Pharmacogenetics Use For Further Treatment Improvement in childreN
Phase: NA
Conditions: Asthma in Children
Sponsor: Academisch Medisch Centrum - Universiteit van Amsterdam (AMC-UvA)

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RESEARCH PROTOCOL 
PUFFIN trial  
(September 2019, version 8) 
 
 

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PROTOCOL TITLE ‘Pharmacogenetics Use For Further treatment Improvement in childreN’ 
 
Protocol ID 
 
Short title 
PUFFIN trial  
EudraCT number  
2017-004424-29 
Version 
8 
Date 
26-09-2019 
Coordinating 
investigator/project leader 
 
Prof. dr. A.H. Maitland – van der Zee 
Academisch Medisch Centrum (AMC) 
Dept. of Respiratory Disease, F5-259 
Postbus 22660 
1100 DD Amsterdam 
Tel. +31 (20) 5668137 
Principal investigator(s) (in 
Dutch: hoofdonderzoeker/ 
uitvoerder) 
<Multicenter research: per site> 
 
1. UMCG, Beatrix Kinderziekenhuis 
G.H. Koppelman 
2. Erasmus MC - Sophia Kinderziekenhuis 
M.W. Pijnenburg 
3. Academisch Medisch Centrum (AMC), Emma 
ziekenhuis 
A.H. Maitland – van der Zee  
N.W.F. Rutjes 
4. Tergooi Ziekenhuis 
B. van Ewijk  
5. Rijnstate ziekenhuis 
J. Wesseling 
6. Hagaziekenhuis 
M. Nuijsink 
7. Catharinaziekenhuis 
H.J. Brackel 
8. Medisch Spectrum Twente 
B. Thio 
9. Canisius Wilhelmina Ziekenhuis 
M.L. Brouwer 
10. Maasstad ziekenhuis 

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M. Verwaal 
11. Medisch Centrum Leeuwarden 
T.W. de Vries 
12. Zuyderland Medisch Centrum 
H. Hendriks 
13. Amphia ziekenhuis 
A.A.P.H. Vaessen-Verberne 
14. RadboudUMC 
P.J.F.M. Merkus 
15. Martini ziekenhuis 
A.W.A. Kamps 
16. OLVG 
Q.W. van Dellen 
17. Spaarne Gasthuis 
M.E. Mérelle 
18. Reinier de Graaf ziekenhuis  
L. van Veen 
      19. VUmc locatie Boelelaan 
            E.G. Haarman 
      20. St. Franciscus Gasthuis  
            I. de Kleer 
     21. Dijklander ziekenhuis 
           G. ten Tusscher 
       
 
 
   
 
Sponsor (in Dutch: 
verrichter/opdrachtgever) 
AMC 
 
 
Subsidising party 
Longfonds 

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Independent expert (s) 
Drs. D.H.F. Geurts 
Dept. Of  Paediatrics 
Erasmus MC – Sophia Kinderziekenhuis 
Wytemaweg  80 
3000 CB Rotterdam 
Tel. +31107034454 
d.geurts@erasmusmc.nl 
 
 
 
 
Laboratory sites <if applicable> 
Prof. dr. R.H.N. van Schaik 
 
Erasmus MC 
Afd. Klinische Chemie  
's-Gravendijkwal 230 
3015 CE Rotterdam  
Tel: +31 (0)10-70 33119 
 
 
 
 

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PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Sponsor or legal 
representative: 
Prof. dr. A. Vonk 
Noordegraaf 
Head Respiratory Medicine, 
AMC 
 
 
[Coordinating Investigator]: 
Prof. dr. A.H. Maitland – van 
der Zee 
 
 
26-09-2019 
 
 

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TABLE OF CONTENTS 
 
1. 
INTRODUCTION AND RATIONALE .............................................................................11 
2. 
OBJECTIVES ................................................................................................................16 
3. 
STUDY DESIGN ...........................................................................................................17 
4. 
STUDY POPULATION ..................................................................................................20 
4.1 
Population (base) ...................................................................................................20 
4.2 
Inclusion criteria .....................................................................................................20 
4.3 
Exclusion criteria ....................................................................................................21 
4.4 
Sample size calculation ..........................................................................................21 
5. TREATMENT OF SUBJECTS ..........................................................................................22 
INVESTIGATIONAL PRODUCT ...........................................................................................22 
5.1 
Name and description of investigational  product(s) ...............................................22 
5.2 
Summary of findings from non-clinical studies ........................................................24 
5.3 
Summary of findings from clinical studies ...............................................................25 
5.4 
Summary of known and potential risks and benefits ...............................................26 
5.5 
Description and justification of route of administration and dosage .........................26 
5.6 
Dosages, dosage modifications and method of administration ...............................26 
5.7 
Preparation and labelling of Investigational Medicinal Product ...............................26 
5.8 
Drug accountability .................................................................................................27 
6. 
METHODS ....................................................................................................................28 
6.1 
Study parameters/endpoints ...................................................................................28 
6.1.1 
Main study parameter/endpoint .......................................................................28 
6.1.2 
Secondary study parameters/endpoints (if applicable) ....................................28 
6.2 
Randomisation, blinding and treatment allocation ..................................................28 
6.1 
Study procedures ...................................................................................................29 
6.2 
Withdrawal of individual subjects ............................................................................33 
6.2.1 
Specific criteria for withdrawal (if applicable) ...................................................33 
6.3 
Replacement of individual subjects after withdrawal ...............................................33 
6.4 
Follow-up of subjects withdrawn from treatment .....................................................33 
6.5 
Premature termination of the study .........................................................................33 
7. 
SAFETY REPORTING ..................................................................................................34 
7.1 
Temporary halt for reasons of subject safety ..........................................................34 
7.2 
AEs, SAEs and SUSARs ........................................................................................34 
7.2.1 
Adverse events (AEs) ......................................................................................34 
7.2.2 
Serious adverse events (SAEs) .......................................................................34 
7.2.3 
Suspected unexpected serious adverse reactions (SUSARs) .........................35 
7.3 
Annual safety report ...............................................................................................36 
7.4 
Follow-up of adverse events ...................................................................................37 
7.5 
Data Safety Monitoring Board (DSMB) ...................................................................37 
8. 
STATISTICAL ANALYSIS .............................................................................................38 
8.1 
Primary study parameter(s) ....................................................................................38 
8.2 
Secondary study parameter(s) ...............................................................................39 

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8.3 
Other study parameters ..........................................................................................41 
8.4 
Regulation statement .............................................................................................42 
8.5 
Recruitment and consent........................................................................................42 
8.6 
Objection by minors or incapacitated subjects (if applicable) ..................................42 
8.7 
Benefits and risks assessment, group relatedness .................................................45 
8.8 
Compensation for injury .........................................................................................45 
8.9 
Incentives (if applicable) .........................................................................................45 
9.1 Handling and storage of data and documents .............................................................46 
9.2 Monitoring and Quality Assurance ..............................................................................46 
9.3 Amendments ...............................................................................................................46 
9.4 Annual progress report ................................................................................................46 
9.5 Temporary halt and (prematurely) end of study report ................................................47 
9.6 Public disclosure and publication policy ......................................................................47 
10. STRUCTURED RISK ANALYSIS ...................................................................................48 
10.1 Potential issues of concern .......................................................................................48 
10.2 Synthesis ..................................................................................................................48 
11. REFERENCES ...............................................................................................................48 
 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
 
 
 
 
ABR 
ABR form, General Assessment and Registration form, is the 
application form that is required for submission to the 
accredited Ethics Committee (In Dutch, ABR = Algemene 
Beoordeling en Registratie) 
ADRB2 
gene encoding beta-2-adrenergic receptor 
AE 
Adverse Event 
AR 
Adverse Reaction 
(c-)ACT 
(childhood-) Asthma Control Test 
CCMO 
Central Committee on Research Involving Human Subjects; 
in Dutch: Centrale Commissie Mensgebonden Onderzoek 
CV 
Curriculum Vitae 
DSMB 
Data Safety Monitoring Board 
FDA 
Food and Drug Administration 
FeNO 
Fraction exhaled Nitric Oxide 
FEV1 
Forced Expiratory: Volume per second 
GCP 
Good Clinical Practice 
IC 
Informed Consent 
ICS 
 
Inhaled corticosteroids 
ICU 
Intensive Care Unit 
IgE 
Immunoglobulin E 
LABA 
Long acting beta2 agonists 
LTRA 
Leukotriene Receptor Antagonists 
NVK 
Nederlandse Vereniging voor Kindergeneeskunde (Dutch 
Society for Paediatrics) 
MARS 
Medication Adherence Reporting Scale 
METC  
Medical research ethics committee (MREC); in Dutch: 
medisch ethische toetsing commissie (METC) 
 

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OCS 
Oral corticosteroids 
PAQLQ 
Paediatric Asthma-related Quality of Life Questionnaire 
PACQLQ Paediatric Asthma-related Caregiver Quality of Life 
Questionnaire 
PedSQL 
Paediatric Quality of Life InventoryTM 
PCQ 
QALY 
Productivity Cost Questionnaire 
Quality Adjusted Life Year 
QoL 
Quality of Life 
RAST 
Radioallergosorbent-test (blood test to measure allergy) 
SABA 
Short acting beta2 agonists 
(S)AE 
(Serious) Adverse Event  
Sponsor 
The sponsor is the party that commissions the organisation 
or performance of the research, for example a 
pharmaceutical 
company, academic hospital, scientific organisation or 
investigator. A party that provides funding for a study but 
does not commission it is not regarded as the sponsor, but 
referred to as a subsidising party. 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
Wbp 
Personal Data Protection Act (in Dutch: Wet Bescherming 
Persoonsgevens) 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: 
Wet Medisch-wetenschappelijk Onderzoek met Mensen 
 
 
 

