Multispectral Optoacoustic Tomography for Advanced Imaging of Centronuclear Myopathy
Conditions: Centronuclear Myopathy
Sponsor: University of Erlangen-Nürnberg Medical School

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MOSAIC: Multispectral Optoacoustic Tomography for Advanced Imaging of 
Centronuclear Myopathy 
 
16.04.2025 

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Table of contents 
 
1. Study Title, Version, Date of document ........................................................................ 3 
2. Project Summary ....................................................................................................... 4 
3. Responsibilities ......................................................................................................... 6 
4. Scientific background ................................................................................................. 7 
5. Study objectives .......................................................................................................11 
6. Study design ............................................................................................................13 
7. Target values ...........................................................................................................14 
8. Study Population ......................................................................................................15 
9. Course of study ........................................................................................................18 
10. Benefit Risk Assessment .........................................................................................20 
11. Biometrie ...............................................................................................................22 
12. Data management and data protection .....................................................................23 
13. Telemedic Procedures .............................................................................................24 
14. Biological samples ..................................................................................................25 
 

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1. Study Title, Version, Date of document 
Study title 
Multispectral Optoacoustic Tomography for Advanced Imaging of Centronuclear Myopathy 
Acronym: MOSAIC 
 
Version 
1.2 
 
Date of document 
16.04.2025 
 
Protocol version 
Date 
Version Status 
Changes 
21.03.2025 
1.0 
First draft  
Lina Tan 
21.03.2025 
1.1 
Corrected figure  
Lina Tan 
16.04.2025 
1.2 
Amendment 
Lina Tan 
 

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2. Project Summary 
Centronuclear myopathy (CNM) belongs to the group of congenital myopathies. It is named after 
its histopathological feature: the nucleus is located in the center of the muscle cell instead of 
its physiological position at the periphery. CNM is extremely rare; only epidemiological data for 
the broader group of congenital myopathies are available. The incidence is estimated at 0.06 
per 1,000 live births. 
 
CNM is genetically and clinically heterogeneous. Identified gene mutations affect, among 
others, genes encoding proteins involved in membrane remodeling, transport, and excitation-
contraction coupling (Reumers et al. 2021).  
Patients with CNM typically present in early childhood with muscle weakness and hypotonia. 
The severity of the disease varies depending on the underlying genotype and ranges from 
reduced exercise tolerance and ptosis to floppy infant syndrome and respiratory failure. One 
example of a severe disease course is the X-linked form caused by an MTM1 nonsense 
mutation (XL-MTM). Affected individuals become symptomatic in the neonatal period and usually 
die during childhood or adolescence. In contrast, patients with DNM2 mutations show a later 
onset and a milder progression. Among the few reported patients with BIN1 mutations, the 
onset occurs in infancy or adulthood, with a moderate disease course. RYR1-associated CNMs 
also vary in terms of age of onset and disease severity (Reumers et al. 2021; Fattori et al. 
2015).  
 
Diagnosis is based on molecular genetic testing and muscle biopsy. However, these methods 
are not widely available, time-consuming, and invasive. In young patients, anesthesia is usually 
required. Multispectral optoacoustic tomography (MSOT) allows the detection of specific 
endogenous chromophores such as collagen, myoglobin, and hemoglobin using a non-invasive 
approach comparable to conventional ultrasound. Instead of sound waves, MSOT uses near-
infrared light pulses, which are absorbed by the tissue, leading to thermoelastic expansion of 
specific molecules. This expansion generates ultrasound waves that are detected by the same 
device. Multispectral illumination and signal unmixing enable precise localization and 
quantification of muscle-specific subcellular structures. MSOT has already demonstrated the 
potential to visualize muscle structure and the clinical extent of muscle disease in patients with 
Duchenne muscular dystrophy, spinal muscular atrophy, and late-onset Pompe disease (LOPD), 
and to differentiate these patients from healthy volunteers. To date, no optoacoustic data on 
CNM are available. 

