Targeting the Epigenetic Regulators Suv420h1/2 in Hepatocytes to Treat Nonalcoholic Fatty Liver Disease
Phase: NA
Conditions: NAFLD
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

 
 
 
 
 
 
Dipartimento Area dei Servizi 
SC Medicina Trasfusionale 
 
 
 
 
- Direttore: Dr. Daniele Prati 
Tel. 02 5503.4687   
E-mail: daniele.prati@policlinico.mi.it 
 
 
 
 
 
 
Targeting the Epigenetic Regulators Suv420h1/2 in 
hepatocytes to treat nonalcoholic fatty liver disease 
 
Acronym: TERS 
 
Project code: RF-2021-12373889 
 
Version: v.1.0 
Date: 26/01/2023 
 
 
Study Sponsor: Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,  
Via Sforza 28, 20122 Milano, Italia 
 
Study Coordinator: S.C. Medicina Trasfusionale 
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico,  
Via F. Sforza 35, 20122 Milano, Italia 
 
Principal Investigator: Prof. Luca Valenti 
 
 
 
 
         
                                        ______________________________ 
 
 
 
 
DECLARATION OF CONFIDENTIALITY 
All the information presented in this document must be considered confidential and will be property of Fondazione 
IRCCS Ca 'Granda Ospedale Maggiore Policlinico. The use of this confidential information is restricted to recipient for 
the aim agreed and must not be disclosed, published or communicated to unauthorised persons, for any reason, in any 
form, without the prior written consent of Fondazione IRCCS Ca 'Granda Ospedale Maggiore Policlinico.

 
 
 
FLOWCHART 
 
Activities 
Months 
 
T (1) 
Enrolment 
T(2) 
Follow-up 
Enrollment: exclusion criteria check 
X 
 
Informed consent 
X 
 
Clinical evaluation 
X 
X 
Intervention: Isolation, generation 
and characterization of organoids  
X 
 
Data analysis 
X 
X 
T (1)=basal or routine follow-up visits (REASON/PERSPECTIVE)    
LIST OF ABBREVIATIONS 
 
A1AT: Alpha-1 antitrypsin 
AFP: Alpha fetoprotein 
Alpha-SMA: Alpha smooth muscle actin 
ASOs: Antisense oligonucleotides 
CI: Informed Consent 
CRF: Case Report Form 
GCP: Good Clinica Practice 
GWAS: Genome Wide Association Studies 
HLA: Human liver assembloids 
HLO: Human liver organoids 
HNF4: Hepatocyte nuclear factor 4 
HSC: Hepatic stellate cells 
IEC: Independent Ethics Committee 
IF: Immunofluorescence 
IHC: Immunohistochemistry 
KC: Kupffer cells 
KMT5B/C: Lysine methyltransferase 5B/C 
LSEC: Liver sinusoidal endothelial cells 
MAF: Minor allele frequency 
MAFLD: Metabolic associated fatty liver disease 

 
 
MRI-PDFF: Magnetic resonance imaging proton density fat fraction 
NAFLD: Nonalcoholic fatty liver disease 
NASH: Nonalcoholic steatohepatitis 
NPC: Non-parenchymal cells 
 
RESPONSABILITIES (role of promotor and collaborators) 
 
The Promoter of the study is Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico. The 
Coordinating Center will be the SC Medicina Trasfusionale unit of Fondazione IRCCS Ca’ Granda 
Ospedale Maggiore Policlinico. The PI of the study will be Prof. Luca Valenti, responsible for the 
patients enrollment and coordination of the project. 
 
Unità operativa 
Nome partecipante 
Ruolo e funzioni nello studio 
SC 
Medicina 
Trasfusionale, 
Fondazione IRCCS Ca' Granda 
Ospedale Maggiore Policlinico 
Prof. Luca Valenti (PI) 
Dr. Daniele Prati 
Dr. Alessandro Cherubini 
Dr. Serena Pelusi 
 
Sample/data recruitment and 
characterization; 
Isolation and generation of or-
ganoids, spheroids and tissues; 
Characterization of organoids 
by gene and protein expression; 
Data analysis 
Study coordination 
 
Internal Collaborations 
Unità operativa 
Nome partecipante 
Ruolo e funzioni nello studio 
SC Chirurgia Generale E 
Trapianti Di Fegato, 
Fondazione IRCCS Ca' Granda 
Ospedale Maggiore Policlinico 
Dr. Daniele Dondossola 
Referral of eligible patients and 
characterization of samples (as 
part of the REASON study) 
 