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SUMMARY 
 
Rationale: Asthma is the most common chronic disease in children. There is a large 
variability in treatment response to asthma medication and a one-size fits all approach might 
not be optimal for all paediatric patients. In children who are not well controlled on inhaled 
corticosteroids (ICS), guidelines suggest to double the dose of ICS or add a long acting beta-
agonist (LABA). In children with asthma variation in the gene encoding the beta-2 adrenergic 
receptor (ADRB2), has been associated with poor response to long-acting beta-2 agonists 
(LABA). Children with asthma carrying a risk variant might therefore benefit more from 
doubling inhaled corticosteroids (ICS) than from adding LABAs.  
Objective: To assess whether ADRB2 genotype-guided asthma treatment in children with 
persistent asthma symptoms despite ICS treatment leads to better asthma control compared 
to non-genotype-guided asthma treatment. 
Study design: National, multi-centre randomized controlled double blind trial  
Study population: 310 children (6-17 years of age) with a doctor’s diagnosis of asthma, who 
require a step-up in asthma treatment because of uncontrolled asthma symptoms despite 
adherent and adequate use of low dose ICS. 
Intervention (if applicable): Participants will be randomized to 1) a genotype-guided 
treatment arm or 2) a usual care (non-genotype guided) control arm. In the genotype-guided 
arm, children will be treated based on their genotype of ADRB2 (single nucleotide 
polymorphism rs1042713). Children homozygous for the risk variant (Arg16Arg) and 
heterozygotes (Arg16Gly) will be treated with doubling dosages of their ICS. Children 
homozygous for the wild type allele (Gly16Gly) will receive LABA as add-on to low dose ICS. 
In the control arm, children will be randomized between doubling ICS dosage or adding 
LABA, the two most common chosen options among respiratory paediatricians in the 
Netherlands when children are uncontrolled despite low dosages of ICS.  
Main study parameter: Improvement of asthma control based on repeated measurement 
analysis of (childhood)-Asthma Control Test scores after 3 months.  
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: The burden of the children and parents is moderate. The trial duration is 
6 months, with 3 visits in the hospital/at home and 4 additional online measurements (online 
diary). Biosamples will be taken (feces, saliva, nasal swabs) with limited burden to the 
children. There are no additional risks for participation in this study as only registered 
medication will be used. In our opinion a delay of 7-10 days before stepping up treatment in 
children with uncontrolled or poorly controlled symptoms is acceptable. In the future, the 
results of this study could lead to improved treatment for paediatric patients.  
 
 

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1. INTRODUCTION AND RATIONALE 
 
The burden of asthma 
Asthma is a chronic respiratory disease that affects approximately 300 million people 
worldwide [1]. It is characterized by recurrent respiratory symptoms and variable airflow 
limitation due to bronchial obstruction. Asthma is associated with a substantial health impact 
for the patient and large healthcare expenditures for society. In 2005, the total costs of 
childhood asthma for the 25 countries of the European Union were estimated at three billion 
euros, with the Netherlands contributing approximately 106 million euros and the UK 359 
million euros [2]. These costs include medical and non-medical direct costs (e.g. GP visits, 
hospitalization and medication, diagnostics) as well as indirect costs (e.g. school days loss, 
caregiver productivity loss). One of the main drivers of direct medical costs is urgent asthma 
care [3]. Severe attacks of asthma symptoms (exacerbations) may lead to hospitalizations and 
admissions to the Intensive Care Unit. In the Netherlands, approximately 11% of the asthma 
patients are hospitalized each year [4]. Almost half of the costs associated with asthma 
management arise from hospital admission and unscheduled health care visits [5]. Despite 
progress in understanding this chronic disease, the burden of asthma due to hospitalizations 
and unscheduled visits is not decreasing.  
 
Stepwise asthma treatment 
Childhood asthma is treated in a stepwise approach. The first step of asthma treatment 
consists of short acting beta-2 agonists (SABA) as needed to relieve asthma symptoms. These 
drugs act as bronchodilators. Inhaled corticosteroids (ICS) are added to the treatment regime 
if asthma symptoms persist despite SABA use (step 2). ICS suppress the airway inflammation 
and are considered to be the cornerstone of maintenance asthma treatment. If a child’s asthma 
remains uncontrolled, ICS dosage may be increased, or long-acting beta-2 agonists (LABA) or 
leukotriene receptor agonists (LTRA) can be added (step 3) [1]. However, the addition of a 
LTRA is not a very common chosen treatment option among respiratory paediatricians in the 
Netherlands.  
 
The influence of genetic variation on asthma treatment response 
There is large variability between patients in the level of symptom control or lung function 
improvement upon asthma maintenance treatment. In patients with poorly controlled asthma, 
improving adherence and inhaler technique are the first steps to improve asthma. However, 
even in clinical trials in which adherence to treatment is closely monitored, subgroups of 
patients remain symptomatic despite maintenance treatment [5]. Already in 2000, Drazen et 
al. suggested that up to 80% of the interindividual variance in lung function response upon 

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treatment in asthmatic patients could be due to genetic variations [6]. Since then, several 
candidate gene studies and a handful of genome-wide association studies (GWAS) have 
described genetic variants associated with response to asthma treatment.  
One of these variants in a gene encoding for the beta-2 adrenergic receptor (ADRB2), has 
been positively associated poor response to long-acting beta-2 agonists [7-9]. This variant 
(rs1042713) is known as Arg16Gly since the 16th amino acid of the receptor is changed from 
glycine into arginine and the homozygous Arg16 variant is present in approximately 1 in 6 
children [7]. A recent meta-analysis in the Pharmacogenomics in Childhood Asthma 
Consortium (PiCA) of 5 populations with 4,226 children and young adults of white Northern 
European and Latino origin showed that this variant was associated with an increased risk of 
asthma exacerbations when treated with LABA as add on treatment [7]. Per copy of the risk 
allele patients exposed to LABA had an increased risk of 52% for a severe asthma 
exacerbation. There was no increased risk for severe asthma exacerbations if patients were 
not exposed to LABA. An important observation from this meta-analysis is that the adverse 
effects of LABA were also observed in heterozygote carriers of the Arg16 variant, thereby 
providing evidence that over 60 percent of the population (17% homozygotes and 50% 
heterozygotes) is potentially at risk. This observational study indicated that a large group of 
paediatric asthma patients might not benefit from LABA and even suffer from more 
exacerbations, which may be preventable if they would have been treated differently. Besides 
that this leads to the research question whether genotyping is effective in children that are not 
under control on step 2 of asthma treatment, there is another question that remains 
unanswered. It is unclear how to treat the patients with the heterozygous variant. This is why 
it is very important to include heterozygous patients in our study.  
 
Other clinical trials 
One previous trial has been performed to address the effects of LABA in relation to ADRB2 
genotype, but only with children homozygous for the risk variant [10]. Sixty-two asthmatic 
children with the Arg16Arg genotype were randomized to treatment with ICS plus LABA or ICS 
plus LTRA and followed for 1 year. The trial showed that children treated with LTRA had fewer 
school absences, used less rescue medication, had less symptoms and a better quality of life 
compared to the group treated with LABA, with no effect on lung function scores between both 
study arms. The difference between both treatment groups could already be observed after 3 
months (figure 1).  
 

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Why should children be studied? 
It is important to study children with asthma instead of adults since ample data indicate that 
this genetic risk effect is mainly observed in the paediatric population. The adverse effects of 
LABA might be more prominent in children than in adults, which was clearly shown in a meta-
analysis on the risk of LABA of 110 controlled clinical trials with 60.954 patients performed by 
the Food and Drug Administration (FDA) [11]. It could be that adults with asthma are less 
vulnerable to the negative effects of LABA due to the influence of other modifying factors such 
as increased airway wall rigidity (caused by airway remodeling over time), long-term 
inflammation or a different affinity of the beta-2 adrenergic receptors to their agonists [12]. 
However, a subsequent safety trial mandated by the FDA found no significant difference 
between the risk of serious asthma events in children receiving a combination of LABA and 
ICS compared to children who only received an ICS [13]. 
Therefore, it is important to study the effect of this genetic variant on the treatment outcome 
in children. This study will include children with asthma to test whether ADRB2-genotype 
guided treatment will lead to better and faster asthma control.   
 
Why should other –omics markers also be included in this study?  
There are some first results pointing in the direction that –omics markers are predictive in 
responsiveness to asthma medication, for example in microbiomics, metabolomics, 
transcriptomics and epigenomics. A genomic susceptibility alone does not seem to be 
enough to drive asthma phenotypes. The interaction with the environment might play a major 
role in driving a predisposed genetic background towards the breakage of immune tolerance 
Figure 1. Change in asthma-related school 
absences and use of salbutamol reliever 
use 
in 
62 
asthmatic 
children 
with 
Arg16Arg genotype treated with LABA or 
alternative drug. Change in asthma-related 
school absences and the use of salbutamol 
reliever over a 1-year study period with 3-
monthly visits between groups treated with 
ICS plus oral montelukast (ML) or ICS+LABA 
plus 
placebo 
montelukast 
(SM). 
The 
differences 
in 
school 
absences 
and 
bronchodilator 
usage 
can 
already 
be 
observed early in the trial (after 3 months, visit 
1) [10].  

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and the development of asthma, likely by influencing microbiotic, bacterial metabolomics 
and/or the epigenome and the transcriptome. Obtaining more information about the whole 
phenotype of the asthmatic children via nose swabs and feces sampling is one of our 
secondary outcomes.  
 
Recent data has shown that the nasal epithelium is an excellent proxy tissue for –omics’ 
studies of the lower airways [14]. In addition to the primary analyses, we therefore aim to 
perform an in-depth integrative -omics analysis of children unresponsive to therapy with 
double dosage of ICS.  
 
