DEFINING THE GENETIC DRIVERS OF ADULT-ONSET CHOLESTATIC LIVER DISEASE
Phase: NA
Conditions: Cholestatic Liver Disease, Progressive Familial Intrahepatic Cholestasis
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

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- Direttore: Daniele Prati 
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“DEFINING THE GENETIC DRIVERS OF ADULT-ONSET 
CHOLESTATIC LIVER DISEASE” 
 
Acronym: FIRST 
 Version Number: v.2.0 
Date: 30/10/2025  
 
Promoter: Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Via Sforza 28, 20122 
Milano, Italia 
 
Coordinating center: SC Medicina Trasfusionale - Fondazione IRCCS Ca’ Granda Ospedale 
Maggiore Policlinico 
 
Principal Investigator: Dr.ssa Luisa Ronzoni 
 
 
 
 
 
 
 
 
CONFIDENTIALITY STATEMENT 
All information presented in this document will be considered confidential and will remain the exclusive property of 
Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. The use of such confidential information must be limited 
to the recipient for the agreed upon purpose and must not be disclosed, published or otherwise communicated to 
unauthorized persons, for any reason, in any form without the prior written consent of Fondazione IRCCS Ca' Granda 
Ospedale Maggiore Policlinico. 

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FLOWCHART 
Period 
Visit 1 
(T0) 
Enrollment 
 
Informed consent 
X 
Inclusion/Exclusion criteria 
X 
Study procedures 
 
   Demographic and clinical data   
collection * 
X 
   Blood sample collection*  
X 
   Biochemical analysis* 
X 
   Genetic analysis° 
X 
Outcomes 
 
 Presence of rare pathogenic      
variants at WGS analysis 
X 
 
* standard of care; ° extra standard of care 
 
 
 
 
 
 
 
 

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ABBREVIATIONS  
ALP: alkaline phosphatase 
ALT: alanine aminotransferase 
ANA: antinuclear antibody 
cANCA: cytoplasmatic antineutrophil cytoplasmic antibodies 
pANCA: perinuclear antineutrophil cytoplasmic antibodies 
AMA: antimitochondrial antibody 
AST: aspartate aminotransferase 
BMI: body mass index 
CE: ethical committe  
CI: Informed consent 
CRF: case report form 
ENA: extractable nuclear antigen  
Extra-SOC: extra standard of care 
GCP: good clinical practice 
GGT: gamma-glutamyltransferase  
Hb: hemoglobin 
LKM: liver kidney microsome  
LPAC: low-phospholipid-associated cholelithiasis  
MRC: magnetic resonance cholangiopancreatography 
PBC: primary biliary cholangitis 
PFIC: progressive familial intra-hepatic cholestasis  
PSC: primary sclerosing cholangitis  
SOC: standard of care 
WGS: Whole Genome Sequencing 
 

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RESPONSIBILITIES (ROLE OF THE PROMOTER AND INVESTIGATORS) 
Promoter: Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico 
Coordinating center: SC Medicina Trasfusionale 
Principal Investigator (PI): Dr.ssa Luisa Ronzoni, PI will be responsible for coordinating the study. 
 
Internal collaborators 
Center 
Participants 
Roles and functions 
SC Medicina Trasfusionale, 
Fondazione IRCCS Ca' 
Granda 
Ospedale Maggiore Policlinico 
Prof. Luca Valenti 
Dott. Daniele Prati 
Dott.ssa Giulia Periti 
Dott.ssa Serena Pelusi 
Dott.ssa Elena Sinopoli 
Dott. Lorenzo Miano 
 
Co-investigator: results interpretation 
Co-investigator: results interpretation 
Co-investigator: patient enrollment 
Co-investigator: patient enrollment 
Co-investigator: sample processing 
Co-investigator: bioinformatic and 
statistical analysis 
 
 
External collaboration  
 
Institution 
Units 
Roles and functions 
Human Technopole 
National Facility for 
Genomics 
 
