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Towards genetic prediction of non-alcoholic fatty liver disease trajectories: PNPLA3

and beyond

Marcin Krawczyk, Roman Liebe, Frank Lammert

PII: S0016-5085(20)30229-8
DOI: https://doi.org/10.1053/j.gastro.2020.01.053
Reference: YGAST 63217

To appear in: Gastroenterology

Accepted Date: 29 January 2020

Please cite this article as: Krawczyk M, Liebe R, Lammert F, Towards genetic prediction of non-
alcoholic fatty liver disease trajectories: PNPLA3 and beyond, Gastroenterology (2020), doi: https://
doi.org/10.1053/j.gastro.2020.01.053.

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Towards genetic prediction of non-alcoholic fatty liver disease trajectories:

PNPLA3 and beyond

Marcin Krawczyk1, Roman Liebe1,2 & Frank Lammert1

1
Department of Medicine II (Gastroenterology & Endocrinology), Saarland University

Medical Center, Saarland University, Homburg, Germany

2
Department of Department of Gastroenterology, Hepatology and Infectious Diseases,

University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany

Address for correspondence:

Univ.-Prof. Dr. med. Frank Lammert

Department of Medicine II (Gastroenterology and Endocrinology)
Saarland University Medical Center
Saarland University
Kirrberger Str. 100
D 66421 Homburg
Germany
Phone: +49-6841-16-15021
Fax: +49-6841-16-15022
Email: frank.lammert@uks.eu
Abstract

Non-alcoholic fatty liver disease (NAFLD) is on the verge of becoming the leading

cause of liver disease. NAFLD develops at the interface between environmental

factors and inherited predisposition. Genome-wide association studies, followed by

exome-wide analyses, led to identification of genetic risk variants (e.g. PNPLA3,

TM6SF2, SERPINA1) and key pathways involved in fatty liver disease pathobiology.

Functional studies improved our understanding of these genetic factors and the

molecular mechanisms underlying the trajectories from fat accumulation to fibrosis,

cirrhosis and cancer over time. Here, we summarize key NAFLD risk genes and

illustrate their interactions in a 3D "risk space". Although NAFLD genomics sometimes

appears to be "lost in translation", we envision clinical utility in better trial design,

outcome prediction, and NAFLD surveillance.

..

Keywords

Adiponutrin, genetic risk score, genomics, non-alcoholic steatohepatitis, risk factor,

risk prediction

2
I. Introduction

The increasing prevalence of overnutrition and sedentary lifestyle, resulting in excess

hepatic fat deposition, has revealed the detrimental impact of several genetic factors

that were previously not considered pathogenic. Non-alcoholic fatty liver disease

(NAFLD) actually presents the perfect storm of a rapid and profound environmental

change - in the shape of massive, epidemic overnutrition - that requires a fulminant

change in metabolic strategy relative to the previous evolutionary pressures and

constraints of cyclical fame and famine over thousands of generations. Familial

clustering, inter-ethnic differences in susceptibility and twin studies indicate there is a

significant heritable component to NAFLD1. In the early years of research into the

genetics of NAFLD, most information was provided by rare, high-impact variants that

predisposed small subgroups of patients for diabetes (e.g. hepatocyte nuclear factor

mutations in maturity onset diabetes in the young, MODY) or dyslipidemia (e.g.

variants of apolipoprotein B, APOB). These high-impact variants are in themselves a

cause of disease due to their high penetrance and metabolic disturbance caused.

Prior to the advent of technology for genome-wide association studies (GWAS),

research on polymorphisms in known metabolic genes (e.g. redox handling, fatty acid

oxidation or chain elongation) or their regulators, e.g. peroxisome proliferator-activated

receptors (PPAR, a.k.a. nuclear receptor 1C), did not yield much insight into the role of

high-frequency, low-impact risk factors for progressive liver disease that only show an

impact under excess metabolic load. The arrival of affordable oligonucleotide scanning

arrays facilitating large scale GWAS in extended patient cohorts with NAFLD and its

more severe clinical variety, non-alcoholic steatohepatitis (NASH), has enabled

3
researchers to identify key molecules that predispose individuals for clinically relevant

consequences such as hepatic cirrhosis.

The success story of GWAS in metabolic disease began with patatin-like

phospholipase domain containing 3 (PNPLA3) gene: Previously unknown, it took

almost 10 years of functional studies to yield the information how a isoleucine-to-

methionine exchange mediates the highest impact of any frequent variant on chronic

liver disease progression known to date, approaching Mendelian inheritance2. But

even without knowing its detailed function, a plethora of GWAS revealed its

involvement in fatty liver disease of various etiologies. Other genes of higher

(membrane bound O-acyltransferase domain containing 7, MBOAT7) or lower risk

allele frequency (transmembrane 6 superfamily member 2, TM6SF2) and higher

impact were subsequently identified, studied, and will be described in their effect on

biochemistry and their value as predictors of liver disease progression in this review.

Apart from the profound knowledge of lipid handling in liver cells that has been

generated by the studies of the GWAS-identified disease modifiers, the identification of

these "weak spots" under metabolic pressure highlights potential intervention targets.

Amelioration of the impact of e.g. PNPLA3 gene variation by silencing of the risk allele

with liver-targeted antisense oligonucleotides would no doubt have a beneficial impact

on carierrs with NAFLD, who are more prone to develop cirrhosis and subsequently

hepatocellular carcinoma (HCC). This effect was shown in mice3, but the strategy

might not be directly translatable to humans due to the pleiotropic effects of the

PNPLA3 risk variant, which shows protective effects against acne, gallstones, gout,

and hypercholesterolemia4. Of note, a recent Mendelian randomization study in

cohorts of NAFLD patients who underwent liver biopsy suggested that the causal

4
association between long-term hepatic fat accumulation and fibrosis is independent of

disease activity, while the causal effect of NAFLD on insulin resistance and risk of type

2 diabetes is mediated by fibrosis and not the presence of fat in the liver5.

