Published online: 2021-07-07
THIEME
421
Insights into Nonalcoholic Fatty-Liver Disease
Heterogeneity
Marco Arrese, MD1,2 Juan P. Arab, MD1,2 Francisco Barrera, MD1,2
Luca Valenti, MD4 Ariel E. Feldstein, MD3
1 Department of Gastroenterology, Escuela de Medicina, Pontificia
Universidad Católica de Chile, Santiago, Chile
2 Centro de Envejecimiento y Regeneración (CARE), Departamento de
Biología Celular y Molecular, Facultad de Ciencias Biologicas,
Pontificia Universidad Catolica de Chile, Santiago, Chile
3 Department of Pediatric Gastroenterology, Rady Children’s
Hospital, University of California San Diego, California
4 Department of Pathophysiology and Transplantation, Universita
degli Studi di Milano, Translational Medicine, Department of
Transfusion, Medicine and Hematology, Fondazione IRCCS Ca’
Granda, Pad Marangoni, Milan, Italy
Benedikt Kaufmann, MD3
Address for correspondence Marco Arrese, MD, Department of
Gastroenterology, Escuela de Medicina, Pontificia Universidad
Católica de Chile, Diagonal Paraguay #362, 8330077 Santiago, Chile
(e-mail: marrese@uc.cl).
Ariel E. Feldstein, MD, Division of Pediatric Gastroenterology
Hepatology and Nutrition, UCSD 3020 Children’s Way, MC 5030, San
Diego, CA 92103-8450
(e-mail: afeldstein@ucsd.edu).
Semin Liver Dis 2021;41:421–434.
Abstract
Keywords
► nonalcoholic fattyliver disease
► steatosis
► cirrhosis
► fibrosis
► nonalcoholic
► steatohepatitis
► NAFLD
► NASH
► metabolic syndrome
► heterogeneity
The acronym nonalcoholic fatty-liver disease (NAFLD) groups a heterogeneous patient
population. Although in many patients the primary driver is metabolic dysfunction, a
complex and dynamic interaction of different factors (i.e., sex, presence of one or more
genetic variants, coexistence of different comorbidities, diverse microbiota composition, and various degrees of alcohol consumption among others) takes place to
determine disease subphenotypes with distinct natural history and prognosis and,
eventually, different response to therapy. This review aims to address this topic
through the analysis of existing data on the differential contribution of known factors
to the pathogenesis and clinical expression of NAFLD, thus determining the different
clinical subphenotypes observed in practice. To improve our understanding of NAFLD
heterogeneity and the dominant drivers of disease in patient subgroups would
predictably impact on the development of more precision-targeted therapies for
NAFLD.
Nonalcoholic fatty-liver disease (NAFLD) is currently considered the commonest liver disease worldwide with an
estimated global prevalence of 25%.1,2 The acronym refers
to a range of hepatic histological alterations including
isolated steatosis (also referred as NAFL), non-alcoholic
streatohepatitis (NASH which is hallmarked by the presence of hepatocellular injury accompanied by inflammation and variable degrees of fibrosis), and cirrhosis.3
Importantly, NAFLD is a risk factor for hepatocellular
carcinoma (HCC) development even in the absence of
cirrhosis.4 NAFLD relevance is underscored by data pointing to NAFLD as the most fast-growing cause of cirrhosis
published online
July 7, 2021
DOI https://doi.org/
10.1055/s-0041-1730927.
ISSN 0272-8087.
and the need liver transplantation in the United States.5
Moreover, epidemiological figures are worrisome as,
according to epidemiological modeling, NAFLD burden is
expected to grow in the coming decades burdening health
care systems and causing substantial economic costs and
compromising individual health, determining significant
morbidity and mortality.6
To date, dietary caloric restriction and exercise are the
cornerstone of NAFLD therapy, and although these measures
have been proven to be efficacious, therapeutic goals are
difficult to achieve or sustain.7–9 Unfortunately, no Food and
Drug Administration (FDA)-approved therapies for NAFLD
© 2021. The Author(s).
This is an open access article published by Thieme under the terms of the
Creative Commons Attribution-NonDerivative-NonCommercial-License,
permitting copying and reproduction so long as the original work is given
appropriate credit. Contents may not be used for commercial purposes, or
adapted, remixed, transformed or built upon. (https://creativecommons.org/
licenses/by-nc-nd/4.0/)
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Arrese et al.
are currently available. Clinical trials conducted so far with a
myriad of investigational agents have shown relatively low
rates of response compared with placebo.10 The latter may
be related to several factors11 with disease heterogeneity
being a relevant aspect. In fact, inclusion criteria for most of
clinical studies is usually based mainly on histological diagnosis of NAFLD which result in mixed population of
patients.12 Thus, more granular data on the effectiveness
of new therapies in NAFLD subgroups is needed to create the
proper grounds for personalized or precision medicine
approaches in the near future.13–15
As mentioned above, the NAFLD acronym groups a heterogeneous patient population. NAFLD clinical expression
results from a complex and multilayered dynamic interaction of different factors including sex,16 presence of several
genetic variants,17 coexistence of different comorbidities,18
diverse microbiota composition,19–21 and various degrees of
alcohol consumption22 among others. This complex interaction results in several disease subphenotypes with distinct
natural history and prognosis and, eventually, different
response to therapy (►Fig. 1). Of note, NAFLD heterogeneity
was one of the reasons argued by an expert panel that
suggested to replace NAFLD by a new acronym derived
from the initial letters of metabolic (dysfunction)-associated
fatty-liver disease (i.e., MAFLD). NAFLD renaming was proposed aiming to better capture the main driver of the disease
in the majority of patients.23,24 However, this suggestion had
not been universally accepted and a debate on the matter is
ongoing.25,26 In the present review, we aim to address the
issue of NAFLD heterogeneity through the analysis of existing
data on the differential contribution of known factors to the
clinical expression of NAFLD. The impact of better deciphering NAFLD heterogeneity and the dominant drivers of disease
in patient subgroups on the development of more targeted
therapies for NAFLD is also highlighted.
