Nasal Brushing for the Diagnosis and Understanding of Telomeropathies
Phase: NA
Conditions: Telomere Disease, Telomere Shortening
Sponsor: Cliniques universitaires Saint-Luc- Université Catholique de Louvain

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EXPERIMENTAL PROTOCOL 
 
 
 
 
 
STUDY TITLE 
 
“Nasal brushing for the diagnosis and understanding of telomeropathies” 
 
 
 
 
 
Principal investigator:  
Prof. Caroline Huart 
Otorhinolaryngology 
Cliniques universitaires Saint-Luc 
Avenue Hippocrate, 10 
1200 Brussels 
 
Co-investigators: 
Prof. Anabelle Decottignies 
de Duve Institute, GEPI-Telo group 
Avenue Hippocrate, 75+1 
1200 Brussels 
 
Prof. Bénédicte Brichard 
Pediatric hemato-oncology 
Cliniques universitaires Saint-Luc 
Avenue Hippocrate, 10 
1200 Brussels 
 
Prof. Anne de Leener 
Human genetics 
Cliniques universitaires Saint-Luc 
Avenue Hippocrate, 10 
1200 Brussels 
 
Prof. Bénédicte Delire 
Hepathology and gastroenterology 
Cliniques universitaires Saint-Luc 
Avenue Hippocrate, 10 
1200 Brussels 
 
 
Prof. Antoine Froidure 
Pneumology 
Cliniques universitaires Saint-Luc 
Avenue Hippocrate, 10 
1200 Brussels 
 
 
 

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Prof. Valérie Hox 
Otorhinolaryngology 
Cliniques universitaires Saint-Luc 
Avenue Hippocrate, 10 
1200 Brussels 
 
Prof. Xavier Poiré 
Hematology 
Cliniques universitaires Saint-Luc 
Avenue Hippocrate, 10 
1200 Brussels 
 
 
 
 

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TABLE OF CONTENTS 
 
1. INTRODUCTION  
 
2. OUTLINE OF OBJECTIVES AND PROJECT DESIGN 
 
2.1. 
OBJECTIVES 
2.2.  STUDY DESIGN 
 
2.2.1.   
Study population 
2.2.2.  
Study endpoints 
 
3. EXPERIMENTAL PROCEDURES AND DATA ACQUISITION 
3.1.  BLOOD SAMPLE  
3.2.   NASAL BRUSHING 
3.3.  OLFACTORY ASSESSMENT 
 
4. RECRUITMENT OF PARTICIPANTS 
 
5. INFORMED CONSENT 
 
6. PRE-STUDY SCREENING 
 
7. SELECTION AND WITHDRAWAL OF VOLUNTEERS 
 
7.1.  Inclusion and exclusion criteria   
7.2.  Discontinuation criteria 
 
8. SAFETY 
 
8.1  
Adverse event 
8.2  
Serious adverse events 
8.3  
Potential adverse events 
8.4  
Reporting procedures for all adverse events 
8.5  
Serious adverse event reporting procedures 
 
9. DATA ANALYSIS  
 
10. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
 
11. ETHICS 
 
11.1  Declaration of Helsinki 
11.2  Informed consent 
11.3  Independent ethics committee 
11.4  Subject confidentiality 
 
12. DATA HANDLING AND RECORD KEEPING 
 
13. FINANCING AND INSURANCE 

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14. PUBLICATION POLICY 
 
15. BIBLIOGRAPHY 
 
 
 
 
 
 
 

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1. INTRODUCTION  
 
Telomere biology is a growing research field. New genes and/or genomic 
variants of established telomere-related genes are still being identified as 
modulators of telomere length. While long telomeres are considered as a sign 
of good cellular health associated with a better regenerative potential of the 
tissues, abnormally short telomeres are responsible for premature ageing 
syndromes, collectively known as telomeropathies, that can be very severe and 
life threatening. The diagnosis of telomeropathy is complicated by the fact that 
patients can present with a variety of symptoms at various ages, including 
aplastic anemia, pulmonary fibrosis, liver cirrhosis, unexplained liver 
regenerative nodular hyperplasia or myelodysplasia. If, to date, there is no 
treatment yet to these genetic diseases, identifying the telomeric origin of the 
disease is important to avoid inappropriate therapeutic treatments. Developing 
new and poorly invasive diagnostic tools for telomeropathy is one of our 
research objectives. Our second objective is to better understand the genetics 
of telomeropathies through functional studies of new disease-causing 
mutations. Together, these two objectives will contribute to a better detection 
and understanding of telomere-associated pathologies.  
 
