Diagnostic Yield of ACTG2 and FLNA Gene Assessment in Adult Patients With Idiopathic Chronic Intestinal Pseudo-Obstruction and Reduced or Absent Distal Esophageal Contractility
Conditions: Pseudo Obstruction
Sponsor: Marina Coletta

----------------- Sistema Socio Sanitario 
~ 
Fondazione IRCCS ca· Gronda fl'I Regione 
~ 
Ospedale Maggiore Policlinico I.lii Lombardia 
Dipartimento Area Medica 
SC Gastroenterologia ed Endoscopia 
Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
-Direttore: Prof. Maurizio Vecchi 
Diagnostic yield of ACTG2 and FLNA gene assessment in adult patients 
with Idiopathic Chronic Intestina/ Pseudo-Ohstruction and Reduced or 
Absent Distai Esophageal Contractility 
Acronimo: CIPO c;EN 
Promoting center: Fondazione IRCCS Ca' Granda Ospedale Maggiore 
Policlinico, Via Sforza 28, 20122 Milano, Italia 
Coordinator center: SC Gastroenterologia cd Endoscopia, 
Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, 
Via Francesco Sforza 35, 20122 Milano, Italia 
Principal investigator: Dott.ssa Marina Colett 
Finna ---1----1------~-H--l---"1&---
Type of document: non-interventional study protocol 
Version number: v.1.0 
Date: 21/07/2025 
CONFIDENTIALITY STATEMENT 
Ali information contained in this documcnt shall be considered confidential and shall remain thc exclusive property of 
Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. Thc use of such confidential information must be limited to 
thc recipient for the agrccd purpose and must not be discloscd, published, or othcrwise communicatcd to unauthorized 
pcrsons, for any reason, in any form, without the prior written consent of Fondazione IRCCS Ca' Granda Ospedale Maggiore 
Policlinico. 
ISTITUTO DI RICOVERO E CURA A CARATTERE SCIENTIFICO DI NATURA PUBBLICA 
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lei. 02 5503. 1 
www .policlinico.mi.il 
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PAGINA 1 01 19 

PAGINA 2 DI 19 
 
 
 
 
Dipartimento Area Medica 
SC Gastroenterologia ed Endoscopia              -Direttore: Maurizio Vecchi 
Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
 
 
TABLE OF CONTENTS 
1. Title Page 
2. List of Abbreviations 
3. Responsibilities (Role of the Sponsor and Collaborators) 
4. Amendments and Other Protocol Changes 
5. Timelines 
6. Rationale and Background 
7. Research Question and Objectives 
8. Methods 
8.1. Study Design 
8.1.1. Primary Endpoints 
8.1.2. Secondary Endpoints 
8.2. Setting 
8.2.1. Study Population 
8.2.2. Inclusion Criteria 
8.2.3. Exclusion Criteria 
8.2.4. Description of the Study Object 
8.2.5. Scheduled Visits and Assessments 
8.3. Variables 
8.4. Source Documents 
8.5. Sample Size 
8.6. Data Management 
8.7. Data Analysis 
8.8. Quality Control 
8.9. Study Limitations 
9. Protection of Study Subjects 
9.1. Subject Information Sheet and Consent Form for Personal Data Processing 

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Dipartimento Area Medica 
SC Gastroenterologia ed Endoscopia              -Direttore: Maurizio Vecchi 
Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
 
 
9.2. Insurance Coverage 
10. Dissemination and Communication Plan for Study Results 
11. Publications and intellectual property rights on the study result 
12. Funding 
13. References 
 
2. List of abbreviations  
CIPO: chronic intestinal pseudo-obstruction 
MNGIE: Mitochondrial neurogastrointestinal encephalomyopathy 
ACTG2: smooth muscle actin gamma 2 
FLNA: filamin A 
HRM: high resolution manometry 
DCI: distal contractile integral 
GAD: glutamic acid decarboxylase 
ASMA: anti-smooth muscle antibodies 
gAChR: ganglionic acetylcholine receptor 
 