Whole Genome Sequencing (WGS)  
 
 
 
 
 
 
 
 

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SUMMARY 
1. INTRODUCTION 
1.1 Background and rationale 
2. STUDY OBJECTIVES 
2.1 Primary objective 
2.2 Secondary objective 
3. STUDY DESIGN 
3.1 Study design and setting 
3.2 Inclusion criteria 
3.3 Exclusion criteria 
3.4 Withdrawal criteria 
4. STUDY DESCRIPTION 
4.1 Procedures and timepoints 
5. ENDPOINT 
5.1 Primary endpoint 
5.2 Secondary endpoint  
6. STUDY TIMELINES 
7. STATISTICAL ANALYSIS 
7.1 Sample size 
7.2 Data analysis 
8. ADVERSE EVENTS 
9. EVALUATION OF RISKS/BENEFITS 
10. STUDY MANAGEMENT  
10.1 Data collection and management   
10.2 Regulatory aspects and ethical considerations  

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10.2.1 Authority Approval 
10.2.2 Ethical Committe Approval 
10.2.3 Informed consent 
10.3 Investigators’ responsabilities  
10.4 Monitoring 
10.5 Quality of the study assurance 
10.6 Study conclusion 
10.7 Document archiving 
10.8 Dissemination of information regarding the scientific results 
10.8.1 Confidentiality 
10.8.2 Publications and intellectual property  
11. INDEMNITY AND COMPENSATION IN THE EVENT OF DAMAGE 
12 CONFLICT OF INTEREST DISCLOSURES  
13 FUNDING 
14 REFERENCES 
 

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1.BACKGROUND AND RATIONALE 
Cholestatic liver diseases are a heterogenous group of conditions, including progressive familial 
intra-hepatic cholestasis (PFIC), low-phospholipid-associated cholelithiasis (LPAC), primary 
biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC), characterized by impaired bile 
flow at the intra- or extra-hepatic level, leading to fibrosis and end-stage liver disease.  
Diagnosis is often challenging due to the heterogeneous and broad overlapping presentations. 
Reliance upon clinical data, serum chemistries, histological and instrumental assessments 
(abdominal ultrasound and magnetic resonance cholangiopancreatography [MRC]) is frequently 
insufficient to make a diagnosis. The diagnostic delay is still more evident in individuals with subtle 
or atypical phenotypes [1-4]. 
In this context, genetic analyses through next-generation sequencing technologies have a key role 
to make an early diagnosis in patients who remained unexplained despite extensive clinical and 
instrumental evaluations, as demonstrate for other liver diseases [5-7]. The utility of genetic testing 
has been proven in pediatric cohorts, unrevealing rare damaging variants in genes involved in bile 
synthesis and secretion contributing to up 25% of unexplained child-onset cholestatic liver disease 
[8,9] However, despite existing recommendations [1], genetic testing in adults with unexplained 
cholestasis remains uncommon or targeted to a limited number of genes, mainly involved in PFIC 
[10,11]. The role of other cholestasis or liver-related genes, as well as the contribution of genetic 
variants to PSC or PBC pathogenesis is still largely unknown [12-15].  
The implementation of genetic testing in the diagnostic algorithm of adult-onset cholestatic 
disorders will improve diagnostic accuracy, reducing the diagnostic odyssey and allowing to set-up 
personalized follow-up strategies and to optimize the clinical management of these complex 
conditions. 

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2. STUDY OBJECTIVES 
We hypothesize that a subset of patients with unexplained cholestatic liver disease or with atypical 
presentations harbors rare damaging variants in genes involved in bile homeostasis or related to a 
wider extent to genetic liver disorders. 
 
2.1 Primary objective: 
To evaluate the diagnostic yield of whole genome sequence (WGS) in adult patients with unexplained 
or atypical cholestatic liver disease. A genetic diagnosis will be defined as the presence of pathogenic 
variants in genes known to be causative of genetic cholestasis, consistent with the clinical phenotype 
and mode of inheritance. 
 