Supporting this concept, several studies have shown associations between epigenetic 
markers and asthma or asthma-related outcomes [15]. A new pool of biomarkers may be 
found in the gut (faeces) of asthmatic patients. It has been  shown that the microbial 
composition changes in the airways and gut of asthmatic patients compared to healthy 
individuals. Levels of short chain fatty acids (SCFA) and branched chain fatty acids (BCFA) 
are an indication of changed carbohydrate and protein fermentation, respectively, by 
commensal bacteria, such as bifidobacteria and bacteriodes [16]. SCFA and BCFA have 
been demonstrated to be important for regulation of asthma-associated inflammatory 
responses [17]. The human microbiome is acquired during life, and it is a very important 
changeable property. Several studies have identified molecules and mechanisms that 
connect diet, the gut microbiota and immune-related diseases such as asthma [18].  
 
Methylation of inflammatory genes changes over time in asthmatics starting at the early 
stages of disease development. Differences in methylation patterns between asthmatics and 
healthy individuals may be detectable as early as in cord blood [9]. In asthmatics methylation 
patterns may determine the dynamics of disease from progression to remission. On the other 
hand treatment and disease control may also affect methylation in a feedback loop. 
Identification of key epigenetic markers can lead to a change in treatment strategies from 
palliative to preventive [19]. Furthermore, recent literature suggests that patients with severe 
asthma carry methylation changes in specific genes compared to non-severe patients [20].  
 
Taken together, -omics dimensions (such as microbiomics & bacterial metabolomics, 
breathomics, transcriptomics and epigenomics) may constitute the final bridge between 
genetic-predisposition and actual disease development. Single-dimension biomarker 
approaches to phenotype asthma are increasingly regarded to be inaccurate and outdated, 
especially since different dimensions can interact (Figure 1). 
 

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Figure 1. Host (epi)genomics, immunology and microbiota likely collectively influence asthma treatment response (Huang et al, 
JACI 135:25-30, 2015). 
 
Precision Medicine (also known as Personalized Medicine) is the field that aims to optimize 
effectiveness of treatment for individual patients or patient groups by taking into account 
individual patient characteristics. Many different factors can play a role in determining the 
best treatment for a particular patient. Paediatric asthma patients differ considerably from 
adult patients in the nature of the disease, its triggers, the course of disease and therefore 
the response to therapy. In paediatric asthma the collection of airway biopsy specimens and 
invasive procedures in general are difficult. Therefore we consider it of utmost importance to 
target tissues and materials for biomonitoring that can be easily and non-invasively gathered 
in routine clinical practice, such as saliva, exhaled breath, and faeces. Rather than using 
specimens that could never be part of a clinical strategy for optimising drug therapy. 
Biomarkers that will be studied in this proposal are (epi)genetic factors (nose swabs), 
microbiome (feces) and metabolomic markers in exhaled breath.  
 
 
Assessing the costs and benefits of ADRB2-genotyping 
Alongside this RCT, a cost-utility analysis will be conducted in order to quantify the incremental 
costs and benefits of introducing a genetic-based diagnostic tool into clinical asthma practice 
from a societal perspective. A key research question that we will consider is if ADRB2-
prospective genotyping is found to be clinically effective, is whether this translates into 
economically important differences in patients’ health, their quality of life, healthcare resource 
utilization and costs. 

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2. OBJECTIVES 
 
Primary Objective:  
 To assess whether ADRB2 genotype-guided treatment leads to better asthma control 
after 3 months compared to usual care in children who are uncontrolled despite 
adherent and adequate use of ICS  
 
Secondary Objectives: 
 To assess whether ADRB2 genotype-guided treatment leads to better asthma control 
at 6 months. 
 To assess whether ADRB2 genotype-guided treatment leads to improved quality of 
life (QoL), fewer school absences, fewer exacerbations, and better lung function  
compared to usual care in children at 3 and 6 months 
 To assess whether ADRB2 genotype-guided treatment leads to fewer changes in 
asthma therapy at 3 months, compared to usual care.  
 To assess whether ADRB2 genotype-guided treatment leads to a shorter time to 
reach asthma control, compared to usual care 
 To assess the cost-utility of ADRB2-genotype guided treatment 
 To identify –omics-biomarkers for non-response to ICS treatment   
 
 

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3. STUDY DESIGN 
 
Study design: national, multi-centre, double-blind randomized controlled trial 
 
Duration: 6 months, with 3 visits in the hospitals/at home (at t=0, t=3 months and t=6 
months) 
 
Setting: Patients are recruited at out-patient asthma clinics in secondary and tertiary care 
hospitals in the Netherlands.  
 
Description: Three hundred ten children (6 to 17 years of age) with a doctor’s diagnosis of 
asthma and uncontrolled asthma symptoms despite adherent and adequate use of ICS for at 
least three months (step 2 asthma treatment) will be recruited by secondary and tertiary care 
centers in the Netherlands. All participants are eligible for step-up asthma treatment (from 
step 2 to step 3) as assessed by the treating paediatrician/paediatric pulmonologist 
Participants will be randomized to a genotype-guided treatment arm (n=155) or to a usual 
care, non-genotype guided, arm (n=155) (Figure 2) and followed for 6 months.  
 
 
Figure 2. PUFFIN study design 
 
Genotyping before start treatment 
During the baseline visit in the hospital/at home, clinical data and biological samples 
(including a DNA sample) will be collected. Upon this visit, the DNA sample will be send to 
the Clinical Chemistry department of the Erasmus MC (Head: Prof. R. van Schaik) to perform 

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genotyping of the ADRB2 gene within one week. The treating physician will adapt the 
treatment regime of the participant based on the treatment advice of the study coordinator 
(Table 1). For the children in the genotype-arm, this will be based on the genotype. The 
treating physician will not know (be blinded) whether the treatment advice was based on the 
genotype (intervention arm) or based on randomization (control arm).  The participant will be 
followed for 6 months. If the participant is still uncontrolled at t=3 months, treatment will be 
adapted according to Table 1.  All children will be genotyped, in order to assess the influence 
of the genotype on treatment outcome in the usual arm group retrospectively. The children 
should use the same inhalation device during the study to avoid confusion on how they 
should inhale their medication.  
 
Table 1. Treatment regimes 
 
 
Intervention arm: ADRB2 genotype-guided treatment arm  
In the genotype-stratified arm, children will be treated based on their ADRB2 genotype. 
Children homozygous for the risk variant Arg16 and heterozygotes (Arg16Gly) will be treated 
with doubling dosages of their ICS. Children homozygous for the wild type allele (Gly16Gly) 
will receive LABA.  
 
Control arm: Non genotype-guided treatment arm  
In the control arm, genotyping will be performed for retrospective analysis, but the genotype 
information will not be used to guide treatment. Children in this study arm will be randomized 
again between doubling ICS dosage (n=75) or LABA treatment (n=75), the two most 
commonly preferred add-on options among paediatric pulmonologists in the Netherlands. We 
choose to randomize between both treatments options, since international guidelines do not 
agree on the preferred treatment option [1,15].  
Furthermore, to test our hypothesis it is necessary to have enough children in the control 
group with Arg16Arg or Arg16Gly to be treated with LABA. The amount of children 
treated with LABA and ICS should be equal in the control group. Therefore we decided to 
randomise children in the control group over doubling ICS (n=77) and adding LABA 
(n=77). This will lead to an estimated number of children with Arg16Arg or Arg16Gly of 
 
Arg16Arg or Arg16Gly 
Gly16Gly 
Therapy: month 0- 3  Double ICS 
Double ICS 
ICS+LABA 
Therapy for month 
4-6 if still 
uncontrolled at 3 
months 
Normal dosage ICS and 
LTRA 
Normal dosage of ICS 
and LABA 
Double ICS 

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51 who will get LABA add on. In this way the power is high enough to determine the 
effectivity of both treatment options in the three genotypes. We find it important to 
define effectivity next to the question whether genotyping benefits children with asthma. 
In the control group DNA samples will be obtained for retrospective analysis. 
It is safe to randomise the children again who are randomised within our control arm, 
because treatment with a double dose of ICS and adding a LABA are both standard of 
care. A Cochrane review from 2009 has shown that both treatments have proven to be 
equally effective in both children and adults 
Randomisation in the control arm is important because it would be futile if the children in 
this arm would be treated with the same therapy by accident. Randomisation is 
necessary to make the trial as small and effective as possible. At this moment physicians 
do not have the tools to determine which therapy is the best for every child. This is why 
we think it is correct to randomise in the control arm. 
 
Based on the previous studies of Lipworth et al. [10], and Turner et al. [7], we hypothesize 
that children with one or two Arg16 alleles in ADRB2 will experience less asthma control, 
thus randomization in the control arm will ensure sufficient children with these ADRB2 
genotypes to be exposed to LABA in order to test our hypothesis. We will perform interim 
genetic analysis to verify that we include sufficient children with these risk genotypes in our 
study. Our trial is designed to reflect clinical practice as closely as possible, therefore the 
choice of the type of ICS or LABA will be done by the treating physician. 

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4. STUDY POPULATION 
4.1 Population (base)  
Children will be selected from the asthma clinic of at least 21 participating Dutchhospitals. In 
general, children (6-18 years) who are well-controlled on step 2 treatment will be treated by 
general practitioners (GPs). GP guidelines suggest to refer children with asthma not well 
controlled on step 2 treatment. Therefore, children treated in the hospitals will be at least on 
step 2 treatment. We expect that approximately 25% of these children are poorly controlled 
or uncontrolled on step 2 treatment. The hospitals that expressed their intent to participate in 
the study were confident that they were able to recruit enough patients in the study.    
 
Plan B 
The contact with other Dutch hospitals is good, and in case the current sites fail to include 
enough patients, we will extent the inclusion sites to other hospitals. The novel initiated 
Dutch consortium Paediatric Pulmonology will also play an active role in stimulating hospitals 
to participate.  
Another option is to let general practitioners play a role in our patient inclusion. 
 