Role of Genetic Determinants
NAFLD has a strong heritability and during the last year’s
genome-wide association studies (GWAS) have allowed the
discovery of the main common genetic determinants of this
condition and of the susceptibility to hepatic fat
Fig. 1 The acronym nonalcoholic fatty-liver disease (NAFLD) groups a heterogeneous patient population. The different phenotypes observed in
clinical practice stem from a complex and dynamic interaction of different factors (age, sex, reproductive status, presence of one or more
genetic variants and epigenetic factors, a diverse microbiota composition, coexistence of different comorbidities, the degree of alcohol
consumption, and muscle mass and physical activity among other). These variables critically influence NAFLD development and progression.
Disease heterogeneity imply that pathophysiological mechanisms underlying NAFLD may have different hierarchy or trajectory in different
patients defining different subphenotypes which may be relevant to molecular pathway-based therapies. NAFLD heterogeneity may also
influence natural history and prognosis and, eventually, response to therapy.
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accumulation.17 Their common pathogenic mechanism
turned out to be the alteration of hepatocellular lipid handling, favoring on one hand retention in lipid droplets over
secretion within very-low density lipoproteins and on the
other hand synthesis over catabolism.27 Increased quantity
and altered composition of lipids determines lipotoxicity
and potentially progressive liver disease.27
Identification of patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M and transmembrane 6 superfamily member 2 (TM6SF2) p.E167K, as the main risk
variants, allowed soon to pinpoint a subset of patients
carrying multiple variants who are at higher risk of progression to the hepatic rather than to the cardiometabolic
complications of NAFLD.14,17,28,29 Due to the increased
power of studies conducted in large population-based
cohorts, the number of NAFLD risk variants is now increasing by the day.30–33 We are therefore presently moving to a
model where, across the continuum of the distribution of
the inherited predisposition to NAFLD in the population, the
development of polygenic risk scores (PRS) can stratify the
risk of this condition and of liver-related complications.30,31,34–36 For example, “high” PRS can predict the
evolution to cirrhosis and HCC independently of classical
risk factors in individuals with dysmetabolism and/or
NAFLD, potentially being able to aid in the clinical management and in the design of referral pathways.35
On the other hand, it has become clear that the main
common risk variant and PRS cannot discriminate accurately
between “metabolic” and “genetic” NAFLD.28 Vice versa, very
robust evidence has accumulated that increased adiposity, and
dysmetabolism is the main trigger for the phenotypic expression of this genetic predisposition; the higher the body mass
index, the higher the risk of liver disease in carriers of NAFLD
risk variants.30,35,37,38 In addition, genetic predisposition to
NAFLD contributes to insulin resistance via inducing liver
injury.34 Finally, the main genetic risk variants in PNPLA3
and TM6SF2 are similarly enriched in “overweight/obese”
versus “lean” NAFLD.39,40 Indeed, central adipose tissue distribution, genetic causes of insulin resistance,37 and other factors
predisposing to hepatic fat accumulation can vicariate the role
of excess adiposity among the small fraction of lean individuals
who develop NAFLD.
Excess intake of alcohol is another key determinant of the
phenotypic expression of NAFLD risk variants17,30 which are
also the main genetic risk factors of alcoholic liver disease.41
Fructose intake, chronic hepatitis C, and iron accumulation
are additional triggers of liver disease.42–44 This new evidence suggests that several conditions that were previously
considered as separated diseases share major pathogenic
mechanism of liver damage centered on fat accumulation
and lipotoxicity which are initiated by specific but often
concomitant insults. The most frequent is the combination of
obesity, excessive alcohol, and genetic predisposition. Environmental and toxic insults may further push this deleterious mix toward liver disease. As such, genetics has clearly
highlighted that clinical heterogeneity should perhaps be the
best considered in the overall context of fatty-liver diseases
than in NAFLD per se.
Arrese et al.
However, there is initial evidence from the first next
generation sequencing studies that rare genetic variants
with a large impact on hepatic fat can predispose to NAFLD
irrespective of dysmetabolism.14,45 For example, carriage of
apolipoprotein B (APOB) variants predispose to severe
NAFLD, cirrhosis, and HCC in nonobese individuals.45,46
This subset of patients is at low risk of cardiovascular disease
due to reduced circulating lipoproteins and may also possibly
benefit from vitamin E supplementation due to malabsorption of lipophilic vitamins.45–47
There is additional evidence that the genetic setup of
NAFLD can influence the profile of extrahepatic organ
damage and the response to therapy. Thanks to the recent
discoveries in NAFLD genetics,14,17,28,29 the frontier of
disease phenotyping can move away from the “metabolic
vs. genetic” simplistic dichotomy, toward a subclassification
integrated with the clinical risk factors and based on
combined environmental-genetic pathways underlying disease predisposition. The challenge ahead will be to develop
reliable clusters of genetic variants that predict disease
subtypes. Based on the current knowledge at least three
groups can be identified. The first is defined by the presence
of high genetic risk of obesity and insulin resistance37,48 in
the absence of specific risk determinants of liver injury
(“insulin-resistant fatty-liver disease”). This roughly corresponds to the previous classification of “metabolic NAFLD,”
at high risk of cardiometabolic complications, but lower risk
of liver-related events. Among the specific determinants of
liver involvement in individuals with dysmetabolism, a
group of genetic variants including those in glucokinase
regulator (GCKR) that facilitate fat accumulation by diverting carbon substrates from glucose metabolism to lipid
synthesis (de novo lipogenesis [DNL]) can define “enhanced
lipogenesis fatty-liver disease,” at high risk of dyslipidemia.17,49 Insulin-resistant patients carrying these variants
may possibly benefit from approaches reducing lipogenesis,
such as statins and omega-3 fatty acids.14,50 A pathophysiological distinct subset (“impaired lipid remodeling fatty
liver disease”) is represented by those with a high burden of
genetic variants, including those in PNPLA3, TM6SF2, membrane bound O-acyl transferase (MBOAT7), and APOB,
impairing lipid remodeling and secretion and favoring lipotoxicity. These patients are at a particularly high risk of
liver-related complications, including hepatocellular carcinoma before cirrhosis development.14 They develop type-2
diabetes,34,48 but are relatively protected from cardiovascular disease.14,35 From a therapeutic point of view, they
respond well to weight loss, but when they carry the
PNPLA3 variant do not benefit from statins and are at
increased risk of liver injury due to enhanced hepatic lipid
accumulation during insulin therapy (reviewed by Cespiati
et al14).
