DEFINIZIONE DEL PANORAMA GENOMICO DELLA MASLD (DEFINING THE GENOMIC LANDSCAPE OF METABOLIC STEATOTIC LIVER DISEASE)
Phase: NA
Conditions: Cirrhosis, MASLD
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

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DEFINING TH
 
(Title of the study in English “D
S
Pr
 
 
Promoter:IRCCS Ca' Grand
Via Sforza 28, 20122 Milan,
 
Coordinating Center:SC T
IRCCS Ca' Granda Foundati
Via Sforza 35, 20122 Milan,
 
Principal Investigator:Luca
 
 
 
        
 
____
 
 
 
PRIVACY STATEMENT 
All information presented in this docum
Fondazione IRCCS Ca' Granda Ospeda
the intended recipient and for the a
communicated to unauthorized persons
IRCCS Ca' Granda Ospedale Maggiore 
-Director: Daniele Prati 
clinico.mi.it  
HE GENOMIC LANDSC
MASLD 
DEFINING THE GENOMIC LANDSCAP
STEATOTIC LIVER DISEASE”) 
 
Acronym: DETECTIVE 
 
rotocol version number: v.2.0 
Date: 08/18/2025 
da Foundation Maggiore Hospital Poly
, Italy 
ransfusion Medicine 
ion Maggiore Hospital Polyclinic, 
, Italy 
a Vittorio Carlo Valenti 
___________________________ 
ment will be considered confidential and will remain
ale Maggiore Policlinico. Use of such confidential info
agreed-upon purpose and must not be disclosed,
s, for any reason, in any form, without the prior writt
Policlinico. 
 
Translated from Italian
 
 
page1 
APE OF 
PE OF METABOLIC 
yclinic, 
n the exclusive property of 
ormation must be limited to 
 published, or otherwise 
ten consent of Fondazione 
www.onlinedoctranslator.com
 -
n to English 

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page2 
 
FLOW CHART 
 
 
 
 
 
Screening 
Intervention 
Period
(-t1)
(t1)
Enlistment
informed consent
x
inclusion/exclusion criteria 
x 
 
Administration of the intervention 
 
 
biological sample collection * 
 
x 
phenotypic data collection ** 
x
(*) the biological sample has already been previously 
collected in the context of the SERENA, REASON 
or LIVER-BIBLE studies 
(**) These are data previously collected in the 
context of the SERENA, REASON or LIVER-
BIBLE studies 
 
 

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page3 
 
 
LIST OF ABBREVIATIONS 
THERE IS:Ethics Committee 
THERE:Informed Consent 
CRF:Case Report Form, data collection form 
GCP:Good Clinical Practice, good clinical practice 
WGS: Whole Genome Sequencing 
MASLD: Metabolic dysfunction-associated steatotic liver disease 
HCCHepatocellular Carcinoma 
LoFLoss of function 
CHIP: Clonal hematopoiesis of indeterminate potential 
PRSPolygenic risk score 
AST: Aspartate aminotransferase 
ALT: Alanine aminotransferase 
GGTGamma-glutamyl transferase 
 
RESPONSIBILITY (role of the promoter and collaborators) 
Sponsor: IRCCS Ca' Granda Foundation, Ospedale Maggiore Policlinico, Via Sforza 28, 20122 
Milan, Italy 
Coordinating Center: Transfusion Medicine Department, IRCCS Ca' Granda Foundation, Ospedale 
Maggiore Policlinico. 
The Principal Investigator (PI) for this study is Professor Luca Vittorio Carlo Valenti, Head of the 
Biological Resources Center and the Precision Medicine Unit, Department of Transfusion Medicine, 
Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan. As PI, he will be responsible 
for patient enrollment and study coordination. 
 
 
Internal collaborations 
Structure 
Participant name 
Role and functions in the study 

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SC Transfusion Medicine, 
IRCCS Ca' Granda Foundation 
Maggiore Polyclinic Hospital 
Dr. Daniele Prati 
Dr. Alessandro Cherubini 
Dr. Giulia Periti 
Dr. Serena Pelusi 
Dr. Sara Margarita 
Dr. Elia Casirati 
Dr. Francesco Malvestiti 
Dr. Stefania Mira 
Dr. Francesca Iemma 
 
Statistical analysis of data from 
Whole Genome Sequencing 
 
External collaborations (biological sample analysis, data analysis, diagnostic procedures, etc.) 
 