2.2 Secondary objectives: 
To evaluate the presence of rare damaging variants in liver-related genes and their enrichment at 
single gene level to get insight into disease pathogenesis. 
 
3. STUDY DESIGN 
 
3.1 Study design and setting 
Monocentric, non-pharmacological, no-profit interventional study with genetic analysis. 
This study will be conducted under the coordination of the SC Medicina Trasfusionale, Fondazione 
IRCCS Ca' Granda Ospedale Maggiore Policlinico. Genetic analysis will be performed at Human 
Technopole Institution. 
Whole genome sequencing (WGS) will be performed in 60 adults with unexplained cholestatic liver 
disease or with atypical presentations. 

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As a control cohort, we will take advantage of a well-characterized reference group of healthy 
individuals (blood donors with medical history, anthropometric, hematological and biochemical 
evaluation) from the FOGS study (currently n=1025), for whom WGS has already been performed in 
collaboration with Human Technopole. The genetic data from these subjects are available as pseudo-
anonymized data. No additional evaluations or samples will be performed or requested for these 
subjects (FOGS, promoted by Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, CE 
Milano Area2, last approval 21/03/2025).  
 
3.2 Inclusion criteria 
Cases: 
- Adults, aged > 18 years with: 
1) persistent or intermittent elevations in serum alkaline phosphatase (ALP) or gamma-
glutamyltransferase (GGT) for at least six months not explained following standard diagnostic 
assessment adhering to the guidelines of the European Association for the Study of the Liver (EASL), 
or with a positive family history of unexplained cholestasis or hepato-biliary cancer, negative to 
previous genetic tests (targeted panel for PFIC genes or WES); 
2) primary sclerosing cholangitis (PSC) with unusual features: small-duct PSC, non-typical 
radiological findings according to radiological guidelines on PSC, absence of concomitant 
inflammatory bowel disease, negative to previous genetic tests (targeted panel for PFIC genes or 
WES) or who didn’t perform previous genetic test; 
3) primary biliary cholangitis (PBC) without specific anti-mitochondrial antibodies, negative to 
previous genetic tests (targeted panel for PFIC genes or WES) or who didn’t perform previous genetic 
test. 
- Signature of informed consent 
 
 

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Controls: 
-Blood donors (age 18-65 years) without clinical signs of liver diseases based on the collected clinical 
parameters: anthropometric (BMI>18 and <25), haematological (Hb, white blood cells, platelets 
within the reference range), biochemical traits (albumin, bilirubin, AST, ALT, GGT, ALP within the 
reference range), medical history (negative for chronic or concomitant diseases, including 
immunological diseases) 
 
3.3 Exclusion criteria 
Cases: 
Patients who do not possess the above inclusion criteria or have at least one of the following exclusion 
criteria:   
- an already known genetic diagnosis explaining the clinical phenotype  
- affected by other causes of liver disease such as viral or autoimmune hepatitis 
 
Controls: 
Blood donors with clinical signs of liver diseases 
 
3.4 Withdrawal criteria  
Removal of consent of study participation. 
 
4. STUDY DESCRIPTION 
4.1 Procedures and timepoints 
Patient enrolment will be performed during clinical evaluating as per standard of care at SC Medicina 
Trasfusionale, Outpatient Clinical Service, Fondazione IRCCS Ca’ Granda Ospedale Maggiore 

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Policlinico. A informed consent will be obtained prior to performing any study specific procedures. 
Moreover, a unique code will be assigned to each participant in order to deidentify the data. 
After the evaluation of inclusion and exclusion criteria and having obtained patient consent, data 
about demography, clinical characterization, lifestyle, current medical therapy, and comorbidities will 
be collected as per standard of care. Clinical report of liver biopsies and imaging data (abdominal 
ultrasound and magnetic resonance cholangiopancreatography [MRC]), previously performed for 
clinical indication, will be collected.  
Peripheral blood sample will be collected to evaluate biochemical parameters (SOC) such as blood 
count, albumin, bilirubin, liver indices: AST, ALT, GGT, ALP, immunological profiles (ANA, AMA, 
ENA, LKM, p-c-ANCA), and for genetic analysis (extra-SOC; 1 EDTA 7ml vial).  
 