TELOMEROPATHIES: DIAGNOSIS 
Telomeropathies are premature ageing diseases resulting from pathogenic 
germline mutations in telomere biology-related genes. Most telomeropathy 
patients have telomeres that are too short for their age. Telomere length (TL) 
can be measured in blood cells using a technique called Flow-FISH (FF) that 
combines fluorescent in situ hybridization (FISH) with flow cytometry 
(Baerlocher et al. 2006). To provide a diagnostic tool for telomeropathies in 
Belgium, the laboratory of A. Decottignies, in collaboration with CUSL, 
recently implemented the FF technique and measured TL in a cohort of 491 
healthy volunteers aged 0-99 years. In combination with FF, additional genetic 
and phenotypic studies on patients’ cells are needed to tell whether the disease 
is caused by prematurely defective telomeres. Knowing this is important to i) 
exclude any other cause of the disease, ii) avoid the use of inappropriate 
treatments, iii) better manage the immunosuppressive regimens that are 
associated with hematopoietic stem cell or lung transplantation in some 
telomeropathy patients and iv) enable possible targeted therapies. For 
instance, while pulmonary fibrosis carriers with TOLLIP polymorphism may 
benefit from N-acetylcysteine treatment, those with defective telomeres may 
possibly benefit from Danazol, a telomerase-activating drug previously 
associated with increased TL in PBMCs and stability of lung function in 
idiopathic PF (IPF) patients (Townsley et al. 2016).  
 
To date, mutations in 15 telomere biology-related genes have been linked to 
telomeropathies and include TERC and TERT telomerase core components, 
various chaperone genes with roles in TERC maturation and stability, shelterin 
complex genes, the CST complex with important function in telomere 
replication and RTEL1 with multiple roles at telomeres, including replication 
(Savage 2018). There are however many patients with abnormally short 
telomeres and still unidentified mutation. On the other hand, in patients with 
CTC1 or POT1 mutations, presenting with severe telomeropathies, telomeres 

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are defective but not abnormally shortened (Gu et al. 2018), underlying the 
need to combine TL measurements with additional phenotypic and genetic 
analyses for the proper diagnosis of telomeropathies.  
 
One way to evaluate telomere dysfunction, independently of TL measurement 
in blood cells by FF, is to perform a combination of telomeric FISH and 
immunofluorescence (FISH/IF) against DNA damage markers to quantify the 
damaged telomeres, a conserved feature of cells from telomeropathy patients. 
The evaluation of cellular senescence, using the well-established Senescence-
Associated β-Galactosidase (SA-β-Gal) assay (Dimri et al. 1995) is yet another 
assay to evaluate a premature ageing cellular phenotype. Importantly however, 
neither FISH/IF nor SA-β-Gal assays can be performed on blood cells. The 
cellular diagnosis therefore requires other cell types. Usually, a skin biopsy is 
performed that requires to establish a primary culture of fibroblasts for further 
analyses. The skin biopsy is however invasive, leaving permanent scars. A skin 
biopsy can also be used to identify the genetic mutations at the origin of the 
disease. This type of cell is usually preferred over blood cells because of the 
known ability of blood cells to undergo somatic genetic rescue that can offset 
the pathogenic effect of germline mutations (Revy et al. 2019). In this respect, 
although intragenic compensatory mutations are observed in blood cells from 
telomeropathy patients, in the majority of the cases, indirect somatic genetic 
rescue is obtained through the acquisition of well-defined activating point 
mutations in the promoter of hTERT gene that codes for the catalytic subunit of 
telomerase (Revy et al. 2019). Since this has already been observed in cases 
of heterozygous germline mutations in PARN, NHP2 or TERC genes (all 
involved in maturation and activity of telomerase), this suggests that the 
detection of these point mutations in blood cells may be a strong indicator of 
the existence of a telomeropathy, prior to the exome-seq analysis. 
 
Importantly as well, having non-blood cells in culture, like fibroblasts, allows 
functional studies of the mutations to increase our knowledge of the 
telomeropathies at the cellular level.  
 
Altogether, this underlines the interest of having access to non-blood cells from 
telomeropathy patients for proper diagnosis, mutation identification and 
functional studies. In that context, developing alternative and less invasive 
approaches to collect non-blood cells so as to replace skin biopsies would be 
highly beneficial to patients.  
 
 
THE OLFACTORY EPITHELIUM: AN ALTERNATIVE SOURCE OF CELLS 
FOR TELOMEROPATHY DIAGNOSIS AND UNDERSTANDING? 
The olfactory neuroeptithelium (ONE), located at the upper part of nasal fossae 
in a region named the olfactory cleft, is considered as a unique structure owing 
to its sustained regenerative capacity. Indeed, mature olfactory sensory 
neurons are continuously produced from a population of stem cells (horizontal 
and globose cells) throughout adulthood age (Rawson and Ozdener 2013). 
Consequently, ONE is considered as the unique source of regenerating 
neuronal cells that can be easily obtained from living humans.  