External Collaborations (biological samples analysis, data analysis, diagnostic procedures) 
Istituzione 
Unità operativa 
Nome partecipante 
Ruolo e funzioni nello 
studio 
IRCCS Ospedale San 
Raffaele 
Division of Genetics and 
Cell Biology 
Dr.ssa Laura Silvestri  
Preclinical and experi-
mental models in mice; 
Evaluation of the impact 

 
 
of therapy with antisense 
oligonucleotides in pre-
clinical models. 
Università 
Magna 
Graecia Catanzaro  
Dipartimento di Scienze 
Mediche e Chirurgiche 
Prof. Stefano Romeo  
Transcriptomic, lipidomic 
and genetic analyses in 
clinical (in collaboration 
with Milan) and genetic 
analyses in population-
based cohorts (UK Bi-
obank). 
 
 

 
 
 
 
 
 
Dipartimento Area dei Servizi 
SC Medicina Trasfusionale 
 
 
 
 
- Direttore: Dr. Daniele Prati 
Tel. 02 5503.4687   
E-mail: daniele.prati@policlinico.mi.it 
 
 
 
 
 
 
SUMMARY  
1. BACKGROUND 
1.1 Overall Summary 
1.2 Background and State of Art 
2. PURPOSE AND AIMS 
2.1.1 Specific Aim 1 
2.1.2 Specific Aim 2 
3. STUDY DESIGN  
3.1 Study design 
3.2 Inclusion criteria 
3.3 Exclusion criteria 
4. STUDY PROTOCOL, PROCEDURES AND METHODS 
4.1 Procedures 
4.2 Analysis 
5. ENDPOINTS 
5.1 Main Outcome 
5.2 Secondary Outcomes 
6. PROJECT TIMELINE 
6.1 Gantt Chart 
7. STATISTICAL ANALYSIS 
7.1 Sample size 
7.2 Statistical analysis plan 
8. ADVERSE EVENTS 
9. RISK / BENEFIT EVALUATION 
10. PROJECT MANAGEMENT  
10.1 Regulatory and Ethics 
     10.1.1 Patient information 
10.3 Confidentiality 
10.3.1 Publications 
10.4 Intellectual property rights of study results 
11. INSURANCE 
12 REFERENCES 
 
 

 
 
1. BACKGROUND 
 
1.1 Overall Summary 
Nonalcoholic fatty liver disease (NAFLD) is globally the leading cause of liver disease and frequent-
ly progresses to cirrhosis and liver cancer. The identification of effective drugs is the main unmet 
clinical need. Changes in liver histones methylation accompanies the development and progres-
sion of NAFLD. Our preliminary data demonstrate that inactivation of the methyltransferases 
SUV420H1/2 in hepatocytes protects mice against NAFLD. In this project we propose to examine 
the relevance of these findings by evaluating the impact of genetic deletion of hepatic 
SUV420H1/2 in mice fed a steatogenic diet. To further evaluate the potential for clinical transla-
tion of these results, we will next 1) evaluate the expression of SUV420H1/2 in human liver tran-
scriptomic data and 2) analyze the impact of genetic variations on disease outcomes in popula-
tion-based cohorts; 3) test an innovative therapeutic approach based on hepatocyte-targeted an-
tisense oligonucleotides downregulating SUV420H1/2 in human liver organoids/assembloids. 
 