Healthy blood donors data (control cohort) has been already collected and will be re-analyzed under 
the coordination of SC Medicina Trasfusionale, Fondazione IRCCS Ca’ Granda Ospedale Maggiore 
Policlinico. No additional evaluations or samples will be performed or requested for these subjects.  
Given the large number of subjects involved (n = 1025), obtaining informed consent would represent 
a disproportionate effort, potentially making it impossible or seriously compromising the 
achievement of the research objectives. Therefore, data will be used without consent, in accordance 
with Article 110 of Legislative Decree 196/2003, as amended by Legislative Decree 101/2018. 
Among the reasons qualifying as “organizational impossibility” that justify this choice are both the 
large sample size, which makes rocontacting patients a disproportionate effort, and the non-profit 
nature of the study, which limits the financial resources and staff available to support the recontacting 
process. 
In compliance with current legislation, a data protection impact assessment will be prepared and 
published on the Fondazione’s website pursuant to Article 35 of EU Regulation 679/2016, and the 

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Italian Data Protection Authority will be notified. Furthermore, the privacy notice on data processing 
will be made publicly available on the Fondazione’s website. 
 
4.2 Sample processing and analysis 
For genetic analysis, DNA will be extracted from deidentified peripheral blood samples at the 
Genomic platform of the Fondazione IRCCS Ca’ Granda, and quantified by a Qubit 2.0 analyzer 
using the Qubit dsDNA BR Assay Kit (Thermo-Fisher). DNA purity will be evaluated using a 
Nanodrop 1000 spectrophotometer (Thermo-Fisher) and integrity assessed by gel electrophoresis.  A 
rate (concentrations range from 50 to 100 ng/μl in a total volume of 50–100 μl) of DNA will be sent 
to the National Facility - Human Technopole to perform WGS.  
Sequencing and bioinformatic analysis, including annotation and gene-level interpretations and 
diseases association analysis, will be performed at Human Technopole. 
The presence of rare damaging variants, including structural variants, in genes known to be associated 
with cholestasis, starting from genes described in PFIC or other forms of genetic cholestasis (about 
50 genes [15]), will be evaluated. Variants will be filtered according to their frequency in the general 
population (gnomAD) and their consequence, and classified according to published guidelines [16, 
17]. Genetic findings will be integrated with clinical data and a genetic diagnosis will be performed 
in the presence of pathogenic variants consistent with the clinical phenotype and mode of inheritance. 
Moreover, we will evaluate the presence of rare damaging variants, including structural ones, in other 
liver-related genes associated with mitochondrial hepatopathies, metabolic disorders and/or 
ciliopathy to identify new potential genetic determinants of cholestasis [15]. As a local control cohort, 
more similar to the genetic background of patients, we will take advantage from the data derived from 
WGS previously performed in a well-characterized reference group of healthy individuals (blood 
donors with medical history, anthropometric, hematological and biochemical evaluation), analyzed 
with the same criteria used for cases. Rare damaging variants in liver-related genes more frequent in 

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cases vs local controls could be considered as potential contributors to clinical phenotype. Moreover, 
the burden of rare damaging variants at single gene level in patient vs controls will get insight into 
disease pathogenesis. 
All the remaining samples not used by Human Technopole for Genomics will be returned and stored 
at the POLI-MI Biobank, Fondazione IRCCS Ca Granda. These samples could be use, upon specific 
biobanking consent signature, for research purpose for future studies beyond the end of the present 
study.  
Genetic data will be stored in a secure server of Fondazione IRCCS Ca’ Granda, protected through 
WORM technology, as FastQ and VCF files for 2 and 20 years respectively. Data will be not 
systematically reanalyzed, unless explicitly requested, in line with international guidelines [18]; in 
this case, a new informed consent will be required. At the end of the study, data will be submitted in 
anonymous and aggregate form in public database, such as NCBI GEO e/o European Nucleotide 
Archive (ENA) and could be used for research purpose for future studies beyond the end of the present 
study. 
 
