Clinical Determinants of Disease Progression in Patients With Limb Girdle Muscular Distrophy Type 2E
Conditions: LGMD2E
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

CLINICAL DETERMINANTS OF DISEASE 
PROGRESSION IN PATIENTS WITH LIMB GIRDLE 
MUSCULAR DYSTROPHIES TYPE 2E
Version 1.0, 24/04/2020

CLINICAL  DETERMINANTS  OF  DISEASE  PROGRESSION  IN  PATIENTS  WITH  LIMB 
GIRDLE MUSCULAR DYSTROPHIES TYPE 2E
Acronym: NeuroLGMD2E
Sponsor: Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Via Sforza 28, 20122 
Milano, Italia
Coordinator  Center:  Neurology  Unit,  Fondazione  IRCCS  Ca’  Granda  Ospedale  Maggiore 
Policlinico, Via F. Sforza 35, 20122 Milano, Italia 
Main Researcher: Dr. Yvan Torrente
Version number: v.1.0 
Data: 24/04/2020

Index
1. Title 
2. Abbreviations 
3. Responsibility 
4. Amendments and modifications of the protocol
5. Timelines 
6. Rational and background 
7. Research query and objective
8. Methods 
8.1. Study design 
8.1.1. Primary endpoint 
8.1.2. Secondary endpoint
8.2. Setting 
8.2.1. Study population 
8.2.2. Inclusion criteria 
8.2.3. Exclusion criteria
8.3. Variables 
8.4. Source documents
8.5. Cohort dimensions 
8.6. Data management 
8.7. Data analysis 
8.7.1. Primary endpoint: analysis 
8.7.2. Secondary endpoint: analysis
8.7.3. Ad interim analysis 
8.7.4. Further analysis 
8.8. Quality control 
8.9. Limitation to the study 
8.10. Other aspects
9. Protection of included subjects
9.1. Information sheet and data treatment consent 
9.2. Assurance 
10. Plan of scientific divulgation and popularization
11. Funding
12. Bibliography
Annex 1. Other documents not included in the protocol

2. Abbreviations 
6MWT = Six Minutes Walking Test 
AHI = Apnea Hyponea Index 
ALT= Alanina Aminotransferasi 
ALP= Alkaline Phosopatase 
AST= Aspartato Aminotransferase 
BNP= Brain Natriuretic Peptide 
CPK= Creatine phosphokinase 
CPT= Lung Total Capacity 
DMD= Duchenne Muscular Dystrophy 
ECG= Elettrokardiogram 
FC= Cardiac Frequency
FE= Ejection Fraction of left ventricle 
FEF = Forced Expiratory Flux 
FEV1= Forced Expiratory Volume 
FVC = Forced Vital Capacità
GGT= Gamma Glutamiltransferase 
GSGC= Gait Stair Gleason Chair 
INR= International Normalised Ratio 
LDH= Lactic Dehydrogenase 
LGMD= Limb Girdle Muscular Dystrophy  
MEF= Maximal Expiratory Flux 
MEP= Maximal Expiratory Pressure
MFM= Motor Function Measure 
MIP= Maximal Inspiratory Pressure
MMEF= Mean Maximal Expiratory Flux
MOC= Mineralometry Bone Computed 
MVV= Maximal voluntary Ventilation
NSAA= North Star Ambulatory Assessment 
ODI= Oxygen Desaturation Index 
PEF= Peak of Expiratory Flux
PTH= Parathormone 
QMT= Quantitative Muscle Testing 
RM = Magnetic Resonance
RX= radiography
VIT D= Vitamin D 
VR= Residual Volume 
3. Responsibility (role of the study promoter and of researchers) 
Main researcher: Prof. Yvan Torrente Collaborators: Dott.ssa Giulia Marchetti, graduated with Prof. 
Torrente and now attending the first year of school in medical genetics at the Fondazione IRCCS Cà 
Granda, Ospedale Maggiore Policlinico. She will collect clinical charts and report clinical data to 
analyze in a excel file that will be used for the statistics. Prof. Giacomo Comi, Director of the 
Neuromuscular and rare Disease Unit 
4. Emendamenti ed altre modifiche al protocollo Not relevant. 
5. Timelines Planning: start of data collection: June 2020. End of data collection: September 2020 
Final report: December 2020 
6. Rational and background 
LGMD2E is a rare type of a hereditary muscular dystrophy transmitted in an autosomic recessive 
manner. This disease is determined by the presence of mutations involving the Beta Sarcoglycan 
gene, coding for a protein which is involved in the stabilization of the glycoprotein complex 