Institution 
Operational unit 
Role and functions in 
the study 
Human Technopole 
National Facility for 
Genomics 
 
Whole Genome 
Sequencing (WGS) 
 
 
 
 
 
 
 
 
 
 

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INDEX  
1. INTRODUCTION 
1.1 Background and rationale 
2. OBJECTIVE OF THE EXPERIMENTATION 
2.1 Primary objective 
2.2 Secondary objective(s) 
3. STUDY DESIGN 
3.1 Study design 
3.2 Inclusion criteria 
3.3 Exclusion criteria 
4. ENDPOINT 
4.1 Primary endpoint 
4.2 Secondary endpoint 
5. DURATION/TIMELINE OF THE STUDY 
5.1 Gantt Chart 
6. STATISTICAL ANALYSIS 
6.1 Sample size 
6.2 Data analysis 
7. ADVERSE EVENTS 
8. RISK/BENEFIT ASSESSMENT 
9. STUDIO MANAGEMENT  
9.1 Data collection and management   
9.2 Regulatory aspects and ethical considerations 
     9.2.1 Approval by the competent authority 
     9.2.2 Ethics Committee Approval 
     9.2.3 Informed consent 
 
9.3 Duties of the investigator 
9.4 Study monitoring   

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9.5 Study quality assurance 
9.6 Closure of the study 
9.7 Document storage 
9.8 Disclosure of information regarding scientific discovery 
      9.8.1 Confidentiality 
      9.8.2 Publications and Intellectual Property Rights on the Results of the Study 
10. Indemnity and compensation in case of damages 
11. Financial agreements 
12. Disclosure on conflicts of interest 
13. References 
 
 

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1. INTRODUCTION 
1.1 Background and rationale 
Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is a leading cause of chronic 
liver disease globally, with a prevalence exceeding 30% in the population. MASLD is closely 
associated with insulin resistance and cardiometabolic conditions and, in 20-30% of cases, can 
progress to steatohepatitis (MASH), which is characterized by progressive liver damage and 
inflammation [1]. In patients at highest risk, the disease can lead to the onset of advanced fibrosis, 
cirrhosis and hepatocellular carcinoma (HCC). One of the main problems in the clinical 
management of MASLD is the absence of specific risk biomarkers and the lack of effective 
treatments, especially for patients with advanced stage disease [2]. MASLD has a large and well-
documented genetic component, with studies identifying several common variants associated with 
this pathology, such as those in the PNPLA3, TM6SF2 and MBOAT7 genes [3]. However, these 
variants identified so far explain only a small part of the heritability of MASLD, also suggesting the 
contribution of rare loss-of-function (LoF) variants [4-5]. Furthermore, scientific evidence indicates 
that the accumulation of somatic variants, both in hepatocytes and myeloid cells, could also play a 
key role in the progression of MASLD [6]. In particular, clonal hematopoiesis of indeterminate 
potential (CHIP), which is a condition characterized by the presence of hematopoietic clones with 
somatic mutations often associated with leukemia and cardiovascular diseases [7], could favor the 
onset of hepatocellular carcinoma [8]. However, the evidence available to date is still limited and 
requires further investigation and studies on larger cohorts. 
The study in question therefore aims to further investigate this aspect by analyzing the genetic 
profile using a Whole-Genome Sequencing (WGS) approach; peripheral blood DNA samples from 
patients with advanced MASLD and peripheral blood DNA samples from controls presenting 
various associated metabolic risk factors will be sequenced. 
Additionally, 80 liver tissue samples from patients with advanced MASLD will be sequenced to 
identify specific somatic mutations. Expected outcomes of this study include the identification of 
novel genetic variants associated with MASLD progression, improved risk stratification through the 
development of polygenic risk scores, and the identification of potential therapeutic targets. This 

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page8 
 
study represents a fundamental step in understanding the biology of MASLD and could have 
important clinical implications for disease management. 
 
2. OBJECTIVE/S/HYPOTHESIS OF THE EXPERIMENTATION 
A careful review of the scientific literature shows that MASLD has a strong genetic component, but 
the evidence available to date only partially explains the predisposition to the advanced form of the 
disease. Furthermore, it is known that somatic variants, particularly clonal hematopoiesis of 
indeterminate potential (CHIP), may contribute to the progression of MASLD to cirrhosis and 
hepatocellular carcinoma (HCC). 
 
2.1 Primary objective 
The primary objective of this study is toto identify genetic variants (germline and/or somatic) 
associated with the risk of developing advanced MASLD (fibrosis ≥2 and/or hepatocellular 
carcinoma) in the Italian population. 
 
2.2 Secondary objectives 
The secondary objectives of this study are: 
 To characterize the contribution of somatic variants (in hepatocytes and hematopoietic cells) 
that are involved in the progression of advanced MASLD; 
 
Evaluate and develop polygenic risk scores (PRS) for risk stratification and identification of 
specific subphenotypes; 
 
To study the associations between genetic variants and metabolic phenotypes related to 
MASLD; 
 
Identify relevant therapeutic targets for the development of precision medicine strategies in 
advanced MASLD. 
 