3. Responsibilities (Role of the Sponsor and Collaborators) 
The sponsor of the study is Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan. 
The coordinating center is the Gastroenterology and Endoscopy Unit of the Foundation. 
The Principal Investigator (PI) is Dr. Marina Coletta, who will oversee the study. 
The following collaborators are affiliated with the same unit and will perform the corresponding roles: 
 
Dr. Beatrice Marinoni: coordinator of all phases of the study, data analysis, manuscript drafting 
and editing (collaborator of the Gastroenterology and Endoscopy Unit, freelance contract) 
 
Dr. Giovanni Aldinio: coordinator of all phases of the study, data analysis, manuscript drafting 
and editing (resident physician, University of Milan) 

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E-mail: gastroenterologia@policlinico.mi.it 
 
 
 
Dr. Beatrice Guzzi: data manager (scholarship holder at the Gastroenterology and Endoscopy 
Unit) 
External collaborations (biological sample analysis, data analysis, diagnostic procedures, etc.) 
Institution 
Operative unit 
Participant name 
 
Role and functions in the 
study 
Fondazione 
IRCCS 
Carlo 
Besta 
Neurological Institute 
Biotechnologist 
at 
Neurology Unit 3 
Dott.ssa 
Margherita 
Marchi 
Biological 
sample 
analysis, data analysis, 
manuscript drafting and 
editing 
 
4.  Amendments and Other Protocol Changes 
Not applicable 
 
5. Timelines 
 
Study status 
Planning  
Start of the study and data collection 
October 2025 
End of the study and data collection 
October 2026 
Final report of the study 
February 2027 
 
6. Rationale and background 
Chronic intestinal pseudo-obstruction (CIPO) is an extremely rare and disabling condition, characterized 
by recurrent symptoms of intestinal obstruction in the absence of any mechanical occlusive lesion ([1], 
[2]). CIPO results from the damage of neuronal, muscular, or mesenchymal structures of the gut wall or 
its extrinsic neuronal control, leading to chronic impairment of tonic and propulsive motor functions in 
one or more segments of the gut ([1], [2]).  
CIPO may be secondary to an underlying disease (Table 1) or it may be idiopathic. 
 
Table 1. Diseases associated with chronic intestinal pseudo-obstruction (CIPO) in adults (2) 

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Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
 
 
Congenital 
Hirschsprung’s disease
Mitochondrial diseases 
Mitochondrial neurogastrointestinal encephalomyopathy 
(MNGIE)
Neurological diseases 
Muscular dystrophy (Myotonic, Duchenne)
Autonomic dysfunctions
Multisystem atrophy
Infiltrative 
Amyloidosis
Autoimmune 
Celiac disease
Systemic lupus erythematosus
Anti-synthetase syndrome (myositis, dermatomyositis)
Connective tissue disor-
ders
Systemic sclerosis
Infectious 
Borrelia burgdorferi (Lyme disease)
Strongyloides stercoralis
Trypanosoma cruzi (Chagas disease)
Paraneoplastic 
Small cell lung carcinoma and other cancers
Primary or Idiopathic 
Sporadic autoimmune/inflammatory
Genetic
  
In the latter group, two distinct subgroups can be identified: one in which CIPO results from an 
autoimmune or inflammatory response targeting the neural or muscular structures of the gut, and one in 
which an inherited or de novo genetic mutation disrupts muscle, neural, or interstitial cell function. 
Among the genes associated with CIPO, mutations in the smooth muscle actin gamma 2 (ACTG2) gene 
are the most common with 139 published cases, 36 of them reaching the adult age ([3] – [27]). More 
rarely, CIPO has been reported to be associated with mutations in filamin A (FLNA) gene. This gene is 
located on the X chromosome and accordingly the disease has been reported only in males.  31 cases of 
CIPO linked to mutations in filamin A (FLNA) gene have been reported so far in pediatric patients ([28] 
– [37]) and in only one adult patient in which the disease was transmitted from the healthy mother [38].  
Both ACTG2 and FLNA mutations impair intestinal smooth muscle cell function, leading to 
heterogeneous phenotypes of visceral myopathy [39]. The functional impairment of the smooth muscle 
can be suggested by an abnormal esophageal motility as shown by the genotype/phenotype correlation in 
patients with ACTG2 and FLNA mutations ([40] – [42]). Kocoshis et al showed that esophageal motility 
is reduced or absent in children with severe impairment of gut motility due to ACTG2 gene mutations [5]. 
Topa et al reported that the esophageal distal contractile integral was reduced (DCI <450 mmHg*s*cm) 