associated to dystrophin (1). The main role of beta Sarcoglycan only partly explains the severity of 
the clinical phenotype reported in these patients compared with other LGMD (2). LGMD 2E debuts 
in early childhood, involving skeletal muscles with a progressive centripetal trend that in last stages 
affects also respiratory and cardiac muscles (3). The early onset and the severity of the disease 
affecting cardiopulmonary apparatus, make this disease very close to the more famous and studied 
Duchenne Muscular Dystrophy (4). More than 60% of LGMD 2E patients suffer from heart failure 
(5), not less common is the involvement of respiratory system which insufficiency represents the 
main cause of death in these patients (6). Unfortunately, because of its rareness, literature is poor of 
data about this disorder and no natural history study have ever been reported until now. Lacking 
similar studies, therapeutical and clinical management is not easy and often based on studies 
investigating other subtypes of muscular dystrophies, often more benign. 
Furthermore, we have little knowledge about possible genotype-phenotype correlations for this 
disease (7). We do believe that a natural history study might help us to improve our knowledge of 
this condition’s evolution, leading to the design of specific guidelines for the follow up of these 
patients, both under the therapeutical and clinical point of view. 
Guidelines  will  uniform  the  clinical  approach  to  this  disease, both on the  National  and the 
international  scenario,  and  will  improve  the  use  of  sanitary  resources.  Furthermore,  with  a 
phenotype-genotype correlation study, it will be possible to recognize those patients that are more at 
risk to develop a more severe disease.  
7. Research query and objective 
The lack of data on natural history of this rare disease and the need to define guidelines for the best 
follow up of these patients, compelled us to set up this study. In this study any investigation 
performed in patients affected by LGMD 2E will be considered with the aim to recognize the 
progression of the disease and those variables that are more useful and precise in describing its 
evolution. Primary objective of the study is to obtain more and better knowledge of this rare group 
of LGMD, describing its natural history through the analysis of clinical charts of the more affected 
patients possible. 
To select clinical variables that are more useful to define the evolution of the disease and to identify 
any possible differences in clinical presentation that might be due to differences in genotype-
phenotype correlation. Secondary objective of the study is to demonstrate the real impact of 
cardiopulmonary impairment on the prognosis of these patients. 
Thank to these knowledges, to lead to a better therapeutic approach to this disease, stimulating the 
development of new drugs.
8. Methods
8.1 Study design
Retrospective observational monocentric study, aimed to investigate the natural history of a rare 
disease like the LGMD 2E 
8.1.1 Primary endpoints 
To describe the natural history of the LGMD 2E 
To define of the clinical variables that more strongly relate with the evolution of the disease
To identify any possible correlation between variables
To identify any possible correlation between variables and clinical outcomes 
To identify any possible correlation between phenotype and genotype
8.1.2 Secondary endpoints 
To define the better follow up routine for LGMD 2E patients in view of possible genetic differences 
To define those therapies that are able to slow the progression of the disease 
To ameliorate the use of sanitary resources for the management of these chronic patients.  
To gain new knowledges that could be used for the development and the management of future new 
therapies. 
8.2 Setting 

