Luca ValentiDepartment of Pathophysiology and Transplantation, Università degli Studi di Milano

Internal Medicine, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico

luca.valenti@unimi.it

Genetics & Epigenetics

Monotematica AISF 8 Ottobre 2015

Steatosis

Peri-cellular/venular fibrosis Cirrhosis

Histological spectrum of NAFLD:From simple steatosis to NASH and HCC

HCC

Lobular inflammationBallooning

Liver histology in 351 consecutive severely obese individuals

Normal liver 14%

Simple steatosis 31.5%

NASH w/o Significant fibrosis

31%

NASH & Significant fibrosis

11.5%

Browning, Hepatology 2004

Prevalence of steatosis varies with ethnicity

Hispanics Whites Blacks

Fat

ty li

ver

%

45%

M F

24%

M F

42%

M F

24%

LYPAL1

Speliotes

PPP1R3B

GCKR

NCAN

TRIB1

Chambers

HSD17B13

CPN1

GWA studies in NAFLD: an overview

PNPLA3rs738409

C>G

Romeo

Anstee

Kitamoto

1 48117910

I148MDGGVGASAG

16647

PATATIN-LIKE PHOSPHOLIPASE DOMAIN-CONTAINING 3 (PNPLA3)

II

IM

MM

0

2

4

6

Hep

atic

Fat

(%

)

p = 7 x 10-14

0

10

20

30

40

BM

I (kg

/m2 )

0

1

2

3

4

HO

MA

-IR

0

25

50

75

100

Pla

sma

TG

(m

g/d

l)PNPLA3 rs738409 C>G (I148M)

always confirmed top hit with larger effect size

Tg

Endoplasmic Reticulum

Lipid droplets

Obesity and insulin resistance favor lipid droplets accumulation in hepatocytes

FFAs

Extracellularspace

VLDLsecretion

Early Golgi

TM6SF2167E Nascent

VLDL

ObesityInsulin resistance

PNPLA3148I

HFC: 0-5%

Dongiovanni, BMC Research International, 2015

Endoplasmic Reticulum

Lipid droplets

Romeo, Nat Genet 2008, He, J Biol Chem 2010; Ruhanen, J Lipid Res 2014; Dongiovanni, World J Gastroentorol2013; Dongiovanni, Hepatology 2014

Impaired lipid droplets remodeling causes NASH in PNPLA3 I148M carriers

FFAs

Extracellularspace

VLDLsecretion

Early Golgi

TM6SF2167E Nascent

VLDL

PNPLA3148M

ObesityInsulin resistance

Tg

HFC: 6% �

Interaction between PNPLA3 I148M and diet

The NASH score

PNPLA3 I148M and fibrosis risk in patients with NAFLD

Sookoian, Hepatology 2011

Alle

licO

dd

s R

atio

0

0,5

1

1,5

2

2,51.77

[1.42-2.19]p= 2.8 x 10-7

1.55[1.03-2.34]

p= 3.5 x 10-2

2.20[1.80-2.67]

p=4.7 x 10-15

945 1,374n= 2,503

Trépo E, Hepatology 2014

PNPLA3 I148M and HCC risk in cirrhosisan individual patient data meta-analysis

Obesity andinsulin resistance

Excess alcoholFructose

Hepatitis C virus

PNPLA3 148I

Mild uncomplicated steatosis

PNPLA3 148M

Steatohepatitis &

fibrogenesis

Cirrhosis

Valenti, Hepatology 2012Valenti, Dig Liver Dis 2013

PNPLA3 I148M and progressive liver disease:a new paradigm in hepatology

Hepatocellularcarcinoma

PNPLA3 148M/M

Directcarcinogenic

activity

Endoplasmic Reticulum

Lipid droplets

Kozlitina, Nat Genet 2014; Holmen, Nat Genet 2014; Mahdessian, PNAS 2014; Dongiovanni, Hepatology 2014

Impaired VLDL secretion in E167K TM6SF2carriers causes NASH

FFAs

Extracellularspace

VLDLsecretion

Early Golgi

TM6SF2167K Nascent

VLDL

ObesityInsulin resistance

PNPLA3148I

HFC: 6% �

E167K TM6SF2

0

20

40

60

80

100

NASH Advanced fibrosis Carotid plaques

Pre

vale

nce

%

EE

EK + KK‡

p=0.003 p=0.008 p=0.031

n = 1,044

n = 157

TM6SF2 E167K variant disentangles NASH from cardiovascular disease

Endoplasmic Reticulum

Fattyacids

Extracellularspace

VLDLsecretion

Early Golgi

TM6SF2Nascent

VLDL

PNPLA3

ObesityInsulin resistance

APOB

Lipid droplets

GCKR

HyperglycemiaHyperinsulinemia

TRIB1

MTTP

Mitochondria

Lipotoxicity FFAR4IL28B

UCP2SOD2

IRS1ENPP1

KLF1FABP5

LYPLAL1

Maternal fat intake primes NASH

Bruce, Hepatology 2009Anderson, J Hepatol 2014; Breji, JCEM 2014

Control of gene expression by DNA methylation and chromatin remodeling

Maternal fat intake alters gene expression and DNA methylation

Dudley, PONE 2011

p21 demethylation correlates with:• increased expression

• cell senescence

Differential methylation in NAFLD impacts on gene expression and disease

severity

Murphy, Gastroenterology 2013

PNPLA3 expression is regulated by methylation at CpG99

Kitamoto, J Hepatol 2015

micro-RNAs are small noncoding RNAs that regulate gene expression at post-

transcriptional level

NASH is associated with deregulation of hepatic miRNAs

Cheung, Hepatology 2008

miR-122 deletion favors NASH and HCC

Hsu, J Clin Invest 2012

NASH results from the interaction among genes, environment & diet

Epigenetics

Key points :� NASH is a strongly heritable disease

� PNPLA3 I148M variant is the major common genetic riskfactor, but the phenotypic expression triggered byenvironment and diet (obesity, fructose, alcohol)

� Most genetic risk variants regulate hepatic lipidmetabolism

� NASH is associated with altered chromatin remodeling,mainly characterized by DNA hypomethylation, andaltered miRNA expression

� Epigenetic modifications mediate the effect ofenvironment, diet-insulin resistance, aging andheritability on NAFLD phenotype

Thank you for your attention!

AcknowledgementsMetabolic Liver Diseases Lab

Benedetta DonatiMarta MilanoMarica MeroniOscar BorsaniGuido BaselliClaudia Lanti

Paola DongiovanniRaffaela RamettaAlessandro Pietrelli

Clinical centerSilvia FargionAnna FracanzaniCristina BertelliErika FattaGiuseppina PisanoSerena PelusiMarianna PorzioRosa LombardiVittorio Borroni

UdineGiorgio Soardo

Migliavacca CenterMassimo Colombo

Alessio AghemoPietro Lampertico

INGMRaffaele Defrancesco

Cristina Cheroni

PathologyValentina VairaMarco MaggioniSilvano Bosari

NewcastleQuentin Anstee

Chris Day

GothenburgStefano Romeo

New YorkDomenico Accili

Utpal Pajvani

SurgeryStefano GattiEnrico Mozzi

TorinoElisabetta Bugianesi

Ester VanniRoma

Valerio NobiliLuca Miele, Anna Alisi

PalermoSalvo Petta

Zurich/DresdenFelix Stickel

Jochen Hampe

DallasJulia Kozlitina