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Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
 
 
in one adult patient with CIPO due to ACTG2 mutation [4]. In the above-mentioned adult patient with 
CIPO due to FLNA mutation, the esophageal distal contractility was absent (DCI = 0 mmHg*s*cm).   
According to these observations it can be hypothesized that at least some of the patients with idiopathic 
CIPO and ineffective or absent esophageal contractility might underlie a mutation in genes that control 
smooth muscle function. Thus, the aim of this study will be to assess the diagnostic yield of Sanger 
sequencing for ACTG2 and FLNA gene mutations (the latter tested only in males) in patients with 
idiopathic CIPO in which high-resolution esophageal manometry (HRM) shows reduced (DCI <450 
mmHg*s*cm) or absent distal esophageal contractility.  
The results of this study will allow a better classification of idiopathic cases of CIPO in which the detection 
of a genetic cause of the disease should prompt genetic counselling and in future genetic therapy. 
Moreover, the precise definition of a myopathic phenotype of CIPO, that is unrelated to the genes that are 
known to cause this condition, will allow further studies of whole exome sequencing aimed to identify 
new gene mutations that may cause the smooth muscle impairment.  
 
7. Research question and objectives 
The study object is to assess the diagnostic yield of Sanger sequencing for ACTG2 and FLNA genes 
evaluation on blood or saliva sample in patients with idiopathic CIPO in which high-resolution 
esophageal manometry (HRM) shows reduced (DCI <450 mmHg*s*cm) or absent distal esophageal 
contractility.  
 
8. Methods 
8.1 Design of the study 
No profit multicenter prospective observational study. 
 
8.1.1 Primary endpoint 
The prevalence of mutations of ACTG2 or FLNA genes in a subgroup of patients with idiopathic CIPO 
with a myopathic phenotype revealed by reduced or absent distal esophageal contractility evaluated with 
high resolution esophageal manometry   
 

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SC Gastroenterologia ed Endoscopia              -Direttore: Maurizio Vecchi 
Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
 
 
 
8.2 Setting 
The CIPO-GEN study is a multicenter investigation conducted at one center in Italy and one in the 
United Kingdom (Nottingham), with the Gastroenterology and Endoscopy Unit of the Policlinico of 
Milan serving as the coordinating center. Patient recruitment will take place through the participating 
hospital facilities, using medical records, endoscopic and radiological reports, and manometric 
evaluations as data sources. The study will begin in October 2025 and conclude in June 2026, with a 
12-month enrollment period. 
 
8.2.1 Study population 
Patients with primary CIPO enrolled between October 2025 and October 2026. 
 
8.2.2 Inclusion criteria 
Patients who simultaneously meet the following criteria will be included: 
 Age ≥18 years  
 Both male and females 
 Primary CIPO (2) 
 Reduced (DCI <450 mmHg*s*cm) or absent distal esophageal contractility at HRM.  
 
8.2.3 Exclusion criteria  
Patients who do not meet the inclusion criteria and who have the following exclusion criteria will be 
excluded: 
 Diagnosis of secondary CIPO (Table 1) 
 Diagnosis of primary CIPO caused by an autoimmune/inflammatory disorder revealed by the 
presence of anti-Hu, anti-GAD, ASMA or anti-gAChR antibodies.  
8.2.4 Description of study object 
The CIPO-GEN study aims to investigate possible genetic alterations in the ACTG2 and FLNA genes in 
patients diagnosed with primary CIPO. The study will be conducted according to standard clinical 

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E-mail: gastroenterologia@policlinico.mi.it 
 
 
practice. Patients will be appropriately informed about the study’s objectives, the planned procedures, and 
the potential benefits and risks. Each participant will receive a detailed informed consent form, outlining 
the study rationale, the assessments to be performed (such as blood or saliva collection and, if necessary, 
high-resolution esophageal manometry), the duration of follow-up, and any associated discomfort. Full 
compliance with ethical standards and good clinical practice will be ensured, safeguarding the voluntary 
nature of participation and the right to withdraw from the study at any time without any impact on clinical 
care. 
 