1.2 Background and State of Art 
Nonalcoholic fatty liver disease (NAFLD), the leading cause of liver damage worldwide, is 
characterized by liver fat accumulation and association with insulin resistance, and frequently 
progresses to steatohepatitis (NASH), ultimately leading to cirrhosis and liver cancer1-3. 
NAFLD/NASH is becoming globally a leading cause of liver-related morbidity and mortality and by 
altering liver function contributes more widely to the burden of cardiometabolic, renal and 
neoplastic diseases 4,5. In the presence of concomitant hepatotoxic factors such as in particular at-
risk alcohol intake and some medications (defined in this case as metabolic dysfunction associated 
fatty liver disease or MAFLD) synergizes with them representing a major driver of liver disease 
progression 6. However, for those with advanced disease or who cannot successfully change their 
diet and lifestyle, no effective treatment is yet available to prevent or treat this condition, which is 
therefore projected to become a major public health threat in the next decade. Although 
understanding the mechanisms of NAFLD development and progression is essential for its 
prevention/treatment, molecular players involved in its progression are poorly defined. Recently, 
an epigenetic component is recognized in this disorder and the histone methyltransferases 
SUV420H1/2 are promising candidates for this function. Multiple evidences connect lipid and iron 
metabolism in the hepatocyte: 1) Genome Wide Association Studies (GWAS) in human populations 
showed an overlap of loci affecting iron and lipid metabolism, and excess iron in hepatocytes 
favors dyslipidemia and NAFLD 7; 2) upregulation of the liver hormone hepcidin, under the control 
by the BMP-SMAD pathway, is protective against NAFLD-NASH 8,9. Interestingly, a GWAS 
performed in mouse strains kept on high-iron diet identified a shared association between liver 
iron and triglyceride levels at a region of chromosome 7 encompassing the histone 

 
 
methyltransferase Suv420h2. Our preliminary data show that mice with Suv420h inactivation in 
adipose tissue are resistant to diet-induced liver steatosis due to increased PPAR  signaling. Since 
mice with liver BMP-SMAD pathway upregulation showed Suv420h downregulation, we 
hypothesize that the protective effect of increased hepatocyte BMP-SMAD signaling on NAFLD 
development is due to Suv420h. In agreement, Suv420h inactivation in hepatocytes counteracts 
diet-induced NAFLD, as highlighted by our preliminary results. 
 
The present study is part of the RF project (Ricerca Finalizzata) – Project code: RF-2021-12373889 
funded by the grant call of the Ministery of Health (2020-2021). In the attached project we 
proposed 3 different aims: 
1) To characterize the disease progression in Suv420h1/2-liver conditional KO mice and to identify 
the molecular pathways/genes involved in the protection against NAFLD-NASH development. 
2) To examine the impact of SUV420H1/2 genetic, epigenetic and transcriptional variability on 
clinical outcomes for the identification of novel biomarkers of NAFLD-NASH. 
3) To propose and test an innovative therapeutic approach based on hepatocyte-targeted 
antisense oligonucleotides (ASOs) against Suv420h1/2 in preclinical models in mice and in human 
liver organoids and assembloids. 
 
 
The present clinical study protocol will focus on the clinical aspects and procedures that will regard 
an intervention on data and sample of clinical cohorts. Further details about the analyses 
conducted in animal models, for which a specific Authorization has been requested, can be found 
in the attached project.  
 
2. PURPOSE AND AIMS 
 
As previously reported the general aim of the project is to study the cellular and molecular mecha-
nisms of correlation between the SUV420H1/H2 methyltransferase and the progression of liver 
damage in mice (San Raffaele Institute) and to confirm these findings in human samples and in vitro 
3D liver models.  
 
2.1 Primary Aim 
 
A)  To identify the main genes and pathways differentially expressed and the main factors associat-
ed with SUV420H1/H2 expression by comparing the transcriptomic and lipidomic profile of individ-
uals with low hepatic SUV420H1/H2 mRNA expression levels (lowest expression quartile) to that 
from individuals with high expression levels (top quartile). 

 
 
In order to evaluate the role of SUV420H1/H2 in the regulation of gene expression in NAFLD pa-
tients, we will exploit already available transcriptomic and lipidomic data by analyzing the gene ex-
pression databases deriving from ongoing studies conducted at the Fondazione and by the PI of the 
Catanzaro collaborating centre.  Data have been generated from liver and visceral adipose biopsy of 
obese individuals at high risk of developing NASH (PERSPECTIVE and REASON studies at Fonda-
zione, and MAFALDA Study, coordinated by prof Stefano Romeo, PI of the Università Magna Grae-
cia Catanzaro).  
 
2.2 Secondary Aims  
 
B) To evaluate whether there is a causal association between SUV420H1/2 activity and NAFLD. 
 
In order to address this aim, we will examine the impact of both common naturally occurring ge-
netic variants in the SUV420H1/2 genes (minor allele frequency (MAF) >0.01), rare (MAF< 0.01) on 
the risk of developing liver disease. This will be achieved by analyzing in pseudo-anonymised and 
aggregated form the genetic databases deriving from previously characterized human cohorts 
comprising patients with severe NAFLD and controls in NGS studies conducted at the Fondazione 
(PERSPECTIVE study) and by analyzing the public population-based UK Biobank cohort (N>200,000 
with complete genomic data available), for which the PI of the Catanzaro group has approved au-
thorisation. 
 