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The aim of this study is to gain molecular insights into muscle degeneration in CNM patients 
for the first time. To obtain a comprehensive picture of the optoacoustic characteristics of CNM, 
patients of different ages and disease severities will be recruited. Due to the rarity and small 
number of CNM cases, recruitment is challenging. Therefore, the upcoming patient meeting in 
Bad Nauheim (May 29 – June 1, 2025) will be used as an opportunity to offer study participation 
to as many patients as possible. 
As part of the study, in addition to non-invasive, radiation-free imaging, clinical-functional tests 
will be performed. These include muscle strength testing using the Medical Research Council 
(MRC) sum score and the Timed Up and Go test (TUAG; measured in seconds). For MSOT 
imaging, various accessible muscle groups will be scanned, including the paraspinal muscles 
and trapezius muscle, as well as the following bilateral limb muscles: deltoid muscle, biceps 
brachii, forearm flexors, quadriceps femoris, adductor muscles, hamstrings (ischiocrural 
muscles), triceps surae, and tibialis anterior. 
 

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3. Responsibilities 
Principal Investigator 
PD Dr. med. Ferdinand Knieling, E-Mail: ferdinand.knieling@uk-erlangen.de  
Department of Pediatrics and Adolescent Medicine 
Universitätsklinikum Erlangen 
Loschgestr. 15  
91054 Erlangen 
 
Other Participants 
Lina Tan, E-Mail: lina.tan@uk-erlangen.de 
Department of Pediatrics and Adolescent Medicine 
Universitätsklinikum Erlangen 
Loschgestr. 15  
91054 Erlangen 
 
Funding 
Internal funds of the Department of Pediatrics and Adolescent Medicine Erlangen until 
application for third-party funding. 
Provision of the examination device by Ithera Medical GmbH 
Screen4Care 

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4. Scientific background 
CNM is a neuromuscular disease characterized by the clinical features of congenital myopathy. 
They have the histopathological feature of a central nucleus and are characterized by generally 
progressive muscle weakness and atrophy (Reumers et al. 2021).  
There is genetic and clinical heterogeneity, as well as a genotype-phenotype correlation (Fattori 
et al. 2015). Mutations are known in the key genes MTM1 gene, which codes for myotubularin, 
DNM2 gene, which codes for dynamin-2 gene, BIN1 gene, which codes for amphiphysin-2 (or 
bridging integrator-1), as well as RYR1 gene, which codes for the ryanodine receptor of skeletal 
muscle, and TTN gene, which codes for titin. The pathogenesis of the disease is still poorly 
understood. It is currently assumed that the mutations lead to impaired membrane transport 
and deficient autophagy (Jungbluth and Gautel 2014). 
Depending on the genotype, CNM presents as a spectrum disease with different onset, course 
and clinical features. Onset is possible at any age: more than half of patients become 
symptomatic within the first year of life, a quarter develop the disease in childhood or 
adolescence, and around a fifth in adulthood. The main symptom of this disease is muscle 
weakness, which can either be generalized or isolated to the respiratory, swallowing and eye 
muscles. Patients with a mutation in the MTM1 gene usually have an early onset of the disease, 
a rapidly progressive course and a high mortality rate. A common symptom in these patients is 
ophthalmoparesis. In contrast, DNM2 patients show a milder course overall. These patients 
usually reach all motor milestones. The first symptoms include exercise-induced muscle pain 
and slowly progressive muscle weakness. As the disease progresses, orthopaedic 
complications as a result of the long-term muscle weakness come to the fore: scoliosis, pes 
cavus and contractures. RYR1 patients, on the other hand, are characterized by delayed motor 
milestones, whereby the severity of muscle and respiratory involvement varies greatly. 
Mutations in the TTN gene are rare, but affected patients often suffer from severe 
cardiomyopathy (Fattori et al. 2015). Respiratory muscle involvement is common and initially 
occurs at night. In addition, respiratory complications are the main cause of morbidity (Smith 
et al. 2016). 
The diagnosis of central nucleus myopathy (CNM) is based on typical histopathologic findings 
in muscle biopsy, complemented by characteristic clinical features. Magnetic resonance 
imaging (MRI) of the muscles can support clinical assessment and suggest genetic testing in 
cases with non-specific features. Genetic counseling should be offered to all patients and 
families diagnosed with CNM. Muscle biopsy shows centronuclear myopathy, i.e. centrally 