Patients followed by the ambulatory of neuromuscular disorders of Centro Dino Ferrari, Padiglione 
Monteggia of Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico (Unit of Neurology), 
will be recruited during the ordinary follow up routine or will be reached out to participate to this 
study. Furthermore, thank to the sponsorship of Gruppo familiari beta sarcoglicanopatie (GFB), 
more patients that are in the network of this association will be reached out from the PI of the study 
and eventually included.  Data will be provided by patients themselves during the visit at the room 
number  17  of  Padiglione  Monteggia  of  Fondazione  IRCCS  Ca’  Granda  Ospedale  Maggiore 
Policlinico (Unit of Neurology). If the patients won’t be able to move and will be interested in 
participating to the study a collaborator of the main researcher will reach the patients at his 
residence to recruit him. Main source of data will be the clinical chart of the patient and all his 
clinical documents assessing any operation, ascertain, visits performer through the years until this 
moment. Main centre will be the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico 
(Unit of Neurology). Data collection will last six months and later data will be studied. 
8.2.1 Study population 
Studied  population.
 The  association  GFB  (GRUPPO  FAMILIARI  BETA-
SARCOGLICANOPATIE) is a group of families of patients affected by beta-sarcoglycanopathy 
and other muscular dystrophies. This association has been created by few families resident in 
Lombardia and now it includes people from all over Italy. The study will be sponsored by GFB that 
will inform its associated about this study. Patients interested in participating to the study will reach 
out the PI of the study to be eventually included. 
Furthermore, patients affected by LGMD 2E followed at the Fondazione IRCCS Ca’ Granda 
Ospedale Maggiore Policlinico (Unit of Neurology) and that have previously signed an informed 
consent for the use of their data for scientific research will be included. Data from this second group 
of patients will already be available in databases created in our hospital for previous scientific 
researches.
8.2.2 Inclusion criteria 
Will be included only those patients, both adults and minors, that have a genetical diagnosis of 
LGMD 2E and that will sign the specific informed consent for data management. 
8.2.3 Exclusion criteria 
Th lacking subscription of informed consent and or the lack of genetic diagnosis represent the only 
exclusion criteria. 
8.2.4 Patient participation 
Patients might be asked t ogive to their physician their clinical charts in order to evaluate the visits 
and investigations performed through the years, also in other hospitals. 
8.3 Variables 
Will be extract from the clinical charts of these patients, after the sign of the informed consent for 
the data management, only retrospective data (therefore all those data older than the day of the 
recruiting visit). 
These data will be reported in a excel file specifically created. On this file data will be anonymized 
through a patient identification code that will be decrypted in another separate file.  For this file will 
be considered the following data: 
a) Dati anagrafici: 
1. Anonymized name
2. Sex
3. Age
Form 2- DIAGNOSIS: 
1) Age at onset
a) clinical presentation
b) clinical progression
2) CK at onset
3) EMG

4) Biopsy:
a) Histology
b) Immunohistochemistry
c) Western Blot
5) Genetic:
a) In frame del/dupl
b) Out of frame del/dupl
c) Missense
d) Nonsense
Form 3- CLINICAL DATA AND TREATMENTS:
Hospitalization: Data – Cause – Frequency  Surgery: Data – Complications  Fractures: Data – 
Frequency – Location  Fell: Data – Frequency  Emergency Room Accesses: Data – Cause
APPARATUS
Skeletal-Muscle
1) Clinical tests:
NSAA  MFM  QMT  Brook Scale  Egen Klassification  Barthel Index  Walton and 
Gardner &  Medwin   GSGC  Grip  Strength  (quantitative)  Handheld  Dynamometry 
Performance of the Upper Limb Module for DMD 2.0  10m Walk: 1- normal, 2- slightly impaired- 
3 moderately impaired- 4- impaired- 5 with device - 6 not walking but stands- 7 wheelchair 
6MWT  time to rise from the floor  Climb 4 stairs (1 no help - 2 one hand on the leg – 3 two 
hands on the legs - 4 stand with help -5 Handrail - 6 hardly climbs- 7 Not able). 10m Run  
Time to rise from a chair (1 Normal - 2 with difficulties -3 helping with one hand - 4 Helping with 
two hands - 5 other helps - 6 Impossible). Time to stand from the floor (1 Normal - 2 first rise the 
pelvis and then one hand on the floor - 3 First rise the pelvis and then two hands on the floor - 4 
One hand on the leg - 5 Two hands on the legs - 6 Other help - 7 Impossible). 
Neuromuscular evaluation (date):  maximal voluntary strength: bilateral knees extension, Isometric 
contraction test, bilateral knee flexion, Workspace volume  
2) Exams:
CK*Muscular RMN
3) Symptoms:
Loss  of  independent  ambulation*  Calf  hypertrophy  (Unilateral/Bilateral,  if  bilateral  → 
Symmetrical/Asymmetrical)Calf
 
atrophy :
 