5. ENDPOINT 
5.1 Primary Endpoint 
The prevalence of pathogenic variants in genes known to be involved in genetic cholestasis. 
5.2. Secondary Endpoints 
The prevalence of rare damaging variants in liver-related genes associated with mitochondrial 
hepatopathies, metabolic disorders and/or ciliophaties  
 

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6. STUDY TIMELINES 
Study starting date: November 2025 
Closing of Enrolment: January 2026  
Study analysis closing date: July 2026 
Study end date and final report: October 2026 
 
7. STATISTICAL ANALYSIS  
7.1 Sample size  
We plan to include 60 adults with unexplained or atypical cholestasis, according to the clinical criteria 
described above. For each patient genetic analysis through WGS will be performed. Based on 
preliminary study [15], assuming that 15% of the patients have pathogenic variants, and 15% of drop-
out, the sample size is estimated to detect the expected proportion with 9.8% absolute precision and 
95% confidence. 
 
To descriptive aim, we will also evaluate the data derived from WGS previously performed in a well-
characterized reference group of healthy individuals (blood donors with medical history, 
anthropometric, hematological and biochemical evaluation) from the FOGS study (n=1025). Based 
on preliminary study [15], the sample size is estimated to allow a statical power >95% with an alpha 
error of 0.05 to detect a five-fold impact of rare variants on cholestatic phenotypes, as calculated 
using the G* Power software (Universität Dusseldorf). 
  
7.2 Data analysis 
Categorical variables will be expressed as frequencies and percentages; continuous variables as mean 
±standard deviation or median and interquartile range (IQR), as appropriate. The prevalence of rare 
damaging variants will be calculated assuming a confidence interval of 95%. Variant frequencies will 

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be compared between patients and controls using chi-squared tests adjusted for false discovery rate 
(FDR). The enrichment in rare variants in cases vs controls will be evaluated by Burden test (SKAT 
and SKAT-O methods), adjusted for multiplicity of testing. Statistical analyzes will be performed 
using JMP Pro (SAS Institute) and the latest version of R statistical analysis software 
(http://www.Rproject. org/).  
 
8. ADVERSE EVENTS 
This study does not involve administration of drugs or other substances or invasive clinical practices. 
Therefore, no adverse events are expected. 
During genetic analysis, incidental or secondary findings, defined as results unrelated to the original 
reason for testing, unintentionally discovered or actively searched for respectively, could be 
generated. This possibility will be discussed with the patient prior to obtained the informed consent 
for genetic analysis and the patient will be able to decide whether or not to be informed of these 
results. Only incidental findings related to serious genetic diseases actionable of early or 
presymptomatic medical intervention will be considered and refer to the patient, in accordance with 
recommendations endorsed by the European Society of Human Genetics (ESHG) [18]; secondary 
findings will be not actively searched for. Geneticists will deliver the final report and provide post-
test counselling to the patients. 
 
9. EVALUATION OF RISKS/BENEFITS 
By integrating clinical and genomic data, employing a WGS approach in a well-characterized patient 
cohort, we expect to improve the diagnostic accuracy of these complex rare conditions, reclassifying 
unexplained cholestatic liver disease and conditions currently diagnosed as immune-mediated. The 
benefits include the possibility to set-up personalized follow-up strategies, optimizing the clinical 

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management, paving the way to a personalized medicine approach. The identification of a genetic 
diagnosis also allows for screening of first-degree relatives and early treatments or prevention 
whenever required. 
 