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located nuclei surrounded by a perinuclear halo of glycogen accumulations. These central nuclei 
are present in all muscles. Another common feature is type 1 fiber hypotrophy, which may 
precede the development of central nuclei. In a few fibers, there may be compensatory 
hypertrophy of type 2 fibers. Histopathologic changes may worsen over time, with marked 
increases in adipose and connective tissue described as prominent features (Jungbluth, 
Wallgren-Pettersson, and Laporte 2008).  
DNA sequencing of the exons and exon-intron boundaries of the affected genes is used for 
molecular confirmation of the diagnosis. Depending on whether a particular gene is suspected, 
the mutation analysis may first examine these suspected gene regions. If genetic segregation 
is uncertain and in cases where there is no clear genotype-phenotype correlation, all genes 
should be analyzed. RNA sequencing can be used if tissue or cultured cells from the patient 
are available, as the genes in question are ubiquitously expressed in most cell types. In 
addition, examination of RNA integrity or protein levels could reveal mutations in introns or 
regulatory sequences that may not be detected by DNA sequencing (Jungbluth, Wallgren-
Pettersson, and Laporte 2008). It is important to note that in some patients with 
histopathological findings, no causative gene mutation has yet been discovered (Smith et al. 
2016). 
In MRI studies, the musculature in CNM with DNM2 and RYR1 gene was examined with regard 
to muscular involvement. A characteristic progressive sequence was found in patients with 
DNM2 mutations: early involvement of the plantar flexor muscles (plantar flexors) subsequently 
leads to changes in the hamstrings and anterior thigh muscles (Jungbluth, Wallgren-Pettersson, 
and Laporte 2008). This sequence does not occur in cases with mutations in the RYR1 gene. 
Instead, there is earlier involvement of the muscles of the anterior thigh compartment and 
relatively selective changes in the soleus muscle (Fischer et al. 2006). Few to no MRI findings 
have been published for the other CNM mutations.  
Multispectral optoacoustic tomography (MSOT) is a promising imaging method that offers better 
availability and scan time compared to MRI.  
For MSOT, an ultrasound transducer is applied to the patient's skin, similar to a conventional 
ultrasound. Instead of sound waves, MSOT illuminates the tissue with light of transient energy, 
typically light pulses in the near infrared range, which are absorbed by the tissue and lead to 
thermoelastic expansion. This expansion generates ultrasound waves that are detected by the 
same device. Studies have already shown that MSOT-based assessment of hemoglobin levels 
in the intestinal wall has the potential to assess Crohn's disease activity (Knieling 2017, 

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Waldner 2016). With a newly configured device (Acuity Echo, iThera Medical GmbH, Munich, 
customized platform), an extended spectrum of laser light can be used, which enables the 
detection not only of hemoglobin, but also of other biomarkers such as collagen or lipids. In 
previous study, we were able to show the molecular composition of muscles in Duchenne 
muscular dystrophy using MSOT and propose to use the non-invasively measured collagen 
content as a new biomarker for disease severity (Regensburger et al. 2019). In a study on 
Pompe's disease, we were able to show that glycogen and the corresponding water binding can 
be visualized with the help of MSOT, as well as a change in the collagen and lipid profile of the 
muscle (Tan et al. 2024)  
In this study we want to investigate the use of MSOT imaging for disease detection and 
monitoring of patients with CNM for the first time and compare it with healthy volunteers. 
 