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ONE cells can be easily collected using a technique called nasal brushing. In 
this technique, ONE is sampled using a cytology brush that is introduced in the 
olfactory cleft at the level of the head of the middle turbinate and gently moved 
by circular movements to collect epithelial cells. It can easily be performed 
under endoscopic control and has no known adverse effect (Benitez-King et al. 
2011; Orru et al. 2014). This non-invasive technique was found to be efficient 
in obtaining mature olfactory sensory neurons as well as neuronal cell 
precursors and stem cells from healthy subjects and patients (Benitez-King et 
al. 2011). Importantly, neuronal precursors can be propagated in culture 
(Benitez-King et al. 2011). Studies have shown that the collected neuronal 
precursors possess an undifferentiated state, the capability of self-renewal and 
the capacity to proliferate and differentiate into neuronal and glial cells without 
genetic reprogramming (Idotta et al. 2019; Jimenez-Vaca et al. 2018; Matigian 
et al. 2010; McCurdy et al. 2006). Moreover, despite a superficial exfoliation, it 
is important to mention that nasal brushing allows the collection of a significant 
number of cells, Brozzetti et al. reporting that the number of cells ranged up to 
2 millions, with 40% of those being neuronal cells (Brozzetti et al. 2020). 
Although non-invasive, the brushing technique thus represents a valuable tool 
to collect cells from the ONE and is thought to be an excellent surrogate model 
to study pathophysiological neural mechanisms.  
 
Besides that, it is also important to underline the link between aging and 
dysfunction of the olfactory system. Indeed, from a behavioral point of view, it 
is well known that aging is associated to a progressive impairment of olfactory 
function (Huart et al. 2013). Although the decrease of olfaction is physiological 
with age, aged people having a more severe olfactory dysfunction are at higher 
risk to develop neurodegenerative disease, such as Alzheimer’s and 
Parkinson’s disease (Mesholam et al. 1998), and are also at higher risk of 5-
years mortality (Devanand et al. 2015; Pinto et al. 2014). This relationship being 
specific to the sense of smell, these findings suggest that the olfactory system 
is particularly sensitive to aging. Currently, olfactory impairment is thus 
considered as an emerging biomarker of biological aging. Among the diverse 
hypotheses explaining the link between olfactory impairment and aging, it has 
been postulated that olfactory impairment might reflect a decline of this 
continuous neurogenesis, through environmental exposure or brain aging. Loss 
of this necessary process could be seen more globally as an indicator of 
lowered physiologic repair function which may reflect senescence.   
 
Therefore, we think that ONE could provide an interesting cellular model for the 
diagnosis and study of telomeropathies and replace the invasive skin biopsies 
that are currently being performed for genetic and phenotypic analyses of 
telomere dysfunction and senescence. Moreover, assessment of olfactory 
function in patients with telomeropathy could bring new interesting insights. 
 
 
2. OUTLINE OF OBJECTIVES AND PROJECT DESIGN 
 
2.1  
OBJECTIVES 
 
 

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The main objective of this study is to develop new tools for the diagnosis and 
understanding of telomeropathies. Our more specific aims are: 
• To assess the suitability of nasal brushing analyses and blood tests for the 
diagnosis of telomeropathies 
• To assess whether primary cell cultures obtained from nasal brushing could 
constitute a valuable tool for functional studies of new germline mutations 
 
 
2.2  
STUDY DESIGN 
 
 
This study is a monocentric prospective interventional study on 250 subjects 
(150 patients and 100 controls). The promotor of this study is the Cliniques 
universitaires Saint-Luc (Brussels, Belgium).  
 
2.2.1   
Study population  
 
This study will be performed in 150 subjects with confirmed or suspected 
telomeropathy and recruited through Cliniques universitaires Saint-Luc (CUSL) 
(Brussels, Belgium).  
We will include patients with suspicion of telomeropathy: patients with idiopathic 
pulmonary fibrosis, adult or pediatric medullar dysplasia or myelodysplasia, 
unexplained liver cirrhosis or unexplained liver regenerative nodular 
hyperplasia.  
100 age-matched healthy controls will be recruited from CUSL. 
 
2.2.2   
Study endpoints   
 
Endpoint #1: To assess the suitability of nasal brushing analyses and 
blood tests for the diagnosis of telomeropathies.  
 
Rationale  
To date, the complete diagnosis of telomeropathies, including the identification 
of responsible mutations, is based on blood samples and fibroblast cultures 
obtained through skin biopsies. Cells obtained through nasal brushing offer the 
opportunity to detect mutations involved in telomeropathies, in a mildly invasive 
way. We will thus assess a population of patients with a suspicion of 
telomeropathy, using a combination of blood tests and nasal brushings, and will 
compare their nasal brushing results to those of age-matched healthy controls. 
If we confirm that the nasal brushing offers the opportunity to i) detect damaged 
telomeres and premature cellular senescence and ii) identify mutations related 
to telomeropathies, this technique could become a non-invasive clinical tool for 
the diagnosis work-up of telomeropathies 
 
 
Assessments 
We will collect blood samples in each patient and will perform a nasal brushing 
to each subject (patients and controls) included in this study. One sample of 
blood will be collected for each patient and processed following the protocol 
further described (see chapter 3.1.). 