8.2.5 Scheduled Visits and Assessments 
The study includes a single outpatient visit during which the patient’s clinical history will be collected, a 
blood (two 3ml test tubes) sample will be taken, and high-resolution esophageal manometry (HRM) will 
be performed if indicated. A second outpatient visit will take place three months later to communicate 
the results of the genetic analysis. 
DNA will be extracted from peripheral blood samples or saliva. Genomic DNA targeted regions for 
ACTG2 and FLNA will be sequenced using the Sanger method. 
 
8.3 Variables 
Data collection will include: 
 Demographic data (age, sex, date of diagnosis, family history of CIPO, age of symptoms onset, 
site of gut dilatation, involvement of extra intestinal organs (bladder, heart, blood vessels)  
 Clinical presentation and symptoms (constipation, nausea/vomiting, abdominal distension and 
pain, recurrent episodes of acute intestinal obstruction, urinary retention and need for 
catheterization, neurological and musculoskeletal symptoms) 
 Diagnostic tests (radiological and endoscopic findings, esophageal and anorectal manometry, 
transit studies, full thickness gut biopsies when available) 
 Pharmacological therapies (prokinetics, laxatives, analgesics)   
 Surgery (decompressive procedures, ostomies, resections)  
 Long-term follow-up (complications, need for parenteral nutrition)  

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8.4 Source Documents 
The study is based on clinical data from patients who have already been followed on an outpatient basis 
at the Foundation, as well as previous instrumental investigations (HRM, EGDS). 
Data derived from HRM will be stored in digital format and analyzed using dedicated software for trace 
processing. 
 
8.5 Sample Size 
Given the extreme rarity of idiopathic CIPO with reduced or absent distal esophageal contractility, a 
formal sample size calculation was not performed. Moreover, this study is designed as an exploratory 
investigation to determine the of mutations of ACTG2 or FLNA genes in this population. Thus, all eligible 
patients meeting the inclusion criteria and providing informed consent at the participating centers between 
October 2025 and October 2026 will be enrolled consecutively. 
Based on the number of patients usually referred to our units and collaborating centers for CIPO, we 
expect to enroll approximately 20 subjects during the 12-month recruitment period. However, the final 
sample size may vary according to actual case availability. 
The results will provide preliminary data on the prevalence of ACTG2 and FLNA mutations in this 
population and may inform the design of future studies. 
 
8.6 Data Management 
At the time of enrollment, each participant will be assigned a unique code. Data de-identification will be 
carried out in such a way that individuals accessing the database will not be able to trace the identity of 
the subjects. Only local investigators will be able to link the code to the subjects' identities. 
The necessary data for the study will be recorded in a dedicated eCRF within a Data Management System 
validated according to national regulations and provided by the Scientific Directorate of the Foundation. 
The platform used will be REDCap (Research Electronic Data Capture). 
The REDCap Consortium includes over 1,000 institutional partners worldwide (research institutions, 
universities, ministries, etc.). The consortium supports a secure web-based application (REDCap) 
designed exclusively to facilitate data collection for research studies. REDCap allows users to quickly and 

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SC Gastroenterologia ed Endoscopia              -Direttore: Maurizio Vecchi 
Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
 
 
securely create and manage online databases and is currently used in more than 110,000 projects by 
approximately 150,000 users across various research domains. 
For this study, REDCap will ensure: 
a) user-level identification, with role-based access restrictions 
b) real-time validation and data integrity checks 
c) patient de-identification prior to data export 
d) centralized data archiving with daily backups, on a secure server within the Foundation's IT 
infrastructure 
 
8.7 Data Analysis 
The statistical analysis will primarily be descriptive. Prevalence will be calculated as the proportion of 
patients with at least one detected mutation over the total number of enrolled patients. Continuous 
variables will be summarized using mean and standard deviation (SD) or median and interquartile range 
(IQR), depending on the distribution. Categorical variables will be presented as absolute frequencies and 
percentages. 
 