 
 
 

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Literaturverzeichnis: 
Fattori, F., L. Maggi, C. Bruno, D. Cassandrini, V. Codemo, M. Catteruccia, G. Tasca, A. 
Berardinelli, F. Magri, M. Pane, A. Rubegni, L. Santoro, L. Ruggiero, P. Fiorini, A. Pini, T. 
Mongini, S. Messina, G. Brisca, I. Colombo, G. Astrea, C. Fiorillo, C. Bragato, I. Moroni, 
E. Pegoraro, M. R. D'Apice, E. Alfei, M. Mora, L. Morandi, A. Donati, A. Evila, A. Vihola, 
B. Udd, P. Bernansconi, E. Mercuri, F. M. Santorelli, E. Bertini, and A. D'Amico. 2015. 
'Centronuclear myopathies: genotype-phenotype correlation and frequency of defined 
genetic forms in an Italian cohort', J Neurol, 262: 1728-40. 
Fischer, D., M. Herasse, M. Bitoun, H. M. Barragan-Campos, J. Chiras, P. Laforet, M. Fardeau, 
B. Eymard, P. Guicheney, and N. B. Romero. 2006. 'Characterization of the muscle 
involvement in dynamin 2-related centronuclear myopathy', Brain, 129: 1463-9. 
Jungbluth, H., and M. Gautel. 2014. 'Pathogenic mechanisms in centronuclear myopathies', 
Front Aging Neurosci, 6: 339. 
Jungbluth, H., C. Wallgren-Pettersson, and J. Laporte. 2008. 'Centronuclear (myotubular) 
myopathy', Orphanet J Rare Dis, 3: 26. 
Regensburger, A. P., L. M. Fonteyne, J. Jungert, A. L. Wagner, T. Gerhalter, A. M. Nagel, R. 
Heiss, F. Flenkenthaler, M. Qurashi, M. F. Neurath, N. Klymiuk, E. Kemter, T. Frohlich, 
M. Uder, J. Woelfle, W. Rascher, R. Trollmann, E. Wolf, M. J. Waldner, and F. Knieling. 
2019. 'Detection of collagens by multispectral optoacoustic tomography as an imaging 
biomarker for Duchenne muscular dystrophy', Nat Med, 25: 1905-15. 
Reumers, S. F. I., C. E. Erasmus, K. Bouman, M. Pennings, M. Schouten, B. Kusters, F. A. M. 
Duijkers, A. van der Kooi, B. Jaeger, C. C. Verschuuren-Bemelmans, C. G. Faber, B. G. 
van Engelen, E. J. Kamsteeg, H. Jungbluth, and N. C. Voermans. 2021. 'Clinical, 
genetic, and histological features of centronuclear myopathy in the Netherlands', Clin 
Genet, 100: 692-702. 
Smith, B. K., M. S. Renno, M. M. Green, T. M. Sexton, L. A. Lawson, A. D. Martin, M. Corti, and 
B. J. Byrne. 2016. 'Respiratory motor function in individuals with centronuclear 
myopathies', Muscle Nerve, 53: 214-21. 
Tan, L., J. Zschuntzsch, S. Meyer, A. Stobbe, H. Bruex, A. P. Regensburger, M. Classen, F. 
Alves, J. Jungert, U. Rother, Y. Li, V. Danko, W. Lang, M. Turk, S. Schmidt, M. Vorgerd, 
L. Schlaffke, J. Woelfle, A. Hahn, A. Mensch, M. Winterholler, R. Trollmann, R. Heiss, A. 
L. Wagner, R. Raming, and F. Knieling. 2024. 'Non-invasive optoacoustic imaging of 
glycogen-storage and muscle degeneration in late-onset Pompe disease', Nat Commun, 
15: 7843. 
 

