedenken of de

hoeveelheid van foliumzuur in de aminozuurmengsels niet verminderd dient te worden.

Aminozuren

De totale eiwitinname in onze patiënten was ruim boven de minimaal vereiste dagelijkse

hoeveelheid. Het onderzoek naar aminozuren in bloed (plasma) toonde aan dat, onder

andere, het aminozuur arginine beneden de referentiewaarde lag. Verder onderzoek is

nodig om te bepalen of het gerechtvaardigd is om de inname van arginine aan te vullen.

Arginine speelt namelijk een belangrijke rol in stikstofoxidevorming en het verwijderen

van ammoniak uit het lichaam. Andere aminozuren die beneden de referentiewaarden

Chapter 9

~ 206 ~

waren, waren asparagine, 2-aminoboterzuur en tyrosine. De klinische implicaties van

deze bevindingen zijn echter onduidelijk.

Essentiële vetzuren

Bij onze patiënten met PKU bleken de vetinname via de voeding en de meeste essentiële

vetzuren in rode bloedcellen over het algemeen normaal. Het vetzuur genaamd

eicosapentaeenzuur (EPA, C20: 5ω3) bleek echter beneden de referentiewaarden.

Omdat EPA een voorloper is van prostaglandinen en een positief effect heeft op hart- en

vaatziekten kan eventueel overwogen worden om EPA in de aminozuurmengsels te

verhogen. Een sterke aanbeveling kan echter niet worden gemaakt op basis van deze

bevindingen omdat andere bestaande studies in relatief kleine groepen patiënten zijn

verricht. Om gerichtere aanbevelingen te kunnen doen aangaande voedingsstoffen in

patiënten met PKU, dienen grotere studies te worden verricht.

Dutch Summary

~ 207 ~

~ 208 ~

Addendum

~ 210 ~

List of Publications

List of Publications

~ 211 ~

LIST OF PUBLICATIONS

1. Demirdas S, Coakley KE, Bisschop PH, Hollak CE, Bosch AM, Singh RH:

Bone health in phenylketonuria: a systematic review and meta-

analysis. Orphanet J Rare Dis 2015, 10: 17.

2. Demirdas S, Maurice-Stam H, Boelen CC, Hofstede FC, Janssen MC, Langendonk

JG, Mulder MF, Rubio-Gozalbo ME, van Spronsen FJ, de Vries M, Grootenhuis

MA, Bosch AM: Evaluation of quality of life in PKU before and after

introducing tetrahydrobiopterin (BH4); a prospective multi-center

cohort study. Mol Genet Metab 2013, 110 Suppl: S49-S56.

3. Demirdas S, Eijgelshoven I, Smith TA, van Loon JM, Latour S, Bosch AM:

The time consuming nature of phenylketonuria: a cross-sectional

study investigating time burden and costs of phenylketonuria in the

Netherlands. Mol Genet Metab 2013, 109: 237-242.

4. Demirdas S, van Kessel IN, Korndewal MJ, Hollak CE, Meutgeert H, Klaren A,

van Rijn M, van Spronsen FJ, Bosch AM; Dutch working Group: Clinical

pathways for inborn errors of metabolism: warranted and feasible.


5. Demirdas S, Schroder CH: An infant with orange-colored urine. Pediatr

Nephrol 2010, 25: 381.

Addendum

~ 212 ~

Co-Authors and Affiliations


~ 213 ~

CO-AUTHORS AND AFFILIATIONS

Co-authors are listed in alphabetical order

Bisschop, P.H.

Department of Internal Medicine, Division of Endocrinology and Metabolism,

Academic Medical Center, University of Amsterdam, Amsterdam, The

Netherlands

Boelen, C.C.

Department of Pediatrics, Division of Metabolic Diseases, Leiden University

Medical Center, Leiden, The Netherlands.

Bosch, A.M.

Department of Paediatrics, Emma Children's Hospital, Academic Medical Center,

University of Amsterdam, Amsterdam, The Netherlands

Coakley, K.E.

Nutrition and Health Sciences and Molecules to Mankind Programs, Laney

Graduate School, and Department of Human Genetics, Emory University Atlanta

GA United States

Eijgelshoven, I.