C) To validate the protective effect of SUV420H1-H2 downregulation in human cells and to propose 
an innovative therapeutic approach based on hepatocyte-targeted antisense oligonucleotides 
(ASOs) by evaluating the intracellular fat reduction and the fibrosis and inflammation regulation. 
 
In order to fulfil this aim, we will take advantage of human liver organoids (HLO) and assembloids 
(HLA) isolated from surgical specimens from an ongoing study at the Fondazione (REASON study). 
 
 
3. STUDY DESIGN  
3.1 Study design 
Interventional, genetic, biological, non-pharmacological and multicentric. 
 
3.2 Inclusion Criteria 
We will analyse data and samples from subjects with the following criteria: 
 

 
 
- Subjects aged>18; 
- Subjects who have already given their consent to genetic analysis and whose samples and data 
have already been collected as part of the PERSPECTIVE, REASON and MAFALDA studies; 
- Subjects who have given their consent to participate in this study. 
 
In particular, subjects with the following characteristics were included respectively: 
 
- in the PERSPECTIVE study: 
1. Diagnosis of NAFLD  
2. Age between 45 and 75 years old 
3. Any of the following criteria: 
a. F3-F4 fibrosis, determined histologically, or by non-invasive techniques, or evidence of cirrhosis 
deriving from biochemical tests or imaging methods; 
b. Family history of related first-degree primary liver cancer, or carrier status of rare mutations 
associated with the development of HCC (such as mutations in APOB and TERT) 
c. Male patient with type 2 diabetes or obesity carrying at least three genetic variants in PNPLA3, 
TM6SF2, MBOAT7. 
 
- in the REASON study: 
Patients aged>18, who have given their consent to participate in the study, who underwent the fol-
lowing procedures: 
- liver biopsy for suspected non-alcoholic steatohepatitis (NASH) at the time of diagnosis; 
- liver resection for hepatocarcinoma, other liver lesions (including secondaries from other neo-
plasms and benign focal lesions, which will allow obtaining healthy starting liver tissue), biopsies 
of whole liver explants obtained at the time of liver transplantation AND cholecystectomies. 
 
- In the MAFALDA study: 
Patients undergoing bariatric surgery for grade 3 obesity (BMI g40 Kg/m2) or grade 2 obesity plus: 
- metabolic comorbidities (uncontrolled hypertension, diabetes, dyslipidemia); 
- lack of contraindication to surgery (e.g. advanced liver disease with portal hypertension); 
- willingness to sign an informed consent. 
 
3.3 Exclusion Criteria 
Individuals not reporting one of the inclusion criteria listed above or reporting at-risk alcohol intake 
(>30/20 g/day in M/F), viral and autoimmune hepatitis or other causes of liver disease will be ex-
cluded. 
 

 
 
4. STUDY PROTOCOL, PROCEDURES AND METHODS  
 
4.1 Procedures 
 
An informed consent to the use of previously collected data and samples, to carry out new specific 
analysis related to this study, will be signed by the study participants during the basal or routine fol-
low-up visits already planned by the normal clinical practice. 
 
Patients enrolled in the REASON study will be asked their consent to the use of the biological sam-
ples already collected in the context of the study, to conduct the experiments and analysis related 
to this specific study. 
 
For patients in the PERSPECTIVE study, anamnestic data, family history, lifestyle, demographic and 
anthropometric parameters, and clinical data (Glucose, HbA1c, Insulin, Tot. Chol, HDL, TGL, AST, 
ALT, GGT, Ferritin, Albumin, PLTs, HBcAb, AFP) will be collected during the basal or routine annual 
follow-up visit, as normal clinical practice 
 
All raw data and biological samples are available respectively from the ongoing PERSPECTIVE and 
REASON studies (Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico - Milano) and MAFAL-
DA and UK Biobank cohorts (Prof. Romeo, Università della Magna Grecia Catanzaro). 
 