3. STUDY DESIGN 
 
3.1 Study design 

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The proposed study is a single-center, national, non-pharmacological interventional study with 
genetic analysis and is non-profit. 
The total number of samples for which Whole Genome Sequencing (WGS) is planned is 2880, 
distributed as follows: 
- 800 peripheral DNA samples from patients with advanced MASLD attending the Fondazione 
IRCCS Ca' Granda Ospedale Maggiore Policlinico and collected as part of the SERENA study 
(study promoted by the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, approved by 
the CE Milano Area2 on 07.26.2017); 
- 2000 peripheral DNA samples from controls presenting metabolic risk factors collected as part of 
the Liver-BIBLE study (a study promoted by the Fondazione IRCCS Ca' Granda Ospedale Maggiore 
Policlinico, approved by the CE Milano Area2 on 01.07.2020); 
- 80 pathological liver tissue samples isolated from patients with advanced MASLD in the context of 
the REASON clinical study (study promoted by the Fondazione IRCCS Ca' Granda Ospedale 
Maggiore Policlinico, approved by the CE Milano Area2 on 07.07.2021); 
 
3.2 Inclusion criteria 
Specific inclusion criteria for patients with advanced MASLD: 
- Patients with advanced MASLD defined as liver fibrosis ≥2 and/or development of HCC; 
- patients enrolled in the SERENA study and where applicable also in the REASON study; 
- Liver biopsy for suspected non-alcoholic steatohepatitis (NASH) at the time of diagnosis; 
- Cholecstectomies 
- age [40-70 years] 
- patients who have signed the informed consent 
 
Specific inclusion criteria for control group: 
Blood donors from the LIVER-BIBLE study aged between 40 and 70 years in the presence of 
overweight or obesity (Body mass index-BMI>25Kg/m2) and at least two of the following risk 
factors: 
- impaired fasting blood glucose or diabetes mellitus (blood glucose ≥100 mg/dl) 

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- dyslipidemia (triglycerides≥150mg/dl, HDL<45/55 in M/F) 
- arterial hypertension 
 
 
3.3 Exclusion criteria 
Specific exclusion criteria for patients with advanced MASLD: 
- Positivity for chronic viral hepatitis (HCV-RNA and/or HBsAg); 
- Positivity for other liver diseases such as autoimmune and viral hepatitis (hepatitis B and C), 
hereditary hemochromatosis, alpha-1-antitrypsin deficiency, Wilson's disease. 
 
Specific exclusion criteria for the control group: 
Subjects suffering from chronic degenerative diseases will be excluded, with the exception of well-
controlled hypertension and type 2 diabetes mellitus not requiring pharmacological therapy (as is 
already common practice for eligibility to donate blood) and donors aged > 65 and < 40. 
 
4. PROCEDURES RELATING TO THE STUDY 
4.1 Patient recruitment 
Patient enrollment will take place at the outpatient clinics of the Transfusion Medicine Unit of the 
Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico during routinely scheduled visits. 
If obtaining consent for the clinical study is not compatible with the conduct of the study, consent 
will be obtained remotely via telephone, as described in chapter 9.2.3. 
In all other cases, the informed consent process will be managed in person. 
Patients who enroll in the DETECTIVE study will not undergo any additional procedures, but will 
consent to the use of data and biological samples previously collected in the SERENA, REASON, 
and Liver-Bible studies. 
 
4.2 Sample and data collection 

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Our research is based on two fundamental pillars, represented by rigorously selected study cohorts. 
The SERENA project involves 800 patients with advanced MASLD, whose peripheral blood DNA 
will be sequenced (WGS, 20x coverage). To further deepen our understanding of disease 
progression, a subset of 35 of these patients will undergo even more detailed analysis: we will 
sequence at 50x 80 pathological liver tissue samples from the REASON study, including samples 
with advanced fibrosis and, where present, hepatocellular carcinoma (HCC). 
On the control side, the Liver-BIBLE Milano project offers us a valuable cohort of 2,000 
individuals matched by sex and metabolic risk factors. These participants are selected from blood 
donors aged 40 to 70 years who meet at least two criteria for metabolic syndrome. Their phenotypic 
characterization is extremely thorough, ranging from anthropometric assessment to metabolic and 
inflammatory profiles, from noninvasive liver injury diagnostics to OMICs analysis. This integrated 
approach, combining genetic and clinical data from well-defined cohorts, will allow us to conduct 
robust comparisons between cases and controls, significantly advancing our understanding of 
advanced MASLD. 
Specifically, biological samples will be collected as follows: 
 Peripheral Blood:Peripheral blood DNA samples will be collected from all 800 MASLD 
patients and all 2000 metabolic controls. 
 Pathological Liver Tissue:A total of 80 pathological liver tissue samples will be collected 
from a subset of 35 patients with advanced MASLD. These samples will be derived from 
the REASON study and will include tissue with advanced fibrosis (n=30) and, when 
present, hepatocellular carcinoma (HCC) samples (n=12). 
Clinical and phenotypic data associated with the biological samples collected: 
 For Patients with Advanced MASLD, aimed at understanding the progression of the disease, 
its complications and related factors: 