There is also the rather considerable aspect of cardiometabolic pleiotropy:

Individuals bearing the PNPLA3 risk allele have a higher risk of liver disease via

impaired fatty acid handling, resulting in hepatocellular accumulation, but as a

consequence of decreased export, bear a lower risk of cardiovascular disease,

probably due to a lower serum lipid levels6 Potential cardioprotective effects of the

PNPLA3 risk allele were observed in 60,801 patients with coronary artery diseases in

comparison with 123,401 controls: Carriers of the p.I148M variant had a lower

incidence of coronary artery disease7. The association between the PNPLA3 variant

and lower serum triglyceride concentrations as well as lower odds of coronary artery

disease was also reported 2017 in a large exome-wide study involving > 300,000

participants, with validation > 280,000 individuals, showing that PNPLA3 and TM6SF2

variation is associated with lower plasma lipid levels and lower risk for coronary artery

disease, but increased risk of fatty liver and type 2 diabetes8. Predating this

observation, an opposing effect of the NAFLD-predisposing allele on total cholesterol

9, 10
and myocardial infarction risk had been described for the TM6SF2 . This illustrates

how the partition of lipids between liver and blood shifts the impact of excess

metabolic load from one organ to another and underscores the role of hepatic

modulation of lipid metabolism in cardiovascular diseases. Thus any treatment

strategy for fatty liver under metabolic stress needs to consider this shift in risk from

the liver to the heart, which has been observed in several clinical studies in

steatohepatitis. The anti-NASH effect of the farnesoid X receptor agonist obeticholic

5
acid is accompanied by increased serum lipid concentrations that require additional

treatment with lipid-lowering drugs11. A similarly negative impact on serum lipids and

thus potential cardiovascular risk was observed in the phase 2 clinical trial of an

acetyl-CoA carboxylase 1 inhibitor in NASH12.

While the risk of using GWAS-identified "metabolic bottlenecks" as treatment

targets in patients with prior cardiometabolic risk factors is prohibitive for drug

development, they may still serve as signposts that guide treatment with drugs that

show better cardioprotective profiles. In patients that appear to be unable to maintain

their weight and are refractory to dietary or lifestyle interventions, targeting those with

the highest risk of progression to cirrhosis and HCC might be necessary to channel

limited healthcare resources13.

II. Our favorite NAFLD risk genes

The results of candidate gene studies and GWAS of phenotypes of both NAFLD and

alcohol-related liver disease were summarized in the comprehensive review by Anstee

et al.14 in this journal in tabular format, listing all the genes potentially contributing to

the interindividual variation in the progression and outcomes of non-alcoholic and

alcohol-related liver diseases. In this section, we summarize the findings of those

genes that in our opinion display the strongest impact and potential relevance for

clinical applications. We opted to illustrate the results of the genetic studies and the

associations with the various subphenotypes of fatty liver diseases using "tree

diagrams" that apply the hierarchical structure of disease classifications and also

6
reflect the temporal disease progression (a.k.a. trajectories), from fatty liver to

steatohepatitis and cirrhosis. This approach has been termed TreeWAS15 and takes

advantage of these diagnostic hierarchies to illustrate the underlying shared genetic

architecture of fatty liver diseases and their trajectories (Figure 1 & Supplementary

Material 1).

Since the detected genetic variants account for only a small fraction (Table 1) of

the heritability of NAFLD found by biometrical analysis (20 - 50%)16, it has often been

speculated that other mechanisms including environmental and epigenetic effects

contribute to the evolution of NAFLD traits. As pointed out by Walsh & Lynch recently,

this "crisis of apparently missing heritability" has been largely resolved by

demonstrating that "such a pattern of missing heritability is exactly what is expected

17, 18
when each underlying locus only explains a small fraction of trait variation." .

Walsh & Lynch also point to the implausibility of a long-term contribution of non-

genetic effects to phenotypic evolution by recalling that "there is no permanent

response to selection within populations unless there is variation at the DNA level",

with several lines of experimental dating back to the beginning of the twentieth

century17.

PNPLA3 (adiponutrin)

Adiponutrin, the enzyme encoded by the PNPLA3 gene (a.k.a. calcium-independent

phospholipase A2ε), is a 481-amino acid member of the patatin-like phospholipase

domain-containing family (PNPLA). This domain was originally discovered in lipid

hydrolases of potato and named after the most abundant protein of the potato tuber,

patatin. PNPLA3 is expressed predominantly in the liver, retina, skin, and adipose

7
tissue19. In a series of seminal genetic studies, the common non-synonymous variant

p.I148M (c.444C>G, rs738409) of the PNPLA3 gene has emerged as the key genetic

determinant of fatty liver disease in pediatric and adult patients20. The prevalence

differs among ethnic groups, and these differences generally parallel those for NASH

and its sequelae. The variant is most prevalent in Hispanics (49%), less common in

non-Hispanic Europeans (23%), and the least common in African Americans (17%)21,
22
.

The first GWAS in the population-based Dallas heart study comprising 2111

individuals with different ethnic backgrounds demonstrated the p.I148M variant to be

associated (P = 5.9 × 10−10) with increased liver fat content, as determined by 1H-

magnetic resonance spectrometry21. There was a stepwise increase of hepatic

triglyceride content with the number of p.I148M risk alleles. The association was most

prominent in Hispanic patients, who are in general at a greater risk of developing fatty

liver as compared to European and African Americans21.

Of note, the PNPLA3 variant p.I148M is also associated with serum activities of

liver enzymes. The analyses of two large population-based cohorts demonstrated that

PNPLA3 polymorphisms are associated with serum alanine aminotransferase (ALT)

and γ-glutamyl transpeptidase activities in healthy individuals23, 24

. The genetic

association between the PNPLA3 mutation p.I148M and fatty liver disease was

subsequently replicated in many studies25-28.

Further studies showed that the PNPLA3 variant not only increases the odds of

developing fatty liver itself but it also determines the degree of hepatic injury and the

full spectrum of histopathological consequences of NAFLD29. Valenti et al.30 reported

that the PNPLA3 variant was associated with hepatic steatosis and NAFLD severity as

8
determined by liver biopsy, in particular the presence of NASH, steatosis grade > S1,

and fibrosis stage > F1, independent of age, body mass index (BMI), or diabetes.