4.2 Analysis  
 
Analyses related to aim A 
 
As previously reported, in order to address the aim A the transcriptomic and lipidomic profile of 
SUV420H1/2 will be analyzed in ongoing studies (PERSPECTIVE and REASON) and characterized co-
hort of ~500 individuals at high risk of NASH who underwent liver and visceral adipose tissue biopsy 
during metabolic surgery10 (Transcriptomic cohort, n= 250 individuals in whom liver and visceral ad-
ipose are paired from the same individual; MAFALDA cohort, a cross-sectional study comprising a 
total of 264 severely obese adult participants (BMI > 40 kg/m2) undergoing bariatric surgery en-
rolled at the Campus Bio-Medico University of Rome (study  PI: Prof. Romeo).  
The main genes and pathways differentially expressed and the main factors associated with 
SUV420H1/H2 expression will be identified as previously described 10. Lipidomic profile will be ana-
lyzed as previously described 11. Specifically, we will compare transcriptomic and lipidomic profile 
from individuals with low hepatic SUV420H1/H2 mRNA expression levels (f25 centile) to that from 
individuals with high expression levels (g75 centile). Through Gene Set Enrichment Analysis (GSEA) 

 
 
and Ingenuity Pathway Analysis (IPA), the pathways differentially expressed and the main factors 
associated with SUV420H1/H2 expression will be identified. 
Then, genes significantly regulated in the two groups (high versus low SUV420H1/2 expression) will 
be confirmed at protein level (Western Blotting and immunohistochemistry assays).  Finally, a CHIP-
Seq analysis will be performed in a subset of liver samples (n=25 from PERSPECTIVE and REASON 
Studies) to evaluate the direct involvement of histone methylation regulation on expression modu-
lation of the main SUV420H1/H2 targets. This aim will be conducted in collaboration between the 
Milan and Catanzaro Units. 
 
 Analyses related to aim B 
 
To support a causal association between impairment of SUV420H1/2 activity and the risk of NAFLD, 
we will examine the impact of both common (MAF >0.01), rare (MAF <0.01) variants altering 
SUV420H1 (also known as KMT5B) and SUV420H2 (also known as KMT5C) genes expression and 
predicted protein activity and those of genes differentially expressed on the risk of NAFLD-NASH 
and liver-related and metabolic outcomes in two cohorts we previously exploited to uncover NAFLD 
genetics: patients with severe NAFLD (that is with advanced fibrosis and/or hepatocellular carcino-
ma) and controls (patients from the PERSPECTIVE cohort not carrying the variants)11 and the popu-
lation-based UK Biobank cohort (n>350,000).  
The common variant analyses will be done by assuming an additive genetic model and using a bina-
ry logistic (for categorical traits) or a liner regression model (for linear traits) adjusting for age, gen-
der and body mass index. The rare variant analyses will be conducted by using two models: a) in 
nonsense genetic variation (splicing, frameshift, premature stop codon) burden test will be used 
and b) missense (nonsynonymous) SKAT-O test will be used assuming bidirectionality of the effect 
association with the genetic variants.  
We will consider as outcomes liver-related traits (cirrhosis, hepatocellular carcinoma, severe liver 
disease, hepatic fat content measured by magnetic resonance imaging proton density fat fraction 
(MRI-PDFF) and liver enzymes) and metabolic traits (adiposity, diabetes, circulating lipids). We will 
assess the interaction between genetic traits and environmental triggers (adiposity and insulin re-
sistance) 12. All these variables are regularly collected for clinical practice except for the MRI-PDFF 
which is however available for the participants in the UKBB cohort study. This aim will be conducted 
in collaboration between the Milan and Catanzaro Units. 
 
Analyses related to aim C 
 
As previously reported, to validate the protective effect of SUV420H1-H2 downregulation in human 
cells we will take advantage of human liver organoids (HLO) isolated from surgical specimens from 