 
4.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
- 
Between 6-18 years of age 
- 
Doctor’s diagnosis of asthma (ever) based on patient history, FEV1 reversibility ≥ 
12 % and/or bronchial hyperresponsiveness.  
- 
Current asthma symptoms (based on ACT (≥12 years) or C-ACT (<12 years) 
score ≤ 19 
- 
ICS use ≥ 3 months before inclusion (start dosage ICS, treatment step 2 
according to childhood asthma guideline NVK, Table 3) 
- 
Adequate inhalation technique (based on validated checklist score [21]) 
- 
Self-assessed good adherence to maintenance asthma treatment 
- 
Understanding of Dutch or German language 
- 
Internet access a home, willing to fill in internet questionnaires 
 
Table 3. ICS dosing step 2 
ICS 
Dosage (µg) 
Beclomethason 
2 dd 200 

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Beclomethason (extra fine) 
2 dd 100 
Budesonide 
2 dd 200 
Fluticason 
2 dd 100-125 
Ciclesonide 
1 dd 160 
 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from participation 
in this study: 
- 
Active smoking 
- 
Congenital heart disease 
- 
Serious lung disease other than asthma (Cystic Fibrosis,  Primary Ciliary Dyskinesia, 
congenital lung disorders, severe immune disorders) 
- 
LABA use in past 6 months  
- 
Omalizumab use 
- 
ICU admission in the previous year  
 
4.4 Sample size calculation 
 
A sample size analysis was performed for Arg16 homozygotes double dose ICS vs. LABA 
treatment (our main research question), with power 80%, α=0.05 2-sided, and based on the 
ACT scores and correlation between repeated measurements in a previous performed RCT 
in children with asthma (the BATMAN study) (SD=3.6, r=0.58) [22]. Taking into account a 
minimal clinically relevant significant difference of 3 points on the ACT scores, that 16% of 
the children in the PACMAN study were carrying Arg16Arg, and that ACT will be measured 4 
times (baseline t=0, t=1, t=2 and t=3) before the primary endpoint (at t=3 months), a 
minimum of 153 children need to be included in each study arm. When the analysis will be 
adjusted for the baseline value, this will reduce the variance of the estimate of difference 
between the group by a factor 1-r^2, and thereby increase the power. Because not all 
children might be willing to complete the trial we will include a total of 310 children. The 
power is based on the inclusion of 16% of children carrying Arg16Arg. We will count the 
amount of children with this genotype when 310 children have been included. If this amount 
is not reached after 310 children we will continue the trial until we have included the 
expected 49 children with the Arg16Arg genotype. 
 
 

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5. TREATMENT OF SUBJECTS 
 
Intervention: ADRB2 genotype-guided treatment  
The intervention consists of genotype-guided treatment. All children in the trial require 
additional asthma treatment (step 3). There are two preferred treatment options among 
paediatric pulmonologists in the Netherlands: 1) doubling ICS dosage or 2) adding LABA to 
the standard dose of ICS. In the intervention group, the choice of treatment will be based on 
the child’s ADRB2 genotype (rs1042713). This variant is known as Arg16Gly since the 16th 
amino acid of the receptor is changed from glycine into arginine. Children homozygous for 
the risk variant Arg16 and heterozygotes (Arg16Gly) will be treated with doubling dosages of 
their ICS. Children homozygous for the wild type allele (Gly16Gly) will receive LABA.  
 
Comparator: Non genotype-guided treatment  
In the comparator group choice of treatment will be based on randomization (1:1) to the two 
treatment options (doubling ICS dosage or adding LABA treatment) and will not be based on 
ADRB2 genotype. This arm reflects usual care. Genotyping will be performed for 
retrospective analysis, but the genotype information will not be used to guide treatment. 
 
This study meets the definition of a “clinical study with limited intervention” as is defined in 
the new EU regulation Article 2, Clause 3. Children with asthma are treated in this study with 
drugs that are approved by the European Medicines Agency and are used according to the 
approved Summary of Product Characteristics. Children with asthma who are uncontrolled 
asthma on treatment step 2 will be treated according to step 3, which is double dose ICS or 
adding a LABA. It is not clear which of the two treatment options in step 3 is most effective in 
the different phenotypes of children.  In this study the choice for the drugs will be determined 
by a genetical test or by randomisation. The procedures in this study are minimal invasive. 
They will consist of three visits in the hospital/at home, monthly questionnaires and obtaining 
samples (saliva, feces, nose swabs). 
 
INVESTIGATIONAL PRODUCT  
 
5.1 Name and description of investigational  product(s) 
Product and dose in mcg  
Active substance 
AirFluSal aerosol 25/125, 25/250 Sandoz 
Salmeterol/fluticason 
AirFluSal Forspiro 50/500 Sandoz 
Salmeterol/fluticason 
Salmeterol 25 aerosol Fisher  
Salmeterol  
Salmeterol 25 aerosol GSK 
salmeterol 
Salmeterol/Fluticason 25/125, 25/250 
aerosol Vincion 
Salmeterol/fluticason 
Salmeterol/Fluticason 25/125, 25/250, 
50/500 aerosol GSK 
Salmeterol/fluticason 

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Seretide 25/50, 25/125, 25/250 aerosol 
GSK  
Salmeterol/fluticason 
Seretide Diskus 50/100, 50/250, 50/500 
inhalatiepoeder GSK 
Salmeterol/fluticason  
Serevent 25 aerosol GSK 
Salmeterol 
Serevent Diskus 50 GSK  
Salmeterol  
Aerivio Spiromax 50/50 
Salmeterol/fluticason 
Fluticasonproprionaat 125, 250 aerosol 
Vincion 
fluticason 
Fluticasonproprionaat 50, 125, 250 aerosol 
GSK  
Fluticason  
Budesonide 200 aerosol Allgen 
Budesonide 
Budesonide 200 aerosol Mylan 
Budesonide 
Budesonide Novolizer 200, 400  
budesonide 
Budesonide Easyhaler 100, 200, 400 
budesonide 
Formoterol Novolizer 6, 12  
Formoterol  
Formoterol Easyhaler 12 Sandoz 
Formoterol  
Atimos 12 Chiesi 
Formoterol  
Oxis turbuhaler 12 
Formoterol  
Oxis turbuhaler 6 
Formoterol  
 
 
Alvesco dosisaerosol 160 
ciclesonide 
Beclometason 100 aerosol TEVA 
Beclometason 
Beclometason 250 aerosol TEVA 
Beclometason 
Beclometason 50 aerosol TEVA 
beclometason 
Beclometason 50 aerosol Allgen 
beclometason 
Beclometason 100, 250, 500 aerosol Mylan 
Beclometason 
Beclometason 100, 250, 500 aerosol 
Sandoz 
beclometason 
Qvar 50, 100 aerosol  
beclometason 
Foster 100/6, 200/6 aerosol Chiesi 
Beclometason/formoterol 
Qvar 50, 100 redihaler 
beclometason 
Bufoler Easyhaler 320/9, 160/4.5 
Budesonide/formoterol  
Duoresp Spiromax 160/4.5 
Budesonide/formoterol  
Flutiform 50/5, 125/5, 250/10 aerosol 
Fluticason/formoterol  
Foster NEXThaler 100/6, 200/6 Chiesi 
Beclometason/formoterol 
Relvar Ellipta 92/22, 184/22 GSK 
Fluticasonfuroaat/vilanterol  
Symbicort 200/6 aerosol 
Budesonide/formoterol  
Symbicort Turbuhaler 100/6, 200/6, 400/12 
Budesonide/formoterol 
 
A  meta-analysis of 25 clinical trials with a total of 5572 children has shown that on average 
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ICS combination [23].  
 
5.2 Summary of findings from non-clinical studies 
A summary of findings from non-clinical studies can be found in the Summary of Product 
Characteristics (SPC) on the pages defined below:  
 
Product and dose in mcg 
Location of findings from non-clinical 
studies:  
AirFluSal aerosol 25/125, 25/250 Sandoz 
Section 5.3, page 18 
AirFluSal Forspiro 50/500 Sandoz 
Section 5.3, page 20 
Salmeterol 25 aerosol Fisher  
Section 5.3, page 11  
Salmeterol 25 aerosol GSK 
Section 5.3, page 12  
Salmeterol/Fluticason 25/125, 25/250 
aerosol Vincion 
Section 5.3, page 13 
Salmeterol/Fluticason 50/100, 50/250, 
50/500 aerosol GSK 
Section 5.3, page 18 
Seretide 25/50, 25/125, 25/250 aerosol GSK  Section 5.3, page 17 
Seretide Diskus 50/100, 50/250, 50/500 
inhalatiepoeder GSK 
Section 5.3, page 17 
Serevent 25 aerosol GSK 
Section 5.3, page 11 
Serevent Diskus 50 GSK  
Section 5.3, page 9  
Aerivio Spiromax 50/50 
Section 5.3, page 19 
Fluticasonproprionaat 125, 250 aerosol 
Vincion 
Section 5.3, page 12  
Fluticasonproprionaat 50, 125, 250 aerosol 
GSK  
Section 5.3, page 12  
Budesonide 200 aerosol Allgen 
Section 5.3, page 11  
Budesonide 200 aerosol Mylan 
Section 5.3, page 8  
Budesonide Novolizer 200, 400  
Section 5.3, page 11  
Budesonide Easyhaler 100, 200, 400 
Section 5.3, page 11 
Formoterol Novolizer 6  
Section 5.3, page 8 
Formoterol Novolizer 12 
Section 5.3, page 8 
Formoterol Easyhaler 12 Sandoz 
Section 5.3, page 7 
Atimos 12 Chiesi 
Section 5.3, page 7 
Oxis turbuhaler 12 
Section 5.3, page 7 
Oxis turbuhaler 6 
Section 5.3, page 7 
 