Another detailed analysis of histopathological severity of NAFLD was performed by

Rotman et al.31, demonstrating that the PNPLA3 variant is associated with steatosis,

portal and lobular inflammation, NAFLD activity score (NAS), and fibrosis. The first

meta-analysis by Sookoian and Pirola20 included data from 16 studies and concluded

that the PNPLA3 p.I148M variant is associated with fatty liver (odds ratio [OR] for

homozygous carriers 3.3 and heterozygous carriers 1.9), NASH (OR 3.3 and 2.7,

respectively), necroinflammation (OR 3.2 and 2.6, respectively), and fibrosis (OR 3.3

and 2.1, respectively); the association with necroinflammation and fibrosis was

independent of the severity of steatosis. The recent meta-analysis by Xu et al.32

confirmed these results, with subgroup and sensitivity analyses showing that the

results were neither influenced by ethnicities nor the age of subjects or the source of

controls. Using two histopathologically characterized cohorts encompassing steatosis,

steatohepatitis, fibrosis, and cirrhosis (N = 1,074), Liu et al.33 confirmed that the

PNPLA3 variant is associated with advanced fibrosis/cirrhosis, which is independent of

age, BMI, and type 2 diabetes as confounders. Availing of transient elastography to

quantify liver fibrosis in 899 patients with chronic liver diseases, an association

between the PNPLA3 mutation and enhanced liver stiffness was identified in a wide

spectrum of chronic liver diseases34. Sensitivity analysis showed that the association

was present across stiffness values between 12 kPa and 40 kPa34, indicating that the

variant affects not only fibrogenesis but also cirrhosis severity.

Non-obese NAFLD is defined as NAFLD that develops in patients with a BMI

less than 25 kg m−2 35

. In patients with non-obese NAFLD, the PNPLA3 p.I148M allele

9
is more frequent than in other NAFLD patients22, 36-38 and independently associated

with both NASH and fibrosis stage ≥ F239.

Carriers of the PNPLA3 mutation who are obese40 or present with NASH41 are

predisposed to HCC development. In multivariate analysis adjusted for age, sex, BMI,

diabetes, and cirrhosis, carriage of each copy of the PNPLA3 risk allele conferred an

additive risk for HCC (OR 2.3), with homozygotes exhibiting a fivefold increased risk

relative to carriers of only wild-type alleles. When compared to the UK general

population, the risk-effect was more pronounced (OR 12.2)41. Further analysis of the

data found the positive predictive value of p.I148M to be weak, but the negative

predictive value of the absence of p.I148M to be strong (97%). Hence, prospective

studies are warranted to validate the clinical utility of PNPLA3 genotyping to select the

patients who are least likely to develop HCC and therefore least likely to benefit from

surveillance. In a series of newly diagnosed patients with HCC42, homozygosity for the

genotype p.I148M represented even an independent risk factor for death; HCC

patients with this PNPLA3 genotype displayed reduced median survival (16.8 months)

in comparison to carriers of the wild-type allele (25.9 months).

Overall the above studies document that the PNPLA3 mutation p.I148M

increases the risk of developing severe hepatic fat accumulation, progressive

inflammation, advanced fibrosis, and HCC across different ethnicities worldwide.

Accordingly, PNPLA3 p.I148M was associated with increased liver disease mortality in

the population-based United States National Health and Nutrition Examination

Survey43. In particular when fully adjusting for age, sex, ethnicity and alcohol,

homozygosity conferred a hazard ratio of 18.2 (95% confidence interval 3.5 - 94) for

liver-related death. Quantitative analyses showed that adiposity amplifies the genetic

10
risk conferred by PNPLA3 across the full spectrum of NAFLD, ranging from increased

hepatic triglyceride content to cirrhosis. The increase of hepatic triglyceride content is

amplified by increasing BMI, as is the risk of cirrhosis44. Additional studies are needed

to determine the cost effectiveness and utility of multifactorial risk stratification that

incorporates PNPLA3 genotype and other risk factors for HCC.

Variant PNPLA3 and metabolic traits

Patients with fatty liver disease often present with dyslipidemia and insulin

resistance45. However, the association of PNPLA3 with metabolic traits in humans is

more complex. Several studies did not detect a relationship between the p.I148M

mutation and serum glucose or lipid concentrations28, 30 46, 47

. In contrast, a Danish

analysis of > 4,000 individuals with normal glucose tolerance demonstrated an

association of the risk allele with increased fasting glucose levels, but the same allele

was associated with lower serum concentrations of triglycerides and cholesterol in

patients with impaired glucose tolerance48. Lower fasting triglyceride levels were also

observed in severely overweight patients carrying the risk variant49. In line with these

results, Krawczyk et al.50 and other groups51, 52

identified a possible association

between higher fasting glucose levels and the PNPLA3 p.I148M mutation. These

results contradict the general paradigm that insulin resistance represents the main

driver of common NAFLD and pointed to the presence of a potentially different

mechanism of hepatic fat accumulation in the carriers of the PNPLA3 minor allele.

Indeed, a dissociation between the presence of fatty liver and insulin resistance

appears to be present among carriers of the PNPLA3 risk variant46. On the other hand,

the coexistence of increased systemic insulin concentrations seems to further magnify

11
the risk for liver steatosis in individuals carrying the PNPLA3 p.148M allele53. Specific

circulating triacylglycerol signatures were observed in carriers of the variant, which

resembled those of obese subjects with NAFLD54. PNPLA3-associated NAFLD is

associated with a relative deficiency of triacylglycerols, supporting the idea that the

variant impedes intrahepatocellular lipolysis rather than stimulating lipid synthesis.

NAFLD in obese patients is associated with multiple changes in triacylglycerols, which

can be attributed to obesity and insulin resistance but not increased liver fat content

per se.

Functional analyses of the PNPLA3 variant

The close similarity to adipose triglyceride lipase and the presence of typical structural

motifs (ααβαααsandwich structure, consensus serine lipase GXSXG motif, catalytic

dyad S-D) indicate a lipase function of PNPLA355. Furthermore, Pirazzi et al.56

demonstrated that PNPLA3 has retinyl-palmitate lipase activity in hepatic stellate cells.

The expression of human PNPLA3 is induced by carbohydrates and fatty acids via

sterol response element binding protein 1c57-60.

Livers from NAFLD patients are characterized by an increased number and size

of lipid droplets within hepatocytes. Of note, PNPLA3 is predominantly localized in the

endoplasmic reticulum and on the surface of lipid droplets61. The rs738409 variant of

PNPLA3 results in an isoleucine to methionine substitution at amino acid residue 148.