 
 
an ongoing study at the Fondazione (REASON). 
In the contest of the REASON study, in collaboration with Unità dei Trapianti di Fegato of IRCCS Ca’ 
Granda Foundation, we collect liver samples starting from waste tissue of patients who underwent 
cholecystectomy, both intra-tumoral and extra-tumoral hepatocellular carcinoma resection, or from 
transplanted liver. 
Liver samples are collected in perfusion solution at 4°C and processed to generate liver organoids 
(HLO) and assembloids (HLA) as soon as possible. We will exploit already available data generated 
by previously collected samples and collect new samples, after informed consent. 
HLO will be generated, expanded and differentiated as previously described 13 and reported in the 
attached project.  
To generate HLA, single HLO will be combined with NPC (physiologic ratio of 30:8:6:5 
HC:HSC:KC:LSEC) 14 in a small drop of extracellular matrix and cultured in a spinning bioreactor (95 
rpm) as previously reported 15. HLA structure will be analyzed by IHC/IF and compared to the start-
ing liver tissue.  
To induce NASH in vitro, both HLO and HLA will be treated for 7 days with a steatogenic insults: 
high-fat (palmitic and oleic acid 300 mcM), high cholesterol (50 mcM), high fructose (10 mM d-
fructose) and high-insulin (300 nM) or a mix of them. Concentrations will be optimized to maximize 
the impact avoiding excessive lipotoxicity and cell death. 
ASO-mediated SUV420H1/H2 knock-down will be performed via transfection-free uptake of SUV-
specific or scramble ASO in HLO/A culture medium for 6 days. To confirm the ASO- mediated 
SUV420H1/H2 knock-down at the RNA and protein level we will perform respectively a quantitative 
real time PCR and a Western blot analysis as detailed in the attached project.  
Finally, to measure the impact of SUV420H1/H2 reduction in fatty liver disease pathophysiology, 
HLO/A will be exposed for long period to the most effective steatogenic environment (palmitic and 
oleic acid both 300 μM), and genes involved in lipid metabolism (identified in aim A) will be ana-
lysed by gene expression by qRT-PCR as previously described. Furthermore, intracellular fat reduc-
tion will be detected by BODIPY™ 493/503  staining and by using a quantitative Triglyceride Assay 
Kit (Abcam), TNFalpha  mRNA expression will be evaluated by in situ hybridization assays, type 1 
collagen will be quantified by immunofluorescence assays (as detailed in the project). This aim will 
be conducted by the Milan Unit. 
 
5.ENDPOINTS 
  
5.1 Primary endpoint 
 
A) The primary endpoint of this study is the identification of biological pathways differentially ex-
pressed in NAFLD individuals, a complementary analysis for replicating the findings of the main aim 

 
 
of the RF project (which is a Theory Enhancing, mostly basic study project).  
 
 5.2 Secondary endpoints 
B) The identification of a correlation between genotype influencing the SUV420H1/2 pathway and 
NAFLD phenotype. 
 
C) Reduction of intracellular fat accumulation, collagen accumulation and inflammation in terms of 
TNFalpha mRNA expression levels.  
 
In case of positive results from SUV420H1/H2 downregulation by antisense technology, both in 
mouse models than in human liver organoids and assembloids, we will submit a patent request for 
the human anti-SUV420H1-H2 ASO for the treatment of steatohepatitis (Fondazione IRCCS Ca' 
Granda and San Raffaele Institute). 
 
6. PROJECT TIMELINE 
The clinical cohorts for transcriptomic and genomic analysis are being recruited and data are 
stored in consolidated database available for gene expression and genomic studies. Initial anal-
yses have already led to peer-reviewed publications in international journals 10, 12, 16. 
 
Study start: March 2023 
Enrolment and data collection start: March 2023 
Enrolment and data collection closure: February 2025 
End of study: February 2026 
 
 
6.1 Gantt Chart  
 
Aims 
Activities 
Months 
01-06 07-12 13-18 19-24 25-30 31-36 
A 
Characterize the disease progression in Suv420h1/2 liver conditional KO 
mice and HLO 
 
 
 
 
 
 
  
Identification of the main genes and pathways associated with 
SUV420H1/2 expression in mice (HSR Institute) 
 
 
 
 
 
 
  
Human Transriptomic data analysis and identification of the SUV420H1-
H2 gene signature 
 
 
 
 
 
 
  
Evaluation of human lipidomic fingerprint associated with different levels 
of SUV420H1-H2 expression 
 
 
 
 
 
 

 
 
  
ChIP-seq analysis 
 
 
 
 
 
 
B 
Identification of novel biomarkers of NAFLD-NASH 
 
 
 
 
 
 
  
Genomic data analysis to evaluate the impact of rare and common vari-
ants altering SUV420H1-H2 pathway expression on NAFLD progression 
 
 
 
 
 
 
C 
Innovative therapeutic approach in preclinical models and in human 
liver organoids and assembloids 
 
 
 
 
 
 
 