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- Detailed Clinical Phenotype: Includes a detailed medical history focused on the history of 
MASLD, including diagnosis, progression, presence and severity of specific symptoms, 
history of pharmacological or surgical treatments, and relevant comorbidities, particularly 
metabolic and cardiovascular ones. 
- Biomarkers of Liver Injury and Function: Liver enzymes such as alanine aminotransferase 
(ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and 
alkaline phosphatase (ALP) levels, indicators of hepatocellular injury or cholestasis. 
- Liver synthetic function: Bilirubin, albumin, prothrombin time/INR, which reflect the 
liver's synthetic capacity. 
- Non-invasive biomarkers of liver damage: data from tools such as Fibroscan or predictive 
score calculations (e.g., FIB-4, NAFLD Fibrosis Score) to assess the presence and degree 
of steatosis, steatohepatitis (NASH), fibrosis, and cirrhosis. 
- Metabolic Risk Factors: such as data on glucose, lipids (total cholesterol, HDL, LDL, 
triglycerides), insulin, HbA1c and indices of insulin resistance (e.g. HOMA-IR), which are 
essential for outlining the metabolic profile underlying MASLD. 
- Inflammatory Biomarkers: Such as C-reactive protein (CRP) and ferritin, useful for 
assessing systemic and/or hepatic inflammatory status. 
- Hepatocellular Carcinoma (HCC) Data: For patients who have developed HCC, specific 
information on diagnosis, staging, oncological treatments and follow-up is collected, 
essential for studying clonal evolution. 
 
For Metabolic Controls, aimed at providing an extremely in-depth baseline characterization 
of their general health and metabolic risk profile, in the absence of advanced MASLD, to 
serve as a robust comparison: 
- Detailed Anthropometric Assessment: Height, weight, waist circumference, BMI, for a 
precise quantification of adiposity. 
- Detailed Medical History: Lifestyle, eating habits (diet), complete medical history, with 
particular attention to the presence of metabolic risk factors (hyperglycemia, hypertension, 
dyslipidemia, increased adiposity) which are inclusion criteria. 

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- Complete Blood Count: Evaluation of the cellular components of the blood. 
- Coagulation Parameters: Includes factors such as FVIII, VWF, protein C (PC) and D-
dimer. 
- Standard Clinical Biochemistry: Includes assessments of general liver and renal function, 
as well as inflammatory biomarkers. 
- Complete Metabolic Profiles: Glucose, lipids (total cholesterol, HDL, LDL, triglycerides), 
insulin and HbA1c, to quantify metabolic risk factors. 
- Liver Biomarkers: Although not targeted to progression of advanced MASLD, baseline 
biomarkers of liver function are included to ensure the absence of significant non-MASLD 
liver disease. 
- Non-Invasive Liver Injury Assessment (Fibroscan): Essential to confirm the absence of 
significant liver fibrosis in these controls, although they may have steatosis or early 
NASH. 
- OMIC characterization (in a subset): This is a distinctive feature of this cohort, which 
includes circulating lipidomics, metabolomics, and metagenomics. It provides a very high 
level of molecular detail on their metabolic profiles and gut microbiome, crucial for 
understanding gene-environment-metabolism interactions. 
- Prospective Follow-up Assessments: The longitudinal nature of the Liver-BIBLE Milano 
study means that follow-up data will be updated over time, providing information on the 
evolution of their metabolic and health status. 
4.3 Sample analysis 
The genetic analyses for this study will be conducted in close collaboration and under the 
supervision of Human Technopole. 
As anticipated, the methodology envisaged for conducting this study involves whole-genome 
sequencing on DNA derived from peripheral blood (20x coverage) and pathological liver tissue 
(50x coverage) to characterize the genetic and somatic variants that influence disease progression. 