Structural modeling indicates that the longer methionine side chain blocks the access

of substrates to the catalytic site62, 63. PNPLA3 is induced with increased free fatty acid

availability during hyperinsulinemia but degraded rapidly upon fasting both by

autophagy and proteasome-mediated degradation64. In carriers of the p.I148M

12
mutation, the variant PNPLA3 evades ubiquitylation and subsequent proteosomal

degradation, which results in accumulation of PNPLA3 on hepatic lipid droplets64. The

enzyme interacts with the comparative gene identification-58 (CGI-58; a.k.a. α-β

hydrolase 5, ABHD5) protein, which is causative in Chanarin-Dorfman syndrome, a

rare multisystem neutral lipid-storage disease that also affects the liver65, 66
;

heterozygous ABDH5 mutations may cause NAFLD and/or dyslipidemia in adults67.

Unlike many members of this family, CGI-58 itself lacks lipase activity but serves as

co-activator of the major adipose triacylglycerol lipase ATGL (PNPLA2) and possibly

other PNPLA family members. If the accumulation of mutant PNPLA3148M sequesters

CGI-58, ATGL would lack CGI-58, which results in decreased lipolysis and lipophagy.

These observations explain the increase of lipid droplets in both number and size62, 68-
70
.

Pnpla3-/- mice do not present with steatosis, pointing to the dominant-negative

effect of the variant, i.e. the disease-causing version of the enzyme needs to be

expressed to mediate the observed effect70, 71. Studies performed in transgenic mice

overexpressing variant Pnpla3148M but not wild-type PNPLA3 in liver demonstrated that

these animals develop steatosis due to triacylglycerol accumulation as well as several

alterations of hepatic lipid metabolism, including increased synthesis of fatty acids and

triacylglycerol, impaired hydrolysis of triacylglycerol, and depletion of triacylglycerol

long-chain polyunsaturated fatty acids72. To determine whether the mutant p.148M

allele causes fat accumulation in the liver when expressed at physiological levels,

Pnpla3148M knock-in animals were generated, which displayed normal levels of hepatic

fat on chow diet, but when challenged with a high-sucrose diet the liver fat content

increased two- to threefold compared to wild-type controls without changes in glucose

13
homeostasis. The increased liver fat in the knock-in mice was accompanied by a 40-

fold increase of the PNPLA3 quantity on hepatic lipid droplets with no increase in

hepatic Pnpla3 mRNA73. In line with these observations the analysis of the Pnpla3148M

animals showed that the increased intracellular PNPLA3 content persists after the

high-sucrose diet is terminated70. This observation illustrates the decreased

degradation of adiponutrin in carriers of the PNPLA3 risk allele p.148M. High

quantities of PNPLA3 on lipid droplets might prevent the mobilization of triglycerides70.

The presence of the risk allele is associated with increased hepatic contents of

polyunsaturated fatty acids (PUFA), potentially due to dysfunction of PNPLA3,

because under lipolytic condition, it impairs the transfer of PUFA from diacylglycerols

for the hepatic synthesis of unsaturated phosphatidylcholines74. This study

demonstrates that the PNPLA3 p.I148M variant remodels liver triglycerides in a

polyunsaturated direction and results in PUFA deficiency of VLDL triglycerides

secreted by the liver.

PNPAL3 genotype-guided treatment of NAFLD

The in vitro and in vivo studies indicate that suppression of mutant PNPLA3 would

have beneficial effects in NAFLD and represent a novel therapeutic target for NAFLD.

Until then, lifestyle modification represents the cornerstone of NAFLD treatment.

Although to date large prospective studies concerning long-term effects of the

common PNPLA3 p.I148M variant on liver phenotypes are lacking, the first pilot

studies indicate that weight loss has positive effects in carriers of the risk allele75, 76. To

explore whether the PNPLA3 variant modulates the effects of weight loss on liver fat

and insulin sensitivity, eight homozygous carriers were placed on a hypocaloric low-

14
carbohydrate diet for six days76. Liver fat content, whole-body insulin sensitivity of

glucose metabolism (euglycemic clamp technique), and lipolysis ([2H5]glycerol

infusion) were measured before and after the diet. At baseline, fasting serum insulin

and C-peptide concentrations were lower in mutation carriers. However after a mean

weight loss of 3.1 kg, liver fat decreased by 45% in patients with PNPLA3148M but by

18% only in controls. The PNPLA3 variant also modulated the changes in metabolic

profile and intrahepatic triglycerides (IHTG) in 154 NAFLD patients77. The presence of

the mutation and BMI were independently associated with greater reduction in IHTG.

Although the PNPLA3 risk allele confers a higher risk of NAFLD, these patients seem

to be more sensitive to the beneficial effects of lifestyle modification. In the same line,

obese patients carrying the prosteatotic PNPLA3148M allele lose more weight and liver

fat one year after bariatric surgery, as compared to carriers of PNPLA3 wild-type

alleles78. The PNPLA3 genotype and the initial grade of steatosis were independent

predictors of improvement of steatosis after surgery. In contrast, the PNPLA3 p.I148M

variant has also been reported to reduce the beneficial effects of statins on

steatohepatitis79.

TM6SF2

A GWAS using a dense panel of exonic polymorphic markers in three independent

populations (N > 80,000) identified the TM6SF2 variant p.E167K (c.449C>T,

rs58542926) to confer susceptibility to NAFLD in addition to PNPLA380.The TM6SF2

variant (allele frequency 7% in Europeans) is markedly less frequent than the PNPLA3

p.I148M polymorphism in all ethnic groups (23% in Europeans, 55% in Hispanics).

TM6SF2 encodes a protein of 351 amino acids with 7–10 predicted transmembrane

15
domains. TM6SF2 is localized in the ER and the Golgi complex of hepatocytes and

enterocytes, which synthesize apolipoprotein B-containing lipoproteins69. In contrast to

PNPLA3, the expression of TM6SF2 is not altered by dietary challenges69. The

polymorphism is associated with higher liver fat content but lower serum levels of total

and low-density lipoprotein (LDL), cholesterol, and triglycerides9, 81

. The TM6SF2

p.E167K variant does not exert its effect on hepatic fat content or circulating lipid

profiles by worsening hepatic insulin sensitivity82. In patients with histologically proven

NAFLD, an association of this variant with the degree of steatosis, necroinflammation,

ballooning, and advanced fibrosis was observed, after adjustment for age, sex, BMI,

and diabetes10, 33. Accordingly, the TM6SF2 variant was independently associated with

advanced fibrosis, when assessed noninvasively by transient elastography in 890

individuals83. In a study involving 1,020 patients with HCC, 2,484 healthy individuals

and 2,021 patients with chronic liver diseases both the PNPLA3 and the TM6SF2

polymorphism were associated with the development of HCC in multivariate models

adjusting for confounding factors (OR 1.67 and 1.45, respectively)84. Here, the variants

were independently associated with increased odds of HCC in patients with alcohol-

related liver diseases84, but similar observations have been made for NAFLD-HCC

cohorts as well85.