Unilateral/Bilateral,
 
if
 
bilateral
 
→ 
Symmetrical/Asymmetrical) Tendon  contractures*Scoliosis  Scapular  winging* Tiptoe  gait 
pattern*  Muscle  pain*  Muscular  trophism*  Musvular  wekness  (Symmetrical/Asymmetrical  - 
Proximal/Distal - Progression: chronic/acute)* Muscle contractures (Unilateral/Bilateral - site  
Cramps: Severity – Frequency)  Clubfoot (Unilateral/Bilateral) Spine anomalies Ability to walk 
on  toes Fasciculations Percussion  induced  muscular  contractions Macroglossia  (yes/no)* 
Dysphagia(yes/no,  Data  onset,  Progression  chronic/acute,  Severity->  light/moderate/severe, 
frequent aspirations:yes/no) 
4)Muscules involved: shoulder* arms* forearms* hand* spine pelvic muscules* thigh* leg* feet  
5)Wheelchair: yes/no; if yes → changed through the years – still using yes/no – how long-other 
ambulatory devices yes/no; if yes → name → still using yes/no – how long – has it been changed 
through the years?  
6)Deambulation and motility: general comments  Electromyography
Cardiovascular
1)Evaluations (data): general conditions: using devices to support heart function (eg. pacemakers, 
ICD, etc.) - name still using? – How long? - has it been changed through the years?  
2)Conduction system anomalies: 

EKG   Holter EKG* Echocardiogram* events monitoring 
3)Cardiomyopathy Heart RM  
4) Simptoms: basal rattling* Oedemas* slopes edemas   Holter  Echocardiogram: Description* 
FE*  Left  ventricle  telediastolic  Volume*  Left  ventricle  telediastolic  diameter Arythmies 
Coronaropathy  Valve  Pulmonary hypertension Palpitations* Syncope almost syncope  
Respiratory
1) Evaluations (data):  
2) Respiratory support:  Invasive/ Noninvasive  Cough assist  Cough peak  Overnight 
BiPAP   Other  
3) Respiratory status  Pulsoxymetry/polysomnography (mean SpO2 - SpO2 minimal - FC 
mean - AHI – ODI – % MVV – MIP – MEP.  Dispnoea: supine position– stress induced – 
at rest 
4) Quality of sleep: daily somnolence not restorative sleep frequent overnight awakening 
morning headaches*  daily fatigue 
Laboratory Tests 
1)Imaging tests (data): RX thorax  RX spine* RM muscle  
2) Blood tests: Glucose* Proteins* Hemoglobin* White blood cells* Neutrophils INR* RATIO* 
CPK* LDH* Amilase* Cistatine C  AST * ALT * ALP * GGT * Bilirubin total/direct/ indirect 
Creatinine  Electrolytes  (Sodium  –  Potassium  -  Clorum  –  Calcium  total/ionized)  Urine 
(macroscopic view - blood (Yes/No) – anomalies)  
3) Bone status: MOC - PTH pg/mL - VIT D1,25 pmol/L-VIT D 25 nmol/L  
4)Glycemic state: Insuline - Hb1aC  Pro BNP 
Autoimmune disease  
Name 
Treatment 
Therapy 
1) Name - posology – data – rationale
2) Prescriptions  
3) Pain therapy  
4) Steroids
8.4 Data sources 
Data used to collect variables will include any clinical document provided by patients or available 
in our hospital thank to the server of the Unit of Neurology. Only official clinical charts will be 
considered.  Clinical  outcomes  will be defined as  the age of loss of ambulation,  the age of 
introduction of cardiological supporting drugs and the age of introduction of respiratory supporting 
devices/therapies.   These ages will be directly obtained from the clinical reports ( on report it is 
reported the age of the patient) of indirectly ( according to the data of the report that describes the 
first introduction of the drug/devices or that firstly reports the loss of the ambulation) 
8.5 Cohort dimension 
Expected  cohort dimension is  of 33 patients  that  is the number  of patients  followed at the 
Neurological Unit in the last 10 years. Statistical analysis will be based on generalized linear 
regression models, suitable both for continuous variables both for independent ones. Variables with 
a nonlinear distribution will be used only in logarithmic scale.  Only data corresponding to yearly 
visit (at least two data in two years) will be used for the linear correlation analysis and in 
multivariate  correlation  models.  Furthermore,  to  verify  the  accuracy  of  clinical  variables  in 