10. STUDY MANAGEMENT  
 
10.1 Data collection and management 
The coordinator center has arranged an electronic case report form (eCRF), approved by the ethics 
committee, where will be registered all the data declared in this protocol. Data required in the CRF 
are only those necessary to verify the hypothesis of the study. The CRF system is provided by 
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico using the RedCap (Research 
Electronic Data Capture) platform. The REDCap Consortium is composed >1000 institutional 
partners worldwide (research institutions, universities, etc). The consortium supports a secure web 
application (REDCap) designed exclusively to support data capture for research studies. The 
REDCap application enables users to create and manage online databases quickly and securely, 
and is currently in use for more than 110,000 projects with approximately 150,000 users covering 
numerous research interest areas across the consortium. Through REDCap, the following will be 
implemented for this study: a) user-level identification, with specific restrictions based on role in 
the study; b) real-time data integrity validation and checking; c) de-identification of patients before 
data export; d) centralized data storage with daily backup within a secure server of Fondazione 
IRCCS Ca’ Granda Ospedale Maggiore Policlinico IT service).  
To guarantee data protection, patients will be indicated with a unique code. Only the study 
investigators will have passwords to access that file. Each process to promote data quality will be 
guaranteed. Data insertion in the database will be performed in a correct and accurate way 
according to the source documentation. In case of lack of data, the motivation will be provided. In 
order to ensure adequate control over the quality of the study, the investigator will allow, if 

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required from the regulatory authorities, direct access to all relevant documentation and devote 
part of his time to discuss the study results. 
 
10.2 Regulatory aspects and ethical considerations 
10.2.1 Authority Approval 
All the study phases will be conducted in accordance with ICH (International Conference of 
harmonization)/GCP and all applicable law, including the Declaration of Helsinki of June 1964, 
modified by the last World Medical Association General Assembly (Helsinki, Finland, 2024). The 
PI will obtain the ethical board approval before the trial commencement. 
 
10.2.2 Ethical Committee Approval 
Principal Investigator will make sure that the study protocol had obtained the local ethical board 
approval before the study commencement. 
The local ethical board must verify and approve the informed consent module before the study 
enrollment. Whenever it is necessary to modify the protocol and/or the informed consent along the 
study, the PI will guarantee and perform the protocol revision and approval according to the Ethical 
board requirement. The contents of the above-mentioned changes to the protocol will be included 
only after the ethical board approval. 
 
 
10.2.3 Informed Consent 
The PI or other personnel in charge must inform the patients about all aspects the procedures 
involved by the study. The local PIs and sub-investigators will collect the written informed consent 
to the study participation. 
The process for obtaining informed consent must be in agreement with the current rules. The PIs 
or sub-investigators and the patient must sign and date the consent before starting any procedure 
involved in the study. The PIs and other personnel involved in the study must not force in any way 

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or influence the choice of participating or continuing to participate. 
The choice of participating to the study must be completely free. The PIs must underline during 
the consent collection process that the subject can decline the participation at any time without any 
penalty or loss of benefits for which they have the right. The written or oral information concerning 
the study – including the informed consent module – must not contain any language expression 
that will oblige the subject to neglect their legal rights or relieve PIs or the Institution from any 
responsibility because of negligence. 
The consent form signed by patients will include the consent for data processing and for the 
participation to the study, as well as, for the storage of biological samples (biobanking) and genetic 
data and their use for future studies. 
With regard to the control group, as already specified in the protocol, consent will likewise not be 
obtained, in line with Article 110 of Legislative Decree 196/2003, as amended by Legislative 
Decree 101/2018. The reasons underlying this choice are related to “organizational impossibility,” 
whereby recontacting patients would represent a disproportionate effort, potentially hindering or 
even preventing the achievement of the research objectives. 
 
10.3 Investigators’ responsibilities 
According to the currently applicable rules, the PI will send reports about the study enrollment and 
progression to the ethical board every year and will notify the end of the study. The year report and 
the communication of the study end are the investigator’s responsibilities. 
 