Impact of Suv420h1-h2 downregulation on hepatic fat accumulation, 
inflammation, fibrosis and targets engagement in mice after 10 weeks of 
the FPC diet 
 
 
 
 
 
 
 
ASOs downregulate SUV420H1/2 specifically in human hepatocytes 
 
 
 
 
 
 
 
SUV420H1/H2 target genes modulation 
 
 
 
 
 
 
 
Parenchymal and NPC (LSEC, KC, and HSC) co-colture in 3D structures 
 
 
 
 
 
 
 
ASO beneficial impact on intracellular fat accumulation and on the 
expression of genes involved in the induction of inflammation and 
fibrogenesis in steatogenic HLO 
 
 
 
 
 
 
 
ASO beneficial impact on intracellular fat accumulation and on the 
expression of genes involved in the induction of inflammation and 
fibrogenesis in steatogenic HLA 
 
 
 
 
 
 
 
 
7. STATISTICAL ANALYSIS 
 
7.1 Sample size 
Considering the large number of individuals with severe liver disease in the NAFLD PERSPECTIVE 
and MAFALDA cohorts (n= 500) and the large size of UKBB population-based cohort (n=350,000 for 
common variants and n=200,000 for rare variants), for variants with a minor allele frequency (MAF) 
>0.01, we estimated that the study has a power >80% to detect six-fold difference in the risk of se-
vere NAFLD in the PERSPECTIVE/MAFALDA, a 1.8% difference in the NAFLD prevalence (UK Bi-
obank), and 4.4% difference in the liver fat content as measured by PDFF (UK Biobank) between 
carriers and non–carriers (Aim A and B). 
The high number of HLOs (>10^3), HSCs (>10^5), KC (>10^6) and LSEC (>10^5) isolated from a single 
donor and the high efficiency of isolation, will confer >80% power to detect and analyze distinct cell 
subsets representing >1% of overall liver cell population. The variable monitored to define the 
group size is the level of intracellular fat reduction in treated or untreated HLO or HLA with 
SUV420H1-H2-GalNAc-ASO (Aim C). Then, 9 HLO or HLA will be analysed to determine an effect size 
of 1.5 between the two groups, with a power of 80% and an alpha error of 0.05 (effect size = 1.5). 
This sample size is determined by Student's t-test to compare the means of two independent 

 
 
groups, using the G* Power software. 1-3: Student's t test (for 2 groups) or ANOVA (for multiple 
comparisons) will be used. Statistical significance will be considered for P<.05. 
 
7.2 Statistical analysis plan 
Statistical 
analyses 
will 
be 
carried 
out 
using 
both 
JMP 
(SA, 
Cary, 
NC) 
and 
R 
(http://www.Rproject.org/) softwares. For descriptive statistics, continuous variables will be shown 
as mean and SD. Categorical variables will be tested by chisquare test and presented as number and 
proportion. In all transcriptome-wide unsupervised analysis and GSEA, p values will be corrected for 
multiplicity by Benjamini-Hochberg false discovery rate method, and adjusted p values 0.01) anal-
yses will be performed assuming an additive genetic model and using a binary logistic (for categori-
cal traits), liner regression (continuous traits), or ordinal regression (ordinal traits) model as appro-
priate, adjusted for confounding factors including age, gender, and body mass index. The rare vari-
ant (MAF >0.01) analyses will be performed assuming an additive genetic model and using a binary 
logistic (for categorical traits), liner regression (continuous traits), or ordinal regression (ordinal 
traits) model as appropriate, adjusted for confounding factors including age, gender, and body mass 
index. The rare variant (MAF <0.01) analyses will be performed using burden, sequence kernel as-
sociation test (SKAT) and SKAT-O gene-based tests as implemented in SAIGE-GENE to identify cod-
ing variants associated with PDFF used as a linear trait, alternatively with cirrhosis or chronic liver 
disease (defined by using ICD-10 codes), as categorical traits in the analyses. 
Interaction between genetic traits and environmental triggers in determining liver disease will be 
assessed by including the interaction term into each statistical model. 
 
8. ADVERSE EVENTS 
The project does not include the administration of drugs or other substances or invasive clinical 
practices. Therefore, no adverse events are expected. 
 