MAPI Consultancy, Houten, The Netherlands

Grootenhuis, M.A.

Psychosocial Department, Emma Children's Hospital, Academic Medical Center,


Addendum

~ 214 ~

Hofstede, F.C.

Department of Metabolic Diseases, Wilhelmina Children's Hospital Utrecht,

University Medical Center, Utrecht, The Netherlands

Hollak, C.E.

Department of Internal Medicine, Division of Endocrinology and Metabolism,

Academic Medical Center, University of Amsterdam, Amsterdam, The

Netherlands

ter Horst, N. M.

Department of Dietetics, Academic Medical Center, (AMC), University of

Amsterdam, Amsterdam, The Netherlands.

Janssen, M.C.

Department of Internal Medicine, Nijmegen Center for Mitochondrial Disorders,

Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands

van Kessel, I.N.

Department of Pediatrics, Emma Children's Hospital, Academic Medical Center,


Klaren, A.

The Dutch Society for Adults and Children with an Inborn Error of Metabolism

(VKS), Zwolle

Korndewal, M.J.

Department of Pediatrics, Emma Children's Hospital, Academic Medical Center,



~ 215 ~

Langendonk, J.G.

Center for Lysosomal and Metabolic diseases, Department of Internal Medicine,

Erasmus MC, Rotterdam, The Netherlands

Latour, S.

Merck Serono S.A., Genève, Switzerland

van der Lee, J.H.

Clinical Research Unit, Woman-Child Center, Academic Medical Center,


van Loon, J.M.

MAPI Consultancy, Houten, The Netherlands

Maurice-Stam, H.

Psychosocial Department, Emma Children's Hospital, Academic Medical Center,


Meutgeert, H.

The Dutch Society for Adults and Children with an Inborn Error of Metabolism

(VKS), Zwolle

Mulder, M.F.

Department of Pediatrics, Free University Medical Center (VUMC), Amsterdam,

The Netherlands

van Rijn, M.

Division of Metabolic Diseases, Beatrix Children’s Hospital, University Medical

Center Groningen, University of Groningen, Groningen, The Netherlands

Addendum

~ 216 ~

Rubio-Gozalbo, M.E.

Department of Pediatrics and Laboratory Genetic Metabolic Diseases, Maastricht

University Medical Center, Maastricht, The Netherlands

Singh, R.H.

Metabolic Nutrition and Genetics Program Department of Human Genetics,

Emory University Atlanta GA United States

Smith, T.A.

MAPI Consultancy, London, UK

van Spronsen, F.J.

Division of Metabolic Diseases, Beatrix Children's Hospital, University Medical

Center, Groningen, University of Groningen, Groningen, The Netherlands

Vaz, F.M.

Laboratory Genetic Metabolic Disease, Academic Medical Center, Amsterdam,

The Netherland.

de Vries, M.

Institute for Genetic and Metabolic Disease, Department of Pediatrics, Radboud

University Nijmegen Medical Center, Nijmegen, The Netherlands

Wijburg, F.A.