10.4 Monitoring 
According to the currently applicable rules of good clinical practice, monitoring visits will be 
performed on line and/or in person in each participating centre. The duration, nature and frequency 
of the visits will depend on the actual recruitment rate of each centre, the amount and quality of 

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documents and adherence to the protocol. 
Through the monitoring visit, the monitor will: 
- Check and evaluate the progression of the study 
- Examine the data collected 
- Verify the source documents 
- Identify problems and find solutions 
The objectives of the monitoring activity will be verifying that: 
- Patients’ rights and wellbeing is respected 
- The data are accurate, complete and verifiable by the source documents 
- The study follows the protocol and eventual approved amendments, GCP and applicable rules. 
The investigator must: 
- Provide access to all the documents related to the patients to the monitor 
- Dedicate portion of his/her own time and staff to discuss the results of the monitoring visit and any 
other possible aspects related to the study. 
The monitor must contact the center before the beginning of the study to discuss the protocol and data 
collection with the staff. 
 
10.5 Quality of the study assurance 
As promoter, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico could apply a 
quality control to the study. In this case, the PI must allow the monitor to verify directly to all the 
study documents and to spend his/her time and staff to discuss the monitoring results and other 
aspects of the study. 
Moreover, the Regulatory Authorities can perform inspections. In this case, the PI must allow the 
authorities to directly verify all the documents related to the study and spend the residual volume 
of his/her time and staff to allow the inspector to discuss the monitoring and eventually other 
aspects of the study. 

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10.6 Study Conclusion 
At the study conclusion, the monitor and the PI must: 
- review all the documents related to the study 
- reconcile the data 
- harmonize all the clarification reports. 
 
10.7 Document archiving 
According to the current national rules, the PI must keep a copy of all the study documents and 
store it in a dry and safe place after the study end. 
 
10.8 Dissemination of information regarding the scientific results 
10.8.1 Confidentiality 
The PI or other personnel involved in the study must deal with all the information related to the study 
(including the protocol, data and documents produced during the study), and must not use the study 
information, study data or reports for any purpose different from those described by the protocol. The 
above-mentioned restrictions are not applicable to 
1) information that will become available in public, not because of negligence of the PI and his/her 
personnel. 
2) information that requires communication to the ethical board only because of evaluating the 
protocol 
3) information that are communicated with the purpose of getting adequate medical care for the study 
subject. 
 

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10.8.2 Publications and intellectual property 
 
• 
Monocentric study with collaboration 
Publications: 
As the Sponsor, Fondazione will ensure the dissemination and publication of the study results, even 
in the event of negative results, without any restrictions and ensuring the collaborating center's 
visibility proportionate to its actual participation. Each scientific journal or publication containing the 
data and study results must indicate the role and participation of the collaborating center and of 
Fondazione, according to their actual contribution to the study and their role. The data may be 
published in aggregate form or otherwise anonymized, to avoid the identification of the subject to 
whom the data refers. 
Intellectual property: 
The Parties acknowledge that, in conducting the collaboration within the scope of the study, data, 
information, know-how, and inventions (whether patentable or not) owned by each party may be used 
and shared. Each party retains exclusive ownership of the data, information, know-how, and 
inventions, even if it grants the other a non-exclusive, free right of access and use, solely for the 
purposes of carrying out the activities covered by the study and limited to the duration of the study. 
It is understood that this right of use does not include the right to sublicense to third parties. 
In accordance with applicable law, the data and results generated within the scope of the Study will 
be the property of the Sponsor, unless otherwise agreed between the Sponsor and the collaborating 
center(s). 
 
11. INDEMNITY AND COMPENSATION IN THE EVENT OF DAMAGE 
In the event of unwanted events or any damages that may derive from participation in the research, 

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the Insurance Policy of our Institute is also extended to cover the subjects participating in the research 
projects. 
 