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5. Study objectives 
Hypotheses 
The optoacoustic spectrum of the muscles of patients with CNM differs compared to healthy 
subjects. 
The quantitative proportion of hemoglobin/myoglobin in muscles, determined by MSOT, differs 
in patients with CNM compared to healthy subjects. 
The quantitative proportion of oxygenated/deoxygenated hemoglobin in muscle, as determined 
by MSOT, differs in patients with CNM compared to healthy volunteers. 
The quantitative proportion of lipid signal in muscle, as determined by MSOT, differs in patients 
with CNM compared to healthy subjects. 
The quantitative part of the collagen signal in the muscles, determined by MSOT, differs in 
patients with CNM compared to healthy subjects. 
There is a correlation between the signal derived by MSOT and the clinical condition of patients 
with CNM. 
Primary objectives 
- 
Comparison of the optoacoustic spectrum of MSOT measurements in different 
subgroups of patients with CNM and healthy controls 
Secondary objectives 
• 
Comparison of the quantitative fraction of hemo/myoglobin signal determined by MSOT 
in patients with CNM compared to healthy subjects and between subgroups. 
• 
Comparison of the quantitative proportion of oxygenated/deoxygenated hemoglobin 
determined by MSOT in patients with CNM compared to healthy subjects and between 
subgroups. 
• 
Comparison of the quantitative lipid signal determined by MSOT in patients with CNM 
compared to healthy subjects and between subgroups. 
• 
Comparison of the quantitative part of the collagen signal determined by MSOT in 
patients with CNM compared to healthy subjects and between subgroups. 
• 
Correlation of hemoglobin/myoglobin content determined by MSOT with disease 
duration/patient age. 
• 
Correlation of oxygenated/deoxygenated hemoglobin determined by MSOT with disease 
duration/patient age. 
• 
Correlation of lipid content determined with MSOT with disease duration/patient age. 

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• 
Correlation of collagen content determined with MSOT with disease duration/patient 
age. 
• 
Correlation of the hemoglobin/myoglobin content determined with MSOT with age-
dependent functional muscle function tests (MRC). 
• 
Correlation of the oxygenated/deoxygenated hemoglobin content determined with MSOT 
with age-dependent functional muscle function tests (MRC). 
• 
Correlation of the lipid content determined with MSOT with age-dependent functional 
muscle function tests (MRC). 
• 
Correlation of the collagen content determined with MSOT with age-dependent functional 
muscle function tests (MRC).  
 

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6. Study design 
Study type 
This is a cross-sectional study with a control group. 
Study arms 
Control groups 
No interventions are planned. In the evaluation, a distinction is made between the control 
group and subgroups of disease severity. All groups will receive the examinations described 
above. 
Randomization 
No randomization is planned. 
Blinding 
Due to the study setting, blinding of the study is not possible. The ¬evaluation of the data will 
subsequently be carried out in a blinded manner. Blinding of the test subjects is not 
necessary. 
 
 
 
 
Abbildung 1: course of study. 

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7. Target values  
All measurements with the MSOT and ultrasound are performed on the paraspinal muscles, 
trapezius muscle and on both sides of the deltoid muscle, biceps brachii, forearm flexors, 
quadriceps femoris muscle, ischiocrural muscles, triceps surae and tibialis anterior.8. 
Studienpopulation 
 

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8. Study Population  
Inclusion Criteria 
Patients with CNM 
- 
Confirmed diagnosis  
- 
age: at least 2 years of age 
Healthy controls  
- 
age: at least 2 years of age 
The inclusion of minors is necessary because centronuclear myopathy is very rare and usually 
begins in childhood. The onset of the disease in childhood is associated with a different and 
rapidly progressing course of the disease compared to adults. Therefore, the inclusion of 
children and adolescents is important in order to do justice to this age-specific course of 
centronuclear myopathies. The study will predominantly examine adolescents. The MSOT has 
CE certification from the age of 2 years.  
The healthy control group will be recruited from patients of the Pediatric and Adolescent Clinic 
Erlangen who have been hospitalized for a non-muscular disease. These patients are clinically 
stable. 
Exclusion criteria 
CNM patients and healthy controls 
- 
pregnancy 
- 
breastfeeding mothers 
- 
Tattoo in the area of the examination field 
- 
Subcutaneous fat > 3 cm 
Additionally for healthy controls  
- 
Other neuromuscular diseases (anamnestic) 
Number of patients  
As this is a pilot study, it is not possible to calculate the exact number of patients. The plan is 
to include a total of around 20 patients with CNM (adults, adolescents and children). However, 
this number is only a rough estimate. All patients who wish to participate should be included, 