TM6SF2 protein expression is markedly decreased in livers of NAFLD patients

compared to controls86. siRNA knockdown of Tm6sf2 in mice decreases very-low-

density lipoprotein (VLDL) secretion and increases cellular triglyceride concentration

and lipid droplet content, whereas overexpression reduces liver cell steatosis80, 87
.

Chronic inactivation of Tm6sf2 in mice is associated with hepatic steatosis and

inflammation as well as decreased plasma levels of total and LDL cholesterol, thus

16
recapitulating the phenotype observed in humans69, 88
. These observations indicate

that TM6SF2 activity is required for normal VLDL assembly and that carriers of the

TM6SF2 p.E167K variant have fatty liver as a result of reduced VLDL lipidation.

Holmen et al.9 and Dongiovanni et al.10 reported that these patients have lower

myocardial infarction risk as well as reduced risk of developing carotid plaques and

cardiovascular events, respectively. In addition, the effect of insulin on glucose

production and lipolysis is significantly better in patients carrying the TM6SF2 p.E167K

variant. Hence, they display a distinct subtype of NAFLD, which is characterized by

preserved insulin sensitivity with regard to lipolysis, hepatic glucose production, and

lack of hypertriglyceridemia despite clearly increased hepatic fat content82. The

dichotomic role of the TM6SF2 variant in protecting against cardiovascular disease

and conferring risk for NAFLD was confirmed in a meta-analysis of 10 studies. Pooled

estimates of random effects in > 100,000 individuals showed that homozygous or

heterozygous carriers of the minor T allele are protected from cardiovascular disease,

showing lower levels of total and LDL cholesterol as well as triglycerides, while hepatic

lipid fat content is about 2% higher89. Accordingly, there is a moderate overall effect on

the risk of NAFLD (OR 2.1). Hence, the variant confers protection against

cardiovascular disease at the expense of an increased risk of NAFLD81.

Interestingly, the PNPLA3 and TM6SF2 variants are not only associated with

clinical phenotypes but also with health services utilization in the general population90.

In 3759 participants from study of health in Pomerania (SHIP), homozygous carriers of

the PNPLA3 risk allele had an increased odds of hospitalization (OR 1.5) as compared

to major allele homozygous subjects, and carriers of the TM6SF2 risk allele had 68%

higher outpatient utilization and inpatient days than major allele homozygous subjects.

17
These findings highlight the strong effects of these variants that can be detected even

in health economic analysis. Recently the PNPLA3 p.I148M polymorphism was

studied in four large cohorts with extensive health information (23andMe, UK Biobank,

FINRISK, CHOP) for association with 1683 binary endpoints in up to 700,000

individuals4. This study design has been termed phenome-wide association study

(PheWAS), representing an unbiased approach to test for associations between

genetic variants and a wide range of phenotypes in large population cohorts91.

PheWAS can help in identifying shared genetic variants affecting two or more

phenotypes92. Of note, the PNPLA3 variant, which predisposes to fatty liver disease,

was associated with diabetes and lower serum cholesterol levels, while an inverse

association was detected for gallstones, acne, and gout, with the latter association

being confirmed in a recent smaller PheWAS93. Beyond that, the analysis also

indicated that carriers of the risk allele are prone to develop drug-induced liver injury

(OR 1.5) when treated with non-steroidal anti-inflammatory drugs (NSAID) such as

ibuprofen or aspirin4. These associations were replicated in the UK biobank cohort and

remained when adjusting for elevated liver tests4.

MBOAT7

After a GWAS had reported that the rs641738 variant linked to the 3' untranslated

region of the gene encoding membrane-bound O-acyltransferase domain-containing 7

gene (MBOAT7, a.k.a. lysophosphatidylinositol-acyltransferase 1, LPIAT1) increases

the risk for alcoholic cirrhosis94, 2,736 participants from the Dallas heart study who had

undergone proton magnetic resonance spectroscopy to measure IHTG and 1,149

European individuals from the liver biopsy cross-sectional cohort were genotyped for

18
rs64173895. The rs641738 variant (g.54173068 C>T/c.50 G>A), which encodes

p.G17E in the transmembrane channel-like 4 (TMC4), is associated with suppression

of MBOAT7 at the mRNA and protein levels. It was also associated with increased

hepatic fat contents, more severe liver damage, and increased stages of fibrosis

compared to subjects without the variant. Furthermore, it might predispose to HCC in

patients without cirrhosis85. Figure 2 illustrates the combined effects of the three risk

gene variants of PNPLA3, TM6SF2 and MBOAT7 on a patient's individual risk of

increased hepatic triglyceride content in and elevated serum liver enzyme activities.

Their impact at the population level can be quantified by the population attributable

fraction (PAF), which is the proportional reduction in population morbidity that would

occur if exposure to a risk factor were reduced to an alternative ideal exposure

scenario (Table 1).

Notably, MBOAT7 deficiency has been implicated in neurological disorders

leading to intellectual disabilities and development delay96. Interestingly, the analysis

of the Nutrition Examination Survey (NHANES) database showed that homozygpus

carriers of the PNPLA3 p.148M allele might have an increased risk of bipolar disorder

(OR 4.6) as compared to carriers of the common allele97. Still the data on the

association between PNPLA3 and neurological disorders are less solid than for the

MBOAT7 polymorphism. MBOAT7 is anchored to endomebranes by six

transmembrane domains and transfers polyunsaturated fatty acids (PUFA) such as

arachidonic acid to lysophospholipids98. The MBOAT7 rs641738 risk allele was

associated with altered plasma phosphatidylinositol (PI) species, consistent with

decreased MBOAT7 function. Metabolic profiling indicates changes of PI side-chain

remodeling, in particular a lack of transfer of arachidonoyl-CoA to lyso-PI (Lands

19
cycle)99. Interestingly, PNPLA3 promotes the transfer of PUFA, especially arachidonic

acid, from triglycerides to phospholipids in hepatic lipid droplets100. These findings

together indicate a potential role of arachidonic acid incorporation in phospholipids

during NAFLD pathogenesis.