12. CONFLICT OF INTEREST DISCLOSURES 
The PI and collaborators of the study have no conflict of interest to disclose. 
 
13. FUNDING 
Samples analysis and related data will be carried out free of charge at the HUMAN TECHNOPOLE 
NATIONAL FACILITY, as foreseen in the “HUMAN TECHNOPOLE NATIONAL FACILITY 
FOR GENOMICS CALL FOR ACCESS 25-G-ROUND1 (project code ID 2166994)” 
 
14. REFERENCES 
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Hepatol 81(2):303-32 (2024)  
2. Bull LN & Thompson RJ. Progressive Familial Intrahepatic Cholestasis. Clinics in Liver Disease 
22:657-669 (2018) 
3. Manns MP et al. Primary sclerosing cholangitis. Nat Rev Dis Prim 11(1):17 (2025) 
4. Chazouilleres O et al. EASL Clinical Practice Guidelines on sclerosing cholangitis. J Hepatol 
77:761-806 (2022) 
5. Valenti L, Ronzoni L. Genetics: A new clinical tool for the hepatologist. Liver Int 42(4):724-726 
(2022) 
6. Pelusi S, Ronzoni L et al. Clinical exome sequencing for diagnosing severe cryptogenic liver 
disease in adults: A case series. Liver Int. 42(4):864-870 (2022) 
7. Ronzoni L et al. Diagnostic Uptake of Targeted Sequencing in Adults With Steatotic Liver 
Disease and a Suspected Genetic Contribution. Liver Int. 45(3):e70010 (2025) 

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- Direttore: Daniele Prati 
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8. Zheng Y et al. Diagnostic yield and novel candidate genes by next generation sequencing in 166 
children with intrahepatic cholestasis. Hepatol Int 18(2):661-672 (2023)  
9. Nicastro E et al. Diagnostic Yield of an Algorithm for Neonatal and Infantile Cholestasis 
Integrating Next-Generation Sequencing. J Pediat 211:54-62.e4 (2019) 
10. Vitale G et al. Cryptogenic cholestasis in young and adults: ATP8B1, ABCB11, ABCB4, and 
TJP2 gene variants analysis by high-throughput sequencing. J Gastroenterol 53:945-958 (2018) 
11. Nayagam JS et al. Clinical phenotype of adult‐onset liver disease in patients with variants in 
ABCB4, ABCB11, and ATP8B1. Hepatol Commun 6(10):2654-2664 (2022)  
12. The UK-PSC Consortium et al. Genome-wide association study of primary sclerosing cholangitis 
identifies new risk loci and quantifies the genetic relationship with inflammatory bowel disease. 
Nat Genet 49(2):269-273 (2017) 
13. The UK-PSCSC Consortium et al. Dense genotyping of immune-related disease regions 
identifies nine new risk loci for primary sclerosing cholangitis. Nat Genet 45:670-675 (2013) 
14. Karpen SJ et al. Use of a Comprehensive 66-Gene Cholestasis Sequencing Panel in 2171 Cholestatic 
Infants, Children, and Young Adults. JPGN 72:654-660 (2021) 
15. Scaravaglio M, Ronzoni L et al. Diagnostic yield of whole exome sequencing in adult-onset 
cholestatic liver disease. Clin Gastroenterol Hepatol. 2025 Aug 4:S1542-3565(25)00649-4.  
16. Richards S, Aziz N, Bale S, et al. Standards and Guidelines for the Interpretation of Sequence 
Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and 
Genomics and the Association for Molecular Pathology. Genetics in Medicine 17, no. 5 405–424 
(2015) 
17. Riggs ER, Andersen EF, Cherry AM, et al. Technical Standards for the Interpretation and 
Reporting of Constitutional Copy-Number Variants: A Joint Consensus Recommendation of the 
American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome 
Resource (ClinGen). Genetics in Medicine 22, no. 2 245–257 (2020) 

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18. Souche E, Beltran S, Brosens E, et  al. Recommendations for Whole Genome Sequencing in 
Diagnostics for Rare Diseases. European Journal of Human Genetics 30, no. 9:1017–1021 (2022)