Overall, the newest research developments have suggested new possibilities to integrate, rather than oppose
the genetic markers with clinical information to improve
fatty-liver disease classification and management. The efficacy and cost effectiveness of this approach remains to be
proven before wide implementation can be advised.
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Differential Contribution of Pathogenetic
Pathways
NAFLD pathogenesis is associated with a wide spectrum of
aberrant cellular and molecular mechanisms.51–53 As described above, genetic54 and metabolic55 alterations are key
drivers of the development of NAFLD resulting in an environment that favors hepatic fat accumulation and toxic lipid
metabolites via an increase in circulating free fatty acids
released from a dysfunctional inflamed adipose tissue56,57
and an increased DNL in hepatocytes.58,59 These changes
may lead to a state of “lipotoxicity” that triggers hepatocellular stress and consequently may result in liver injury and
cell death, activating a sterile liver inflammatory response51,53,60 and ultimately liver fibrogenesis contributing
to disease progression. The mechanisms thought to be at play
in NAFLD pathogenesis may act in a sequential or parallel
fashion and with different hierarchies within the spectrum of
disease and over time.57,60 Thus, it is likely that the observed
heterogeneity of NAFLD in the clinic, where some patients
develop isolated steatosis, whereas others develop NASH and
advanced chronic liver disease stems from a differential
contribution of pathogenetic pathways,60,61 as well as
from the activation or deactivation of redundant mechanisms. Also, it is likely that, besides genes, many factors
including diet, exercise, sex, age, and coexistence of obesity
and type 2 diabetes mellitus (T2DM) among others, critically
influence disease pathogenesis in a given patient.15 However, to dissect the precise contribution of each pathway to
NAFLD development and progression is difficult due to
limitations of available animal models62,63 or lack of robust
human data. Remarks on potentially relevant pathogenic
differences among NAFLD patients are provided below.
One important mechanism underlying NAFLD development is an upregulated DNL which has been reported to be
three-fold increased compared with control patients.58 Interestingly, DNL rates are influenced by age and sex, and this
pathway may be pathogenically more relevant in older and
male patients than others.64,65
A previously overlooked player in NAFLD pathogenesis
is the skeletal muscle. Several studies have underscored
that sarcopenia (i.e., decreased muscle mass) or adverse
muscle composition (i.e., low muscle volume and muscle
fat infiltration or myosteatosis) may influence both NAFLD
development and progression.66–68 Although important
methodological differences exist across published studies,
not all patients with NAFLD exhibit muscle alterations and it
is likely that this pathogenic factor be more relevant in
particular populations, such as nonobese or lean NAFLD, as
well as older patients in which sarcopenia is more frequent.69
Activation of the innate immune system has been implicated in NAFLD progression.70,71 After liver damage, a physiological immune response is key for resolution of the
injurious process, liver regeneration and restauration of a
healthy liver state, while an exuberant and persistent innate
immune response may lead to chronic liver inflammation. In
the context of NAFLD, an imbalance of the cross-talk of the
gut microbiome and the liver, known as the gut-liver axis, as
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well as occurrence of hepatocyte cell injury and death are
two key upstream triggers of a persistent pathogenic innate
immune response.57,71 Intestinal dysbiosis, defined as the
imbalance between resident microbial communities that
disturb the symbiotic relationship between gut microbes
and the host, and an increased intestinal permeability may
lead to a pathological translocation of microbial products to
the liver via the portal vein.19,20 In this setting, pathogenassociated molecular patterns (PAMPs) are recognized by
selective pattern recognition receptors, mainly toll-like
receptors, in the liver and cause a chronic innate immune
response. In addition, microbiota-derived metabolites such
as modified bile acids, choline, and ethanol alter hepatic
metabolism and trigger an inflammatory response.72 The
above-mentioned mechanisms may be at play in only a
fraction of individuals with NAFLD. In fact, clinical studies
have uncovered that only a subset of NAFLD patients present
with dysbiosis and/or a dysfunction in the gut barrier.19,72
These findings point that in addition to the genetic and
metabolic modifiers, assessment of microbiome profile,
and or intestinal permeability may allow for a more precise
classification and risk stratification of NAFLD patients to
define an adequate therapeutic intervention.72–74
In certain settings, the immune system may be activated
by other stimuli and contribute to NAFLD development and
progression. This may be the case for the association of
NAFLD with some immune-mediated inflammatory disorders, such as psoriasis75,76 and suppurative hidradenitis,77
which are associated to a low-grade systemic inflammatory
state. Of note, Psoriasis patients exhibit significantly increased serum levels of proinflammatory cytokines such as
tumor necrosis factor-a (TNF-a) interleukin (IL)-6 and reduced levels of the anti-inflammatory adipokine, adiponectin than matched controls.78 Also, psoriasis patients with
NAFLD have higher serum levels of C-reactive protein (CRP)
and have an increased prevalence of advanced liver fibrosis.79 In the same line, patients with suppurative hidradenitis
exhibit a high prevalence of NAFLD independently of classic
metabolic risk factors.80 However, it remains difficult to
dissect whether the underlying systemic inflammation observed in psoriasis and suppurative hidradenitis is a major
contributor to NAFLD development and progression given
the frequent coexistence of obesity in these patient populations.76,81 Interestingly, an imbalance in T-cell subtypes, like
Th17/Treg, leading to an IL-17–mediated inflammation may
be a common pathogenic link in this regard.76,82
Hepatocyte cell death as a result of lipotoxicity is another
key driver of the progression of NAFLD. Cellular release of
damage-associated molecular patterns (DAMPs) by damaged71 or stressed hepatocytes are recognized by the innate
immune system and may trigger chronic liver inflammation.60,83 Recently pyroptosis has been identified as a novel
form of programmed cell death in NAFLD and NASH. Pyroptosis is characterized by the activation and assembly of
multiprotein complexes, called inflammasomes. The most
studied one is the nucleotide-binding domain, leucine-rich–
repeat (NLR) family, pyrin domain containing 3 (NLRP3)
inflammasome. Once activated, the NLRP3 inflammasome
Nonalcoholic Fatty-Liver Disease Heterogeneity
cleaves procaspase-1 to its mature form caspase-1 that
subsequently cleaves IL-1β, IL-18, and gasdermin D. IL-1b
and IL-18 are highly proinflammatory cytokines that are
released into the extracellular space partly through gasdermin D formed transmembrane pores.60,84 NLRP3 was shown
to promote NASH and liver fibrosis in various mouse models.