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The DNA will be isolated at the Foundation and shipped to the National Facility (NF) in batches of 
3-4 plates at a time. 
To identify associations between individual genetic variants and the burden of likely loss-of-
function (LoF) variants at specific genetic loci, comparing participants with advanced MASLD to 
controls, analyses will be performed using a genome-wide regression model implemented in 
REGENIE, with additive genetic models accounting for age, sex, body mass index (BMI), and 
ancestry (BWI 1-10). To ensure the robustness of the results, the genome-wide significance 
threshold will be set at p<5E−8. If the study is not powered to identify new significant associations, 
suggestive hits (p<5E−6) will be replicated in additional cohorts. 
In addition to defining the risk of advanced MASLD compared to controls as the primary outcome, 
we will thoroughly examine the genetic contribution to various endophenotypes. These will include 
metabolic risk factors (such as insulin resistance indices), liver enzymes (AST, ALT, GGT), and 
indicators of liver damage (noninvasive predictors of steatosis, steatohepatitis, fibrosis, cirrhosis, 
and HCC). The results of these analyses will be crucial for improving risk stratification and 
identifying specific disease subphenotypes through the development of polygenic risk scores, which 
have previously proven effective in identifying subsets of patients at specific risk for liver or 
cardiovascular events. This approach will also aim to identify therapeutic targets with a high 
probability of clinical success. 
As regards CHIP (Clonal Hematopoiesis of Indeterminate Potential) and somatic variants, their 
identification will take place through a semi-automated pipeline. CHIP assignment will be assessed 
in a panel of specific genes (including NADK, GNB1, CBL, KMT2D, RHEBL1, PPM1D, JMJD6, 
MFSD11, DNMT3A, SF3B1, ASXL1, NOL4L, GNAS, RUNX1, AF015262.1, RPL34P3, U2AF1, 
TET2, CUX1, SH2B2, BCOR, BCORL1, MCAM, TP53, TLMET23, SRSF2, NPM1P46, 
LINC01426, EZH2P1, F4, AT1PB2, ZNF316, ANAPC1, JAK2), while for somatic variants in 
hepatocytes, relevant scientific literature will be consulted. Variant identification will be performed 
using three callers: Mutect, Vardict, and Freebayes. The impact of inherited genetic variation on 
both CHIP risk and related phenotypes will also be assessed. 

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All unused material at the National Facility for Genomics will be returned and stored at the 
Fondazione IRCCS Ca Granda. Subject to the patient's specific consent for biobanking, these 
residues may be retained beyond the study's conclusion for future research purposes at the 
Foundation's POLI-MI Biobank. 
 
5. ENDPOINT 
5.1 Primary Endpoint 
Identifying the association between germline variants and the presence of advanced MASLD in 
cases versus controls. 
 
5.2 Secondary Endpoints 
Other main endpoints of the study in question are: 
1) Identification of somatic mutations in hepatocytes and hematopoietic cells in order to study their 
association with the development of HCC. 
2) Use of PRS (existing in the literature and new) in predicting patterns of advanced MASLD among 
cases and controls 
3) Analysis of the association between genetic variants and clinical and biological phenotypes of 
MASLD, considering metabolic markers, liver enzymes (AST, ALT, GGT) and non-invasive 
indicators of steatosis and fibrosis (Fibroscan) 
4) Identification of functional variants (e.g. LoF) in genes involved in lipid metabolism, liver 
inflammation, oxidative stress associated with advanced MASLD. 
 
5. DURATION / TIMELINE OF THE STUDY 
Study start date: 09/2025 
Enlistment End Date: December 2025 
Data analysis end date: 07/2026 
Final Study Report: 08/2026 
 
 

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6. STATISTICAL ANALYSIS 
6.1 Sample size 
The total number of samples for which Whole Genome Sequencing (WGS) is planned is 2,880, 
including 800 subjects with advanced MASLD, 2,000 controls with at least two characteristic 
criteria of metabolic syndrome but free of advanced liver disease, and 80 liver tissue samples for 
somatic variant analysis. The sample size estimate is based on the study's primary objective, which 
is the identification of genetic variants (germline and/or somatic) associated with MASLD. 
 
The study is descriptive with a power proportional to the number of subjects included. 
This number was defined on the basis of the maximum availability of subjects that could be enrolled 
in the clinical cohorts with the aim of maximizing the statistical power and scientific value of the 
results.Preliminary data on common variants are available that confirm the statistical power of the 
cohort to identify phenotype-associated variants at the genome-wide level.. 
To confirm the adequate statistical power of the study, an analysis was performed using the R 
statistical analysis software package called “genpwr” by setting the error at a significance level 
of 5x10−8 conventionally used to deϐine signiϐicance at the genomic level (https://cran.r-
project.org/web/packages/genpwr/index.html) and the number of cases and controls in the detective 
cohort. This method is based on an extension of that reported by Gauderman (2002) 
(doi:10.1093/aje/155.5.478) and Gauderman (2002) (doi:10.1002/sim.973) and described in: Moore 
CM, Jacobson S, Fingerlin TE. Power and Sample Size Calculations for Genetic Association Studies in the 
Presence of Genetic Model Misspecification. American Society of Human Genetics. October 2018, San 
Diego. For common variants, combinations with variant allelic frequency (MAF) ≥ 0.1 and OR ≥ 2.0 
were considered, while for low-frequency and rare variants with a greater impact on protein function, 
for which the focus is on identifying variants with a greater impact on disease risk, MAF values between 
0.01 and 0.05 and ORs up to 15 were explored. The results confirm that based on the expected number 
of 800 cases and 2000 new controls (without considering the 1000 controls already available) the 
study will have a power greater than 95% to detect ORs > 2 for common variants with a frequency of 
0.1 (power >80% for OR 1.3 for variants even with a frequency of 0.1, and much greater power for even 
more frequent variants, Graph 1). Furthermore, for low-frequency and rare variants, it will have a 