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For nasal brushing, one collection will be performed in the most accessible 
nostril. We will evaluate if it is possible to detect damaged telomeres and 
premature cellular senescence in patient, in comparison to healthy controls.  
In the patients group, collected cells will be split into three parts, two will be 
devoted to Endpoint#1, and the other for the Endpoint #2, following the protocol 
further described (see chapter 3.2.). 
For patients recruited from the pediatric consultation, if possible, this sample 
will be collected during another medical procedure planned under general 
anesthesia (eg. marrow puncture). Similarly, pediatric controls will be recruited 
among patients undergoing elective otorhinolaryngological surgery (eg. 
grommets, tonsillectomy).  
 
Endpoint #2: To develop primary cell cultures for the functional study of 
new germline mutations.  
 
Rationale 
To date, various germline mutations have already been identified in 
telomeropathy patients, in a total of 15 genes. Understanding how these 
mutations were affecting telomere biology relied on in vitro studies with either 
patient-derived fibroblasts or engineered human cell lines recapitulating the 
mutation. This was a mandatory step towards the molecular understanding of 
these pathologies. Because olfactory neural precursors have the capacity to 
grow in culture, this offers the additional possibility to perform functional studies 
on primary cultures of nasal brushing-derived cells for novel mutations, with still 
unknown impact on telomeres, that would be identified. Again, this could 
advantageously replace patients’ fibroblast cultures. 
 
Assessments 
The second part of cells harvested from the olfactory epithelium will be cultured, 
according to protocol described in chapter 3.2. 
 
Endpoint #3: To evaluate whether patients with telomeropathies have 
impaired olfactory function.  
 
Rationale 
Olfactory function is decreased in several diseases and is increasingly 
recognized as an indicator of biological aging. To date, no data exist regarding 
the impact of telomeropathies on olfactory function. Therefore, we aim to 
psychophysically assess olfactory function in patients with telomeropathies, in 
comparison to age-matched healthy controls. This study will be perfomed in a 
subset of subjects (see section 7.1). 
 
Assessment 
For each participant older than 6 years old, olfactory function will be assessed 
with the validated test.   
 
3. EXPERIMENTAL PROCEDURES AND DATA ACQUISITION  
 
3.1.  BLOOD SAMPLES 
 

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In patients with suspicion of telomeropathy, Flow-FISH analyses are already 
used for the diagnosis in routine. These standard of care procedures will 
therefore not be associated with any additional cost.  
Blood tests will be performed by nurses at the blood collection center of the 
CUSL. One EDTA tube of 7.5 ml is sufficient for Flow-FISH. Blood tests will be 
performed while the patient is at the clinic and will not require a supplementary 
consultation.  
We will measure leukocyte telomere length using a validated Flow-FISH 
technique, developed by Prof A. Decottignies (UCL), and available at CUSL.  
Blood sample for Flow-Fish analysis is performed as standard of care 
procedure for the patients involved in the study. Therefore, these analyses will 
not be associated to additional costs, neither in term of material of personal.  
A Figure is enclosed to this document to summarize experimental procedures 
and samples flow.  
Residual material will be stored in the UCLouvain biobank, at Prof. A. 
Decottignies’ lab. They will be kept for 10 years and then will be destroyed.  
 
 
3.2.  NASAL BRUSHINGS 
 
The nasal brushings will be performed by Prof. C. Huart or Prof. Valérie Hox, 
trained ENT specialists, during their scientific time. For patients and controls 
recruited among patients from the clinic, nasal brushings will be performed 
while these subjects are at the clinic for their standard cares. For controls 
recruited outside the clinic, the nasal brushing will require a specific study 
consultation.  
A local vasoconstrictor (Xylometazolin, 1mg/ml) will be applied at the level of 
the head of the inferior turbinate with the use of a nasal tampon to facilitate the 
visibility and the access to the olfactory cleft. Each nostril will be assessed, 
using a 30° rigid endoscope of 4mm (Karl Storz®, Tuttlingen, Germany), in order 
to identify the nostril having the most accessible olfactory cleft. This nostril will 
be chosen to receive the brushing. A sterile disposable cytology brush (i.e. 
FLOQSwabs, Copan®, Italy) will be gently rolled on the mucosal surface, at the 
level of the head of the middle turbinate, as described previously (Orrù et al., 
2014). Two brushings will be done for each subject. The brush will then be 
immerged in a conical centrifuge tube containing DMEM-F12 medium 
supplemented with GlutaMAX. Cellular material will then be dissociated from 
the brush using disposable micropipette tips. 
In the patients group, cellular material will be split into three parts for specific 
processing. A first part will be cultured (Prof A. Decottignies’ lab, de Duve 
Institute) (Endpoint #2), a second one will be processed by Cytospin for future 
stainings (Endpoint #1) (Prof A. Decottignies’ lab, de Duve Institute) and the 
last part will be kept frozen and further used for whole exome sequencing 
(Endpoint #1), if the suspicion of telomeropathy has been confirmed. This latter 
step will be performed at the laboratory of genetics of CUSL (Prof. A de Leener) 
In the control group, cellular material will be exclusively used for the staining 
evaluating the degree of senescence and damages to telomeres.  
A Figure is enclosed to this document to summarize experimental procedures 
and samples flow.  
 