Genetic constitutive overactivation of the NLRP3 inflammasome causes pyroptosis, inflammation, and fibrosis in the
liver, while global NLRP3 knock out mice show less inflammation and liver fibrosis in models of diet induced
NASH.85,86 Human data on the contribution of NLRP3 to
NAFLD development and progression is scarce; thus, future
translational studies to examine the human relevance of
these pathways to NAFLD heterogeneity are warranted.
As described above, PAMPs and DAMPs activate and
sustain a pattern recognition receptor-mediated innate immune response characteristic for NAFLD. Once activated, a
complex intercellular cross-talk between the different innate
immune cells, mainly macrophages, neutrophils, natural
killer cells, and T-cells, as well as hepatocytes and hepatic
stellate cells (HSCs), defines the progression to steatohepatitis and fibrosis. Macrophages are known to play a crucial
role in orchestrating immune response and the recruitment
of further immune cells.87 Kupffer’s cells, the resident liver
macrophages, are dominant over monocytes and monocytederived macrophages (MoMFs) in a healthy liver. However,
after liver injury, both monocytes and subsequently MoMFs
are rapidly recruited to the liver.88 The functional plasticity
of macrophages was historically subclassified in the two
polarizations, proinflammatory-type M1 and anti-inflammatory-type M2. This classification was shown to lack
accuracy of the macrophage activation program. Instead,
during liver injury, macrophages display wide gene expression profiles that led to the identification of different subpopulations of macrophages. The new technology of singlecell RNA sequencing (scRNA-seq) revealed an accumulation
of MoMF in murine livers in a diet-induced NASH model.89
The population of MoMF was further subclassified in the
three clusters MoMF I to III with each cluster characterized
by particular marker genes. Interestingly, similar subpopulations of bone marrow monocytes were also detected. These
findings indicate a NAFLD gene signature in myeloid leukocytes in both liver and bone marrow.88 Another study using
scRNA-seq identified further subpopulations of macrophages in NASH characterized by the expression of Trem2,
termed NASH-associated macrophages. In human samples,
Trem2 expression correlated with the severity of NAFLD
activity score and the extent of fibrosis. Therefore, markers
of NASH-associated macrophages may function as potential
novel key classifiers of NAFLD/NASH phenotypes.90 The
specific role of Kupffer’s cells during the progression of
NAFLD to NASH has continued to evolve. Recent evidence
has uncovered a reduction of mature Kupffer’s cell population during this process due to increased Kupffer’s cell death.
Monocyte-derived macrophages are then able to enter the
stage of differentiation into Kupffer’s cells. While Kupffer’s
cell acts mainly by facilitating hepatocyte triglyceride accumulation, MoMFs are more proinflammatory.91,92 A better
Arrese et al.
understanding and profiling of the immune cell niche during
NAFLD and NASH may provide a precision medicine approach to the current pathological scoring of inflammation
and allow for risk stratification and identify patients more
likely to have a progressive disease versus those who will
have a nonprogressive liver phenotype.
Liver fibrosis is the critical pathological feature that predicts liver-related outcome in NASH.93,94 Various cytokines
activate HSCs and their transformation toward collagen secreting myofibroblasts (MFB) which are key for triggering and
progression of liver fibrosis as in other hepatic diseases.95
However, considering the disturbed metabolic milieu present
in NAFLD, it is likely, although not certain that a myriad of
disease-specific mechanisms may be at play (recently
reviewed by Schwabe et al).96 Interestingly, a recent study
investigating the population of activated HSCs in liver fibrosis
revealed various clusters of MFBs, suggesting the existence of
heterogeneity within the fibrogenic cell population. While all
clusters showed a high expression of collagen, the ability to
secret chemokines to modulate inflammation was limited to
certain clusters.97 Another study uncovered specific liver
zonation of HSCs, classifying them into portal vein-associated
HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs).
Notably, CaHSCs were identified as the dominant pathogenic
collagen-producing cells. The lysophosphatidic acid receptor 1
(LPAR1) is upregulated in NASH patients and selectively
expressed in CaHSCs but not PaHSCs and NPCs. Pharmacological inhibition of LPAR1 in a murine NASH model has shown to
ameliorate liver fibrosis.98 These findings underline the complexity and diversity of HSCs in the context of the pathogenesis
of liver fibrosis in general and in NAFLD in particular.96 Further
studies are warranted to investigate whether certain subpopulations of HSCs favor remodeling after liver injury or triggering liver fibrosis. These studies may allow to identify
patients with fibrotic NASH that are at particular risk of
worsening of fibrosis versus those that are more likely to
show a robust wound healing response with potential for
spontaneous improvement of fibrosis. In summary, these
novel studies dissecting the distinct immune cell and HSC
niche may allow to provide two additional groups to the ones
described in the previous section including an “Inflammatory
fatty liver disease” and a “profibrotic fatty-liver disease.” This
classification may be central to identify patients with
NAFLD/NASH that are at particular risk for disease progression
or “rapid progressors,” a subphenotype that represents the
central target for therapeutic intervention.