8.8 Quality Control 
To ensure adequate quality control of the study, the investigator will allow, if requested, direct access to 
all relevant documentation and dedicate time to discussing study results. In addition, Regulatory 
Authorities may conduct inspections. In such cases, the investigator must authorize inspectors to access 
all pertinent documentation and dedicate time and staff to discuss the monitoring outcomes and any other 
aspects of the study. For all manual data entries, the presence of two operators will be ensured to minimize 
potential errors. 
 
8.9 Study Limitations 
The main limitations of the study include the small sample size, which may limit the generalizability of 
the results and reduce the statistical power to detect smaller effects. Moreover, the non-randomized and 
uncontrolled nature of patient enrollment may introduce selection bias. 
 

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E-mail: gastroenterologia@policlinico.mi.it 
 
 
9. Protection of Study Subjects 
The study will be conducted in accordance with Good Clinical Practice (GCP) guidelines, the ethical 
principles of the Declaration of Helsinki, and current regulations on observational studies. 
The observational study and related documentation will be submitted to the competent Ethics Committee. 
The study will begin only after receiving all required authorizations in accordance with the institution’s 
internal procedures. 
The Ethics Committee must also approve any protocol amendments and any recruitment advertising, in 
compliance with local regulations. 
 
9.1 Subject Information Sheet and Consent Form for Personal Data Processing 
It is the responsibility of the physicians to obtain consent to the information sheet through a signed and 
dated privacy consent form from each patient before data collection begins. The signature certifies the 
patient’s understanding of the consent and the information contained therein. Additionally, the 
investigator must sign and date the informed consent form. 
Signed documents must be archived by the investigator, and a copy must be provided to the patient.  
 
The study supervisor ensures that the data of enrolled patients will be stored, archived, and processed in 
full compliance with privacy regulations in accordance with Article 13 of Regulation (EU) 2016/679, 
current national privacy legislation, the Code of Ethics for data processing for statistical and scientific 
purposes, and the “Guidelines for the processing of personal data in the context of clinical drug trials” 
published in the Official Gazette No. 190 of August 14, 2008. 
 
Patient data will be collected in a pseudo-anonymized format and will only be accessible to internal and 
external personnel who are specifically authorized and bound by a confidentiality obligation regarding 
any information learned during the study. 
The study supervisor also guarantees that the minimum-security measures prescribed by the 
aforementioned regulations have been implemented for data processing using both electronic and non-
electronic means to prevent any unlawful data processing. 

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SC Gastroenterologia ed Endoscopia              -Direttore: Maurizio Vecchi 
Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
 
 
 
9.2 Insurance Coverage 
Given the observational nature of the proposed studies, no additional insurance policies are required 
beyond those already in place for standard clinical practice. 
 
10. Dissemination and Communication Plan for Study Results 
The study’s scientific supervisor commits to preparing a final report and to making the results publicly 
available at the end of the study. The data will be published anonymously and, when required, presented 
in aggregated form. 
 
11. Publications and intellectual property rights on the study results 
Publications: 
The Foundation, as the Sponsor, shall ensure the dissemination and publication of the study results, 
including negative results, without any restrictions, and shall guarantee the collaborating center 
visibility 
proportional 
to 
its 
actual 
participation. 
Any journal or scientific publication containing the results and data of the study must indicate the 
role and participation of the Centers and the Foundation, in proportion to their actual contribution to 
the 
study 
and 
the 
role 
played 
by 
each 
party. 
Data will be published in aggregate or otherwise anonymized form, so as not to allow in any way the 
identification of the individual to whom the data refer. 
Intellectual Property Rights: 
The Parties acknowledge that, for the purpose of conducting the collaboration within the framework 
of the study, data, information, know-how, and inventions (whether patentable or not) owned by 
each party may be used and shared. Each party shall remain the exclusive owner of such assets, 
even if it grants the other party a non-exclusive and free-of-charge right of access and use, solely for 
the purpose of carrying out the activities covered by the study and only for its duration. 
It is understood that this right of use does not include the right to sublicense to third parties. 
In accordance with applicable laws, the data and results generated within the framework of the 

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Study shall be the property of the Sponsor, unless otherwise agreed between the Sponsor and the 
Centers. 
 