9. RISK / BENEFIT EVALUATION 
The study does not foresee an immediate benefit for patients, but the results of this trial will have 
the potential to lead to a decoding of the mechanisms underlying the development of NASH, to 
resolve the temporal order of the events that regulate its evolutionary trajectory, to understand 
progression, characterization and improve the therapeutic management of patients. 
 
10. PROJECT MANAGEMENT 
 
10.1 Regulatory and Ethics  
This study will be conducted by Good Clinical Practice (GCP) rules; in accordance with the ethical 

 
 
principles that have their origin in the Declaration of Helsinki and with the respect to the Euro-
pean clinical practice, in compliance with all international guidelines and national law regulation 
in Italy. 
The protocol and the informed consent document must be submitted to the Independent Ethics 
Committee (IEC) for review and will receive IEC approval/favourable opinion before initiation of 
the study. During the study, any amendments to the protocol must also be approved by IEC, be-
fore their implementation. A progress report is sent to the IEC at least annually, and a summary 
of the study’s outcome is sent at the end of the study. 
 
10.1.1 Patient information 
Participants enrolled will be required to consent to the deidentified use of personal data for the 
study. Information form and the module for the acquisition of informed consent for the han-
dling of sensitive data will be given to the patient.  
The investigator will fulfil the current regulations for research and documentation of informed 
consent, the standards of Good Clinical Practice and the ethical principles derived from the Dec-
laration of Helsinki. The approval by the Ethics Committee will be required whether an update 
of the informed consent form will be needed during the study. 
According to the recommendations of the Declaration of Helsinki and local regulations, each pa-
tient will be adequately informed about the aims, methods, expected benefits, potential risks 
and problems related to the study. Moreover, patients will be informed of their right to refuse 
consent to the use of their sensitive data or to withdraw it at any time, without having any ef-
fect on their medical care. 
The patient will have all the time necessary for the evaluation of the information received be-
fore providing their informed consent to the use of sensitive data. The investigator will have to 
obtain spontaneous informed consent in writing by the patient, before using them in any way 
for the study. The written consent to the handling of sensitive data must be subscribed by the 
date and signature of the patient and by the investigator's or his representative's ones. 
The investigator has to give to the patient a signed copy of informed consent; the original form 
will be retained with the other documents of the study protocol; the module for the acquisition 
of informed consent to the treatment of sensitive data will be attached to the clinic folder. The 
collaborator will be appointed to review the original forms of all patients’ informed consent.  
 
10.3 Confidentiality 
According to the ICH guidelines for the Good Clinical Practice, the monitoring team must check the 
CRF entries against source documents. The personnel bound by professional secret must maintain 
the confidentiality of all personal identity or personal medical information (according to the confi-
dentiality and personal data protection rules). The confidentiality of records that could identify sub-

 
 
jects should be protected, only initials of the name and the first name will be registered with an in-
clusion coded number for the study (no name nor address nor identifying data). 
 
10.3.1 Publications  
The Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico is the only owner of the data. 
Communications, reports, and publication of the results of the study will be under the responsi-
bility of the principal investigator of the study. A summary of the results of the study will written 
and provided on request of the participating patients  
 
10.4 Intellectual property rights of study results 
The Promoter of the study and the Participating Centres acknowledge and accept that during the 
implementation of the Project, to the extent strictly necessary to the performance of the Project, 
know-how, technical material and/or goods protected by industrial and/or intellectual property 
rights or susceptible to protection, developed prior to the commencement of the Project by the 
Promoter and the Participating Centres, shall be and shall remain their exclusive ownership.  Noth-
ing in this protocol shall be construed as a grant of rights under such intellectual property.  
The results arising from the research activities will be jointly owned by the Promoter of the study 
and the Participating Centres in proportion to their respective contribution, being understood that 
the provision of biological samples underlying the research project, related clinical information and 
related medical know-how by the Study Promoter will be considered as an essential contribution. 
In cases of innovative results, which are subject to patent protection (or similar rights) and/or eco-
nomic exploitation, ownership of such Invention shall be owned based on inventorship contribu-
tion. In case of joint ownership, the Promoter of the study and the Participating Centres will regu-
late, in fair and good condition, the protection and the exploitation of the results. 
 
 
11. INSURANCE 
No additional blood sampling, biological sampling or clinical data will be required in addition to 
those already collected during the regular clinical practice or ongoing studies. The internal institu-
tional insurance policy will cover for any undesirable effects due to the participation in the study. 
 
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