Heterogeneity of Clinical Disease
In the clinical arena, NAFLD patients present with
diverse features. Sources of heterogeneity that can be
considered in the clinic at present time are multiple and
include age, sex, cardiometabolic comorbidities, ethnicity,
endocrine status, as well as alcohol and tobacco
consumption.23
Considerations on the influence of age and sex in NAFLD
clinical presentation and prognosis has been reviewed elsewhere,99–101 underscoring that while pediatric patients may
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have a different natural history and unique susceptibilities,
as well as more severe long-term consequences,101 older
patients exhibit an increased prevalence and severity of
NAFLD.102 With regard to sex, NAFLD is considered a sexually dimorphic disease with clear differences between male
and females with the latter being protected of severe fibrotic
disease during premenopausal stage.103
Being a multisystem disease, NAFLD is commonly associated with metabolic features, with a prevalence of 80% of
obesity, 72% dyslipidemia, and 44% of T2DM.1,104 Although
the combination of these features acts in an additive manner
to promote NAFLD progression, T2DM is the feature most
strongly and consistently associated with a more severe
disease and progression.105–107 Considering the ominous
effect of T2DM on liver disease progression and extrahepatic
complications (i.e., cardiovascular disease, cancer risk, and
chronic kidney disease), most guidelines recommend
patients that NAFLD need to be screened and treated for
metabolic comorbidities.3,108,109 In addition, while obesity is
also considered a risk factor for disease progression,107,110
lean patients with NAFLD have been increasingly recognized
as being at risk of progression to advanced liver disease, as
well as of developing metabolic comorbidities, and cardiovascular disease with impact in overall and liver-related
mortality.111,112 Lean patients account for 20 to 30% of
NAFLD patients.113 The same as obese NAFLD, its presence
is associated with presence of metabolic comorbidities,
increased visceral adiposity and unfavorable genetic polymorphisms.114 Of note, lean NAFLD is not a benign condition,
since it is independently associated with increased overall
and cardiovascular mortality.115
Ethnicity is an important aspect in NAFLD heterogeneity.
Several studies describe Hispanic Latino population to be at
the highest risk for development of NAFLD, whereas nonHispanic Black and non-Hispanic Asian population are at
lowest risk.116 In the United States, most of this risk is
explained specifically by Mexican American population
(50% NAFLD prevalence, compared with only 30% of prevalence in other Hispanic populations). The cause for these
ethnicity differences is probably explained by genetic and
sociocultural factors including diet, exercise, alcohol consumption, education, family income, and quality of life.
Interestingly, novel data suggest that advanced fibrosis
assessed by noninvasive serum test is more prevalent in
non-Hispanic black (28.5%) and non-Hispanic white populations (25.8%) compared with Mexican American (10.8%)
and Asian non-Hispanic population (2.65%).117 Indeed, further studies are needed to validate and explore these
findings.
Sex differences is another important factor to be considered in NAFLD heterogeneity. It has been repeatedly shown
that female patients at reproductive age have a significantly
lower NAFLD prevalence and severity118 which may be
explained by a myriad of sex differences in the pathobiology
of NAFLD (e.g., different storing of surplus calories, differential activation of hepatic DNL, and sex-specific in mitochondrial function and immune responses among others).99 After
menopause, sex differences are reduced in NAFLD and, in
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fact, women have a higher risk of advanced fibrosis than men,
especially after age 50 years,119 suggesting that estrogen
could be a protector factor for NAFLD.99,118 Again, sociocultural characteristics are likely to be involved in sex differences in NAFLD risk.
Obstructive sleep apnea (OSA) is a common complication of obesity, inducing repetitive cycles of hypoxia and
reoxygenation. Presence of OSA is associated with increased liver steatosis, inflammation, and fibrosis.120,121
Unfortunately, trials evaluating OSA therapy with continuous positive airway pressure (CPAP) have shown nonconclusive results in reducing liver fibrosis of NAFLD
patients.120,122
Alcohol consumption has classically been considered an
exclusion criterion for NAFLD. However, it has been increasingly recognized that a group of patients may present a dual
etiology (i.e., alcoholic and nonalcoholic) of their fatty-liver
disease.22 Recent population based longitudinal studies
demonstrate that even mild consumption of alcohol can
have a synergistic effect when combined with obesity, insulin resistance, and metabolic dysfunction to increase risk of
cirrhosis and hepatocellular carcinoma.22 In this context, the
renaming of NAFLD into MAFLD proposes a set of positive
criteria for diagnosis and does not consider the exclusion of
alcohol consumption as a prerequisite criterion for
diagnosis.23,24
Among other relevant factors that may modulate disease
development and severity are presence of skin inflammatory
disorders, such as psoriasis and suppurative hidradenitis,
hypothyroidism, polycystic ovarian syndrome, and periodontitis, all of which have been associated with increased
prevalence of NAFLD.123
Besides systemic comorbidities, patients with NAFLD
represent a wide spectrum of liver disease severity. In the
previous decade, emphasis was made in a dichotomic differentiation between those patients with isolated steatosis and
those with NASH. More recently, emphasis is made on
patients with NASH and liver fibrosis as the latter histological
feature has been shown to be a major prognostic determinant in NAFLD patients.94 Natural history studies, as well as
studies examining patients on the placebo arms of drug
clinical trials, using paired biopsies, showed that evolution
of NAFLD/NASH overtime is highly dynamic and that, although NASH has a higher risk of fibrosis progression and
development of cirrhosis than non-NASH patients, some
individuals will have spontaneous improvement while
some patients with isolated steatosis could also progress
to NASH and cirrhosis.124–131 Current data support the idea
that approximately 20 to 25% of patients with isolated
steatosis will progress to NASH, and from those, approximately 20 to 40% will progress to advanced fibrosis and
cirrhosis. Additionally, 5 to 7% of NASH-cirrhotic patients
will develop HCC.56 Thus, identifying the group of patients
who most likely will progress to advanced fibrosis is a
primary goal of NAFLD/NASH-related research. In this
regard, those at higher risk are patients with metabolic
abnormalities, such as T2DM, metabolic syndrome, and
obesity.2,132
Nonalcoholic Fatty-Liver Disease Heterogeneity
Most natural history studies of NAFLD have assessed liver-related morbidity and mortality in relation to a single
time-point evaluation of histology to identify risk
factors.124,131,133–135 In spite of their intrinsic selection bias,
paired-biopsy studies are informative regarding identification
of factors associated with disease progression.125,126,128–130
In these studies, the main risk factors for presence of NAFLD
are metabolic abnormalities such as T2DM, metabolic syndrome, and obesity.136–138 Regarding predictors of fibrosis
progression, studies have shown that the best predictors are
histological parameters including hepatocellular ballooning
degeneration and inflammation associated with age in
patients with NASH.130 As mentioned above, the presence of
fibrosis on initial biopsy or progression of fibrosis on serial
biopsies appears to be the most prominent histologic predictor
of overall mortality and liver-related outcomes.139–141 During
the last decade, efforts have been made to identify noninvasive
surrogates of liver biopsy that can be followed over time.