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Figure summarizing experimental procedures and samples flow in 
patients (A) and controls (B) 
 
 
 

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3.3.  SNIFFIN’ STICKS TEST 
 
Olfactory function will be evaluated in the department of Otorhinolaryngology of 
CUSL (Prof. C. Huart). 
For patients and controls recruited among patients from the clinic, olfactory 
tests will be performed while these subjects are at the clinic for their standard 
cares. For controls recruited outside the clinic, olfactory testing will require a 
specific study consultation (at the same time than the nasal brushing). 
For adult participants, we will use the Sniffin’ Sticks test which is a validated 
psychophysical testing method (Hummel et al. 2007). Sniffin’ Sticks test is 
based on pen-like odor dispensing devices that will be presented to the 
patients. The extended version of this test will be used and consists of three 
tests of olfactory function, namely tests for odor threshold, odor discrimination 
and odor identification. Odor threshold for n-butanol will be assessed using a 
single-staircase, three alternative forced choice procedure. Odor discrimination 
will be assessed using again a three alternative forced choice procedure. 
Triplets of pens will be presented to the patients, with two containing the same 
odorant and one a different odorant. Odor identification will be assessed for 16 
common odors using a multiple-choice identification from lists of four 
descriptors each. In order to quantify olfactory function, results from the three 
subtests will be presented as a composite “TDI score” (/48) which is the sum of 
the results obtained for each subtest: threshold (/16), discrimination (/16), 
identification (/16). TDI score will be used to classify patients in three 
categories: normosmic, hyposmic or anosmic.  
For the pediatric population, we will use an adapted version of the Sniffin’ Sticks 
test, the Sniffin’ Kids test. This test is a 14-item odor identification test and has 
been validated in children between 6-17 years of age (Schriever et al. 2014). 
Therefore, considering Endpoint #3, olfactory function will be assessed only in 
participants >6 years old. Olfactory function will be assessed during the 
hospitalization, before the general anesthesia. 
 
4. RECRUITMENT OF PARTICIPANTS  
 
Patients with a suspicion of telomeropathy (i.e. idiopathic pulmonary fibrosis, 
medullar aplasia, myelodysplasia, unexplained liver cirrhosis, unexplained liver 
regenerative nodular hyperplasia) will be recruited through pneumology (Prof. 
A. Froidure), adult hematology (Prof. X. Poiré), pediatric hematology (Prof. B. 
Brichard) and adult hepathology (Prof. B. Delire) consultations.  
 
Adult healthy controls will be recruited through the consultation of 
otorhinolaryngology (Prof. C. Huart, Prof. V. Hox) or through advertising. 
Notably, pediatric controls will be recruited from children patients planned for 
elective otorhinolaryngologic surgery under general anesthesia.   
 
The physician in charge of the patients will briefly outline the objectives of the 
study to the potential participants and/or their parents. With their permission, 
the potential participants will then be contacted by one of the investigators who 
will provide detailed explanation of the project’s objectives. If interested, the 
patients and/or their parents will receive a letter of invitation and information 
sheet will be given to the putative participants. They will be free to contact us 

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should they require further clarification or information concerning inclusion and 
exclusion criteria. It will be clearly explained that accepting or refusing to 
participate will have absolutely no impact on present or future care. Subject 
and/or their parents will benefit from at least 24h and enough time to agree to 
participate or not to the study. 
 
If the subject and/or his parents agree to participate to the study, an 
appointment will be fixed, if possible, on the same day that a routine medical 
visit to the CUSL. Written informed consent will be collected that day by the 
principal investigator or one of the medical co-investigator. After giving their 
written informed consent, subjects will be enrolled in the study. The informed 
consent sheet will list the specific experimental procedures and tests required 
to conduct the study, as well as the maximum duration of the 
experiment/testing.  
 
 
5. INFORMED CONSENT 
 
Before any screening or study-specific procedure is performed, the subject will 
be asked to sign and date the subject information sheet and accompanying 
informed consent form. Importantly, since the present study involves genetic 
testing in patients, patients will be explicitly informed that they are able to 
participate to the study although refusing to take part to the genetic testing. A 
specific informed consent form for genetic testing will be signed as well. The 
informed consent will also mention if volunteers will have access or not to their 
own results. Specifically, for genetic analysis, we will ask the patients whether 
they want to be informed about the results or not.     
For minor subjects, both parents will be asked to sign and date a specific 
information sheet and accompanying informed consent. Pediatric patients older 
than 6 years will also be asked whether they agree to participate to this study. 
Pediatric patients will receive adapted information, both through oral 
explanations and through age-adapted informed consent. 
Therefore, we will propose specific informed consents to (1) adult patients, (2) 
adult controls, (3) patients’ parents, (4) controls’ parents, (5) 6-11 years old 
patients, (6) 6-11 years old controls, (7) 12-14 years old patients, (8) 12-14 
years old controls, (9) 15-17 years old patients, (10) 15-17 years old controls.  
 