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power greater than 80% to detect
with array-based methods (see Ch
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Figure 1. Statistical power to id
OR for different minor allelic fr
 
-Director: Daniele Prati 
clinico.mi.it  
t ORs already > 5 for variants with MAF 0.01 t
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page17 
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he Detective cohort 

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Figure 2. Statistical power to id
OR for different MAFs of low-fr
Graph 3. Manhattan plot showi
(GWAS) for common variants i
outcome the risk of fibrosing s
using genomic regression models
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6.2 Data analysis 
-Director: Daniele Prati 
clinico.mi.it  
 
dentify significant genomewide association
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ing the preliminary results of a genomewi
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page18 
ns as a function of the 
hort. 
ide association study 
GS Study) using as an 
results were obtained 
), presence of diabetes 

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The statistical analysis of this study will be conducted in collaboration with Human Technopole. 
The primary analysis will follow a rigorous approach based on genome-wide regression models 
between germline variants and the presence of advanced MASLD. REGENIE software will be used, 
which allows for the evaluation of large-scale whole-genome regression in large genome-wide 
association studies. The secondary analysis involves the development and risk stratification by 
constructing polygenic risk scores specific to advanced MASLD and its subphenotypes. 
The statistical analyses of the study will be conducted using R and Python software. 
 
 
 
7. ADVERSE EVENTS 
The project does not involve the administration of drugs or other substances, nor does it involve 
invasive clinical procedures. Therefore, no adverse events are expected. 
 
8. RISK/BENEFIT ASSESSMENT 
This study does not anticipate an immediate benefit to participants, but the results will be 
fundamental for the identification of specific therapeutic targets and for the development of new 
personalized medicine strategies. 
 
9. STUDIO MANAGEMENT 
  
9.1 Data collection and management 
Each participant will be assigned a unique code upon enrollment. Data de-identification will be 
performed in such a way that people accessing the database will not be able to trace the subjects' 
identities in any way. Only local investigators will be able to trace the identities of enrolled subjects. 
The data required for the study will be recorded in a dedicated eCRF in a Data Management System 
validated according to national regulations, provided by the Foundation's Scientific Directorate. The 
platform used will be RedCap (Research Electronic Data Capture). 
The REDCap Consortium is composed of >1,000 institutional partners worldwide (research 
institutions, universities, ministries, etc.). The consortium supports a secure web application 

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-Director: Daniele Prati 
Tel. 02 5503.4687   
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(REDCap) designed exclusively to support data acquisition for research studies. The REDCap 
application allows users to quickly and securely create and manage online databases and is 
currently in use for more than 110,000 projects with approximately 150,000 users covering 
numerous research areas across the consortium. 
Through REDCap, this study will implement: a) user-level identification, with specific restrictions 
based on role in the study b) real-time data validation and integrity checking c) patient de-
identification before data export d) centralized data storage with daily backup, on a secure server 
within the Foundation's IT infrastructure. 
 
 
9.2 Regulatory aspects and ethical considerations 
9.2.1 Approval by the Competent Authority 
In accordance with applicable regulations, the principal investigator must obtain approval from 
the appropriate Competent Authority before initiating the clinical study. 
 
This study will be conducted in accordance with ICH/GCP (International Conference on 
Harmonization/Good Clinical Practice) regulations and all applicable laws, including the 
Helsinki Declaration of June 1964, as amended by the last World Medical Association General 
Assembly in Seoul, 2008. 
 
9.2.2 Ethics Committee Approval 
The investigator must ensure that the protocol has been reviewed and approved by the local 
independent Ethics Committee (EC) before starting the study. 
The CE must also review and approve the informed consent (IC) form and all written 
information received from the patient prior to enrollment in the study. 
Should it be necessary to modify the protocol and/or the IC during the study, the investigator will 
be the guarantor and therefore the person responsible for ensuring the review and approval of 
such modified document as requested by the CE. 
 