Serum aminotransferase levels are good predictors of improvement of NASH in clinical trials.10 Likewise, markers of
cell death including soluble cytokeratin-18 (CK-18) forms
(total and fragmented) have been shown to be good diagnostic
markers of NASH and predict response to therapeutic interventions but currently remain a research tool and are not
clinically available.142,143 Other circulating markers such as
proinflammatory markers, including circulating levels of cytokines (onlinTNF-a, IL-6, IL-8, and CRP), or other molecules, such
as adiponectin, have shown inconsistent results in part due to
the challenges with the sensitivity of the assays used to detect
changes in blood.143–146 To identify patients who already are
progressing and patients at early stages of fibrosis, noninvasive
scores have been developed. Among them, the NAFLD Fibrosis
Score, Fibrosis-4 (FIB-4), and Hepamet are nonexpensive,
noncommercial, and easily available.147–152 The enhanced
liver fibrosis (ELF) test is a commercial panel that combines
various constituents of collagen matrix deposition and turnover.153 Additionally, imaging assessment of steatosis and
fibrosis has been developed as a noninvasive method and
has focused mainly on transient elastography (TE) with controlled attenuation parameter (CAP) and magnetic resonance
imaging (MRI)-based technologies.153–158 Details on modern
concepts about noninvasive assessment of hepatic fibrosis can
be found in recent in-depth reviews on the topic.159,160
Implications for Clinical Practice and
Research
The differential contribution of genetic/epigenetic, environmental, and metabolic factors deters a significant interpatient variation regarding the major driver of disease
(►Fig. 2). Proper consideration of NAFLD heterogeneity is
relevant for both clinical practice and research. In the clinical
arena, a more precise patient phenotyping could allow to a
more granular grouping of patients to better stratify them
into those at higher risk of adverse outcomes. Also, a better
phenotyping would allow the implementation of tailored
treatment approaches in line with concepts of precision
medicine or individualized care.13,15,161 Since the degree
Arrese et al.
of liver fibrosis has been shown to be closely related to liverrelated mortality,93,94 assessment of this variable is now
considered crucial in patient stratification and should be
performed with the available noninvasive tools.159 However,
most of these tools are not able to distinguish those patients
that show a strong wound healing response and thus more
likely to have a resolutive phenotype with spontaneous
regression of fibrosis versus those that will have a strong
fibrogenic response and will more likely show progression of
fibrosis. The challenge for the future will be to develop deep
phenotyping approaches that also takes into consideration
these processes in a given patient, and incorporates associated comorbidities, race, genetic and epigenetic influences,
and environmental factors.10,162 Thus, clinical variables
known to be associated with rapid disease progression,
such as T2DM, arterial hypertension, severe obesity, and
worsening metabolic health (i.e., presence of disglycemia and
having one or more components of metabolic syndrome),
should be factor in association with molecular and cellular
profiling of liver phenotypes and if present, may confer the
label of “high-risk” NAFLD/NASH163,164 which may imply
higher risk of all-cause mortality, as well as of fibrosis,
cirrhosis, and HCC.165 In addition, consideration on differences linked to age, sex, and hormonal status may be proven
relevant given that these factors may influence NAFLD
natural history, as well as the performance of several diagnostic tools.15 With regard to genetic factors, routine genotyping of genetic variants known to influence NAFLD has not
yet been proven to be cost effective but likely will contribute
to individualized management when used integrated with
all pertinent clinical information14,166 (see section Role of
Genetic Determinants). Finally, detailed phenotyping of behavioral (i.e., physical activity and diet), alcohol use, and
socioeconomic factors, as well as of muscle mass and composition (i.e., using MRI techniques), could aid in better
defining NAFLD patient cohorts.
From a therapeutic standpoint, NAFLD heterogeneity
should be considered when structuring a therapeutic plan
for disease management. As previously outlined in several
clinical guidelines or position papers released and endorsed
by several scientific societies or expert panels,3,108,167 lifestyle modifications remain the cornerstone of NAFLD management and are indicated in all patients.7 Of note, sex
differences in the response to lifestyle changes have been
observed in several studies. Interestingly in the most-cited
study assessing lifestyle interventions in NAFLD,168 men
showed a greater histological improvement than women
after weight loss. In this study, male sex was one of the
factors predicting beneficial histological response following
a relatively modest weight loss (between 7 and 10%), while in
women, a more substantial weight loss (> 10%) was required
to achieve a significant histological improvement.168,169 Thus,
based in these observations, women may require additional
support to achieve the goals to be achieved after introduction
of lifestyle changes. In contrast, patients carrying the p.I148M
variant of the PNAPL3 gene exhibit a better response to
lifestyle modification and bariatric surgery than patients
not carrying the variant.170–172 This information may be of
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Nonalcoholic Fatty-Liver Disease Heterogeneity
Arrese et al.