6. PRE-STUDY SCREENING 
 
After signing the informed contents, investigators will screen the subject to 
ensure the eligibility of the subject. 
 
Demographics. The date of birth and gender of the subject will be recorded.  
 
Medical and surgical history. Subjects will be asked whether or not they suffer 
from any known disease, notably from sinonasal disorders, psychiatric or 
neurological disease, history of olfactory dysfunction (i.e. following upper 
respiratory tract infection, head trauma). We will also record medical and 
surgical ENT history. Additional questions will be asked to ensure correct 
screening for inclusion and exclusion criteria, as detailed in section 7.  

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Physical Examination. Subjects will undergo a rhinological endoscopic 
examination to assess olfactory cleft, notably to rule out any skull base 
abnormality such as meningocele or abnormalities that may lead to bleeding.  
 
At the end of the screening visit, and taking into consideration the inclusion and 
exclusion criteria, the eligibility of the subject will be decided.  
 
7. SELECTION AND WITHDRAWAL OF VOLUNTEERS 
 
7.1.  Inclusion and exclusion criteria 
 
Endpoints #1 and #2 
 
Study population 
Inclusion criteria 
Exclusion criteria 
Adults 
(>18 
years) 
Patients  
- 
Having 
a 
suspicion of or 
a 
confirmed 
telomeropathy
- 
Ability 
to 
provide 
written 
informed 
consent 
 
- 
No access to 
the olfactory 
cleft  
- 
Abnormal 
endoscopic 
finding (i.e. 
meningocele, 
vascular 
ectasia) 
 
 
Controls  
- 
Ability 
to 
provide 
written 
informed 
consent 
- 
No access to 
the olfactory 
cleft  
- 
Abnormal 
endoscopic 
finding (i.e. 
meningocele, 
vascular 
ectasia) 
 
 
Children 
(<18 
years) 
Patients  
- 
Written 
informed 
consent 
obtained from 
the 
parents 
and 
participant 
if 
she/he 
is 
older than 6 
years old 
- 
No access to 
the olfactory 
cleft  
- 
Abnormal 
endoscopic 
finding (i.e. 
meningocele, 
vascular 
ectasia) 
 
 

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Controls 
- 
Written 
informed 
consent 
obtained from 
the 
parents 
and 
participant 
if 
she/he 
is 
older than 6 
years old 
- 
No access to 
the olfactory 
cleft  
- 
Abnormal 
endoscopic 
finding (i.e. 
meningocele, 
vascular 
ectasia) 
 
 
 
Endpoint #3 
Study population 
Inclusion criteria 
Exclusion criteria 
Adults 
Patients  
- 
Having 
a 
suspicion of or 
a 
confirmed 
telomeropathy
- 
Ability 
to 
provide 
written 
informed 
consent 
 
- 
History of 
neurological or 
psychiatric 
disorder known 
to interfere with 
olfactory 
function 
- 
History of 
olfactory trouble 
(postinfectious, 
posttraumatic, 
toxic) or chronic 
rhinosinusitis 
Controls  
- 
Ability 
to 
provide 
written 
informed 
consent 
- 
History of 
neurological or 
psychiatric 
disorder known 
to interfere with 
olfactory 
function 
- 
History of 
olfactory trouble 
(postinfectious, 
posttraumatic, 
toxic) or chronic 
rhinosinusitis 
Children 
Patients  
- 
Age: 6-18 
- 
Written 
informed 
consent 
obtained from 
the 
parents 
and 
participant  
- 
History of 
neurological or 
psychiatric 
disorder known 
to interfere with 
olfactory 
function 
- 
History of 
olfactory trouble 
(postinfectious, 

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posttraumatic, 
toxic) or chronic 
rhinosinusitis 
 
Controls  
- 
Age: 6-18 
- 
Written 
informed 
consent 
obtained from 
the 
parents 
and 
participant  
- 
History of 
neurological or 
psychiatric 
disorder known 
to interfere with 
olfactory 
function 
- 
History of 
olfactory trouble 
(postinfectious, 
posttraumatic, 
toxic) or chronic 
rhinosinusitis 
 
 
 
7.2.  Discontinuation criteria  
 
The subject may be excluded for the reasons given in section 7.1 of this 
protocol. The excluded subjects will be replaced. 
Participants and/or their parents will be informed of the fact that they have the 
right to withdraw from the experiment at any time and for any reason, without 
prejudice, and without being obliged to give the reasons for doing so.  
In the interest of the subject’s health and well-being, the investigator may 
withdraw the subject at any time. In addition, the subject may be withdrawn for 
any of the following reasons: administrative decision by the investigator, 
ineligibility, subject non-compliance with study requirements, and occurrence 
of any adverse event which results in the inability to continue to comply with 
study procedures.  
The reason for withdrawal will be recorded. If the subject is withdrawn due to 
an adverse event, the investigator will arrange for follow-up visits or telephone 
calls until the adverse event has resolved or stabilized. 
 