 
Alvesco dosisaerosol 160 
Section 5.3, page 8 
Beclometason 100 aerosol TEVA 
Section 5.3, page 6 
Beclometason 250 aerosol TEVA 
Section 5.3, page 6 
Beclometason 50 aerosol TEVA 
Section 5.3, page 6 
Beclometason 50 aerosol Allgen 
Section 5.3, page 7 

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Beclometason 100, 250, 500 aerosol Mylan 
Section 5.3, page 7 
Beclometason 100, 250, 500 aerosol 
Sandoz 
Section 5.3, page 7 
Qvar 50, 100 aerosol  
Section 5.3, page 6 
Foster 100/6, 200/6 aerosol Chiesi 
Section 5.3, page 15 
Qvar 50, 100 redihaler 
Section 5.3, page 8 
Bufoler Easyhaler 320/9, 160/4.5 
Section 5.3, page 14 
Duoresp Spiromax 160/4.5 
Section 5.3, page 30 
Flutiform 50/5, 125/5, 250/10 aerosol 
Section 5.3, page 17 
Foster NEXThaler 100/6, 200/6 Chiesi 
Section 5.3, page 16 
Relvar Ellipta 92/22, 184/22 GSK 
Section 5.3, page 41 
Symbicort 200/6 aerosol 
Section 5.3, page 10 
Symbicort Turbuhaler 100/6, 200/6, 400/12 
Section 5.3, page 13 
 
5.3 Summary of findings from clinical studies 
A summary of findings from clinical studies can be found in the Summary of Product 
Characteristics (SPC) on the pages defined below:  
Product and dose in mcg 
Location of findings from clinical 
studies:  
AirFluSal aerosol 25/125, 25/250 Sandoz 
Section 5.1, page 14 
AirFluSal Forspiro 50/500 Sandoz 
Section 5.1, page 12  
Salmeterol 25 aerosol Fisher  
Section 5.1, page 9  
Salmeterol 25 aerosol GSK 
Section 5.1, page 9  
Salmeterol/Fluticason 25/125, 25/250 
aerosol Vincion 
Section 5.1, page 10 
Salmeterol/Fluticason 50/100, 50/250, 
50/500 aerosol GSK 
Section 5.1, page 12  
Seretide 25/50, 25/125, 25/250 aerosol GSK  Section 5.1, page 13 
Seretide Diskus 50/100, 50/250, 50/500 
inhalatiepoeder GSK 
Section 5.1, page 11 
Serevent 25 aerosol GSK 
Section 5.1, page 9  
Serevent Diskus 50 GSK  
Section 5.1, page 6  
Aerivio Spiromax 50/50 
Section 5.1, page 13 
Fluticasonproprionaat 125, 250 aerosol 
Vincion 
Section 5.1, page 10 
Fluticasonproprionaat 50, 125, 250 aerosol 
GSK  
Section 5.1, page 9  
Budesonide 200 aerosol Allgen 
Section 5.1, page 10  
Budesonide 200 aerosol Mylan 
Section 5.1, page 7  
Budesonide Novolizer 200, 400  
Section 5.1, page 10  
Budesonide Easyhaler 100, 200, 400 
Section 5.1, page 10 
Formoterol Novolizer 6 
Section 5.1, page 8 

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Formoterol Novolizer 12 
Section 5.1, page 8 
Formoterol Easyhaler 12 Sandoz 
Section 5.1, page 7 
Atimos 12 Chiesi 
Section 5.1, page 6 
Oxis turbuhaler 12 
Section 5.1, page 6 
Oxis turbuhaler 6 
Section 5.1, page 6 
Striverdi respimat 2,5  
Section 5.1, page 10 
Alvesco dosisaerosol 160 
Section 5.1, page 6 
Beclometason 100 aerosol TEVA 
Section 5.1, page 6 
Beclometason 250 aerosol TEVA 
Section 5.1, page 6 
Beclometason 50 aerosol TEVA 
Section 5.1, page 6 
Beclometason 50 aerosol Allgen 
Section 5.1, page 6 
Beclometason 100, 250, 500 aerosol Mylan 
Section 5.1, page 6 
Beclometason 100, 250, 500 aerosol 
Sandoz 
Section 5.1, page 6 
Qvar 50, 100 aerosol TEVA 
Section 5.1, page 5 
Foster 100/6, 200/6 aerosol Chiesi 
Section 5.1, page 10 
Qvar 50, 100 redihaler 
Section 5.1, page 7 
Bufoler Easyhaler 320/9, 160/4.5 
Section 5.1, page 11 
Duoresp Spiromax 160/4.5 
Section 5.1, page 28 
Flutiform 50/5, 125/5, 250/10 aerosol 
Section 5.1, page 13 
Foster NEXThaler 100/6, 200/6 Chiesi 
Section 5.1, page 11 
Relvar Ellipta 92/22, 184/22 GSK 
Section 5.1, page 36 
Symbicort 200/6 aerosol 
Section 5.1, page 8 
Symbicort Turbuhaler 100/6, 200/6, 400/12 
Section 5.1, page 10 
 
5.4 Summary of known and potential risks and benefits 
Chapter 10 describes our risk analysis.  
 
5.5 Description and justification of route of administration and dosage 
 
Dosage will be according to the SPC’s of the products.  
 
5.6 Dosages, dosage modifications and method of administration 
There will be no dosage modifications or different methods of administration other than 
described in the SPC’s of the products.  
 
5.7 Preparation and labelling of Investigational Medicinal Product 
All investigational medicinal products in this study are used according to the Nederlandse 
Vereninging van Kindergeneeskunde (NVK) and according to registration as described in 
the SPC of each product. The products will be selected and prescribed as standard care. 

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At the moment of inclusion patients are already being treated with other inhalation 
products (SABA and single dose ICS), which will be continued once in the study.  
 
5.8 Drug accountability 
At inclusion the investigators will document the name of the medication (ICS or LABA), the 
manufacturer, batch-number and expiry date of the product in the case report form.  
 
 

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6. METHODS 
6.1 Study parameters/endpoints 
6.1.1 Main study parameter/endpoint 
Change in asthma control based on repeated measurement analysis of ACT or 
C-ACT scores at t=3 months. We will choose t=3 months as a primary outcome, 
since the effect of genotype-guided treatment on asthma control should already 
be visible within this period (based on the Scottish trial in Arg16Arg children [10]). 
Furthermore, in case a child is not controlled within this period, we do not find it 
ethical to continue the same medication. This reflects normal clinical practise at 
the outpatient clinic. However, in order to study the effect on a longer term, 
children will be followed for 6 months in total.    
6.1.2 Secondary study parameters/endpoints (if applicable) 
- 
change in asthma control at t=6 months (repeated measurement analysis) 
- 
time to ACT ≥ 20  
- 
change in asthma-related quality of life scores 
- 
change in fatigue score  
- 
school absences  
- 
exacerbations (oral corticosteroids use, ER visits, hospital admissions)  
- 
time to first exacerbation 
- 
amount of changes in therapy at t=3 months 
- 
change in lung function (FEV1 pre- and postbronchodilator) at t=3 and t=6 
- 
change in FeNO at t=3 and t=6 (in centers where FeNO analysers are 
available) 
- 
change in nasal gene expression and nasal gene methylation in relation to the 
treatment effect at t=3 and t=6 
- 
Incremental cost per Quality Adjusted Life Year (QALY)  
- 
Incremental costs per avoided exacerbation 
 
 
6.2 Randomisation, blinding and treatment allocation 
Randomisation between intervention and control arm 
Participants will be randomized 1:1 to the intervention arm or the control arm. Block 
randomization with randomly chosen block sizes and stratified per center will be applied. 
Randomization software will be used to generate randomization codes.    
 
Randomisation within control arm 

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Children in the control arm will be randomized 1:1 to a) doubling ICS dosage or b) adding 
LABA to the treatment regime. Block randomization with randomly chosen block sizes and 
stratified per center will be applied. Randomization software will be used to generate 
randomization codes.    
 
Blinding  
The study will be double blinded, both patients and paediatricians will be blinded for the 
study arm and will not know whether the treatment advice was based on the ADRB2 
genotype of the child or not. The researchers of the Clinical Trial Unit of the department of 
Respiratory Medicine at the AMC will have access to the randomization arm and according to 
the algorithm (Table 1) will provide the treating physician and research nurses at the 
inclusion sites with a treatment advice for step 3.  
 
Breaking the randomization code 
The randomization code will be broken in case of severe asthma exacerbations requiring ICU 
admission.  
 
6.1 Study procedures 
The study consists of 3 clinical visits/visits at home (t=0, t=3 months (± 2 weeks) , t=6 months 
(± 2 weeks)) and monthly online questionnaires (Figure 3).  
 
Figure 3: Timeline PUFFIN trial  
 
Informing participants 
During a regular care visit, the treating physician will inform the parents and the patient about 
the study. The physician will contact the PUFFIN investigators. Patients and parents will get 
sufficient time to consider participation. A research assistant of the hospital of the child will 

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contact the patients and parents by phone to inform whether they would like to participate. In 
case they are willing to participate, a study visit will be planned within 2 weeks.  
 
Measurements during clinical visit/visit at home 1 
 
Screening 
Upon informed consent, patient will be screened to check whether he/she fulfill the inclusion 
criteria (e.g. current asthma symptoms, adequate inhalation technique (based on validated 
checklist (15), adherence to treatment). In case he/she fulfills these criteria, the following 
study measurements will be performed:  
 
Lung function testing (in case this has not been performed during the last clinical routine visit, 
< 2 weeks preceding the study visit):  
- 
Spirometry: Forced expiratory Volume in 1 second (FEV1), Forced Vital Capacity 
(FVC) and FEV1/FVC ratio, Forced Expiratory Flow after 75% of the exhaled 
volume (FEF75) before and after inhalation of salbutamol will be measured. 
- 
Fraction of exhaled nitric oxide (FeNO) will be measured before spirometry at 
applicable participating sites.  
 