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as refusal would not make sense from an ethical point of view. In addition, approximately 20 
subjects should be included as a control group. 
Patient recruitment 
Patients (and parents) are informed about the possibility of participating in the study. For this 
purpose, they will receive an e-mail from the study coordinator before the start of the study with 
the relevant information sheet and the contact details of the study physicians. Patients willing 
to participate can confirm their participation in advance and send the information sheet to the 
study coordinator or bring it with them on the day of the examination. At the patient day, the 
topic will be presented again by one of the study physicians. During the course of the day, 
patients (and parents) also have the opportunity to register for participation with the study staff. 
The study coordinator will then contact them so that patients (and parents) can discuss the 
study and ask questions. On the day of the examination, there will also be a personal 
information session before each examination with the opportunity to ask questions. If the 
patient is willing to participate, he/she (and parents) will be fully informed about the objectives 
and methods (in particular about the scientific/exploratory nature of the study), the benefits 
and risks as well as the revocability of participation in the study. Patients in childhood and 
adolescence are informed and educated about the study and its procedures in an age-
appropriate manner. 
In the event of incidental findings, the study participants are informed and further diagnostic 
measures are initiated if necessary. 
Healthy control recruitment 
Following patient recruitment, age- and gender-matched subjects (and parents) will be informed 
about the opportunity to participate in the study. Subjects (and parents) willing to participate 
can express their interest in the study in advance. They will then be contacted by the study 
coordinator so that subjects (and parents) can familiarize themselves with the study in advance 
and ask questions. Those interested will then receive an email with the relevant information 
sheet and the contact details of the study doctors. Subjects (and parents) willing to participate 
can confirm their participation in advance and send the information sheet to the study director 
or bring it with them on the day of the study. On the day of the examination, an additional 
personal explanation will take place before each examination. There is always the opportunity 
to ask questions. If the subject is willing to participate, he/she (and parents) will be fully 
informed about the objectives and methods (in particular about the scientific/exploratory nature 
of the study), the benefits and risks as well as the revocability of participation in the study. 

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Subjects in childhood and adolescence are informed and educated about the study and its 
procedures in an age-appropriate manner. 
In the case of incidental findings, the study participants are informed and, if necessary, further 
diagnostic measures are initiated. 

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9. Course of study 
• 
Procedure for providing information and obtaining consent  
Subjects can only be included in the study after written informed consent has been obtained. 
The written declaration of consent requires the subjects and/or parents to be informed verbally 
and in writing about the objectives and methods (including the scientific-exploratory nature of 
the study), the benefits and risks, and the revocability of participation in the study. By giving 
their written consent, the subjects declare that they agree to the collection and storage of study-
relevant data and their review by monitoring or authorities. For participants under the age of 
18, the signature of both parents is a prerequisite for inclusion in the study. If only one parent 
is present, the other parent can sign on behalf of the other parent in exceptional cases. In this 
case, it is important that information about the study has already been made available to the 
participants beforehand, so that the parents have already been able to discuss the study and 
the presumed will of the other parent to participate is therefore known. It will be clearly 
communicated to the study participant that withdrawal of consent is possible at any time and 
without any disadvantage.  
Furthermore, all study participants are informed that this study is a purely scientific study 
without any current diagnostic or therapeutic benefit. The original consent form will be kept in 
the study folder at the study site. Subjects will be given a copy of the study participant 
information and informed consent form. The study participant information and the informed 
consent form can be found in the appendix of this study protocol. In the event of an incidental 
finding, the study participants and parents or legal representatives will be informed and, if 
necessary, further clarification will be initiated. 
• 
Measures and recording of target variables 
After informing the patient/control person and the parent/legal guardian and obtaining consent, 
clinical data will be collected. This includes age, weight, height, genetic findings, date of initial 
diagnosis and clinical course. The clinical assessment scales MRC muscle function test and 
timed up and go test are then collected. The MRC muscle function test and timed up and go 
test assessment scales are then collected. 
MSOT imaging is then performed on 9 anatomical regions in all study participants. This includes 
the paraspinal musculature, as well as the trapezius muscle and additionally 7 anatomical 
regions of the limb muscles on both sides: left and right deltoid muscle, biceps brachii, forearm 
flexors, quadriceps femoris muscle, ischiocrural muscles, triceps surae, tibialis anterior. The 
examination is performed without further invasive procedures. The anatomical region can be 