12. Funding 
No funding. 
 
13. References  
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(accessed February 20, 2025). 
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to identify patient subgroups that are suitable for more specific treatments. Neurogastroenterology & 
Motility 2024;36:e14715. https://doi.org/10.1111/nmo.14715. 
[3] Ravenscroft G, Pannell S, O’Grady G, et al. Variants in ACTG2 underlie a substantial number of 
Australasian patients with primary chronic intestinal pseudo-obstruction. Neurogastroenterol Motil 
2018;30: e13371. https://doi.org/10.1111/nmo.13371. 
[4] Topa M, Porcaro L, Basilisco G. A Young Woman With Chronic Intestinal Pseudo-obstruction Since 
Birth. Gastroenterology 2023;165:1338–41. https://doi.org/10.1053/j.gastro.2023.05.048. 
[5] Kocoshis SA, Goldschmidt ML, Nathan JD, et al. Esophageal dysmotility: An intrinsic feature of 
megacystis, microcolon, hypoperistalsis syndrome (MMIHS). J Pediatr Surg 2019;54:1303–7.  
[6] Mori M, Clause AR, Truxal K, Hagelstrom RT, Manickam K, Kaler SG, Prasad V, Windster J, Alves 
MM, Di Lorenzo C. Autosomal Recessive ACTG2-Related Visceral Myopathy in Brothers. JPGN Rep. 
2022 Oct 20;3(4):e258. Doi: 10.1097/PG9.0000000000000258. PMID: 37168481; PMCID: 
PMC10158422. 
[7] Lehtonen HJ, Sipponen T, Tojkander S, Karikoski R, Järvinen H, Laing NG, Lappalainen P, Aaltonen 
LA, Tuupanen S. Segregation of a missense variant in enteric smooth muscle actin γ-2 with autosomal 
dominant familial visceral myopathy. Gastroenterology. 2012 Dec;143(6):1482-1491.e3. doi: 
10.1053/j.gastro.2012.08.045. Epub 2012 Sep 6. PMID: 22960657. 
[8] Wangler MF, Gonzaga-Jauregui C, Gambin T, Penney S, Moss T, Chopra A, Probst FJ, Xia F, Yang 

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Dipartimento Area Medica 
SC Gastroenterologia ed Endoscopia              -Direttore: Maurizio Vecchi 
Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
 
 
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Muzny DM, Jhangiani S; Baylor-Hopkins Center for Mendelian Genomics; Gibbs RA, Lupski JR, 
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[13] Ravenscroft G, Pannell S, O’Grady G, Ong R, Ee HC, Faiz F, Marns L, Goel H, Kumarasinghe P, 
Sollis E, Sivadorai P, Wilson M, Magoffin A, Nightingale S, Freckmann ML, Kirk EP, Sachdev R, 
Lemberg DA, Delatycki MB, Kamm MA, Basnayake C, Lamont PJ, Amor DJ, Jones K, Schilperoort J, 
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[14] Holla OL, Bock G, Busk OL, Isfoss BL. Familial visceral myopathy diagnosed by exome sequencing 

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SC Gastroenterologia ed Endoscopia              -Direttore: Maurizio Vecchi 
Tel: 02 5503.3418 
E-mail: gastroenterologia@policlinico.mi.it 
 
 
of a patient with chronic intestinal pseudo-obstruction. Endoscopy. 2014 Jun;46(6):533-7. Doi: 10.1055/s-
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[15] Matera I, Rusmini M, Guo Y, Lerone M, Li J, Zhang J, Di Duca M, Nozza P, Mosconi M, Pini Prato 
A, Martucciello G, Barabino A, Morandi F, De Giorgio R, Stanghellini V, Ravazzolo R, Devoto M, Ha-
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