Fig. 2 The differential contribution of genetic/epigenetic, environmental and metabolic factors deters a significant interpatient variation
regarding the major driver of disease. Circles depict three hypothetical patients that exhibit a different predominance of the three abovementioned factors. While patient 1 has environmental factors (e.g., poor diet and/or physical activity) as main driver of disease, patients 2 and 3
has a mixed predominance of genetic factors and metabolic derangement as determinants of their phenotypes. Better distinction of main
drivers in each patient may help to implementation of individualized or precision medicine approaches in nonalcoholic fatty-liver disease
(NAFLD) management (adapted from Eslam et al).23
aid when considering obesity surgery in NAFLD patients.
The same applies to the possibility of applying tailored
cardiovascular risk management in patient subgroups as
patients with the p.I148M variant of PNAPL3, as well as
those with the TM6SF2 E167K gene variant, exhibit a lower
risk of cardiovascular disease compared with noncarriers.17
Finally, tailored HCC screening may be envisioned for some
patients using genetic information as use of PRS, as shown by
Bianco et al in a recent paper in which PNPLA3–TM6SF2–
GCKR–MBOAT7 were combined, may improve HCC risk stratification in NAFLD.173
With regard to interpatient variability to therapeutic
agents, available information is scarce. It is likely that
better and more detailed assessment of certain specific
pathways could be of help in selecting therapeutic agents.
This could be made with the use of metabolomics174,175
or other system biology tools. Thus, if DNL is shown to be
overactive,65 specific use of agents targeting this pathway, such as acetyl-CoA carboxylase (ACC) inhibitors,
could be indicated. 174,175 Also, if appropriate tools to
assess intestinal dysbiosis19 are available, gut microbiome-targeted therapies could be also used only in those
with altered microbial composition. Finally, drugs that
improve dyslipidemia or decrease cardiovascular disease
may be more effective for those patients with a more
profound derangement of metabolic health such as
patients with concurrent T2DM and NAFLD. In the latter
population, one should also consider the potential benefits of antidiabetic drugs, such as pioglitazone and sodium-glucose cotransporter-2 (SGLT2) inhibitors, 176,177 or
the preferential use of the glucagon-like peptide 1 (GLP1) receptor agonists, such as liraglutide or semaglutide,
with potentially beneficial impact on liver histology and
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cardiovascular outcomes in this particular patient
population.178–181
Finally, consideration of psychosocial factors in the
evaluation of any given patient is also relevant for NAFLD
management and should be part of a comprehensive
phenotyping approach. These factors might influence motivation to both adopt and sustain lifestyle changes. In this
regard, necessary support to patients’ efforts through
health coaching and/or motivational interventions is key
to successful adherence to any program aimed at naturalizing physical activity in daily life.
The impact of NAFLD heterogeneity in therapeutic clinical
trials has been also emphasized recently.12,23 Precise stratification of patient cohorts, included in a given study, is a key
when assessing therapeutic response in a complex disease
such as NAFLD. The suboptimal responses observed in several clinical trials of different drugs may be in part due to the
fact that disease heterogeneity was not taken into consideration.11,182 In their recent review on the matter Ampuero
and Romero-Gomez concluded that clinical trial reporting
for NAFLD has been suboptimal with no detailed mention of
dysmetabolic comorbidities or proper recording of daily
dietary and exercise habits. These authors suggest that future
trials should carefully consider the presence of comorbidities
expected to impact on the treatment response (trying to
keep similar proportion of patients in the different arms of a
given trial), as well as proper recording of alcohol intake and
exercise both during the trial and not only at entry, which
will improve capturing of the effects of these variables in
treatment responses. Also, adoption of innovative trials
designs to study novel NAFLD treatments, such as the
adaptative, umbrella, or basket strategies, could be useful
to improve trial efficiency.12,23
Nonalcoholic Fatty-Liver Disease Heterogeneity
Conclusion and Outlook
NAFLD groups a heterogeneous patient population. The heterogeneity of NAFLD is reflected by the fact that some patients
develop only steatosis, whereas others develop hepatocyte
injury, steatohepatitis, and progressive liver fibrosis and,
ultimately, cirrhosis. The interaction of different factors (e.g.,
sex, presence of one or more genetic variants, coexistence of
different comorbidities such as obesity and T2DM, diverse
microbiota composition, and various degrees of alcohol consumption) critically influence different disease pathways to
determine disease subphenotypes with distinct natural history and prognosis and potentially different response to treatment. The recent proposal of NAFLD, renaming into MAFLD
that sets positive criteria for diagnosis,24 has been generally
welcomed by the scientific community, although debate is
ongoing.25 The use of MAFLD may be a step forward in
addressing disease heterogeneity by grouping a more homogenous patient population with more severe metabolic and liver
disease (i.e., presence of significant fibrosis).183–185 In addition, differentiation of three subgroups (i.e., obese MAFLD, lean
MAFLD, and diabetic MAFLD) that may have different prognosis and could be appraised differently, may be also relevant.