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localized using the built-in B-scan sonography; the corresponding optoacoustic signals can then 
be measured. The duration per anatomical region is limited to 2 minutes; this corresponds to 
a maximum of 20 minutes. Patients can remain in a relaxed position during the examination 
without the need for breathing maneuvers or similar assistance. 
• 
Time schedule and duration of the study  
The examination of patients with CNM takes place in a designated room at the family's place 
of residence in Bad Nauheim. The examination of the healthy subjects will take place in a room 
provided for this purpose at the Children's and Youth Clinic Erlangen. A 10-minute explanation 
is provided. Approximately 5 minutes are allowed for the clinical function tests. The subsequent 
MSOT examination takes approximately 2 minutes per muscle and therefore 20 minutes in 
total. The total duration of the study is therefore 35 minutes per patient. 
• 
Total study duration 
The expected total duration consists of data collection of the patient collective in the period 
from 29.05.2025 - 01.06.2025, the data collection of the healthy collective in the period from 
01.06.2025 - 31.07.2025 and the data evaluation of approx. 6 months. The first study results 
are expected 8 months after the study day. 

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10. Benefit Risk Assessment 
Study-related risks 
According to the classification criteria for medical devices (Directive 93/42/EEC, Annex IX), the 
optoacoustic system from iThera Medical corresponds to a Class IIa laser system: 
- Active diagnostic device 
- Non-invasive 
- Temporary use (<60 min) 
The system used is CE-certified for adults and children from 2 years of age (TÜV Süd, 
07.10.2024, type designation according to imprint: MSOT Acuity Echo). A conformity 
assessment procedure to extend the use of the system is currently not intended or planned. 
This is therefore a purely scientific pilot study.  
Adherence to energy values 
Laser safety and the maximum permissible radiation dose for irradiation with laser pulses are 
regulated in the ANSI and IEC 60825 laser standards. The MSOT system complies with these 
standards and therefore remains below the MPE (maximum permissible exposure) limits for 
skin irradiation and is therefore considered safe. 
Temperature increase due to MSOT in the tissue 
Optoacoustic imaging does not lead to a significant temperature increase in the tissue. The 
absorption of a laser pulse in the tissue leads to a local temporary temperature increase of a 
few millikelvin. Depending on the duration of the examination and the patient's skin type, the 
temperature increases are typically in the range of less than one degree Kelvin. 
Histological changes in the tissue 
Histological changes in the target tissue and surrounding structures are not expected, nor have 
they been observed in previous preclinical and clinical studies. 
 
Slight, reversible redness or warming may occur on very sensitive skin; such side effects may 
be noticed by the test person or the doctor at any time; the examination may then be interrupted 
or discontinued. In any case, no irreversible damage is to be expected. In general, the near-
infrared light used in MSOT can lead to retinal damage if the eye is irradiated. To prevent this, 
test participants and examiners wear appropriate laser safety goggles during the examination. 