Children will be instructed to stop their short acting bronchodilators at least 8 hours prior to 
lung function testing. Their long acting bronchodilators should be discontinued at least 24 
hours prior to lung function testing.  
 
NB: A lung function measurement could have been assessed during the screening visit. In 
this case, this measurement is eligible to count for visit 1.  
 
Clinical review 
Measure height/weight.  
 
Questionnaires:  
Children and parents are asked to complete several questionnaires (if possible online): 
 
 Asthma control test 
The validated Dutch or German version of the Asthma Control Test (ACT, for children 
of ≥ 12 years) or Childhood Asthma Control Test (C-ACT, for children < 12 years) 
inquires on asthma symptoms in the past 4 weeks.  
 
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Severe exacerbations (asthma-related unscheduled health care visits, use of OCS, 
admissions), as well as mild-to-moderate exacerbations (sudden increase of 
symptoms, asthma attack requiring additional rescue medication, unplanned visits to 
general practitioner for asthma) and school absences (in days) due to asthma 
symptoms will also be recorded.  
 
 Asthma medication use and adherence to maintenance treatment  
Questions on current asthma medication will be included. Furthermore, patients will be 
asked to complete the medication adherence reporting scale (MARS). Parents and 
children are asked for consent to extract medication dispensing data of the child from 
the local pharmacies 
 
 Asthma-Quality of Life 
In children aged 12 years and older, asthma-related quality of life will be measured with 
the 13-item self-reported Dutch/German validated version of the Paediatric Asthma-
Related Quality of Life Questionnaire (PAQLQ) [24] for children and expressed as 
overall asthma-related quality of life. In children aged below 12 years, we use the 
Paediatric Asthma-Related Caregiver Quality of Life Questionnaire (PACQLQ)[25]. 
 
 Productivity loss parents  
Modules of the Productivity Cost Questionnaire (PCQ) [26] to assess the loss of 
productivity of the caregivers will be included in the questionnaire.  
 
 Fatigue 
The PedsQL Multidimensional Fatigue Score [27] will be included to assess symptoms of 
fatigue in the past month. 
 
 Control of Allergic Rhinitis 
In case, children suffer from allergic rhinitis in addition to asthma, The Control of Allergic 
Rhinitis and Asthma Test (CARAT) [28] will be used to assess the level of control of 
allergic rhinitis.  
 
 Other questions 
Furthermore we will include questions on allergy and rhinitis complaints, environmental 
factors, pre- and postnatal factors, demographics and diet (related to the microbiome).  
 
Noninvasive procedures 
Two nasal epithelial swab (Copan Flocked Swabs), nr 56380CS01 per nostril will be taken 
for DNA and RNA isolation. This sample will be taken by gently rotating the flocked swab at 
the site of the lower inferior turbinate. Furthermore, a saliva sample will be taken for genomic 
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the study visit for microbiomics/metabolomics analysis. Within the AMC inclusion site, breath 
will be analyzed using the SpiroNose. For this measurement, children just have to breathe 
into the SpiroNose. 
 
 
Laboratory testing: 
Nasal epithelial swabs: DNA and RNA isolation will be performed. Whole genome gene-
expression and epigenomics analyses will be performed.  
Saliva: DNA will be isolated and genotyped for the ADRB2 gene.  
Feces: short-chain fatty acids will be measured, 16s RNA sequencing will be performed to 
analyze the microbiome.  
 
Clinical visit/visit at home 2 (t=3 months (± 2 weeks)) 
During this visit asthma control is assessed. In case children are still not controlled with 
current treatment, they will receive alternative treatment according to Table 1. 
The following measurements will be performed during this visit:  
- 
clinical review (similar to clinical visit 1) 
- 
lung function measurements (similar to clinical visit 1), FeNO measurement 
- 
online asthma questionnaire (less extensive as clinical visit 1, but including 
questions on current asthma medication use, symptoms, asthma-related quality of 
life, fatigue and exacerbations) 
- 
Nose swabs for DNA and RNA extraction.  
 
Clinical visit/visit at home 3 (t=6 months (± 2 weeks)) 
Same measurements as clinical visit 2.   
 
Online monitoring / e-diary 
A web-based patient filewill be developed to obtain monthly data and allow patients and their 
caregivers to upload data and complete questionnaires. We developped this research 
database by using CastorEDC that meets all safety and privacy legislations. The web-based 
application will be accessible from every device with an internet connection (such as 
computer, tablet, smartphone). Data entered through the electronic questionnaire will be 
directly sent to a secured database, will be time and date logged and can be accessed by 
staff with sufficient level of access rights. Pop-up messages and emails will be used to alert 
the patients to uncompleted questionnaires. When participants do not fill in their 
questionnaire monthly, research staff will contact the parents/patients and try to motivate 
compliance.  

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The questionnaires include questions on current asthma medication use, symptoms, 
asthma-related quality of life, fatigue and exacerbations. Furthermore, children and 
parents can note changes in medication use and symptoms to discuss this with their 
physician during the clinical visit. When the visit will be conducted at home, the symptoms 
will be communicated with the physician and the patient will be contacted by the physician 
afterwards.   
 
6.2 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. These subjects will then be treated according to standardized care. The 
investigator can decide to withdraw a subject from the study for urgent medical reasons.  
6.2.1 Specific criteria for withdrawal (if applicable) 
Children who will be admitted to the ICU during the study will be withdrawn from the 
study.  
 
6.3 Replacement of individual subjects after withdrawal 
A dropout rate of 5% has been foreseen for this study, based on previous experiences 
with a similar study population in the BATMAN study [ZonMW project number: 
171002101] in which patients were followed for 1 year. Therefore, subjects will not be 
replaced if they drop out of the study.  
 
6.4 Follow-up of subjects withdrawn from treatment 
Subjects will be followed up by their own paediatrician and treated according to national 
guidelines.  
 
6.5    Premature termination of the study 
Since all the medication used in this trial is already used in clinical practise, we do not 
foresee safety reasons for premature termination of this study.  
 
 

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7. SAFETY REPORTING 
7.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise subject 
health or safety.  The sponsor will notify the accredited METC without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending a further positive decision by the accredited METC. The investigator will take 
care that all subjects are kept informed. 
 
7.2 AEs, SAEs and SUSARs 
7.2.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to trial procedure. All adverse 
events reported spontaneously by the subject or observed by the investigator or his 
staff will be recorded, except for the following AEs: AEs related to asthma diagnosis 
including exacerbations and hospitalisation for asthma exacerbations but with the 
exception of asthma-related ICU admissions and AEs already reported in the SPC 
documents of the study medication such as: palpitations, nausea, vomiting, poor/loss 
of taste, pain in the abdomen, dyspepsia, oedema in eyes, face, lips, throat or angio-
oedema, hypersensitivity, oral candidiasis (mouth and throat), sleep disorders, 
psychomotor hyperactivity, anxiety, depression, aggression, changes in behaviour, 
headache, dysphonia, coughing, paradoxal bronchospasms, eczema, skin 
irritation/sensitivity (rash, bruises, contact dermatitis, urticaria, pruritus, erythema, 
exanthem), hypertensia, adrenal suppression, cataract, glaucoma, irritation of the 
throat, dyspnoea, growth slowdown, decreased bone density, restlessness, 
nervousness, tremor, loss of sight, dysphagia, glossdynia, burning mouth, dry mouth, 
atrophic skin, muscle spasms, irritability, hypokalaemia, hyperkalaemia, 
hyperglycaemia, agitation, headache, dizziness, tachycardia, heart arrhythmia, 
angina pectoris, prolonged QT interval, changes in blood pressure, oropharyngeal 
irritation, muscular spasms, arthralgia, non-specific angina pectoris, Cushing 
syndrome, common cold, sinusitis, myalgia, fractures caused by traumas.  
 
7.2.2 Serious adverse events (SAEs) 
A serious adverse event occurring during the trial is any untoward medical occurrence 
or effect that  

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- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes listed 
above due to medical or surgical intervention but could have been based upon 
appropriate judgement by the investigator. 
An elective hospital admission will not be considered as a serious adverse event. 
The investigator will report all SAEs to the sponsor without undue delay after 
obtaining knowledge of the events, except for the following SAEs: SAEs related to 
asthma diagnosis including exacerbations and hospitalisation for asthma 
exacerbations but with the exception of asthma-related ICU admissions and AEs 
already reported in the SPC documents of the study medication. 
 
The sponsor will report the SAEs through the web portal ToetsingOnline to the 
accredited METC that approved the protocol, within 7 days of first knowledge for 
SAEs that result in death or are life threatening followed by a period of maximum of 8 
days to complete the initial preliminary report. All other SAEs will be reported within a 
period of maximum 15 days after the sponsor has first knowledge of the serious 
adverse events. The following SAEs will be reported in line listings every 6 months: 
SAEs related to asthma diagnosis.  
7.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
Adverse reactions are all untoward and unintended responses to an investigational 
product related to any dose administered. 
 
Unexpected adverse reactions are SUSARs if the following three conditions are met 
during the trial: 
1. the event must be serious (see chapter 9.2.2); 
2. there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of 
the administered dose; 
3. the adverse reaction must be unexpected, that is to say, the nature and severity 
of the adverse reaction are not in agreement with the product information as 
recorded in: 

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- 
Summary of Product Characteristics (SPC) for an authorised medicinal 
product; 
- 
Investigator’s Brochure for an unauthorised medicinal product. 
 