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The content of these changes will be implemented only after the EC has approved them. Until 
then, it will be necessary to refer to the previous version of the already approved document. 
 
 
9.2.3 Informed consent (IC) 
The investigator or other personnel designated by him are responsible for informing the subjects 
about all aspects and procedures of the study. 
Informed consent will be collected by the doctor during the visit. 
The informed consent process must comply with applicable regulatory procedures. The 
investigator (or designated staff member) and the subject must date and sign the informed 
consent form before the patient begins any study-related procedures. The subject will receive a 
copy of the informed consent, dated and signed by both parties; the original copy will be retained 
in designated study archives. Neither the investigator nor designated staff member may in any 
way coerce or influence a subject to participate or continue participating in the study. A subject's 
decision to participate in the study must be completely voluntary. The investigator and 
designated staff member must emphasize to the subject that they may withdraw their consent at 
any time without penalty or loss of any benefits to which they may be entitled. 
Written or oral information relating to the study, including the written consent form, must not 
contain any language that forces the subject to waive (even apparently) his or her legal rights, or 
that would exonerate the investigator, institution, or sponsor from liability for negligence. 
 
If participant access to the Foundation is not foreseeable within a timeframe compatible with the 
conduct of the study, given that this study requires the use of biological samples previously 
biobanked at the Foundation (patients have consented to the preservation of biological material 
for future research activities), consent to participate in the study, and the related data processing, 
may be obtained remotely via telephone. 
During the telephone call, the Investigator, or a delegate, will provide the participant with 
information about the study and data processing methods, answer any questions, and verify the 
email address to which study documentation should be sent. The telephone call will be tracked 
using the document "INSTRUCTIONS AND FORM FOR ACQUIRING CONSENT BY 

Department of Services Area 
SC Transfusion Medicine  
 
-Director: Daniele Prati 
Tel. 02 5503.4687   
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TELEPHONE CALL - PARTICIPATION IN CLINICAL TRIALS," which will be archived 
with the study documentation. 
9.3 Duties of the investigator 
In accordance with applicable local regulations, the investigator must submit periodic reports 
regarding the progress of the study at his/her site to the CE and notify the CE of study closure. 
Periodic reports and closure notification are the investigator's responsibilities. 
 
9.4 Study monitoring 
In accordance with applicable regulations and Good Clinical Practice (GCP), the monitor must 
periodically visit or contact the center. The duration, nature, and frequency of these 
visits/contacts depend on the recruitment rate, the quality of the center's records, and their 
adherence to the protocol. 
 
Through these contacts, the monitor must: 
• monitor and evaluate the progress of the study 
• examine the collected data 
• conduct the verification of the source document 
• identify each problem and related solutions 
 
The purposes of the monitoring activity are to verify that: 
• the rights and well-being of the subject are respected 
• the study data are accurate, complete and verifiable from the original documents 
• the study is conducted in accordance with the protocol and any approved amendments, GCP 
and applicable regulations 
 
The experimenter must: 
• give the monitor direct access to all relevant documentation 
• dedicate part of his time and his staff to the monitor to discuss the monitoring results and any 
other possible aspects. 

Department of Services Area 
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The monitor must also contact the center prior to the start of the study to discuss the protocol and 
data collection procedures with the staff. 
 
9.5 Study quality assurance 
As the sponsor, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico may conduct a 
quality control review of the study at its discretion. In this case, the investigator must allow the 
monitor direct access to all relevant documentation and dedicate part of their time and staff to the 
reviewer to discuss the monitoring results and any other aspects of the study. 
Additionally, Regulatory Authorities may conduct inspections. In this case, the investigator must 
grant the inspector direct access to all relevant documentation and dedicate part of their time and 
personnel to the inspector to discuss the monitoring results and any other aspects of the study. 
9.6 Closure of the study 
Upon study closure, the monitor and the investigator must activate a series of procedures: 
• review all study documentation 
• reconcile study data 
• reconcile all clarifying reports. 
 
 
 
9.7 Document storage 
 
In accordance with current national regulations, the investigator must retain a copy of all 
documentation and store it in a dry, safe place after the study has been closed. 
 
9.8 Disclosure of information regarding scientific discovery 
 
    9.8.1 Confidentiality 
The investigator and other personnel involved in the study must maintain all information related 
to the study (including the protocol, data obtained, and all documentation produced during the 
study) and must not use such information, data, or reports for purposes other than those 

Department of Services Area 
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-Director: Daniele Prati 
Tel. 02 5503.4687   
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page24 
 
described in the protocol. These restrictions do not apply to: 
1) information that becomes publicly available, not due to negligence on the part of the 
investigator or his staff; 
2) information requiring confidential disclosure to CE for the sole purpose of evaluating the 
study; 
3) information that must be disclosed in order to obtain appropriate medical care for a study 
subject. 
 