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As the data obtained will not be used to interpret the diagnostic results, there is no risk of 
possible misdiagnosis or misrepresentation of the data in this exploratory pilot study. 
No other risks are associated with this study and were not observed based on our own 
preliminary data. 
Study-related benefits 
The data obtained in the studies could provide important insights into the course and diagnosis 
of centronuclear myopathies. The potentially quantifiable differences could be used in the future 
as an important marker for the therapeutic response or prognosis of these diseases. Potentially 
invasive, high-risk procedures could be replaced. 
Termination criteria 
Particularly in view of the inclusion of children and adolescents, participation in the study is 
discontinued if there is any noticeable warming or reddening of the skin. The examination time 
is limited to 20 minutes, so that these events are very unlikely. 
Due to the short duration of study participation, no further discontinuation criteria are planned. 
There are no plans to discontinue the entire study. 
Statement on medical justifiability 
Based on previous experience, the risk of adverse events is considered to be extremely low.  
To date, no serious incident has been reported by us or in the literature. Most of the reported 
(foreseeable) problems were related either to the use of ultrasound gel for the examination or 
to the need to wear eye protection. The use of filter glasses also explains the reported red 
vision loss. This phenomenon was reversible within seconds.  
We hope that this method will provide an age-appropriate diagnostic tool, particularly in view of 
the inadequate non-invasive diagnostic options for assessing disease activity to date. In 
particular, it should help to support the diagnosis of TK2d, monitor the course of the disease 
and therapy and enable the initiation of early therapy. As this disease is also often progressive 
in childhood and is associated with major health restrictions, a scientific study of this group is 
essential and can only be compared with data from adults to a limited extent. Inclusion of 
pediatric patients in addition to the adult cohort is therefore necessary and medically justifiable. 

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The content of the study protocol is confidential. 
11. Biometry  
Sample size calculation 
As this is a pilot study and no information is currently available regarding the expected 
differences between the various groups, no sample size calculation was performed. The 
indicated sample size represents an estimate.  
Statistic 
Data will be presented as mean with standard deviation. Correlations will be reported using the 
non-parametric Spearman correlation coefficient (Rs). Depending on the group distribution, 
differences in means will be statistically analyzed using a non-parametric t-test (Mann-Whitney 
test) or ANOVA. For all analyses, a p-value of <0.05 will be considered statistically significant. 

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The content of the study protocol is confidential. 
12. Data management and data protection 
Data collection and storage 
The participation of individual study subjects will be documented. The principal investigator will 
maintain a separate list for identifying the participating subjects. This list will include names, 
dates of birth, examination dates, and pseudonymization codes of the subjects. The principal 
investigator is responsible for the quality of data collection and storage. Data will be stored 
exclusively on computers or specially designated network drives of the University Hospital 
Erlangen. 
Pseudonymization 
Before any scientific analysis of the data from this study, all information will be pseudonymized 
in 
accordance 
with 
the 
guidelines 
of 
the 
Federal 
Data 
Protection 
Act 
(Bundesdatenschutzgesetz). 
Data sharing 
Data sharing in this study is limited to the MSOT raw data. These will be shared in 
pseudonymized form without demographic parameters or any additional information, and only 
in physical form on drives, making it impossible to identify individual patients. The data will not 
be exchanged for analysis purposes. 
The study results will be published in anonymized form; therefore, it will not be possible to draw 
conclusions about the identities of participating individuals. The data will be stored for 10 years 
and then destroyed. 
Withdrawal and data deletion 
In the event of withdrawal of consent, data collected up to that point may still be considered 
and retained. However, study participants have the right to request their destruction, unless 
legal provisions prevent such destruction. 

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The content of the study protocol is confidential. 
13. Telemedic Procedures 
No telemedical procedures will be used. 
 

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The content of the study protocol is confidential. 
14. Biological samples  
No biological samples will be collected.