The sponsor will report expedited the following SUSARs through the web portal 
ToetsingOnline to the METC: 
 SUSARs that have arisen in the clinical trial that was assessed by the METC; 
 SUSARs that have arisen in other clinical trials of the same sponsor and with the 
same medicinal product, and that could have consequences for the safety of the 
subjects involved in the clinical trial that was assessed by the METC. 
The remaining SUSARs are recorded in an overview list (line-listing) that will be 
submitted once every half year to the METC. This line-listing provides an overview 
of all SUSARs from the study medicine, accompanied by a brief report highlighting 
the main points of concern.  
The expedited reporting of SUSARs through the web portal Eudravigilance or 
ToetsingOnline is sufficient as notification to the competent authority. 
 
The sponsor will report expedited all SUSARs to the competent authorities in other 
Member States, according to the requirements of the Member States.  
 
The expedited reporting will occur not later than 15 days after the sponsor has first 
knowledge of the adverse reactions. For fatal or life threatening cases the term will 
be maximal 7 days for a preliminary report with another 8 days for completion of the 
report.  
 
 
7.3 Annual safety report 
 
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year 
throughout the clinical trial, a safety report to the accredited METC, competent authority, 
and competent authorities of the concerned Member States. 
This safety report consists of: 
 a list of all suspected (unexpected or expected) serious adverse reactions, along with 
an aggregated summary table of all reported serious adverse reactions, ordered by 
organ system, per study; 

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 a report concerning the safety of the subjects, consisting of a complete safety analysis 
and an evaluation of the balance between the efficacy and the harmfulness of the 
medicine under investigation. 
 
7.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been 
reached. Depending on the event, follow up may require additional tests or medical 
procedures as indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported till end of study within the Netherlands, as defined in the 
protocol  
 
7.5 Data Safety Monitoring Board (DSMB)  
We consider the risk associated with participating in this study moderate: 
- 
The study includes a vulnerable participant group (children),   
- 
However, the medication in this study is already used in clinical care,  
- 
And, there are no high risk measurements or interventions associated with 
significant safety issues, outside what is considered normal practice for this 
patient population 
Therefore, we do not consider it is necessary to establish a DSMB for this study.    

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8. STATISTICAL ANALYSIS 
8.1 Primary study parameter(s) 
 
The primary study parameter is improvement of asthma control after 3 months. Asthma 
control is measured each month and is represented in a score obtained from 5-item asthma 
control questionnaires (ACT or CACT). The score ranges on a scale from 5 to 25 (continuous 
variable).  
 
 To assess whether children with a variant genotype have a poorer improvement in asthma 
control we will assess:  
1. The improvement in asthma control is different between patients with Arg16Arg 
treated with LABA and treated with double dose ICS  
2. The improvement in asthma control is different between patients with Arg16Gly 
treated with LABA and treated with double dose ICS 
3. The improvement in asthma control is different between patients with Gly16Gly 
treated with LABA and treated with double dose ICS 
To assess the clinical impact of a genotype-guided strategy on improvement in asthma 
control we will assess whether:  
4. The improvement in asthma control is different between patients in the genotype-
guided treatment arm compared to the control arm  
 
Univariate repeated measures mixed models analysis will be used, as well as multivariate 
analysis correcting for the following covariates: age, gender, season, ethnicity, 
academic/non-academic hospital, and baseline value. In case the outcome variable 
follows a normal distribution, parametric tests are applied, in case the outcome variable 

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does not follow a normal distribution, appropriate non-parametric testing will be applied. 
In addition, descriptive statistics will be used to describe the characteristics of the study 
population.  
 
8.2 Secondary study parameter(s)  
The following secondary study parameters will be assessed in this trial. In case a 
continuous outcome variable does not follow a normal distribution, appropriate non-
parametric testing will be applied.   
 
1. change in asthma control score at t=6 months  
Changes in asthma control scores at t=6 months between patients with different 
genotypes treated with LABA or double dose ICS, and between patients in the 
intervention arm and in the control arm will be compared.  
 
2. time to ACT ≥ 20 (time-to-event variable)  
A log-rank or similar statistical test will be used to compare time to reach well 
controlled asthma between patients with different genotypes treated with LABA or 
double dose ICS, and between patients in the intervention arm and in the control arm.  
 
3. change in asthma-related quality of life scores 
Asthma-related quality of life is measured using the questions of the PaQLQ, and is 
measured at various time intervals during the trial. The questions of the PaQLQ are 
divided into three domains (activity limitations, symptoms, emotional function) and 
consist of 23 questions (with a 7 point scale). All questions are equally weighted and 
the overall score is the mean of the responses to all questions. Differences in 
mean/median of asthma-related quality of life scores from the beginning to the end of 
trial (t=6 months) between patients with different genotypes treated with LABA or 
double dose ICS, and between patients in the intervention arm and in the control arm 
are assessed using appropriate testing.  
 
4. change in fatigue score  
Fatigue score will be measured at the beginning and the end of the trial using the 
PedSQL. This 18-item questionnaire measures fatigue in three domains (general 
fatigue; sleep fatigue, cognitive fatigue). Each question can lead to 0-4 points. The 
total fatigue score ranges between 0-72.  The differences in median score will be 
compared between patients with different genotypes treated with LABA or double dose 

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ICS, and between patients in the intervention arm and in the control arm using 
appropriate testing.  
 
5. school absences 
The proportion of patients with school absences, as well as the median/mean amount of 
school absences, will be compared between patients with different genotypes treated with 
LABA or double dose ICS, and between patients in the intervention arm and in the control 
arm using Chi-squared and appropriate testing.  
 
6. exacerbations (oral corticosteroids use, ER visits, hospital admissions)  
Exacerbations will be defined as the use of short courses of oral corticosteroids, asthma-
related ER visits and asthma-related hospital admissions. The proportion of patients with 
exacerbations, as well as the median/mean amount of exacerbations per group, will be 
compared between patients with different genotypes treated with LABA or double dose 
ICS, and between patients in the intervention arm and in the control arm using 
appropriate testing.  
 
7. time to first exacerbation 
A log-rank test will be used to compare time to a new exacerbation between patients 
with different genotypes treated with LABA or double dose ICS, and between patients 
in the intervention arm and in the control arm. For the definition of exacerbation see 
secondary outcome #6.  
 
8. amount of changes in therapy at t=3 months 
At t=3 months physicians can adapt the treatment of participants in case they are still 
not well controlled (see table 1). We will assess whether patients in the intervention 
arm have fewer changes in therapy (continuous variable) at t=3 compared to patients 
in the control arm, using appropriate statistical testing.  
 
9. change in lung function (FEV1 pre- and postbronchodilator) at t=3 and t=6 
Differences in change in lung function at t=3 and t=6 will be assessed between 
patients with different genotypes treated with LABA or double dose ICS, and between 
patients in the intervention arm and in the control arm using ANOVA repeated 
measures mixed models analysis  as appropriate.   
 
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FeNO is measured as a continuous variable in parts per billion. We will assess 
whether the median/mean level of FeNO is different between patients with different 
ADRB2 genotypes treated with LABA or double dose ICS, and between patients in the 
intervention arm and in the control arm using appropriate testing (logFeNO).  
 
 
8.3 Other study parameters 
The following –omics analysis will be performed:  
 
1. change in nasal gene expression and nasal gene methylation in relation to the 
treatment effect at t=3 
For each sample, DNA methylation level at each CpG site will be calculated in 
percentage by Β= (M/M+U) * 100%. Where M is the signal strength of methylated 
CpG given by Illumina HumanMethylation450 BeadChip array, and U is the signal 
strength of unmethylated CpG. For each marker a T-test will be used to assess 
methylation variation between responders and non-responders to treatment at t=3 
months. Multiple comparison adjustment will be performed adopting a Bonferroni 
correction for the number of probes that will be successfully analysed after quality 
control 
 
2. change in microbiome profile and treatment effect at t=3 
Using 16S RNA sequencing, each sample sequence set will be sub-sampled to 8,700 
sequences. Differences in abundance will be detected using a Kruskal-Wallis 
generating a Benjamini-Hochberg false-discovery rate corrected p-value, whereby 
responders and non-responders to treatment at t=3 months are compared.   
 
3. Exhaled air at t=0, t=3 and t=6 only for the AMC inclusion site 
Exhaled air will be sampled using the  SpiroNose (AMC, Amsterdam & Comon Invent, 
Delft, NL). In its current form, the SpiroNose is a spirometry coupled electronic nose 
based on metal-oxide semiconductor (MOS) sensors with high between-sensor 
reproducibility. Patients will perform 5 tidal breaths, then, after a single deep 
inspiratory vital capacity manoeuvre and a 5 second breath hold, the patient exhales 
a vital capacity volume into the measurement setup. The exhaled air is directly 
measured by the SpiroNose, which is connected to an Ethernet cable for immediate 
secure data transmission to the online BreathCloud server. Data can be downloaded 
for further processing and analysis with offline pattern recognition software. 

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ETHICAL CONSIDERATIONS 
8.4 Regulation statement 
The study will be conducted according to the principles of the Declaration of Helsinki 
(version 2013, 19-10-2013) and in accordance with the Medical Research Involving 
Human Subjects Act (WMO) and other guidelines, regulations and Acts. We will comply 
with the principles enshrined in the Council of Europe Convention on human rights and 
biomedicine – known as the Bioethics Convention (Oviedo). Its main purpose is to protect 
individuals against exploitation 
 
8.5 Recruitment and consent 
Eligible asthmatic children will be selected in the participating hospitals. The parents and 
children will be informed orally about the study by the treating physicians who will contact 
the investigators. The treating physician will provide the patient and parents with the 
appropriate patient information forms. They will be given sufficient time to consider their 
decision and are free to reconsider their decision at any moment during the study. A 
research assistant will contact the participants and parents to ask whether they would be 
willing to participate. If they are willing to participate a study visit is scheduled in their own 
hospital/at home within 1 week. Before start of the study visit written consent is obtained.  
 