8. SAFETY 
 
8.1.  Adverse event 
 
An adverse event is defined here as a harmful and unintended occurrence in a 
subject participating to the study. All adverse events will be reported from the 
time a signed and dated informed consent form is obtained, until completion of 
the last study-related procedure.  
 
8.2.  Serious adverse events 
 
A serious adverse event is an unexpected occurrence that (1) results in death, 
(2) is life-threatening, (3) requires hospitalization or prolongation of existing 
hospitalization or (4) results in persistent or significant disability or incapacity. 

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Given this research project consists of blood sample and collection of cells 
originating from the olfactory epithelium through non-invasive nasal brushing, 
we do not expect any prolonged or serious adverse event.  
 
8.3.  Potential adverse events  
 
The blood sample may be perceived as slightly painful by the patient (however 
this feeling is temporary) and may result in a cutaneous hematoma.  
The nasal brushing of the olfactory cleft may be perceived as uncomfortable or 
slightly painful by the patient. However, this feeling is temporary. No adverse 
effect has been reported in the literature.  
Similarly to naso-pharyngeal swabs, it is possible that a slight bleeding occurs 
in some subjects with weakened mucosa. The risk of severe bleeding is 
extremely rare. A recent retrospective study investigating the risk of naso-
pharyngeal swabs found a rate of 8 complications (4 bleeding / 4 foreign body) 
out of a total of 643.284 patients (Koskinen et al. 2021). These complications 
were related to incorrect swabbing technique. Moreover, 1 study report CSF 
leak after naso-pharyngeal swab, occurring in a patient with meningo-
encephalocele (Sullivan et al. 2020). Because of the rarity of such skull base 
malformation and the fact that a nasal endoscopy will systematically be 
performed, we can assume that the risk of CSF leak is negligeable. Importantly, 
brushing will be performed by trained ENT specialists, ensuring a as atraumatic 
as possible brushing; and patients will be screened for factors that may 
predispose to severe bleeding. Moreover, if a bleeding occurs, adequate 
medical intervention will be ensured.  
Additionally, one study demonstrated the safety of biopsy of olfactory 
neuroepithelium for the olfactory function (Andrews et al. 2016). Therefore, we 
can assume that the risk of olfactory dysfunction following a poorly invasive 
nasal brushing of the olfactory cleft is extremely low, especially since the 
procedure will be performed unilaterally. However, in the very unlikely event 
that a subject complains of olfactory dysfunction, specific medical measures 
will be taken. 
Importantly, to avoid the discomfort due to blood sample and nasal brushing in 
pediatric population, these samplings will be performed during a general 
anesthesia that is planned for the routine medical management of these 
participants (eg. telomeropathy patients: marrow puncture; pediatric controls: 
tonsillectomy). 
 
8.4.  Reporting procedures for all adverse events 
 
All adverse events occurring during the study observed by the investigator or 
reported by the subject, whether or not attributed to the study, will be recorded. 
The following information will be assigned by the investigator: description, date 
of onset and resolution, severity, assessment of relatedness to the experiment, 
and action taken. The investigator may be asked to provide follow-up 
information. All related adverse events that result in a subject’s withdrawal from 
the experiment will be followed up until a satisfactorily resolution occurs. 
 
8.5. 
Serious adverse event reporting procedures 
 

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A serious adverse event occurring to a research participant will be reported to 
the Ethics Committee that gave a favorable opinion of the study. Reports of 
related and unexpected serious adverse events will be submitted within 15 days 
after becoming aware of the event. 
All serious adverse events that have not resolved by the end of the study, or 
that have not resolved upon discontinuation of the subject’s participation in the 
study will be followed until any of the following occurs: (1) the event resolves, 
(2) the event stabilizes, (3) the event returns to baseline, if a baseline value is 
available, (4) the event can be attributed to agents other than the experiment, 
(5) when it becomes unlikely that any additional information can be obtained 
(subject or healthcare practitioner refusal to provide additional information, lost 
to follow-up after demonstration of due diligence with follow-up efforts). 
Any event requiring hospitalization that occurs during the course of a subject’s 
participation in a clinical study will be reported as a serious adverse event 
except hospitalizations for (1) social reasons in absence of an adverse event, 
(2) surgery or procedures planned before entry into the study, (3) clinic protocol 
procedures. 
All of the medical investigators of this project are medical practitioners 
registered to l’Ordre des Médecins/Orde der Artsen. It will be left to their 
judgment to decide whether or not an adverse event is of sufficient severity to 
require the subject’s removal from the study. A subject may also voluntarily 
withdraw from the study due to what he or she perceives as an intolerable 
adverse event. 
 