GCKR

GWAS and its meta-analyses have identified additional variants that can be

associated with NAFLD. For example, Speliotes et al.28 reported that variants in or

near glucokinase regulatory protein (GCKR), lysophospholipase-like 1 (LYPLAL1) and

phosphatase 1-regulatory subunit 3b (PPP1R3B) are associated with liver fat content

and/or histopathological NAFLD phenotypes at genome-wide significance levels. In

line with this report, the common GCKR variant rs1260326 (c.1337T>C, p.P446L) was

associated with hepatic triglyceride content in the Dallas Heart Study44. The

glucokinase regulator is encoded by GCKR and regulates the activity of hepatic

glucokinase in the fasting state in the nuclei of hepatocytes101. Postprandially, the

protein translocates into the cytoplasm and phosphorylates glucose, which can then

either be converted to glycogen for storage or be glycosylated, thereby stimulating de

novo lipogenesis (DNL)101. The GCKR variant p.P446L reduces the ability of GCKR to

inhibit glucokinase in response to fructose-6-phosphate, thus increasing glucokinase

activity and hepatic uptake of glucose. The resulting disinhibition of glycolysis in the

liver reduces fasting glucose and insulin levels, but increases the production of

malonyl-CoA, which in turn favors the accumulation of liver fat as it serves as a

substrate for DNL and inhibits mitochondrial fatty acid oxidation102, 103. The NALFD risk

allele of GCKR is associated with increased concentrations of LDL cholesterol and

20
triglycerides,42 but other variants display no uniform association, indicating genetic

heterogeneity of signaling pathways affecting metabolic traits.

In a meta-analysis comprising 2,091 NAFLD patients and 3,003 controls from

five studies, Zain et al.104 could confirm that the intronic GCKR variant rs780094 is

significantly associated with an increased NAFLD risk (OR 1.3). The effect size was

comparable in Asian and non-Asian populations. The distribution among the world

population shows that the variant is rare in Africans (9%), but more common in

Americans of European descent (41%), Latin Americans (36%) and East Asians

(48%)105. In 70 adolescents with biopsy-proven NAFLD, the GCKR

rs1260326/rs780094 polymorphisms predisposed to NAFLD and were associated with

the presence of fibrosis, decreased hepatic protein levels as well as impaired hepatic

retinol metabolism106. Of note, the common GCKR variant p.P446L is also associated

with the number of drinks per week in large GWAS using combined samples from over

1 million individuals for alcohol use and risky behavior, similar to the known

association signal in the alcohol metabolizing gene acetaldehyde dehydrogenease 1B

(ADH1B)107, 108
. This observation reinforces the common genetic predisposition for

fatty liver disease in patients exposed to both alcohol and overnutrition. Further studies

also found associations of this variant with slightly lower plasma glucose

concentrations, reduced risk of diabetes and gallstones, and slightly elevated serum

lipids101, 109, 110 as well as increased NAFLD risk in obese children111.

HFE

Hereditary hemochromatosis is caused by HFE gene mutations and considered to be

a monogenic ("Mendelian") liver disease112. Historically, hemochromatosis served as

21
the poster child of diseases for which genetic testing might present clinical and even

potential socioeconomic benefits, with almost no ethical risks. A large meta-analysis

on hemochromatosis encompassing 66,000 cases and 226,000 controls from Europe

and China113 showed that homozygous carriers of the HFE variant p.282Y (rs1800562)

have a 10-fold increased risk of developing NASH. In contrast, p.C282Y

heterozygosity does not confer an increased risk for any liver disease (except for

porphyria cutanea tarda). Carriers of HFE mutations are at increased HCC risk even

without cirrhosis. Similar observations were made in the population-based UK biobank

cohort114: Among 451,243 participants aged 40 to 70 years, 2,890 (0.6%) were

homozygous and 64,444 (14%) were heterozygous for p.C282Y (0.6%).

Hemochromatosis was diagnosed in 22% of homozygous men and 10% of

homozygous women after a mean follow-up of seven years. The odds ratio for any

liver disease and incident HCC is significantly increased to 4.3 and 8.8 in homozygous

men but does not differ from the population risk in homozygous women or

heterozygous carriers. As HFE p.C282Y-associated iron overload is treatable (and

preventable) if phlebotomy starts early, the findings in the UK biobank justify re-

examination of options for early case assessment and screening in the population. Of

note, the 4- to 10-fold increased risk of liver disease, NASH or HCC conferred by the

HFE mutation p.C282Y is comparable to the effect of the PNPLA3 mutation, which is

considered to be a genetic risk factor but not a monogenic liver disease2. This example

suggests that the historical concept of "monogenic or polygenic disease" needs to be

replaced by a more contemporary concept of a continuous spectrum of disease risk

(Figure 3).

22
SERPINA1

In contrast to HFE, even the mere heterozygous carriage of α1-antitrypsin mutations

encoded by the SERPINA1 gene increases the risk to develop chronic liver disease

and cirrhosis. α1-Antitrypsin deficiency is among the most common genetic diseases

affecting lung and liver. Homozygous carriers of the Pi*Z variant (p.E342K) are not

only highly susceptible to develop lung emphysema but also liver cirrhosis due to gain-

of-function toxicity of the variant protein in hepatocytes. In 1,847 liver biopsies and

1,030 autopsy cases, immunhistochemical α1-antitrypsin deposits, indicating Pi*Z

heterozygosity, were more correlated with the inflammatory activity and fibrosis stage

and were more frequent in patients with concurrent liver disease or cirrhotic livers115;

Pi*Z heterozygotes are also at increased risk of primary liver cancer116 This variant is

carried by 2 - 4% of Caucasians, but by 13% of patients with cirrhotic NAFLD117.

Accordingly, the Pi*Z variant was demonstrated to be associated with higher fibrosis

stages in 1,184 patients with NAFLD (OR 2.3)117. In comparison to hemochromatosis,

genetic testing for α1-antitrypsin deficiency disease presents a more complex clinical

scenario; however, there are clearly potential benefits that result in a positive

risk:benefit ratio. Both homozygous and heterozygous carriers of the mutations are

strongly advised not to smoke and not to drink.