describing the evolution of the dystrophy, variables will be coupled and compared  by a bivariate 
analysis. 
8.6 Data management
Data will be managed on a specific database created in a excel file, defended by a password.  The 
file won’t include any personal data and patients will be identified through a pseudonym that could 
be decrypted only through the access to a second file, defended by a different password.
 8.7 Data analysis 
Analysis will be performer using generalized linear regression models suitable both for continuous 
variables both for independent ones. Variables with a nonlinear distribution will be transformed in 
logarithmic scale before the analysis to be performed.  For any analysis data will be divided 
according to the age of the patient at the moment of the evaluation. Data will be analyzed with 
linear correlation both compared to the variable age both in bivariate models and multivariate 
models.  Results  obtained  will  be  adjusted  for  confounding  factors  not  related  to  muscular 
dystrophy. 
Data of included patients will be globally studied and the divide in two groups homogenous for 
genotype. 
8.7.1 Primary endpoints: analysis
 -To obtain a variables distribution according to patients’ age and to verify possible correlations 
with the disease’s progression. 
-To study possible correlation between variables identified. 
-To evaluate possible connections with clinical outcomes
8.7.2 Secondary endpoints: analysis 
-Identify those variables that should be kept in consideration during the follow up of these patients
-To identify the variables that better describe disease progression to start a promptly therapeutic 
diagnosis
-To possibly identify useless analysis and examinations in these patients
8.8 Quality control 
Data quality will be assessed by the reliability of clinical charts examined that will be divided 
according to type of assessment and examination performed in national clinical centres and/or 
private hospitals.
Thereafter reliability of data according to patients’ history and to normal range values will be 
evaluated.  Statistical analysis will enable to validate also the reliability of these data thanks to the 
use of programs of bi-multivariate analysis, biased for factors that do not relate to dystrophy. 
8.9 Limitation to the study  
The limitations to this study relied in the rareness of this condition and therefore in the risk to have 
so few data that we won’t be able to reach a statistical significance. Whenever few patients will 
participate to the study, we won’t be able to get enough data to perform trustable analysis. 
Another limitation is represented by the little knowledge we have nowadays about the best clinical 
variables to study and about the natural history of this condition. Therefore, we will analyze 
variables that will eventually prove to be not reliable. 
Furthermore, the lack of data about the natural history of this disease won’t allow us to provide 
reliable information about the real impact of therapies on the disease progression. 
9. Protection of included patients 
This study will be performer according to Good clinical practice standards and to ethical principles 
derived  from  the  Helsinki  declaration  and  to  standing  law  for  observational  studies.  The 
observational study and the linked documentation will be presented to Ethical Committee of 
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico. The study will start only once 
received the required authorizations according to the Centre’s procedures. The ethical committee 

will also approve any change to the study protocol and recruiting modalities to involve patients in 
the study, according to local laws. 
9.1 Information sheet and data treatment consent
The present study will follow the law number law number 101 of August 10 2018 regarding the 
standing UE regulation 2016/679 of European Parliament and Council of April 27 2016, describing 
the protection of persons under the point of view of personal data protection and free circulation of 
data. Stands the duty to collect informed consent to data treatment of included patients in any case 
whenever it can be possible to inform patients adequately whenever they arrive to the Hospital 
during control visits. 
9.2 Assurance
As the study is an observational one, no insurance policies are needed beyond those already 
provided for common clinical practice. 
10. Plan of scientific divulgation and popularization
The PI of the study will write a final report and publish the results at the end of the study through 
the publication o fan article or through scientific communication at National or International 
congresses.  Data will be published anonymized and will be presented in digest mode. 
11. Bibliography
1. Ozawa E et al. 1998. From dystrophinopathy to sarcoglycanopathy: evolution of a concept of 
muscular dystrophy. Muscle Nerve. 1998. 
2. Shi W, et al. Specific assembly pathway of Sarcoglycan is dependent on beta- and delta-
Sarcoglycan. Muscle Nerve. 2004, Vol. Mar ;29, (3):409-19. 
3. Thompson R, Straub V. Limb-girdle muscular dystrophies - international collaborations for 
translational research.. Nat. Rev. Neur. 2016 May, Vol. 12(5), 294-309. 
4. Schade van Westrum SM, et al. Cardiac involvement in Dutch patients with sarcoglycanopathy: a 
cross-sectional cohort and follow-up study.. Muscle Nerve. 2014 Dec; Vol. 50(6):909–13. 
5. Helle Petri, et al. Progression of cardiac involvement in patients with limb-girdle type 2 and 
Becker muscular dystrophies: A 9-year follow-up study. International Journal of Cardiology. 2015, 
Vol. Volume 182. 
6. Politano L, Nigro V, Passamano L, Petretta V, Comi LI, Papparella S,et al. Evaluation of cardiac 
and respiratory involvement in sarcoglycanopathies.. Neuromuscul Disord. . 2001 Mar; , Vol. 
11(2):178–85. 
7. Francesca Magri MD et.al The Italian LGMD registry: relative frequency, clinical features, and 
differential diagnosis . Muscle Nerve. . 2017 Jan;, Vol. 55(1):55-68.