9. DATA ANALYSIS  
 
Blood samples collected at CUSL will be analyzed at the laboratory of the 
Cliniques universitaires Saint-Luc, where leukocyte telomere length will be 
analyzed using a validated Flow-FISH technique (Prof. P. Saussoy, head of 
biologogical hematology department), developed by Prof A. Decottignies 
(UCLouvain).  
 
Cellular material harvested through the nasal brushing will be processed at the 
laboratory of Prof. A. Decottignies (UCLouvain) and at the laboratory of 
Genetics of CUSL (Prof. A. De Leener). 
 
 
10. DIRECT ACCESS TO SOURCE DATA/DOCUMENTS 
 
The investigator will allow trial-related monitoring, audits, and regulatory 
inspections, and provide direct access to source data and documents to 
authorized auditors and monitors.  
 
11. ETHICS 
 
11.1.  Declaration of Helsinki 
 
The investigator will ensure that the study is conducted in full conformity with 
the current revision of the Declaration of Helsinki (last amended October 2000, 
with additional footnotes added in 2002 and 2004). 

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11.2.  Informed consent 
 
A written version of informed consent form will be presented to the subject and 
/or her/his parents detailing: the exact nature of the experiment, the implications 
and constraints of the protocol, the known side effects, and any risks involved 
in taking part. It will be clearly stated that the subject is free to withdraw from 
the study at any time for any reason without prejudice to future care, and with 
no obligation to give the reason for withdrawal. 
The subject and/or her/his parents will be allowed as much time as wished to 
consider the information, and the opportunity to question any or all investigators 
listed in the ethics application, their general practitioner or other independent 
parties to decide whether they will participate in the study. 
Written Informed Consent will then be obtained by means of subject’s dated 
signature and/or parent’s dated signature, signature of the person who 
presented the informed consent and, if different, the principal investigator. 
A copy of the signed Informed Consent will be given to the subject. The original 
signed form will be retained at the study site. 
 
11.3.  Independent ethics committee 
 
A copy of the protocol, the informed consent form, subject information sheet, 
and any proposed advertising material will be submitted to an Independent 
Ethics Committee for approval. 
The principal investigator will submit and, when necessary, obtain approval 
from the Committee for all subsequent protocol amendments and changes to 
the informed consent document. 
The investigator will notify substantial amendments to the protocol and will 
notify the Committee of these in accordance with local procedures. 
 
11.4.  Subject confidentiality  
 
The investigator will ensure that the subject’s anonymity is maintained. The 
subject will be identified by a subject ID number. As described in section 14, all 
documents will be stored securely and kept in strict confidence. 
 
12. DATA HANDLING AND RECORD KEEPING 
 
All the data will be pseudonymized thanks to the use of a specific identification 
code. The subject will be identified through this identification code, which will 
depend on the study, on the group of subjects and on the inclusion order. 
Pseudonymized data will be recorded in a protected RedCap database.  
Data protection will be ensured by the law of the July 30, 2018 relating to the 
protection of privacy and by Belgian and European regulations (European 
General regulation on the protection of personal data (GRPD) of May 25, 2018). 
These rights will also be guaranteed by the law of April 22, 2002 relating to the 
rights of the patients. 
 
 
13. FINANCING AND INSURANCE 

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The study will be funded by grants obtained by Prof. A. Decottignies and Prof. 
C. Huart.  
Blood sample analysis and genetic analysis (whole-exome sequencing) will 
only be performed in patients. For patients involved in the current study, Flow-
Fish analysis is already performed in clinical routine as standard of care 
procedure. Similarly, genetic analysis on cellular material is considered as a 
standard of care for patients with suspicion of telomeropathy. Therefore, these 
analyses will not be associated to additional costs, neither in term of material 
of personal.  
Indemnity for negligent and non-negligent harm will be provided by a specific 
insurance provided by the Cliniques universitaires Saint-Luc.  
Company: 
Police number: 
 
14. PUBLICATION POLICY 
 
The Principal Investigator will coordinate dissemination of data from this study 
in the form of publications in peer reviewed scientific journals, conference 
presentations, and access to raw data. In addition, research participants who 
are interested in directly acquiring detailed information will be encouraged to 
contact a named investigator listed on this application. 
 
15. BIOBANKING 
 
After analysis of telomere length in blood at CUSL, human samples (blood and 
cells) will be kept frozen at UCLouvain (de Duve Institute, room 75.01.5817).  
Samples will be kept for a maximal duration of 10 years, and will then be 
destroyed.  
Secondary use of human body material: Patients are informed that if, within 
the course of this study, we want to do additional analyses, not included in the 
present protocol, on the remaining material, this will be done after requesting 
the opinion of the ethical committee.  
  
 
 

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