8.6 Objection by minors or incapacitated subjects (if applicable) 
 We will act according to the code of conduct of objection by minors from the Netherlands 
Association for Paediatric Medicine: Code of conduct relating to expressions of objection 
by minors participating in medical research - Netherlands Association for Paediatric 
Medicine:  
 
“Code of conduct” 
1. Individual children respond differently to diagnostic and treatment procedures and to 
participation in medical research. Various factors help to determine the nature of the 
response: the way the child is prepared for what is going to happen, the parent-child 
relationship, the doctor-patient relationship, the child-friendliness of the environment 
in which the procedure takes place and so on. One child will not be unduly disturbed 
by having an injection (even if he or she winces or makes some other display of pain), 
while another will find the experience distressing. Although responses vary 
considerably from child to child, there is a general correlation between the degree of 
‘invasiveness’ of a procedure and the strength of the response. In some cases, fear 

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regarding participation or a particular procedure will prompt a child to object. Patient 
and understanding explanation and reassurance will generally be sufficient to enable 
the research or the procedure to proceed without problems. Where a newborn child or 
infant is concerned, it is much harder to ascertain whether objection has been 
expressed. As a general rule, however, it is reasonable to suggest that a child may be 
deemed to object if its behaviour clearly differs in nature or degree from that normally 
displayed by the child when confronted with situations not encountered in everyday 
life. In this context, situations not encountered in everyday life may be considered to 
include diagnostic or therapeutic procedures. 
 
2. Before seeking consent for a child’s participation in medical research, an investigator 
must fully inform the child’s custodial parent(s) or guardian about what is proposed. 
Information should be provided orally and in writing. The nature of the procedures 
involved in the research should be discussed with the parents and their views sought 
on the child’s likely response. The possibility of the child objecting to participation and 
the type of behaviour that should be regarded as an expression of objection should 
also be discussed. The investigator should also explain what is to happen in the event 
of the child objecting. The consent obtained from the parents should include 
agreement to the proposed procedure for dealing with expressions of objection by the 
child. 
 
3. The consent statement signed by parents should stipulate that, if the child should 
object to participation in the research, consent for its further participation will be 
invalidated. 
 
4. If prior to the research there is doubt as to whether a child should participate, 
consideration may be given to involving the patient in the research for an agreed pilot 
period. 
 
5. While the research is in progress, the behaviour of the child should be continually 
assessed at the research location to determine whether the child’s behaviour is within 
the bounds normally associated with the child when confronted with situations not 
encountered in everyday life. If a child’s behaviour is not within these bounds, he or 
she should be deemed to have expressed an objection in the sense of the WMO. 
 
6. The parents, the investigator(s) and possibly a behavioural scientist should be 
involved in assessment of a child subject’s behaviour. Assessment of a child subject’s 

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behaviour should not be a one-off exercise, but should continue through all phases of 
the research. 
 
7. The parents of a child subject should be able to withdraw their consent at any point 
during the research. If a child subject expresses an objection, the child’s participation 
should be discontinued. 
 
8. In all medical research involving child subjects, the burden associated with 
participation should be minimised; where non-therapeutic research is concerned, the 
law stipulates that it must be negligible. Medical studies often involve the combination 
of research procedures with diagnostic procedures necessary in connection with the 
subject’s treatment. Where research involves an invasive procedure, such as a finger 
prick or venapunction, this should if possible be combined with a procedure 
necessary for diagnostic or treatment purposes, such as blood sampling. If possible, a 
needle or line that has already been inserted should be utilised, so that the number of 
‘jabs’ is kept to the minimum. The burden can also be reduced by the use of plasters 
with local anaesthetic. The various steps to be taken with a view to minimising the 
burden should be detailed in the research protocol and in the information given to the 
parents and subjects. 
 
9. The following should be noted in the research file or the medical (status) report, as 
appropriate: 
(a) the outcome of any trial participation; 
(b) the consent of the custodial parent(s) or guardian, including the procedure to be 
followed in the event of a possible expression of objection; 
(c) an account of the subject’s participation in the research, stating whether objection 
was expressed; 
(d) an assessment as to whether the subject’s behaviour constitutes objection, as 
referred to above; 
(e) the names of the people responsible for assessing the subject’s behaviour, as 
described above; 
(f) an assessment as to whether the subject’s behaviour in the course of the study 
constitutes objection; 
(g) the steps taken to minimise the burden associated with participation. 
The protocol for a medical research project in which minors are to be used as 
subjects should state that the NVK’s code of conduct for dealing with subjects’ 
expressions of objection in the course of the research will be adhered to. 

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10. This code of conduct will be evaluated in consultation with the research community 
two years after its initial publication and amended as necessary.” 
 
This code of conduct was approved by the Board of the Netherlands Association for 
Paediatric Medicine (NVK) on 21 May 2001 and published in NVK Newsletter no. 3, June 
2001. 
 
8.7 Benefits and risks assessment, group relatedness 
Risks: Nasal swabbing (soft tips swabs, Copan Flocked Swabs), nr 56380CS01 provides 
a minimal burden to the children. We have performed the same method in the MAKI3 trial 
in 6 year old children, and found that all children were able to donate the nasal swabs with 
minimal burden. This soft-flocked method has been developed since nasal brushing with 
cytobrushes is painful and provides a significant burden to the children. Children have 
reported an itchy feeling, and in rare circumstances tearing eyes. We regard the collection 
of saliva and faces as having a minimal burden.    
Repeated lung function measurements can be experienced as uncomfortable in case of 
spirometer induced bronchoconstriction, which happens < 3 % in asthmatic children. 
However, this is part of usual asthma care and we will administer salbutamol 400 mcg 
(pMDI with spacer). The risks associated with participation can be considered negligible 
and the burden minimal.  
This study includes only minors, since the effect of the ADRB2 genotype on response to 
asthma medication seems to be restricted to the paediatric population. This patient group 
is expected to have the most benefit from prospective ADRB2 genotyping before starting 
treatment.   
Benefits: in the intervention arm, children may suffer from fewer asthma symptoms 
compared to usual care.  
 
8.8 Compensation for injury 
The sponsor has a liability insurance which is in accordance with article 7 of the WMO. 
  
8.9 Incentives (if applicable) 
Patients and parents will receive compensation in travel costs based on travelled 
kilometres and parking costs for the hospital visits. Children will receive a small present 
for participation.  

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9. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
9.1 Handling and storage of data and documents 
Collected data and samples will be coded complying with the Dutch Personal Data 
Protection Act. The code will not include patient initials and birth-date, but will include a 
code of the hospital were inclusion took place. The key to link patients and their 
genotypes or biomarkers will be securely stored, and accessible only to the principal 
investigators of each participant centre. 
Information gathered by the study will be used only for aggregate analysis, and will not be 
released with any information that identifies research participants. DNA Genotyping of the 
saliva samples will be performed in one centre (ErasmusMC), and the DNA samples will 
be coded and unlinked to individual respondent identifiers. Microbiome/metabolome feces 
samples will be analysed and stored at the AMC. Transcriptomic and epigenomic 
samples will be stored in Groningen and analysis will be performed at the UMC 
Groningen.  
 
We will conduct a genome wide association study (GWAS) in a later stadium to identify 
possibly other involved SNPs in asthma disease in children. This data may be also useful 
to compare with (and later include in) worldwide databases with children with asthma.  
 
9.2 Monitoring and Quality Assurance  
The study will be monitored by an external monitoring (CRU). For more details see the 
monitoring plan (K).  
9.3 Amendments  
Amendments are changes made to the research after a favourable opinion by the 
accredited METC has been given. All amendments will be notified to the METC that gave 
a favourable opinion.  
 
Non-substantial amendments will not be notified to the accredited METC and the 
competent authority, but will be recorded and filed by the sponsor.  
 
9.4 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the 
accredited METC once a year. Information will be provided on the date of inclusion of the 
first subject, numbers of subjects included and numbers of subjects that have completed 

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the trial, serious adverse events/serious adverse reactions, other problems, and 
amendments.  
 
9.5 Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited METC of the end of the study within a 
period of 8 weeks. The end of the study is defined as the last patient’s last visit.  
 
The sponsor will notify the METC immediately of a temporary halt of the study, including 
the reason of such an action.  
    
In case the study is ended prematurely, the sponsor will notify the accredited METC 
within 15 days, including the reasons for the premature termination. 
 
Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited METC. 
9.6 Public disclosure and publication policy 
 
We will follow the basic principles of the CCMO statement on publication policy. The 
protocol will be published at clinicaltrials.gov and trialregister.nl before the first patient is 
included in the study. The results of research will be submitted for publication to peer-
reviewed scientific journals, and will also be updated at clinicaltrials.gov and 
trialregister.nl.   
 
 

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10. STRUCTURED RISK ANALYSIS  
 
10.1 Potential issues of concern 
 
Not applicable.  
 
10.2 Synthesis 
 
All products described in chapter 5 are registered in The Netherlands. In this study the 
products are used according the registration and there is a wide experience with these 
products in daily care of children.  
 
On basis of these considerations chapter 10.2 was not further completed. 
 
 
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Bahadori K, Doyle-Waters MM, Marra C et al. Economic burden of asthma: a systematic 
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NIVEL. Zorgsituatie van mensen met astma. Rapportage 2010. (Available from: 
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Turner S, Francis B, Vijverberg S et al. Childhood asthma exacerbations and the Arg16 
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Zuurhout MJ, Vijverberg SJ, Raaijmakers JA et al. Arg16 ADRB2 genotype increases the 
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Mcmahon AW, Levenson MS, Mcevoy BW, Mosholder AD, Murphy D. Age and Risks of 
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Kersten ETG, Koppelman GH, Thio BJ. Concerns with beta2-agonists in pediatric asthma 
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nasal methylome: a key to understanding allergic asthma. Am J Respir Crit Care Med 
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Yang IV, Schwartz DA. Epigenetic mechanisms and the development of asthma. JACI 2012; 
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