HSD17B13

Abul-Husn et al.118 reported that the variant rs72613567:TA of hydroxysteroid 17β

dehydrogenase 13 (HSD17B13) is associated with a reduced risk of chronic liver

disease and of progression from steatosis to steatohepatitis. The T to TA insertion

variant is adjacent to a donor splice site and leads to the synthesis of a truncated
23
enzyme. The population frequency of the variant is 22%, ranging from 6% in Africans

to 27% in Europeans and 33% in East Asians

(https://gnomad.broadinstitute.org/variant/4-88231392-T-TA?dataset=gnomad_r2_1).

It has to be noted that the term “protection” represents the flipside of “susceptibility”,

hence it depends on the population frequency of the alleles, with the minor allele

defining the direction of the effect (protection vs. predisposition). Using exome

sequence data and electronic health records from 46,544 participants in the

DiscovEHR human genetics study gene variants that are associated with serum

activities of aminotransferases were identified. Replicated variants were evaluated for

association with clinical diagnoses of chronic liver disease in DiscovEHR study

participants as well as two independent cohorts (N = 37,173) and with

histopathological severity of liver disease in 2,391 human liver biopsies. In

homozygous and heterozygous carriers of the variant rs72613567:TA, the risks of

NAFLD and NASH-cirrhosis were reduced by 30% and 17% and 49% and 26%,

respectively. Of note, the HSD17B13 variant ameliorated liver injury associated with

the PNPLA3 p.I148M risk allele. This study also replicated three of the single

nucleotide variants (SNV) to be associated with NAFLD, the odds ratios for all of them

are summarized in Table 2.

In a second detailed histopathological study of 356 patients with biopsy-proven

disease, the variant HSD17B13 version protected against lobular inflammation,

ballooning degeneration, and fibrosis119. When quantifying the protective effect on

alanine aminotransferase activity as serum surrogate marker, the effect size depends

on both environmental and genetic risk. The protective allele is associated with only

marginally lower ALT activities in lean non-drinkers, but homozygotes displayed 12 -

24
18% lower ALT among the most obese, in heavy drinkers, and in individuals carrying

three or four steatogenic alleles in PNPLA3 and TM6SF2120. In line with these findings,

carriage of HSD17B13 rs72613567:TA has also been reported to reduce the odds of

both cirrhosis and HCC in alcohol misusers in multivariate analyses121, 122.

Hydroxysteroid-17β-dehydrogenases (HSD17B) form an enzyme family

characterized by their ability to catalyze reactions in steroid and lipid metabolism.

Recently Ma et al.123 reported that HSD17B13 is a hepatic retinol dehydrogenase that

is targeted to lipid droplets. Mice deficient in Hsd17b13 develop hepatic steatosis,

while serum steroid concentrations are normal124. In line with these changes, the

expression of key proteins in fatty acid synthesis, such as fatty acid synthase, acetyl-

CoA carboxylase 1, and stearoyl-CoA desaturase, is increased in livers from Hsd17b3

knockout mice, while glucose tolerance does not differ124.

Another recently identified, potentially protective gene, MARC1, is described in

Supplementary Material 2.

III. Future perspectives or lost chances?

What can we learn from the geneti studies? In the last decade our knowledge about

the genetic background of fatty liver disease has expanded substantially. Genes like

PNPLA3, MBOAT7 or GCKR have been implicated in the pathogenesis of hepatic

steatosis. Nevertheless, although the costs of genotyping single gene variants are

plummeting and are now lower than the costs of the butterfly needles used to draw

patients' blood (GWAS assays allowing genotyping of 700k markers cost less than

25
100$ per patient), the results of genetic analyses have not yet been translated into

clinical progress. As the price of sequencing falls, GWAS using whole-genome

sequencing (WGS) in large cohorts are becoming achievable. Until then, GWAS based

on dense SNV arrays combined with deep imputation using WGS reference panels will

identify novel associations of rare variants with large effect sizes causing common

traits in a small subgroup of patients125, 126

, as highlighted by the large-scale WGS

study of type 2 diabetes127. Notwithstanding, we are still lacking prospective genetic

studies with repetitive measurements of markers of liver injury that would define the

"real-life" impact of genetic predisposition on the origin and progression of disease.

One might argue that the detected genetic variants render livers more fragile when

challenged by exposome - obviously the major causative agent of fatty liver diseases.

However, the associations between genotypes and end-stage consequences of

chronic liver diseases, namely cirrhosis and HCC, substantiate the attention that they

have received in the last decade. Empirical research is needed to elucidate whether

the consequences of genetic risk factors are time-varying and to what extent they

reflect the individual-specific, hitherto unexplained heterogeneity.

Maybe we should develop drugs that would modify the function of the proteins

detected in the genetic studies? However, such therapies might have deleterious

effects. For example, presence of the variant PNPLA p.I148M allele increases the risk

of severe hepatic steatosis but it also seems to lower the risk of gallstone disease,

gout, acne, or high serum cholesterol concentrations4. Hence, directly blocking

PNPLA3 in carriers of the risk allele might cause deleterious effects outside the liver.

What about stratification of patients according to genetic predisposition? Yki-Järvinen

and co-workers128 reviewed different follow-up schemes of patients with accidentally

26
diagnosed hepatic steatosis based on the presence of markers of liver injury (fibrosis

tests) and the PNPLA3 variant. Such genotype-guided risk stratification of disease

progression and sub-phenotyping could be implemented in randomized controlled

NAFLD trials. For example, based on increased DNL lipogenesis in carriers of

TM6SF2 and GCKR risk variants, these patients might respond to DNL inhibitors,

whereas PNPLA3 p.I148M carriers would not129.

In addition to clinical trial design, clinical utility of risk genes could be disease

prediction and disease surveillance. Known disease variants can be incorporated in a

weighted genetic risk score (GRS), which provide a quantitative metric of the personal

inherited risk based in the cumulative impact of many common variants (Figure 3); the

weights are generally assigned to each genetic variant according to its effect size on

the phenotypic trait130. At present, the out-of-sample predictive power that can be

obtained from GRS is still too limited to be of practical use for most outcomes.