Department of Paediatrics, Emma Children's Hospital, Academic Medical Center,



~ 217 ~

Addendum

~ 218 ~

List of Abbreviations

List of Abbreviations

~ 219 ~

LIST OF ABBREVIATIONS AA : Amino acids AAM : Amino acid mixtures ALA : α-linolenic acid (C18:3ω3) AMC : Academic Medical Center, Amsterdam AND : The Academy of Nutrition and Dietetics AND EA Process : The Academy of Nutrition and Dietetics Evidence Analysis Process BH4 : tetrahydrobiopterin (BH4) BMC : Bone mineral content BMD : Bone mineral density BTM : Bone turnover markers CDC : Centers for Disease Control and Prevention CTx : Carboxy-terminal collagen crosslinks DHA : Docosahexaenoic acid (c22:6ω3) DISABKIDS : The DISABKIDS chronic generic module DXA : Dual-energy X-ray absorptiometry EFA : The European Food Safety Authority EPA : Eicosapentaenoic acid (C20:5ω3) FA : Fatty acids FBMD : Femoral bone mineral density HRQoL : Health Related Quality of Life IEMs : Inborn errors of metabolism IQR : Interquartile range ISCD : International Society for Clinical Densitometry LA : Linoleic acid (C18:2ω6) LBM : Lean body mass LBMD : Lumbar bone mineral density NHANES : National Health and Nutrition Examination Survey OOPC : out-of-pocket cost PedsQL : Pediatric Quality of Life Inventory Measurement Model PAH : phenylalanine hydroxylase Phe : Phenylalanine PINP : Procollagen type I N-terminal propeptide PKU : Phenylketonuria pQUS : Peripheral quantitative ultrasound PRISMA : Preferred Reporting Items for Systematic Reviews and Meta-Analyses PROSPERO : International prospective register of systematic reviews PTH : Parathyroid hormone QCC : Quality criteria checklist SAR : Safe advised range SD : Standard deviation SF-HLQ : The Short Form Health & Labour Questionnaire SIGN : Scottish Intercollegiate Guidelines Network TAAQOL : TNO-AZL Adult Quality of Life TBMD : Total bone mineral density VKS : Dutch Society for Children and Adults with an Inborn Error of

Metabolism WHO : World Health Organization

Addendum

~ 220 ~

Portfolio

Portfolio

~ 221 ~

PhD PORTFOLIO

PhD period: June 2012 to May 2015

Courses

An introduction to Good Clinical Practice; BROK; Practical Biostatistics; Reference Manager; End Note; Systematic Reviews; Oral Presentation; Presenting is an art; Trans-Cultural medicine; Ethics in research; Research in pediatrics; Applying for a new job; How to publish; Finance in research; Online electronic case report form training

Oral presentations at (inter)national Conferences

year

Amsterdam Kindersymposium (pediatric research symposium), Amsterdam, The Netherlands

Quality of life in PKU

2013

5th European Phenylketonuria Group (EPG) Symposium on “Advances and challenges in PKU”, Istanbul, Turkey

Quality of life in PKU Burden of living with PKU

2013

European society for phenylketonuria and allied disorders treated as phenylketonuria (E.S. PKU) conference, Antwerp, Belgium

Burden of living with PKU

2013

Vereniging tot bevordering onderzoek Erfelijke Stofwisselingsziekten in het Nederlandse taalgebied (ESNLT), Fall symposium, Driebergen, The Nerherlands


2013



2014

European society for phenylketonuria and allied disorders treated as phenylketonuria (E.S. PKU) conference, Zagreb, Croatia

Systematic review on bone health

2014

Nederlandse Vereniging voor Kindergeneeskunde (NVK ) (Dutch Society for Pediatrics) conference, Veldhoven, The Netherlands


2014


Systematic review on bone health

2015

Addendum

~ 222 ~

Presentations

year

Sphynx meeting, AMC, division of metabolic diseases

Introducing the MOR 004 phase III clinical trial

2012

VKS vereniging, praatje over morbus Morquio

Explaining Morquio’s disease

2012

Morquio patient’s society

Morquio and ‘The MOR 004’ phase III clinical trial

2013

Sphynx meeting, AMC, division of metabolic diseases

Literature meeting

2013

Metabolic meeting, AMC, division of metabolic diseases

The dentist’s office: MPS in a nutshell

2014


Morquio: MPS IV-A and IV-B

2014

Sharing MOR 004 study results

4 occasions at the AMC: metabolic meeting, Sphynx meeting, meeting at the lung function department, meeting of nurses at the adolescent pediatric clinic department

Metabolic diseases patient’s society (VKS)

2014


Implementing enzyme therapy in the Netherlands

2015

Attended (inter)national Conferences

year

MPS IV investigator meeting, San Francisco, Florida, The USA

Conference concerning ‘The MOR 004’ phase III clinical trial

2012

WORLD symposium, San Diego, Florida, The USA

Conference concerning lysosomal storage diseases

2012

MPS conference, Noordwijk, The Netherlands

Conference concerning mucopolysaccharidosis

2012

MPS master class, Istanbul, Turkey Master class with a wide variety of topics related to MPS