9.8.2 Publications and Intellectual Property Rights on the Results of the Study 
 
a) SINGLE-CENTER STUDY which includes collaborations: 
Publications: 
As the Promoter, the Foundation will ensure the dissemination and publication of the study results, 
even in the event of negative results, without any restrictions and guaranteeing the collaborating 
center visibility proportional to its actual participation. 
Each scientific journal or publication containing the study results and data must indicate the role 
and participation of the collaborating center and the Foundation, in proportion to their actual 
contribution to the study and their role. The data may be published in aggregate form or otherwise 
anonymized, so as not to allow the identification of the data subject. 
 
 
Intellectual Property Rights: 
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 same, even if it grants the other a 
non-exclusive and free right of access and use, solely for the purposes of carrying out the activities 

Department of Services Area 
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-Director: Daniele Prati 
Tel. 02 5503.4687   
AND-email:centrotrasfusionale@policlinico.mi.it  
 
 
 
 
 
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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 current legislation, the data and results generated within the Study will be the 
property of the Sponsor, unless specific agreements are made between the Sponsor and the 
collaborating center(s). 
 
 
10. COMPENSATION AND DAMAGES IN CASE OF DAMAGES 
In the event of any undesirable events or damages arising from participation in research, our 
Institute's insurance policy also extends to cover the subjects participating in research projects. 
 
 
 
11. FINANCIAL AGREEMENTS 
Study procedures exceeding normal clinical practice described in this study will be covered by 
funding from the HUMAN TECHNOPOLE NATIONAL FACILITY FOR GENOMICS CALL 
FOR ACCESS 24-G-PILOT. 
 
 
 
12. DISCLOSURE ON CONFLICTS OF INTEREST 
The experimenters declare no conflicts of interest.13. REFERENCES 
1. Younossi ZM, Golabi P, Paik JM, Henry A, Van Dongen C, Henry L. The global 
epidemiology of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis 
(NASH): 
a 
systematic 
review. 
Hepatology 
2023;77:1335–
47.https://doi.org/10.1097/HEP.00000000000000004 
 

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-Director: Daniele Prati 
Tel. 02 5503.4687   
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2. European Association for the Study of the Liver (EASL). Electronic address: 
easloffice@easloffice.eu , European Association for the Study of Diabetes (EASD), 
European Association for the Study of Obesity (EASO), European Association for the Study 
of the Liver (EASL). EASL-EASD-EASO Clinical Practice Guidelines on the management 
of metabolic dysfunction-associated steatotic liver disease (MASLD). J Hepatol 
2024:S0168-8278(24)00329-5.https://doi.org/10.1016/j.jhep.2024.04.031. 
 
 
3. Dongiovanni P, Stender S, Pietrelli A, Mancina RM, Cespiati A, Petta S, et al. Causal 
relationship of hepatic fat with liver damage and insulin resistance in nonalcoholic fatty 
liver. J Intern Med 2018;283:356–70.https://doi.org/10.1111/joim.12719 
 
4. Pelusi S, Baselli G, Pietrelli A, Dongiovanni P, Donati B, McCain MV, et al. Rare 
Pathogenic Variants Predispose to Hepatocellular Carcinoma in Nonalcoholic Fatty Liver 
Disease. Sci Rep 2019;9:3682.https://doi.org/10.1038/s41598-019-39998-2. 
 
5. Verweij N, Haas ME, Nielsen JB, Sosina OA, Kim M, Akbari P, et al. Germline Mutations 
in CIDEB and Protection against Liver Disease. N Engl J Med 2022;387:332–
44.https://doi.org/10.1056/NEJMoa2117872. 
 
6. Ng SWK, Rouhani FJ, Brunner SF, Brzozowska N, Aitken SJ, Yang M, et al. Convergent 
somatic mutations in metabolic genes in chronic liver disease. Nature 2021;598:473–
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7. Marnell CS, Bick A, Natarajan P. Clonal hematopoiesis of indeterminate potential (CHIP): 
Linking somatic mutations, hematopoiesis, chronic inflammation and cardiovascular 
disease. J Mol Cell Cardiol. 2021 Dec;161:98-105. doi: 10.1016/j.yjmcc.2021.07.004. Epub 
2021 Jul 21. PMID: 34298011; PMCID: PMC8629838. 
 

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8. Marchetti A, Pelusi S, Marella A, Malvestiti F, Ricchiuti A, Ronzoni L, et al. Impact of 
clonal hematopoiesis of indeterminate potential on hepatocellular carcinoma in individuals 
with 
steatotic 
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