However, the first genome-wide polygenic scores (GPS) have recently been created

that integrate all available common variants into a single quantitative measure of

inherited susceptibility. For obesity, such a score based on 2.1 million variants

genotyped in > 300,000 individuals with risk equivalent to rare mutations, but was still

not deterministic130. The top decile of the genome-wide score conferred a 25-fold

gradient in risk of severe obesity, and the score had substantially greater predictive

power than scores comprising less than 150 variants, which fulfilled genome-wide cut-

offs of significance in prior GWAS130.

In fact, we do not envision genetic risk variants to improve the diagnosis and

follow-up of NAFLD per se, since the latter are based on non-invasive assessments

(e.g. clinical chemistry, elastography), which can be obtained regularly. Thus, receiver

27
operating characteristics and accuracy for NAFLD phenotypes do not benefit markedly

from inclusion of genetic markers131. In contrast, we envision genetic markers to be

more useful for the identification of sub-groups of NAFLD patients at high risk of

unfavorable trajectories132. Here, they can inform decision making and early

intervention (both prevention and interception) trials for NAFLD patients who need to

be cared for differently and for whom, for example, personalized HCC surveillance

might be cost-effective133. Hence, on one hand we have been fairly successful in

detecting variants associated with fatty liver and hepatic injury but at the same time we

are struggling with translating these data into the clinic. This appears to be the task

that needs to be accomplished over the next years to produce the maximum benefit for

patients from the genetic studies of the last decade.

28
 Table 1

Population attributable fractions* of NAFLD risk genes

Steatosis NASH Fibrosis (F2 - F4)

PNPLA3 23% (11 - 36) 19% (12 - 26) 18% (10 - 26)

TM6SF2 4% (0 - 14) 4% (1 - 8) 3% (0 - 7)

MBOAT7 15% (3 - 28) 7% (0 - 14) 11% (2 - 23)

*The contribution of a risk factor to a disease or a death is quantified using PAF (with 95% confidence
intervals). PAF is the proportional reduction in population morbidity that would occur if exposure to a risk
factor were reduced to an alternative ideal exposure scenario.

29
 Table 2

Association of exome-wide significant and replicated single nucleotide variants with

liver disease (data extracted from118)

Gene Chro- Single Minor NAFLD NASH cirrhosis HCC

mo- nucleotide allele fre-
some variant quency
OR 95% CI P OR 95% CI P OR 95% CI P

-5 -4
HSD17B13 4 rs72613567 0.263 0.84 0.78- 1.31×10 0.74 0.62-0.88 4.48×10 0.67 0.45-1.00 0.047
c.704/812+2dup 0.91
T>TA insertion
alternative
splicing and
loss-of-function

SERPINA1 14 rs28929474 0.017 1.50 1.21- 5.29×10-4 2.99 2.11-4.24 9.08×10-8 1.86 0.74-4.67 0.24
c.1096 G>A 1.87
p.E342K

TM6SF2 19 rs58542926 0.0760 1.36 1.21- 2.42×10-7 1.64 1.31-2.05 6.04×10-5 1.93 1.22-3.04 0.011
c.499 G>A 1.52
p.E167K

PNPLA3 22 rs738409 0.235 1.65 1.54- 1.31×10-41 2.05 1.76-2.38 1.70×10-19 2.20 1.60-3.02 5.59×10-6
c.444 C>G 1.78
p.I148M

-3
Bonferroni significance threshold: P < 1.92×10

30
Figure legends

Figure 1

Schematic NAFLD classification trees and genetic risk variants. Each node in a tree

represents a clinical diagnosis or surrogate phenotype and nodes are ordered in a

hierarchical structure. White nodes represent the null state whereby there is no known

genetic association with the clinical phenotype; red and green nodes indicate the

alternative state whereby a genetic association with the clinical phenotype has been

reported, with the different colours corresponding to opposite genetic coefficients of

association (red: susceptibility; green: protection). Supplementary Material 1 lists

further references supporting the classification trees.

Abbreviations: ACLF, acute-on-chronic liver failure; AFP, α1-fetoprotein; AH, alcoholic

hepatitis; ALD, alcohol-related liver disease; ALT, alanine aminotransferase; ASH,

(chronic) alcoholic steatohepatitis; CAP, controlled attenuation parameter; DC,

decompensated cirrhosis; F4, fibrosis stage F4 (cirrhosis); HCC, hepatocellular

carcinoma; LB, liver biopsy; NAFL, non-alcoholic fatty liver (steaosis); NAFLD, non-

alcoholic fatty liver disease; NAS, NAFLD actiity score; NASH, non-alcoholic

steatohepatitis; NFS, NAFLD fibrosis score; PDFF, proton density fat fraction; SSM,

serum surrogate markers; TG, triglycerides (serum); US, ultrasound.

Figure 2

Combined genetic effects in NAFLD. (a) Association between number of PNPLA3,

TM6SF2, and MBOAT7 risk alleles and hepatic triglyceride (TG) content in the Dallas

heart study. The graph shows mean hepatic TG content by the number of risk alleles

31
(error bars: SEM). From Mancina et al.95. (b) Association between number of PNPLA3,

TM6SF2 and MBOAT7 risk alleles and liver function tests in the NAFLD CSG study.

The graphs illustrate median aspartate aminotransferase (AST) and ALT activities by

the number of risk alleles (error bars: range). Analyses were performed using trend

tests. From134.

Figure 3

3D risk space (tensor) as integrated model for NAFLD pathogenesis. The number of

inherited NAFLD risk gene variants, which can be expressed as genetic risk score

(GRS), is presented on the z-axis. The y-axis presents the number (n) of exogenous

risk factors for NAFLD. The interaction of all presented factors over time (t) results in

the NAFLD phenotypes given on the x-axis, which follow the common sequence from

steatosis (NAFL) and steatohepatitis (NASH) to progressive fibrosis and cirrhosis, and

eventually HCC, but progression rates differ between patients. The color code

illustrates susceptibility in red and healthy state in green; the final disease stage is

indicated by the white circle. The number of exogenous and genetic risk factors of

NAFLD and the time during which they can exert their harmful effects on the liver

determine this individual trajectory of temporal NAFLD progression. The inherited

predisposition to NAFLD remains stable over time, which can be visualized by one of

the transversal intersections of the cube (GRS = const.; upper right panel), whereas

the exposome may be changed to modulate the trajectory in carriers of NAFLD-

associated gene variants. If the exposome is fixed, the genotype-to-phenotype

correlations (G2P) can be illustrated in a 2D diagram (lower left panel).

32
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