2015

Portfolio

~ 223 ~

Meetings

year

Weekly multi-disciplinary metabolic meeting

2012-2015

Weekly clinical-Laboratory glycosaminoglycans meeting

until Jan 2014

Weekly sphynx lysosomal meeting

until Nov 2012

Vimizim indication commission

2015

Teaching

The MOR 004’ phase III clinical trial, teaching nurses (total of five times)

2011-2014

Workshop diabetes, yearly 3 classes

2012-2014

Physical examination of the child, yearly 3 classes

2012-2015

Guiding a bachelor student

How to write a systematic review

2014

Boardmembership

Jonge Onderzoekers Kindergeneeskunde (JOK) Boardmember (Abactis)

2012-2014

Addendum

Acknowledgements

Acknowledgements

~ 225 ~

ACKNOWLEDGEMENTS

Foremost, I would like to sincerely thank doctor Annet Bosch for the chance to work

with her and learn from her in the years we worked together. Both during my clinical

work in the AMC and during my PhD. You are a role model in many ways.

Secondly, my genuine appreciation and gratitude to prof. dr. Frits Wijburg who gave me

the opportunity to work in the field of orphan diseases and who allowed me to conduct

an international multi-center clinical trial under his professional guidance.

Thirdly, I would like to acknowledge all patients who participated in my studies, without

you this thesis would not have been possible. Additionally, many thanks to the

professionals involved in cooperating on several of these studies for all the hours spent

on patient inclusion, statistics and co-authoring scientific articles resulting from our

collective research.

Fourthly, a great thank you goes out to all my close friends and to my kind plus gezellig

colleagues on the ‘Rode Luifel’ and ‘A3’ for all their support and allowing me to ventilate

my thoughts and feelings when needed.

Furthermore, I would like to show gratitude to my husband for all his support,

providing the layout of this thesis and being available for medical and genetic

consultation. Ez te pir hezdikim evînêmin. Ez ji we jî pir hezdikim dayê, bavo û xuşka

minî delal, therefore I thank you from the bottom of my hearth for always believing in

me and pushing me to go beyond my limits.

Finally, my dearest para nymphs, Gülşen and Diren, I am much obliged to you both for

accepting to stand by me during my defense and helping me with all the much needed

preparations.

Addendum

~ 226 ~

About the Author

About the Author

~ 227 ~

ABOUT THE AUTHOR

Serwet Demirdas was born on October 29th 1981. As the daughter of Kurdish refugees

she came to the Netherlands at the age of three. She grew up in Leiden where she

graduated from the ‘Stedelijk Gymnasium Leiden’ in the year 2000. Afterwards she

started her medical training at the VU university medical center in Amsterdam,

graduating in 2008. She performed extra-curricular work as a board member of two

student’s associations consecutively, and as a member of the Organization Committee of

the first national medical intern’s conference. As part of her medical training she spent

three months at the Eagan University in Izmir (Turkey) to complete an elective

internship at the departments of pediatric hematology, pediatric endocrinology and

clinical genetics. After finishing her medical training she worked consecutively at the

following pediatric clinics from January 2009 to June 2011: the Gelre hospitals in

Apeldoorn, the Juliana Children’s Hospital in The Hague and the Academic Medical

Center in Amsterdam. Subsequently she proceeded as a sub-investigator and

coordinator on an international multi-center placebo controlled phase III trial

concerning intravenous enzyme therapy in patients with mucopolysaccharidosis type

IV-A or Morquio’s disease (June 2011 – May 2015). During this time she also provided

medical care as a clinician for this group of patients. The study was successfully closed

in 2014 and resulted in the first pharmacological treatment for this patient group. From

June 2012 until May 2015, under the professional guidance of dr. A.M. Bosch and prof.

dr. F.A. Wijburg, she worked as a PhD student in the field of Phenylketonuria resulting

in several scientific publications and this thesis. As of May 2015 she is working as a

resident at the Human Genetics department of the Radboud University Medical Center

to further evolve her career.

uva-dare (digital academic repository) phenylketonuria ...inherited disorder of metabolism that...

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