However, further discussion is necessary and consensus needs
to be reached on this matter.26,184
Taking into account, disease heterogeneity is relevant both
clinically, to apply more individualized approaches in NAFLD
diagnosis and treatment, and to drug development due to the
need of including more homogeneous patient populations in
clinical trials. In addition to clinical factors, availability of
modern tools to define activation of DNL with metabolomics,
development of accurate biomarkers of inflammatory, cell
death or liver fibrosis pathways activation, better detection
of presence of dysbiosis assisted by the use of microbiota
signatures, or evaluation of genetic risk of cirrhosis or HCC with
PRS would surely impact our ability to discriminate patients’
subgroups and stratify them according to their risks of poor
outcomes. This also would help to select the most appropriate
treatment for a given patient following the precision medicine
principles.13,15 Thus, in the future, it can be envisaged that
precise phenotyping of pathogenic pathways would lead to
select a specific drug10 or that a genetic treatment27,186 could
be chosen based on global ethnic-specific genetic information.
Also, it is likely that principles of precision nutrition187 and
precision exercise188 would apply to patient management to
increase the therapeutic efficacy of lifestyles changes in NAFLD
management.
Indeed, further research is needed to improve our understanding of NAFLD heterogeneity. Building of large biobanks
from well-characterized patient populations integrating
clinical, genetic, biomarkers, and OMICs information is key
to this end. The use of artificial intelligence approaches offers
the opportunity to combine and decode this information to
develop useful algorithms for patient stratification and management. A better definition of the dominant drivers of
disease in patient subgroups would predictably impact on
the development of more precision-targeted therapies for
NAFLD.
Arrese et al.
Main Concepts and Learning Points
• Nonalcoholic fatty-liver disease (NAFLD) is an umbrella
term that groups a heterogeneous patient population.
• NAFLD heterogeneity is influenced by multiple variables
including age, sex, presence of one or more genetic
variants, coexistence of different comorbidities, diverse
microbiota composition, and various degrees of alcohol
consumption among other factors.
• The heterogeneous nature of NAFLD results from a different hierarchy or trajectory of pathophysiological mechanisms that determine several disease subphenotypes with
distinct natural history and prognosis and, eventually,
different response to therapy.
• Proper consideration of NAFLD heterogeneity is relevant
for both clinical practice to implement individualized
medicine approaches, as well as for clinical research, to
improve clinical trials efficiency.
• The recent renaming of NAFLD into MAFLD (i.e., metabolic
[dysfunction]-associated fatty-liver disease) is a step forward in taking into account NAFLD heterogeneity by
setting specific diagnostic criteria and grouping patients
with disturbed metabolic health. However, more research
is needed to refine discrimination of subtypes of the
disease.
Abbreviations
ACC, acetyl-CoA carboxylase; APOB, apolipoprotein B;
CaHSCs, central vein-associated HSCs; CRP, C-reactive
protein; DAMPs, damage-associated molecular patterns;
DNL, de novo lipogenesis; GCKR, glucokinase regulator;
GWAS, genome-wide association studies; HCC, hepatocellular carcinoma; HSC, hepatic stellate cells; IL, interleukin; MBOAT7, membrane bound O-acyl transferase;
MoMFs, monocyte-derived macrophages; NAFLD, nonalcoholic fatty-liver disease; NAFL, nonalcoholic fatty-liver;
NASH, nonalcoholic steatohepatitis; NLRP3, NLR family
pyrin domain containing 3; PaHSCs, portal vein-associated HSCs; PAMPs, pathogen-associated molecular patterns; PNPLA3, patatin-like phospholipase domaincontaining 3; PRS, polygenic risk scores; scRNA-seq, single
cell RNA sequencing; TDM2, type 2 diabetes mellitus;
TM6SF2: transmembrane 6 superfamily member 2; TNFa, tumor necrosis factor-a.
Funding Sources
This work was funded, in part, by grants from the Fondo
Nacional De Ciencia y Tecnología de Chile (FONDECYT no.:
1191145 to M.A., no.:1200227 to J.P.A., and no.:1191183
to F.B.), the Comisión Nacional de Investigación, Ciencia y
Tecnología (CONICYT, AFB170005, CARE, Chile, UC). M.A.
is part of the European-Latin American ESCALON consortium funded by the European Union’s Horizon 2020
Research and Innovation Program under grant agreement
no. 825510. A.E.F. was supported was by NIH grants R01
DK113592 and R01 AA024206. L.V. was supported by
project grants from MyFirst Grant AIRC n.16888, Ricerca
Finalizzata Ministero della Salute RF-2016–02364358
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430
Nonalcoholic Fatty-Liver Disease Heterogeneity
Arrese et al.
(“Impact of whole exome sequencing on the clinical
management of patients with advanced nonalcoholic
fatty-liver and cryptogenic-liver disease”), Ricerca corrente Fondazione IRCCS Ca’ Granda Ospedale Maggiore
Policlinico, the European Union (EU) Program Horizon
2020 (under grant agreement No. 777377) for the project
LITMUS- “Liver Investigation: Testing Marker Utility in
Steatohepatitis,” Program “Photonics” under grant agreement “101016726” for the project “REVEAL: Neuronal
microscopy for cell behavioural examination and manipulation,” Gilead_IN-IT-989–5790 “Developing a model of
care for risk stratification and management of diabetic
patients with non-alcoholic fatty-liver disease (NAFLD),”
Fondazione IRCCS Ca’ Granda “Liver BIBLE” PR-0391,
Fondazione IRCCS Ca’ Granda core COVID-19 Biobank
(RC100017A).
11 Ratziu V, Friedman SL. Why do so many NASH trials fail?
Gastroenterology 2020:S0016-5085(20)30680-6
12 Ampuero J, Romero-Gomez M. Stratification of patients in NASH
13
14
15
16
17
18
Conflict of Interest
The authors declare that they have no conflict of interest
relevant to the present study. L.V. has received speaking
fees from MSD, Gilead, AlfaSigma, and AbbVie, served as a
consultant for Gilead, Pfizer, AstraZeneca, Novo Nordisk,
Intercept, Diatech Pharmacogenetics and Ionis Pharmaceuticals, and received research grants from Gilead.
19
20
21
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Seminars in Liver Disease
Vol. 41
No. 4/2021
© 2021. The Author(s).
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